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New studies link cell cycle proteins to immunosurveillance of premalignant cells
Curator: Stephen J. Williams, Ph.D.
The following is from a Perspectives article in the journal Science by Virinder Reen and Jesus Gil called “Clearing Stressed Cells: Cell cycle arrest produces a p21-dependent secretome that initaites immunosurveillance of premalignant cells”. This is a synopsis of the Sturmlechener et al. research article in the same issue (2).
Complex organisms repair stress-induced damage to limit the replication of faulty cells that could drive cancer. When repair is not possible, tissue homeostasis is maintained by the activation of stress response programs such as apoptosis, which eliminates the cells, or senescence, which arrests them (1). Cellular senescence causes the arrest of damaged cells through the induction of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 (2). Senescent cells also produce a bioactive secretome (the senescence-associated secretory phenotype, SASP) that places cells under immunosurveillance, which is key to avoiding the detrimental inflammatory effects caused by lingering senescent cells on surrounding tissues. On page 577 of this issue, Sturmlechner et al. (3) report that induction of p21 not only contributes to the arrest of senescent cells, but is also an early signal that primes stressed cells for immunosurveillance.Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Sturmlechner et al. found that activation of p21 following stress rapidly halted cell cycle progression and triggered an internal biological timer (of ∼4 days in hepatocytes), allowing time to repair and resolve damage (see the figure). In parallel, C-X-C motif chemokine 14 (CXCL14), a component of the PASP, attracted macrophages to surround and closely surveil these damaged cells. Stressed cells that recovered and normalized p21 expression suspended PASP production and circumvented immunosurveillance. However, if the p21-induced stress was unmanageable, the repair timer expired, and the immune cells transitioned from surveillance to clearance mode. Adjacent macrophages mounted a cytotoxic T lymphocyte response that destroyed damaged cells. Notably, the overexpression of p21 alone was sufficient to orchestrate immune killing of stressed cells, without the need of a senescence phenotype. Overexpression of other CDKIs, such as p16 and p27, did not trigger immunosurveillance, likely because they do not induce CXCL14 expression.In the context of cancer, senescent cell clearance was first observed following reactivation of the tumor suppressor p53 in liver cancer cells. Restoring p53 signaling induced senescence and triggered the elimination of senescent cells by the innate immune system, prompting tumor regression (5). Subsequent work has revealed that the SASP alerts the immune system to target preneoplastic senescent cells. Hepatocytes expressing the oncogenic mutant NRASG12V (Gly12→Val) become senescent and secrete chemokines and cytokines that trigger CD4+ T cell–mediated clearance (6). Despite the relevance for tumor suppression, relatively little is known about how immunosurveillance of oncogene-induced senescent cells is initiated and controlled.
Source of image: Reen, V. and Gil, J. Clearing Stressed Cells. Science Perspectives 2021;Vol 374(6567) p 534-535.
References
2. Sturmlechner I, Zhang C, Sine CC, van Deursen EJ, Jeganathan KB, Hamada N, Grasic J, Friedman D, Stutchman JT, Can I, Hamada M, Lim DY, Lee JH, Ordog T, Laberge RM, Shapiro V, Baker DJ, Li H, van Deursen JM. p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science. 2021 Oct 29;374(6567):eabb3420. doi: 10.1126/science.abb3420. Epub 2021 Oct 29. PMID: 34709885.
More Articles on Cancer, Senescence and the Immune System in this Open Access Online Scientific Journal Include
Gender affects the prevalence of the cancer type, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Gender of a person can affect the kinds of cancer-causing mutations they develop, according to a genomic analysis spanning nearly 2,000 tumours and 28 types of cancer. The results show striking differences in the cancer-causing mutations found in people who are biologically male versus those who are biologically female — not only in the number of mutations lurking in their tumours, but also in the kinds of mutations found there.
Liver tumours from women were more likely to carry mutations caused by a faulty system of DNA mending called mismatch repair, for instance. And men with any type of cancer were more likely to exhibit DNA changes thought to be linked to a process that the body uses to repair DNA with two broken strands. These biases could point researchers to key biological differences in how tumours develop and evolve across sexes.
The data add to a growing realization that sex is important in cancer, and not only because of lifestyle differences. Lung and liver cancer, for example, are more common in men than in women — even after researchers control for disparities in smoking or alcohol consumption. The source of that bias, however, has remained unclear.
In 2014, the US National Institutes of Health began encouraging researchers to consider sex differences in preclinical research by, for example, including female animals and cell lines from women in their studies. And some studies have since found sex-linked biases in the frequency of mutations in protein-coding genes in certain cancer types, including some brain cancers and advanced melanoma.
But the present study is the most comprehensive study of sex differences in tumour genomes so far. It looks at mutations not only in genes that code for proteins, but also in the vast expanses of DNA that have other functions, such as controlling when genes are turned on or off. The study also compares male and female genomes across many different cancers, which can allow researchers to pick up on additional patterns of DNA mutations, in part by increasing the sample sizes.
Researchers analysed full genome sequences gathered by the International Cancer Genome Consortium. They looked at differences in the frequency of 174 mutations known to drive cancer, and found that some of these mutations occurred more frequently in men than in women, and vice versa. When they looked more broadly at the loss or duplication of DNA segments in the genome, they found 4,285 sex-biased genes spread across 15 chromosomes.
There were also differences found when some mutations seemed to arise during tumour development, suggesting that some cancers follow different evolutionary paths in men and women. Researchers also looked at particular patterns of DNA changes. Such patterns can, in some cases, reflect the source of the mutation. Tobacco smoke, for example, leaves behind a particular signature in the DNA.
Taken together, the results highlight the importance of accounting for sex, not only in clinical trials but also in preclinical studies. This could eventually allow researchers to pin down the sources of many of the differences found in this study. Liver cancer is roughly three times as common in men as in women in some populations, and its incidence is increasing in some countries. A better understanding of its aetiology may turn out to be really important for prevention strategies and treatments.
Immuno-editing can be a constant defense in the cancer landscape, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
There are many considerations in the cancer immunoediting landscape of defense and regulation in the cancer hallmark biology. The cancer hallmark biology in concert with key controls of the HLA compatibility affinity mechanisms are pivotal in architecting a unique patient-centric therapeutic application. Selection of random immune products including neoantigens, antigens, antibodies and other vital immune elements creates a high level of uncertainty and risk of undesirable immune reactions. Immunoediting is a constant process. The human innate and adaptive forces can either trigger favorable or unfavorable immunoediting features. Cancer is a multi-disease entity. There are multi-factorial initiators in a certain disease process. Namely, environmental exposures, viral and / or microbiome exposure disequilibrium, direct harm to DNA, poor immune adaptability, inherent risk and an individual’s own vibration rhythm in life.
When a human single cell is crippled (Deranged DNA) with mixed up molecular behavior that is the initiator of the problem. A once normal cell now transitioned into full threatening molecular time bomb. In the modeling and creation of a tumor it all begins with the singular molecular crisis and crippling of a normal human cell. At this point it is either chop suey (mixed bit responses) or a productive defensive and regulation response and posture of the immune system. Mixed bits of normal DNA, cancer-laden DNA, circulating tumor DNA, circulating normal cells, circulating tumor cells, circulating immune defense cells, circulating immune inflammatory cells forming a moiety of normal and a moiety of mess. The challenge is to scavenge the mess and amplify the normal.
Immunoediting is a primary push-button feature that is definitely required to be hit when it comes to initiating immune defenses against cancer and an adaptation in favor of regression. As mentioned before that the tumor microenvironment is a “mixed bit” moiety, which includes elements of the immune system that can defend against circulating cancer cells and tumor growth. Personalized (Precision-Based) cancer vaccines must become the primary form of treatment in this case. Current treatment regimens in conventional therapy destroy immune defenses and regulation and create more serious complications observed in tumor progression, metastasis and survival. Commonly resistance to chemotherapeutic agents is observed. These personalized treatments will be developed in concert with cancer hallmark analytics and immunocentrics affinity and selection mapping. This mapping will demonstrate molecular pathway interface and HLA compatibility and adaptation with patientcentricity.
Immunotherapy may help in glioblastoma survival, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Glioblastoma is the most common primary malignant brain tumor in adults and is associated with poor survival. But, in a glimmer of hope, a recent study found that a drug designed to unleash the immune system helped some patients live longer. Glioblastoma powerfully suppresses the immune system, both at the site of the cancer and throughout the body, which has made it difficult to find effective treatments. Such tumors are complex and differ widely in their behavior and characteristics.
A small randomized, multi-institution clinical trial was conducted and led by researchers at the University of California at Los Angeles involved patients who had a recurrence of glioblastoma, the most common central nervous system cancer. The aim was to evaluate immune responses and survival following neoadjuvant and/or adjuvant therapy with pembrolizumab (checkpoint inhibitor) in 35 patients with recurrent, surgically resectable glioblastoma. Patients who were randomized to receive neoadjuvant pembrolizumab, with continued adjuvant therapy following surgery, had significantly extended overall survival compared to patients that were randomized to receive adjuvant, post-surgical programmed cell death protein 1 (PD-1) blockade alone.
Neoadjuvant PD-1 blockade was associated with upregulation of T cell– and interferon-γ-related gene expression, but downregulation of cell-cycle-related gene expression within the tumor, which was not seen in patients that received adjuvant therapy alone. Focal induction of programmed death-ligand 1 in the tumor microenvironment, enhanced clonal expansion of T cells, decreased PD-1 expression on peripheral blood T cells and a decreasing monocytic population was observed more frequently in the neoadjuvant group than in patients treated only in the adjuvant setting. These findings suggest that the neoadjuvant administration of PD-1 blockade enhanced both the local and systemic antitumor immune response and may represent a more efficacious approach to the treatment of this uniformly lethal brain tumor.
Immunotherapy has not proved to be effective against glioblastoma. This small clinical trial explored the effect of PD-1 blockade on recurrent glioblastoma in relation to the timing of administration. A total of 35 patients undergoing resection of recurrent disease were randomized to either neoadjuvant or adjuvant pembrolizumab, and surgical specimens were compared between the two groups. Interestingly, the tumoral gene expression signature varied between the two groups, such that those who received neoadjuvant pembrolizumab displayed an INF-γ gene signature suggestive of T-cell activation as well as suppression of cell-cycle signaling, possibly consistent with growth arrest. Although the study was not powered for efficacy, the group found an increase in overall survival in patients receiving neoadjuvant pembrolizumab compared with adjuvant pembrolizumab of 13.7 months versus 7.5 months, respectively.
In this small pilot study, neoadjuvant PD-1 blockade followed by surgical resection was associated with intratumoral T-cell activation and inhibition of tumor growth as well as longer survival. How the drug works in glioblastoma has not been totally established. The researchers speculated that giving the drug before surgery prompted T-cells within the tumor, which had been impaired, to attack the cancer and extend lives. The drug didn’t spur such anti-cancer activity after the surgery because those T-cells were removed along with the tumor. The results are very important and very promising but would need to be validated in much larger trials.
TWEETS by @pharma_BI and @AVIVA1950 at #IESYMPOSIUM – @kochinstitute 2019 #Immune #Engineering #Symposium, 1/28/2019 – 1/29/2019
Real Time Press Coverage: Aviva Lev-Ari, PhD, RN
2.1.3.4 TWEETS by @pharma_BI and @AVIVA1950 at #IESYMPOSIUM – @kochinstitute 2019 #Immune #Engineering #Symposium, 1/28/2019 – 1/29/2019, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
eProceedings for Day 1 and Day 2
LIVE Day One – Koch Institute 2019 Immune Engineering Symposium, January 28, 2019, Kresge Auditorium, MIT
#IESYMPOSIUM@pharma_BI@AVIVA1950 Aviv Regev @kochinstitute Melanoma: malignant cells with resistance in cold niches in situ cells express the resistance program pre-treatment: resistance UP – cold Predict checkpoint immunotherapy outcomes CDK4/6 abemaciclib in cell lines
#IESYMPOSIUM@pharma_BI@AVIVA1950 Diane Mathis @HMS Age-dependent Treg and mSC changes – Linear with increase in age Sex-dependent Treg and mSC changes – Female Treg loss in cases of Obesity leading to fibrosis Treg keep IL-33-Producing mSCs under rein Lean tissue/Obese tissue
#IESYMPOSIUM@pharma_BI@AVIVA1950 Martin LaFleur @HMS Loss of Ptpn2 enhances CD8+ T cell responses to LCMV and Tumors PTpn2 deletion in the immune system enhanced tumor immunity CHIME enables in vivo screening
#IESYMPOSIUM@pharma_BI@AVIVA1950 Alex Shalek @MIT@kochinstitute Identifying and rationally modulating cellular drivers of enhanced immunity T Cells, Clusters Expression of Peak and Memory Immunotherapy- Identifying Dendritic cells enhanced in HIV-1 Elite Controllers
#IESYMPOSIUM@pharma_BI@AVIVA1950 Glenn Dranoff @Novartis Adenosine level in blood or tissue very difficult to measure in blood even more than in tissue – NIR178 + PDR 001 Monotherapy (NIR178) combine with PD receptor blockage (PDR) show benefit A alone vs A+B in Clinical trial
#IESYMPOSIUM@pharma_BI@AVIVA1950 Glenn Dranoff @Novartis PD-L1 blockade elicits responses in some patients: soft part sarcoma LAG-3 combined with PD-1 – human peripheral blood tumor TIM-3 key regulator of T cell and Myeloid cell function: correlates in the TCGA DB myeloid
#IESYMPOSIUM@pharma_BI@AVIVA1950 Yvonne Chen @UCLA Activation of t Cell use CAR t Engineer CAR-T to respond to soluble form of antigens: CD19 CAR Responds to soluble CD19 GFP MCAR responds to Dimeric GFP “Tumor microenvironment is a scary place”
#IESYMPOSIUM@pharma_BI@AVIVA1950 Yvonne Chen @UCLA “Engineering smarter and stronger T cells for cancer immunotherapy” OR-Gate cause no relapse – Probing limits of modularity in CAR Design Bispecific CARs are superior to DualCAR: One vs DualCAR (some remained single CAR)
Ending the 1st session is Cathy Wu of @DanaFarber detailing some amazing work on vaccination strategies for melanoma and glioblastoma patients. They use long peptides engineered from tumor sequencing data. #iesymposium
Some fancy imaging: Duggan gives a nice demo of how dSTORM imaging works using a micropatterend image of Kennedy Institute for Rheumatology! yay! #iesymposium
Lots of interesting talks in the second session of the #iesymposium – effects of lymphoangiogenesis on anti-tumor immune responses, nanoparticle based strategies to improve bNAbs titers/affinity for HIV therapy, and IAPi cancer immunotherapy
Looking forward to another day of the #iesymposium. One more highlight from yesterday – @nm0min from our own lab showcased her work developing cytokine fusions that bind to collagen, boosting efficacy while drastically reducing toxicities
#IESYMPOSIUM@pharma_BI@AVIVA1950 Preeti Sharma, U Illinois T cell receptor and CAR-T engineering TCR engineering for Targeting glycosylated cancer antigens Nornal glycosylation vs Aberrant Engineering 237-CARs libraries with conjugated (Tn-OTS8) against Tn-antigend In vitro
#IESYMPOSIUM@pharma_BI@AVIVA1950 Bryan Bryson @MIT Loss of polarization potential: scRNAseq reveals transcriptional differences Thioredoxin facilitates immune response to Mtb is a marker of an inflammatory macrophage state functional spectrum of human microphages
#IESYMPOSIUM@pharma_BI@AVIVA1950 Bryan Bryson @MIT macrophage axis in Mycobacterium tuberculosis Building “libraries” – surface marker analysis of Microphages Polarized macrophages are functionally different quant and qual differences History of GM-CSF suppresses IL-10
#IESYMPOSIUM@pharma_BI@AVIVA1950 Jamie Spangler John Hopkins University “Reprogramming anti-cancer immunity RESPONSE through molecular engineering” De novo IL-2 potetiator in therapeutic superior to the natural cytokine by molecular engineering mimicking other cytokines
#IESYMPOSIUM@pharma_BI@AVIVA1950 Michael Dustin @UniofOxford ESCRT pathway associated with synaptic ectosomes Locatization, Microscopy Cytotoxic T cell granules CTLs release extracellular vescicles similar to T Helper with perforin and granzyme – CTL vesicles kill targets
#IESYMPOSIUM@pharma_BI@AVIVA1950 Michael Dustin @Oxford Delivery of T cell Effector function through extracellular vesicles Synaptic ectosome biogenisis Model: T cells: DOpamine cascade in germinal cell delivered to synaptic cleft – Effector CD40 – Transfer is cooperative
#IESYMPOSIUM@pharma_BI@AVIVA1950 Michael Dustin @Oxford Delivery of T cell Effector function through extracellular vesicles Laterally mobile ligands track receptor interaction ICAM-1 Signaling of synapse – Sustain signaling by transient in microclusters TCR related Invadipodia
#IESYMPOSIUM@pharma_BI@AVIVA1950 Mikael Pittet @MGH Myeloid Cells in Cancer Indirect mechanism AFTER a-PD-1 Treatment IFN-gamma Sensing Fosters IL-12 & therapeutic Responses aPD-1-Mediated Activation of Tumor Immunity – Direct activation and the ‘Licensing’ Model
#IESYMPOSIUM@pharma_BI@AVIVA1950 Stefani Spranger @MIT KI Response to checkpoint blockade Non-T cell-inflamed – is LACK OF T CELL INFILTRATION Tumor CD103 dendritic cells – Tumor-residing Batf3-drivenCD103 Tumor-intrinsic Beta-catenin mediates lack of T cell infiltration
#IESYMPOSIUM@pharma_BI@AVIVA1950 Max Krummel @UCSF Gene expression association between two genes: #NK and #cDC1 numbers are tightly linked to response to checkpoint blockage IMMUNE “ACCOMODATION” ARCHYTYPES: MYELOID TUNING OF ARCHITYPES Myeloid function and composition
#IESYMPOSIUM@pharma_BI@AVIVA1950 Noor Momin, MIT Lumican-cytokines improve control of distant lesions – Lumican-fusion potentiates systemic anti-tumor immunity
#IESYMPOSIUM@pharma_BI@AVIVA1950 Noor Momin, MIT Lumican fusion to IL-2 improves treatment efficacy reduce toxicity – Anti-TAA mAb – TA99 vs IL-2 Best efficacy and least toxicity in Lumican-MSA-IL-2 vs MSA-IL2 Lumican synergy with CAR-T
excited to attend the @kochinstitute@MIT immune engineering symposium #iesymposium this week! find me there to chat about @CellCellPress and whether your paper could be a good fit for us!
April Pawluk added,
Koch Institute at MITVerified account@kochinstitute
Join leading immunology researchers at our Immune Engineering Symposium on Jan. 28 & 29. Register now: http://bit.ly/2AOUWH6#iesymposium
Bob Schreiber and Tyler Jacks kicked off the #iesymposium with 2 great talks on the role of Class I and Class II neo-Ag in tumor immunogenicity and how the tumor microenvironment alters T cell responsiveness to tumors in vivo
Scott Wilson from @UChicago gave a fantastic talk on glycopolymer conjugation to antigens to improve trafficking to HAPCs and enhanced tolerization in autoimmunity models. Excited to learn more about his work at his @MITChemE faculty talk! #iesymposium
Spending the (literal) first day of my fellowship at the @kochinstitute#iesymposium! @DanaFarber Cathy Wu talking about the use of neoantigen targeting cancer vaccines for the treatment of ‘cold’ glioblastoma tumors in pts
Tyler Jacks talk was outstanding, Needs be delivered A@TED TALKs, needs become contents in the curriculum of Cell Biology graduate seminar as an Online class. BRAVO @pharma_BI@AVIVA1950
Aviva Lev-Ari added,
Anne E Deconinck@AEDeconinck
My boss, @kochinstitute director Tyler Jacks, presenting beautiful, unpublished work at our 3rd #iesymposium.
#IESYMPOSIUM@pharma_BI@AVIVA1950 Stephanie Dougan (Dana-Farber Cancer Institute) Dept. Virology IAPi outperforms checkpoint blockade in T cell cold tumors reduction of tumor burden gencitabine cross-presenting DCs and CD8 T cells – T cell low 6694c2
#IESYMPOSIUM@pharma_BI@AVIVA1950 Melody Swartz (University of Chicago) Lymphangiogenesis attractive to Native T cells, in VEGF-C tumors T cell homing inhibitors vs block T cell egress inhibitors – Immunotherapy induces T cell killing
#IESYMPOSIUM@pharma_BI@AVIVA1950 Cathy Wu @MGH breakthrough for Brain Tumor #vaccine based neoantigen-specific T cell at intracranial site Single cells brain tissue vs single cells from neoantigen specific T cells – intratumoral neoantigen-specific T cells: mutARGAP35-spacific
#IESYMPOSIUM@pharma_BI@AVIVA1950 Cathy Wu (Massachusetts General Hospital) – CoFounder of NEON Enduring complete radiographic responses after #Neovax + alpha-PD-1 treatment (anti-PD-1) NeoVax vs IVAC Mutanome for melanoma and Glioblastoma clinical trials
#IESYMPOSIUM@pharma_BI@AVIVA1950@TylerJacks@MIT Interrogating markers of T cell dysfunction – chance biology of cells by CRISPR – EGR2 at 2 weeks dysfuntioning is reduced presence of EDR2 mutant class plays role in cell metabolism cell becomes functional regulator CD8 T cell
MISSION The mission of the Koch Institute (KI) is to apply the tools of science and technology to improve the way cancer is detected, monitored, treated and prevented.
APPROACH We bring together scientists and engineers – in collaboration with clinicians and industry partners – to solve the most intractable problems in cancer. Leveraging MIT’s strengths in technology, the life sciences and interdisciplinary research, the KI is pursuing scientific excellence while also directly promoting innovative ways to diagnose, monitor, and treat cancer through advanced technology.
HISTORY The Koch Institute facility was made possible through a $100 million gift from MIT alumnus David H. Koch. Our new building opened in March 2011, coinciding with MIT’s 150th anniversary. Our community has grown out of the MIT Center for Cancer Research (CCR), which was founded in 1974 by Nobel Laureate and MIT Professor Salvador Luria, and is one of seven National Cancer Institute-designated basic (non-clinical) research centers in the U.S.
Biological, chemical, and materials engineers are engaged at the forefront of immunology research. At their disposal is an analytical toolkit honed to solve problems in the petrochemical and materials industries, which share the presence of complex reaction networks, and convective and diffusive molecular transport. Powerful synthetic capabilities have also been crafted: binding proteins can be engineered with effectively arbitrary specificity and affinity, and multifunctional nanoparticles and gels have been designed to interact in highly specific fashions with cells and tissues. Fearless pursuit of knowledge and solutions across disciplinary boundaries characterizes this nascent discipline of immune engineering, synergizing with immunologists and clinicians to put immunotherapy into practice.
The 2019 symposium will include two poster sessions and four abstract-selected talks. Abstracts should be uploaded on the registration page. Abstract submission deadline is November 15, 2018. Registration closes December 14.
Featuring on Day 2, 1/29, 2019:
Session IV
Moderator: Michael Birnbaum, Koch Institute, MIT
Jamie Spangler (John Hopkins University)
“Reprogramming anti-cancer immunity through molecular engineering”
Reprogramming anti-cancer immunity response through molecular engineering”
Cytokines induce receptor dimerization
Clinical Use of cytokines: Pleiotropy, expression and stability isssues
poor pharmacological properties
cytokine therapy: New de novo protein using computational methods
IL-2 signals through a dimeric nad a trimeric receptor complex
IL-2 pleiotropy hinders its therapeutic efficacy
IL-2 activate immunosuppression
potentiation of cytokine activity by anti-IL-2 antibody selectivity
Cytokine binding – Antibodies compete with IL-2 receptor subunits
IL-2Ralpha, IL-2 Rbeta: S4B6 mimickry of alpha allosterically enhances beta
Affinity – molecular eng De Novo design of a hyper-stable, effector biased IL-2
De novo IL-2 poteniator in therapeutic superior to the natural cytokine by molecular engineering
Bryan Bryson (MIT, Department of Biological Engineering)
“Exploiting the macrophage axis in Mycobacterium tuberculosis (Mtb) infection”
TB – who develop Active and why?
Immunological life cycle of Mtb
Global disease Mtb infection outcome varies within individual host
lesion are found by single bacteria
What are the cellular players in immune success
MACROPHAGES – molecular signals enhancing Mtb control of macrophages
modeling the host- macrophages are plastic and polarize
Building “libraries” – surface marker analysis of Microphages
Polarized macrophages are functionally different
quant and qual differences
History of GM-CSF suppresses IL-10
Loss of polarization potential: scRNAseq reveals transcriptional differences Thioredoxin facilitates immune response to Mtb is a marker of an inflammatory macrophage state
functional spectrum of human microphages
Facundo Batista (Ragon Institute (HIV Research) @MGH, MIT and Harvard)
“Vaccine evaluation in rapidly produced custom humanized mouse models”
Effective B cell activation requires 2 signals Antigen and binding to T cell
VDJ UCA (Unmutated common Ancestor)
B Cell Receptor (BCR) co-receptors and cytoskeleton
44% in Women age 24-44
Prototype HIV broadly neutralizing Antibodies (bnAb) do not bind to Env protein – Immunogen design and validation
Human Ig Knock-ins [Light variable 5′ chain length vs 7′ length] decisive to inform immunogenicity – One-Step CRISPR approach does not require ES cell work
Proof of principle with BG18 Germline Heavy Chain (BG18-gH) High-mannose patch – mice exhibit normal B cell development
B cells from naive human germline BG18-gH bind to GT2 immunogen
Interrogate immune response for HIV, Malaria, Zika, Flu
Session V
Moderator: Dane Wittrup, Koch Institute, MIT
Yvonne Chen (University of California, Los Angeles)
“Engineering smarter and stronger T cells for cancer immunotherapy”
Adoptive T-Cell Therapy
Tx for Leukemia – Tumor Antigen escape fro CAR T-cell therapy, CD19/CD20 OR-Gate CARs for prevention of antigen escape – 15 month of development
reduce probability of antigen escape due to two antigen CD19/CD20: Probing limits of modularity in CAR design
In vivo model: 75% wild type & 25% CD19 – relapse occur in the long term, early vs late vs no relapse: Tx with CAR t had no relapse
OR-Gate cause no relapse – Probing limits of modularity in CAR Design
Bispecific CARs are superior to DualCAR: One vs DualCAR (some remained single CAR)
Bispecific CARs exhibit superior antigen-stimulation capacity – OR-Gate CAR Outperforms Single-Input CARs
Lymphoma and Leukemia are 10% of all Cancers
TGF-gamma Rewiring T Cell Response
Activation of t Cell use CAR t
Engineer CAR-T to respond to soluble form of antigens: CD19 CAR Responds to soluble CD19
GFP MCAR responds to Dimeric GFP
“Tumor microenvironment is a scary place”
Michael Birnbaum, MIT, Koch Institute
“A repertoire of protective tumor immunity”
Decoding T and NK cell recognition – understanding immune recognition and signaling function for reprogramming the Immune system – Neoantigen vaccine pipeline
Personal neoantigen vax improve immunotherapy
CLASS I and CLASS II epitomes: MHC prediction performance – more accurate for CLASS I HLA polymorphisms
Immune Epitope DB and Analysis Resources 448,630 Peptide Epitomes
PD-L1 blockade elicits responses in some patients: soft part sarcoma
LAG-3 combined with PD-1 – human peripheral blood tumor
TIM-3 key regulator of T cell and Myeloid cell function: correlates in the TCGA DB with myeloid
Adenosine level in blood or tissue very difficult to measure in blood even more than in tissue – NIR178 + PDR 001 Mono-therapy (NIR178) combine with PD receptor blockage (PDR) – shows benefit
A alone vs A+B in Clinical trial
Session VI
Moderator: Stefani Spranger, Koch Institute, MIT
Tim Springer, Boston Children’s Hospital, HMS
The Milieu Model for TGF-Betta Activation”
Protein Science – Genomics with Protein
Antibody Initiative – new type of antibodies not a monoclonal antibody – a different type
Pro TGF-beta
TGF-beta – not a typical cytokine it is a prodamine for Mature growth factor — 33 genes mono and heterogeneous dimers
Latent TGF-Beta1 crystal structure: prodomaine shields the Growth Factor
Mechanism od activation of pro-TGF-beta – integrin alphaVBeta 6: pro-beta1:2
Simulation in vivo: actin cytoskeleton cytoplasmic domain
blocking antibodies LRRC33 mitigate toxicity on PD-L1 treatment
Alex Shalek, MIT, Department of Chemistry, Koch Institute
“Identifying and rationally modulating cellular drivers of enhanced immunity”
Balance in the Immune system
Profiling Granulomas using Seq-Well 2.0
lung tissue in South Africa of TB patients
Granulomas, linking cell type abundance with burden
Exploring T cells Phenotypes
Cytotoxic & Effector ST@+ Regulatory
Vaccine against TB – 19% effective, only 0 IV BCG vaccination can elicit sterilizing Immunity
Profiling cellular response to vaccination
T cell gene modules across vaccine routes
T Cells, Clusters
Expression of Peak and Memory
Immunotherapy- Identifying Dendritic cells enhanced in HIV-1 Elite Controllers
moving from Observing to Engineering
Cellular signature: NK-kB Signaling
Identifying and testing Cellular Correlates of TB Protection
Beyond Biology: Translation research: Data sets: dosen
Session VII
Moderator: Stefani Spranger, Koch Institute, MIT
Diane Mathis, Harvard Medical School
“Tissue T-regs”
T reg populations in Lymphoid Non–lymphoid Tissues
2009 – Treg tissue homeostasis status – sensitivity to insulin, 5-15% CD4+ T compartment
transcriptome
expanded repertoires TCRs
viceral adipose tissue (VAT) – Insulin
Dependencies: Taget IL-33 its I/1r/1 – encoded Receptor ST2
VAT up-regulate I/1r/1:ST2 Signaling
IL-33 – CD45 negative CD31 negative
mSC Production of IL-33 is Important to Treg
The mesenchyme develops into the tissues of the lymphatic and circulatory systems, as well as the musculoskeletal system. This latter system is characterized as connective tissues throughout the body, such as bone, muscle and cartilage. A malignant cancer of mesenchymal cells is a type of sarcoma.
Age-dependent Treg and mSC changes – Linear with increase in age
Sex-dependent Treg and mSC changes – Female
Treg loss in cases of Obesity leading to fibrosis
Treg keep IL-33-Producing mSCs under rein
Lean tissue vs Obese tissue
Aged mice show poor skeletal muscle repair – it is reverses by IL-33 Injection
Immuno-response: target tissues systemic T reg
Treg and mSC
Aviv Regev, Broad Institute; Koch Institute
“Cell atlases as roadmaps to understand Cancer”
Colon disease UC – genetic underlining risk, – A single cell atlas of healthy and UC colonic mucosa inflammed and non-inflammed: Epithelial, stromal, Immune – fibroblast not observed in UC colon IAFs; IL13RA2 + IL11
Anti TNF responders – epithelial cells
Anti TNF non-responders – inflammatory monocytes fibroblasts
RESISTANCE to anti-cancer therapy: OSM (Inflammatory monocytes-OSMR (IAF)
cell-cell interactions from variations across individuals
Most UC-risk genes are cell type specific
Variation within a cell type helps predict GWAS gene functions – epithelial cell signature – organize US GWAS into cell type specific – genes in associated regions: UC and IBD
Melanoma
malignant cells with resistance in cold niches in situ
cells express the resistance program pre-treatment: resistance UP – cold
Predict checkpoint immunotherapy outcomes
CDK4/6 – computational search predict as program regulators: abemaciclib in cell lines
Poster Presenters
Preeti Sharma, University of Illinois
T cell receptor and CAR-T engineering – T cell therapy
TCR Complex: Vbeta Cbeta P2A Valpha Calpha
CAR-T Aga2 HA scTCR/scFv c-myc
Directed elovution to isolate optimal TCR or CAR
Eng TCR and CARt cell therapy
Use of TCRs against pep/MHC allows targeting a n array of cancer antigens
TCRs are isolated from T cell clones
Conventional TCR identification method vs In Vitro TCR Eng directed evolution
T1 and RD1 TCRs drive activity against MART-1 in CD4+ T cells
CD8+
TCR engineering for Targeting glycosylated cancer antigens
Normal glycosylation vs Aberrant glycosylation
Engineering 237-CARs libraries with conjugated (Tn-OTS8) against multiple human Tn-antigend
In vitro engineering: broaden specificity to multiple peptide backbone
CAR engineering collaborations with U Chicago, U Wash, UPenn, Copenhagen, Germany
Martin LaFleur, HMS
CRISPR- Cas9 Bone marrow stem cells for Cancer Immunotherapy
CHIME: CHimeric IMmune Editing system
sgRNA-Vex
CHIME can be used to KO genes in multiple immune lineages
identify T cell intrinsic effects in the LCMV model Spleen-depleted, Spleen enhanced
Loss of Ptpn2 enhances CD8+ T cell responses to LCMV and Tumors
Ptpn2 deletion in the immune system enhanced tumor immunity
2018 Nobel Prize in Physiology or Medicine for contributions to Cancer Immunotherapy to James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan
NIH grantees win 2018 Nobel Prize in Physiology or Medicine.
The Nobel Prize medallion.Nobel Foundation
The 2018 Nobel Prize in Physiology or Medicine has been awarded to National Institutes of Health grantee James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan, for their discovery of cancer therapy by inhibition of negative immune regulation.
The Royal Swedish Academy of Sciences said, “by stimulating the inherent ability of our immune system to attack tumor cells this year’s Nobel Laureates have established an entirely new principle for cancer therapy.”
Dr. Allison discovered that a particular protein (CTLA-4) acts as a braking system, preventing full activation of the immune system when a cancer is emerging. By delivering an antibody that blocks that protein, Allison showed the brakes could be released. The discovery has led to important developments in cancer drugs called checkpoint inhibitors and dramatic responses to previously untreatable cancers. Dr. Honjo discovered a protein on immune cells and revealed that it also operates as a brake, but with a different mechanism of action.
“Jim’s work was pivotal for cancer therapy by enlisting our own immune systems to launch an attack on cancer and arrest its development,” said NIH Director Francis S. Collins, M.D., Ph.D. “NIH is proud to have supported this groundbreaking research.”
Dr. Allison has received continuous funding from NIH since 1979, receiving more than $13.7 million primarily from NIH’s National Cancer Institute (NCI) and National Institute of Allergy and Infectious Diseases (NIAID).
“This work has led to remarkably effective, sometime curative, therapy for patients with advanced cancer, who we were previously unable to help,” said NCI Director Ned Sharpless, M.D. “Their findings have ushered in the era of cancer immunotherapy, which along with surgery, radiation and cytotoxic chemotherapy, represents a ‘fourth modality’ for treating cancer. A further understanding of the biology underlying the immune system and cancer has the potential to help many more patients.”
“Dr. Allison’s elegant and groundbreaking work in basic immunology over four decades and its important applicability to cancer is a vivid demonstration of the critical nature of interdisciplinary biomedical research supported by NIH,” says NIAID Director Anthony S. Fauci, M.D.
About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.
Dr. Lev-Ari covered in person the following curated articles about James Allison, PhD since his days at University of California, Berkeley, including the prizes awarded prior to the 2018 Nobel Prize in Physiology.
2018 Albany Medical Center Prize in Medicine and Biomedical Research goes to NIH’s Dr. Rosenberg and fellow immunotherapy researchers James P. Allison, Ph.D., and Carl H. June, M.D.
New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells
Immunologist James P. Allison today shared the 2018 Nobel Prize in Physiology or Medicine for groundbreaking work he conducted on cancer immunotherapy at UC Berkeley during his 20 years as director of the campus’s Cancer Research Laboratory.
James Allison, who for 20 years was a UC Berkeley immunologist conducting fundamental research on cancer, is now at the M.D. Anderson Cancer Center in Houston, Texas.
Now at the University of Texas M.D. Anderson Cancer Center in Houston, Allison shared the award with Tasuku Honjo of Kyoto University in Japan “for their discovery of cancer therapy by inhibition of negative immune regulation.”
Allison, 70, conducted basic research on how the immune system – in particular, a cell called a T cell – fights infection. His discoveries led to a fundamentally new strategy for treating malignancies that unleashes the immune system to kill cancer cells. A monoclonal antibody therapy he pioneered was approved by the Food and Drug Administration in 2011 to treat malignant melanoma, and spawned several related therapies now being used against lung, prostate and other cancers.
“Because this approach targets immune cells rather than specific tumors, it holds great promise to thwart diverse cancers,” the Lasker Foundation wrote when it awarded Allison its 2015 Lasker-DeBakey Clinical Medical Research Award.
Allison’s work has already benefited thousands of people with advanced melanoma, a disease that used to be invariably fatal within a year or so of diagnosis. The therapy he conceived has resulted in elimination of cancer in a significant fraction of patients for a decade and counting, and it appears likely that many of these people are cured.
“Targeted therapies don’t cure cancer, but immunotherapy is curative, which is why many consider it the biggest advance in a generation,” Allison said in a 2015 interview. “Clearly, immunotherapy now has taken its place along with surgery, chemotherapy and radiation as a reliable and objective way to treat cancer.”
“We are thrilled to see Jim’s work recognized by the Nobel Committee,” said Russell Vance, the current director of the Cancer Research Laboratory and a UC Berkeley professor of molecular and cell biology. “We congratulate him on this highly deserved honor. This award is a testament to the incredible impact that the fundamental research Jim conducted at Berkeley has had on the lives of cancer patients”
“I don’t know if I could have accomplished this work anywhere else than Berkeley,” Allison said. “There were a lot of smart people to work with, and it felt like we could do almost anything. I always tell people that it was one of the happiest times of my life, with the academic environment, the enthusiasm, the students, the faculty.”
In this video about UC Berkeley’s new Immunotherapeutics and Vaccine Research Initiative (IVRI), Allison discusses his groundbreaking work on cancer immunotherapy.
In fact, Allison was instrumental in creating the research environment of the current Department of Molecular and Cell Biology at UC Berkeley as well as the department’s division of immunology, in which he served stints as chair and division head during his time at Berkeley, said David Raulet, director of Berkeley’s Immunotherapeutics and Vaccine Research Initiative (IVRI).
“His actions helped create the superb research environment here, which is so conducive to making the fundamental discoveries that will be the basis of the next generation of medical breakthroughs,” Raulet said.
Self vs. non-self
Allison joined the UC Berkeley faculty as a professor of molecular and cell biology and director of the Cancer Research Laboratory in 1985. An immunologist with a Ph.D. from the University of Texas, Austin, he focused on a type of immune system cell called the T cell or T lymphocyte, which plays a key role in fighting off bacterial and viral infections as well as cancer.
Supercharging the immune system to cure disease: immunotherapy research at UC Berkeley. (UC Berkeley video by Roxanne Makasdjian and Stephen McNally)
At the time, most doctors and scientists believed that the immune system could not be exploited to fight cancer, because cancer cells look too much like the body’s own cells, and any attack against cancer cells would risk killing normal cells and creating serious side effects.
“The community of cancer biologists was not convinced that you could even use the immune system to alter cancer’s outcome, because cancer was too much like self,” said Matthew “Max” Krummel, who was a graduate student and postdoctoral fellow with Allison in the 1990s and is now a professor of pathology and a member of the joint immunology group at UCSF. “The dogma at the time was, ‘Don’t even bother.’ ”
“What was heady about the moment was that we didn’t really listen to the dogma, we just did it,” Krummel added. Allison, in particular, was a bit “irreverent, but in a productive way. He didn’t suffer fools easily.” This attitude rubbed off on the team.
Trying everything they could in mice to tweak the immune system, Krummel and Allison soon found that a protein receptor called CTLA-4 seemed to be holding T cells back, like a brake in a car.
Postdoctoral fellow Dana Leach then stepped in to see if blocking the receptor would unleash the immune system to actually attack a cancerous tumor. In a landmark paper published in Science in 1996, Allison, Leach and Krummel showed not only that antibodies against CTLA-4 released the brake and allowed the immune system to attack the tumors, but that the technique was effective enough to result in long-term disappearance of the tumors.
“When Dana showed me the results, I was really surprised,” Allison said. “It wasn’t that the anti-CTLA-4 antibodies slowed the tumors down. The tumors went away.”
After Allison himself replicated the experiment, “that’s when I said, OK, we’ve got something here.”
Checkpoint blockade
The discovery led to a concept called “checkpoint blockade.” This holds that the immune system has many checkpoints designed to prevent it from attacking the body’s own cells, which can lead to autoimmune disease. As a result, while attempts to rev up the immune system are like stepping on the gas, they won’t be effective unless you also release the brakes.
James Allison in 1993, when he was conducting research at UC Berkeley on a promising immunotherapy now reaching fruition. (Jane Scherr photo)
“The temporary activation of the immune system though ‘checkpoint blockade’ provides a window of opportunity during which the immune system is mobilized to attack and eliminate tumors,” Vance said.
Allison spent the next few years amassing data in mice to show that anti-CTLA-4 antibodies work, and then, in collaboration with a biotech firm called Medarex, developed human antibodies that showed promise in early clinical trials against melanoma and other cancers. The therapy was acquired by Bristol-Myers Squibb in 2011 and approved by the FDA as ipilimumab (trade name Yervoy), which is now used to treat skin cancers that have metastasized or that cannot be removed surgically.
Meanwhile, Allison left UC Berkeley in 2004 for Memorial Sloan Kettering research center in New York to be closer to the drug companies shepherding his therapy through clinical trials, and to explore in more detail how checkpoint blockade works.
“Berkeley was my favorite place, and if I could have stayed there, I would have,” he said. “But my research got to the point where all the animal work showed that checkpoint blockade had a lot of potential in people, and working with patients at Berkeley wasn’t possible. There’s no hospital, no patients.”
Thanks to Allison’s doggedness, anti-CTLA-4 therapy is now an accepted therapy for cancer and it opened the floodgates for a slew of new immunotherapies, Krummel said. There now are several hundred ongoing clinical trials involving monoclonal antibodies to one or more receptors that inhibit T cell activity, sometimes combined with lower doses of standard chemotherapy.
Antibodies against one such receptor, PD-1, which Honjo discovered in 1992, have given especially impressive results. Allison’s initial findings can be credited for prompting researchers, including Allison himself, to carry out the studies that have demonstrated the potent anti-cancer effects of PD-1 antibodies. In 2015, the FDA approved anti-PD-1 therapy for malignant melanoma, and has since approved it for non-small-cell lung, gastric and several other cancers.
Science magazine named cancer immunotherapy its breakthrough of 2013 because that year, “clinical trials … cemented its potential in patients and swayed even the skeptics. The field hums with stories of lives extended: the woman with a grapefruit-size tumor in her lung from melanoma, alive and healthy 13 years later; the 6-year-old near death from leukemia, now in third grade and in remission; the man with metastatic kidney cancer whose disease continued fading away even after treatment stopped.”
Allison pursued more clinical trials for immunotherapy at Sloan-Kettering and then in 2012 returned to his native Texas.
Born in Alice, Texas, on Aug. 7, 1948, Allison earned a B.S. in microbiology in 1969 and a Ph.D. in biological science in 1973 from the University of Texas, Austin.
The CRISPR-Cas9 system has proven to be a powerful tool for genome editing allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells.
CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.
Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells. Using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), an average insertion or deletion (indel) efficiency greater than 80% was achieved. This high efficiency of insertion or deletion generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs.
The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. These results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. As hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.
CRISPR-based editing of T cells to treat cancer, as scientists at the University of Pennsylvania are studying in a clinical trial, should also not have a p53 problem. Nor should any therapy developed with CRISPR base editing, which does not make the double-stranded breaks that trigger p53. But, there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.
The Immune System, Stress Signaling, Infectious Diseases and Therapeutic Implications: VOLUME 2: Infectious Diseases and Therapeutics and VOLUME 3: The Immune System and Therapeutics (Series D: BioMedicine & Immunology) Kindle Edition – on Amazon.com since September 4, 2017
Stimulating the Immune system not only sustaining it for therapies
K. Dane Wittrup | MIT, Koch Institute
8:30 – 9:45Session I Moderator: Douglas Lauffenburger | MIT, Biological Engineering and Koch Institute
Garry P. Nolan – Stanford University School of Medicine Pathology from the Molecular Scale on Up
Intracellular molecules,
how molecules are organized to create tissue
Meaning from data Heterogeneity is an illusion: Order in Data ?? Cancer is heterogeneous, Cells in suspension – number of molecules
System-wide changes during Immune Response (IR)
Untreated, Ineffective therapy, effective therapy
Days 3-8 Tumor, Lymph node…
Variation is a Feature – not a bug: Effective therapy vs Ineffective – intercellular modules – virtual neighborhoods
ordered by connectivity: very high – CD4 T-cells, CD8 T-cels, moderate, not connected
Landmark nodes, Increase in responders
CODEX: Multiples epitome detection
Adaptable to proteins & mRNA
Rendering antibody staining via removal to neighborhood mapping
Human tonsil – 42 parameters: CD7, CD45, CD86,
Automated Annotations of tissues: F, P, V,
Normal BALBs
Marker expression defined by the niche: B220 vs CD79
Marker expression defines the niche
Learn neighborhoods and Trees
Improving Tissue Classification and staining – Ce3D – Tissue and Immune Cells in 3D
Molecular level cancer imaging
Proteomic Profiles: multi slice combine
Theory is formed to explain 3D nuclear images of cells – Composite Ion Image, DNA replication
Replication loci visualization on DNA backbone – nascent transcriptome – bar code of isotopes – 3D 600 slices
use CRISPR Cas9 for Epigenetics
Susan Napier Thomas – Georgia Institute of Technology Transport Barriers in the Tumor Microenvironment: Drug Carrier Design for Therapeutic Delivery to Sentinel Lymph Nodes
Lymph Nodes important therapeutics target tissue
Lymphatic flow support passive and active antigen transport to lymph nodes
clearance of biomolecules and drug formulations: Interstitial transport barriers influence clearance: Arteriole to Venule –
Molecular tracers to analyze in vivo clearance mechanisms and vascular transport function
quantifying molecular clearance and biodistribution
Lymphatic transport increases tracer concentrations within dLN by orders of magnitude
Melanoma growth results in remodeled tumor vasculature
passive transport via lymphatic to dLN sustained in advanced tumors despite abrogated cell trafficking
Engineered biomaterial drug carriers to enhance sentinel lymph node-drug delivery: facilitated by exploiting lymphatic transport
Sturcutral and Cellular barriers: transport of particles is restriced by
Current drug delivery technology: lymph-node are undrugable
Multistage delivery platform to overcome barriers to lymphatic uptake and LN targeting
nano particles – OND – Oxanorbornade OND Time sensitive Linker synthesized large cargo – NP improve payload
OND release rate from nanoparticles changes retention in lymph nodes – Axilliary-Brachial delivery
Two-stage OND-NP delivery and release system dramatically – OND acumulate in lymphocyte
delivers payload to previously undraggable lymphe tissue
improved drug bioactivity – OND-NP eliminate LN LYMPHOMAS
Engineered Biomaterials
Douglas Lauffenburger – MIT, Biological Engineering and Koch Institute Integrative Multi-Omic Analysis of Tissue Microenvironment in Inflammatory Pathophysiology
How to intervene, in predictive manner, in immunesystem-associated complex diseases
Understand cell communication beteen immune cells and other cells, i.e., tumor cells
Multi-Variate in Vivo – System Approach: Integrative Experiment & COmputational Analysis
Cell COmmunication & Signaling in CHronic inflammation – T-cell transfer model for colitis
COmparison of diffrential Regulation (Tcell transfer-elicited vs control) anong data types – relying solely on mRNA can be misleading
Diparities in differential responses to T cell transfer across data types yield insights concerning broader multi-organ interactions
T cell transfer can be ascertained and validated by successful experimental test
Cell COmmunication in Tumor MIcro-Environment — integration of single-cell transcriptomic data and protein interaction
Standard Cluster Elucidation – Classification of cell population on Full gene expression Profiles using Training sets: Decision Tree for Cell Classification
Wuantification of Pairwise Cell-Cell Receptor/Ligand Interactions: Cell type Pairs vs Receptor/Ligand Interaction
Pairwise Cell-Cell Receptor/Ligand Interactions
Calculate strength of interaction and its statistical significance
How the interaction is related to Phenotypic Behaviors – tumor growth rate, MDSC levels,
Correlated the Interactions translated to Phynotypic behavior for Therapeutic interventions (AXL via macrophage and fibroblasts)
Mouth model translation to Humans – New machine learning approach
Minimal Immune response to KP lung tumors: H&E, T cells (CD3), Bcells (B220) for Lenti-x 8 weeks
Exosome sequencing : Modeling loss-and gain-of-function mutations in Lung Cancer by CRISPR-Cas9 – germline – tolerance in mice, In vivo CRISPR-induced knockout of Msh2
Signatures of MMR deficient
Mutation burden and response to Immunotherapy (IT)
Programmed neoantigen expression – robust infiltration of T cells (evidence of IR)
Immunosuppression – T cell rendered ineffective
Lymphoid infiltration: Acute Treg depletion results in T cell infiltration — this depletion causes autoimmune response
Lung Treg from KP tumor-bearing mice have a distinct transcriptional heterogeneity through single cell mRNA sequencing
KP, FOXP3+, CD4
Treg from no existent to existance, Treg cells increase 20 fold =>>> Treg activation and effectiveness
Single cells cluster by tissue and cell type: Treg, CD4+, CD8+, Tetramer-CD4+
ILrl1/II-33r unregulated in Treg at late time point
Treg-specific deletion of IL-33r results in fewer effector Tregs in Tumor-bearing lungs
CD8+ T cell infiltration
Tetramer-positive T cells cluster according to time point: All Lung CD8+ T cells
IR is not uniform functional differences – Clones show distinct transcriptional profiles
Different phynotypes Exhaustive signature
CRISPR-mediated modulation of CD8 T cell regulatory genes
Genetic dissection of the tumor-immune microenvironment
Single cell analysis, CRISPR – CRISPRa,i, – Drug development
Wendell Lim – University of California, San Francisco
Synthetic Immunology: Hacking Immune Cells
Precision Cell therapies – engineered by synthetic biology
Anti CD19 – drug approved
CAR-T cells still face major problems
success limited to B cells cancers = blood vs solid tumors
adverse effects
OFF-TUMOR effects
Cell engineering for Cancer Therapy: User remote control (drug) – user control safety
Cell Engineering for TX
new sensors – decision making for
tumor recognition – safety,
Cancer is a recognition issue
How do we avoid cross-reaction with bystader tissue (OFF TISSUE effect)
Tumor recognition: More receptors & integration
User Control
synthetic NOTCH receptors (different flavors of synNotch) – New Universal platform for cell-to -cell recognition: Target molecule: Extracellular antigen –>> transciptional instruction to cell
nextgen T cell: Engineer T cell recognition circuit that integrates multiple inputs: Two receptors – two antigen priming circuit
UNARMED: If antigen A THEN receptor A activates CAR
“Bystander” cell single antigen vs “tumor” drug antigen
Selective clearance of combinatorial tumor – Boulian formulation, canonical response
Comparing CD19 CARs for Leukemia – anti-CD19- directed CAR T cells with r/r B-cell ALL – age 3-25 – FDA approved Novartis tisagenlecleucel – for pediatric r/r/ ALL
Phase II in diffuse large B cell lymphoma. Using T cells – increases prospects for cure
Vector retroviral – 30 day expression
measuring cytokines release syndrome: Common toxicity with CAR 19
neurological toxicity, B-cell aplagia
CART issues with heme malignancies
decrease cytokine release
avoid neurological toxicity – homing
new targets address antigene escape variants – Resistance, CD19 is shaded, another target needed
B Cell Maturation Antigen (BCMA) Target
Bluebird Bio: Response duratio up to 54 weeks – Active dose cohort
natural ligand CAR based on April
activated in response to TACI+ target cells – APRIL-based CARs but not BCMA-CAR is able to kill TACI+ target cells
Hurdles for Solid Tumors
Specific antigen targets
tumor heterogeneity
inhibitory microenvironment
CART in Glioblastoma
rationale for EGFRvIII as therapeutic target
Preclinical Studies & Phase 1: CAR t engraft, not as highly as CD19
Upregulation of immunosuppression and Treg infiltrate in CART EGFRvIII as therapeutic target, Marcela Maus
What to do differently?
2:15 – 2:45 Break
2:45 – 4:00 Session IV Moderator: Arup K. Chakraborty | MIT, IMES
Laura Walker – Adimab, LLC Molecular Dissection of the Human Antibody Response to Respiratory Syncytial Virus
prophylactic antibody is available
Barriers for development of Vaccine
Prefusion and Postfusion RSV structures
Six major antigenic sites on RSV F
Blood samples Infants less 6 month of age and over 6 month: High abundance RSV F -specific memory B Cells are group less 6 month
Arup K. Chakraborty – MIT, Institute for Medical Engineering & Science How to Hit HIV Where it Hurts
antibody – Model IN SILICO
Check affinity of each Ab for the Seaman panel of strain
Breadth of coverage
immmunize with cocktail of variant antigens
Mutations on Affinity Maturation: Molecular dynamics
bnAb eveolution: Hypothesis – mutations evolution make the antigen binding region more flexible,
Tested hypothesisi: carrying out affinity maturation – LOW GERMLINE AFFINITY TO CONSERVE RESIDUES IN 10,000 trials, acquire the mutation (generation 300)
William Schief – The Scripps Research Institute HIV Vaccine Design Targeting the Human Naive B Cell Repertoire
HIV Envelope Trimer Glycan): the Target of neutralizing Antibodies (bnAbs)
Proof of principle for germline-targeting: VRC)!-class bnAbs
design of a nanoparticle
can germline -targeting innumogens prime low frequency precursors?
Day 14 day 42 vaccinate
Precursor frequency and affinity are limiting for germline center (GC) entry at day 8
Germline-targeting immunogens can elicit robust, high quality SHM under physiological conditions of precursor frequency and affinity at day 8, 16, 36
Germline-targeting immunogens can lead to production of memory B cells
Outsource a part of the T cell’s immune value chain, propose cancer immunotherapy researchers, from patient T cells to donor T cells. The novel allogeneic approach could rely on T-cell receptor gene transfer to generate broad and tumor-specific T-cell immune responses. [NIAID]
A new cancer immunotherapy approach could essentially outsource a crucial T-cell function. This function, T-cell reactivity to specific cancer antigens, is sometimes lacking in cancer patients. Yet, according to a new proof-of-principle study, these patients could benefit from T cells provided by healthy donors. Specifically, the healthy donors’ T cells could be used to broaden the T-cell receptor repertoires of the cancer patients’ T cells.
Ultimately, this approach relies on a cancer immunotherapy technique called T-cell receptor (TCR) transfer, or the genetic transfer of TCR chains. TCR transfer can be used to outsource the T cell’s learning function, the process by which a T cell acquires the ability to recognize foreign antigens—in this case, the sort of proteins that can be expressed on the surface of cancer cells. Because cancer cells harbor faulty proteins, they can also display foreign protein fragments, also known as neoantigens, on their surface, much in the way virus-infected cells express fragments of viral proteins.
The approach was detailed in a paper that appeared May 19 in the journal Science, in an article entitled, “Targeting of Cancer Neoantigens with Donor-Derived T Cell Receptor Repertoires.” This article, by scientists based at the Netherlands Cancer Institute and the University of Oslo, describes a novel strategy to broaden neoantigen-specific T-cell responses. Such a strategy would be useful in overcoming a common limitation seen in the immune response to cancer: Neoantigen-specific T-cell reactivity is generally limited to just a few mutant epitopes, even though the number of predicted epitopes is large.
“We demonstrate that T cell repertoires from healthy donors provide a rich source of T cells that specifically recognize neoantigens present on human tumors,” the study’s authors wrote. “Responses to 11 epitopes were observed, and for the majority of evaluated epitopes, potent and specific recognition of tumor cells endogenously presenting the neoantigens was detected.”
First, the researchers mapped all possible neoantigens on the surface of melanoma cells from three different patients. In all three patients, the cancer cells seemed to display a large number of different neoantigens. But when the researchers tried to match these to the T cells derived from within the patient’s tumors, most of these aberrant protein fragments on the tumor cells went unnoticed.
Next, the researchers tested whether the same neoantigens could be seen by T cells derived from healthy volunteers. Strikingly, these donor-derived T cells could detect a significant number of neoantigens that had not been seen by the patients’ T cells.
“Many of the T cell reactivities [among donor T cells] involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes,” the authors of the Science article continued. “T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such ‘outsourced’ immune responses in cancer immunotherapy.”
“In a way, our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neoantigens,” said Ton Schumacher, Ph.D., a principal investigator at the Netherlands Cancer Institute. “The receptor that is used by these donor T cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”
“Our study shows that the principle of outsourcing cancer immunity to a donor is sound,” added Johanna Olweus, M.D., Ph.D., who heads a research group at the University of Oslo. “However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput.”
“We are currently exploring high-throughput methods to identify the neoantigens that the T cells can ‘see’ on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”
Targeting of cancer neoantigens with donor-derived T cell receptor repertoires
Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen specific T cell responses are therefore attractive. Here, we demonstrate that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11/57 predicted HLA-A2-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.
Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.
Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5′ AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL.
Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.
Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α
CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.
The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.
Figure 1: Proposed model for the metabolic signatures of various Treg cell subsets.
(a) Activated CD4+ T cells that differentiate into the Teff cell lineage (green) (TH1 or TH17 cells) are dependent mainly on carbon substrates such as glucose and glutamine for their anabolic metabolism. In contrast to that, pTreg cells…
T-bet is a key modulator of IL-23-driven pathogenic CD4+ T cell responses in the intestine
IL-23 is a key driver of pathogenic Th17 cell responses. It has been suggested that the transcription factor T-bet is required to facilitate IL-23-driven pathogenic effector functions; however, the precise role of T-bet in intestinal T cell responses remains elusive. Here, we show that T-bet expression by T cells is not required for the induction of colitis or the differentiation of pathogenic Th17 cells but modifies qualitative features of the IL-23-driven colitogenic response by negatively regulating IL-23R expression. Consequently, absence of T-bet leads to unrestrained Th17 cell differentiation and activation characterized by high amounts of IL-17A and IL-22. The combined increase in IL-17A/IL-22 results in enhanced epithelial cell activation and inhibition of either IL-17A or IL-22 leads to disease amelioration. Our study identifies T-bet as a key modulator of IL-23-driven colitogenic responses in the intestine and has important implications for understanding of heterogeneity among inflammatory bowel disease patients.
Th17 cells are enriched at mucosal sites, produce high amounts of IL-17A, IL-17F and IL-22, and have an essential role in mediating host protective immunity against a variety of extracellular pathogens1. However, on the dark side, Th17 cells have also been implicated in a variety of autoimmune and chronic inflammatory conditions, including inflammatory bowel disease (IBD)2. Despite intense interest, the cellular and molecular cues that drive Th17 cells into a pathogenic state in distinct tissue settings remain poorly defined.
The Th17 cell programme is driven by the transcription factor retinoid-related orphan receptor gamma-t (RORγt) (ref. 3), which is also required for the induction and maintenance of the receptor for IL-23 (refs 4, 5). The pro-inflammatory cytokine IL-23, composed of IL-23p19 and IL-12p40 (ref. 6), has been shown to be a key driver of pathology in various murine models of autoimmune and chronic inflammatory disease such as experimental autoimmune encephalomyelitis (EAE)7, collagen induced arthritis8 and intestinal inflammation9, 10, 11, 12. Several lines of evidence, predominantly derived from EAE, suggest that IL-23 promotes the transition of Th17 cells to pathogenic effector cells9, 10, 11, 12. Elegant fate mapping experiments of IL-17A-producing cells during EAE have shown that the majority of IL-17A+IFN-γ+ and IL-17A−IFN-γ+ effector cells arise from Th17 cell progeny13. This transition of Th17 cells into IFN-γ-producing ‘ex’ Th17 cells required IL-23 and correlated with increased expression of T-bet. The T-box transcription factor T-bet drives the Th1 cell differentiation programme14 and directly transactivates the Ifng gene by binding to its promoter as well as multiple enhancer elements15. Indeed, epigenetic analyses have revealed that the loci for T-bet and IFN-γ are associated with permissive histone modifications in Th17 cells suggesting that Th17 cells are poised to express T-bet which could subsequently drive IFN-γ production16, 17.
A similar picture is emerging in the intestine where IL-23 drives T-cell-mediated intestinal pathology which is thought to be dependent on expression of T-bet18 and RORγt (ref. 19) by T cells. In support of this we have recently shown that IL-23 signalling in T cells drives the emergence of IFN-γ producing Th17 cells in the intestine during chronic inflammation20. Collectively these studies suggest a model whereby RORγt drives differentiation of Th17 cells expressing high amounts of IL-23R, and subsequently, induction of T-bet downstream of IL-23 signalling generates IL-17A+IFN-γ+ T cells that are highly pathogenic. Indeed, acquisition of IFN-γ production by Th17 cells has been linked to their pathogenicity in several models of chronic disease13, 21, 22, 23, 24 and a population of T cells capable of producing both IL-17A and IFN-γ has also been described in intestinal biopsies of IBD patients25, 26.
However, in the context of intestinal inflammation, it remains poorly defined whether the requirement for RORγt and T-bet reflects a contribution of Th17 and Th1 cells to disease progression or whether Th17 cells require T-bet co-expression to exert their pathogenic effector functions. Here, we use two distinct models of chronic intestinal inflammation and make the unexpected finding that T-bet is dispensable for IL-23-driven colitis. Rather the presence of T-bet serves to modify the colitogenic response restraining IL-17 and IL-22 driven pathology. These data identify T-bet as a key modulator of IL–23-driven colitogenic effector responses in the intestine and have important implications for understanding of heterogeneous immune pathogenic mechanisms in IBD patients.
Figure 1: IL-23 signalling is required for bacteria-driven T-cell-dependent colitis and the emergence of IL-17A+IFN-γ+ T cells.
C57BL/6 WT and Il23r−/− mice were infected orally with Hh and received weekly i.p. injections of IL-10R blocking antibody. Mice were killed at 4 weeks post infection and assessed for intestinal inflammation. (a) Colitis scores. (b) Typhlitis sores. (c) Representative photomicrographs of colon and caecum (× 10 magnification; scale bars, 200μM). (d) Representative flow cytometry plots of colonic lamina propria gated on viable CD4+ T cells. (e) Frequencies of IL-17A+ and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from two independent experiments (n=12 for WT, n=10 for Il23r−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, ***P<0.001 as calculated by Mann–Whitney U test.
C57BL/6 Rag1−/− mice were injected i.p. with 4 × 105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/− T cells developed clinical signs of disease (4–6 weeks) and assessed for intestinal inflammation. (a) Colitis scores. (b) Representative photomicrographs of proximal colon sections (× 10 magnification; scale bars, 200μM). (c) Concentration of cytokines released from colon explants into the medium after overnight culture. Data represent pooled results from two independent experiments (n=14 for WT, n=11 for Rorc−/−, n=14 forTbx21−/−). Bars are the mean and each symbol represents an individual mouse. Bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.
C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of IL-17A and IFN-γ expression in colonic CD4+ T cells. (b) Frequencies of IL-17A+ and/or IFN-γ+ cells among colonic CD4+ T cells. (c) Total numbers of IL-17A+and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from three independent experiments (n=20 for WT, n=18 for Tbx21−/−, n=12 for Rorc−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.
T-bet deficiency promotes an exacerbated Th17-type response
Our transfer of Tbx21−/− T cells revealed a striking increase in the frequency of IL-17A+IFN-γ−cells (Fig. 3) and we reasoned that T-bet-deficiency could impact on Th17 cell cytokine production. Therefore, we transferred WT or Tbx21−/− CD4+ T cells into Rag1−/− recipients and measured the expression of RORγt, IL-17A, IL-17F and IL-22 by CD4+ T cells isolated from the colon. In agreement with our earlier findings, Tbx21−/− T cells gave rise to significantly increased frequencies of RORγt-expressing T cells capable of producing IL-17A (Fig. 4a). Furthermore, T-bet deficiency also led to a dramatic expansion of IL-17F and IL-22-expressing cells, which constituted only a minor fraction in WT T cells (Fig. 4a,b). By contrast, the frequency of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ producing cells was significantly reduced in T-bet-deficient T cells as compared with WT T cells. When analysed in more detail we noted that the production of IL-17A, IL-17F and IL-22 increased specifically in T-bet-deficient IL-17A+IFN-γ+ T cells as compared with WT T cells whereas IFN-γ production decreased overall in the absence of T-bet as expected (Supplementary Fig. 4A). Similarly, GM-CSF production was also generally reduced in Tbx21−/− CD4+ T cells further suggesting a shift in the qualitative nature of the T cell response.
Figure 4: T-bet-deficient CD4+ T cells promote an exacerbated Th17-type inflammatory response.
C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT orTbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of cytokines and transcription factors in WT or Tbx21−/− colonic CD4+ T cells. (b) Frequency of IL-17A+, IL-17F+, IL-22+, GM-CSF+ or IFN-γ+ colonic T cells in WT orTbx21−/−. (c) quantitative reverse transcription PCR (qRT-PCR) analysis of mRNA levels of indicated genes in colon tissue homogenates. (d) Total number of neutrophils (CD11b+ Gr1high) in the colon. (e) Primary epithelial cells were isolated from the colon of steady state C57BL/6 Rag1−/− mice and stimulated with 10ngml−1 cytokines for 4h after which cells were harvested and analysed by qRT-PCR for the indicated genes. Data in b–d represent pooled results from two independent experiments (n=14 for WT, n=11 for Tbx21−/−). Bars are the mean and error bars represent s.e.m. Data in e are pooled results from four independent experiments, bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01,***P<0.001 as calculated by Mann–Whitney U test.
T-bet-deficient colitis depends on IL-23, IL-17A and IL-22
In the present study we show that bacteria-driven colitis is associated with the IL-23-dependent emergence of IFN-γ-producing Th17 cells co-expressing RORγt and T-bet. Strikingly, while RORγt is required for the differentiation of IFN-γ-producing Th17 cells and induction of colitis, T-bet is dispensable for the emergence of IL-17A+IFN-γ+ T cells and intestinal pathology. Our results show that instead of a mandatory role in the colitogenic response, the presence of T-bet modulates the qualitative nature of the IL-23-driven intestinal inflammatory response. In the presence of T-bet, IL-23-driven colitis is multifunctional in nature and not functionally dependent on either IL-17A or IL-22. By contrast, in the absence of T-bet a highly polarized colitogenic Th17 cell response ensues which is functionally dependent on both IL-17A and IL-22. T-bet-deficient T cells are hyper-responsive to IL-23 resulting in enhanced STAT3 activation and downstream cytokine secretion providing a mechanistic basis for the functional changes. These data newly identify T-bet as a key modulator of IL-23-driven colitogenic CD4+ T cell responses.
Contrary to our expectations T-bet expression by CD4 T cells was not required for their pathogenicity. In keeping with the negative effect of T-bet on Th17 differentiation40, 41, 42, we observed highly polarized Th17 responses in T-bet-deficient intestinal T cells. Early studies demonstrated that IFN-γ could suppress the differentiation of Th17 cells40 and thus the reduced IFN-γ production by Tbx21−/−T cells could facilitate Th17 cell generation. However, our co-transfer studies revealed unrestrained Th17 differentiation of Tbx21−/− T cells even in the presence of WT T cells, suggesting a cell autonomous role for T-bet-mediated suppression of the Th17 programme. Indeed, the role of T-bet as a transcriptional repressor of the Th17 cell fate has been described recently. For example, T-bet physically interacts with and sequesters Runx1, thereby preventing Runx1-mediated induction of RORγt and Th17 cell differentiation43. In addition, T-bet binds directly to and negatively regulates expression of many Th17-related genes15, 34 and we identified IL23r to be repressed in a T-bet-dependent manner. In line with this we show here that T-bet-deficient intestinal T cells express higher amounts of Il23r as well as Rorc. This resulted in enhanced IL-23-mediated STAT3 activation and increased production of IL-17A and IL-22. It has also been suggested that T-bet activation downstream of IL-23R signalling is required for pathogenic IL-23-driven T cell responses43, 44. However, we did not find a role for IL-23 in the induction and/or maintenance of T-bet expression and colitis induced by T-bet-deficient T cells was IL-23 dependent. Collectively, these findings demonstrate that T-bet deficiency leads to unrestrained expansion of colitogenic Th17 cells, which is likely mediated through enhanced activation of the IL-23R-STAT3 pathway.
The observation that T-bet-deficient T cells retain their colitogenic potential is in stark contrast to earlier studies. Neurath et al.18 convincingly showed that adoptive transfer of Tbx21−/− CD4+ T cells into severe combined immunodeficiency (SCID) recipients failed to induce colitis and this correlated with reduced IFN-γ and increased IL-4 production. Another study revealed that IL-4 plays a functional role in inhibiting the colitogenic potential of Tbx21−/− T cells, as recipients ofStat6−/−Tbx21−/− T cells developed severe colitis37. Importantly, the intestinal inflammation that developed in recipients of Stat6−/−Tbx21−/− T cells could be blocked by administration of IL-17A neutralizing antibody, suggesting that the potent inhibitory effect of IL-4/STAT6 signals on Th17 differentiation normally prevent colitis induced by Tbx21−/− T cells37. Various explanations could account for the discrepancy between our study and those earlier findings. First, in contrast to the published reports, we used naïve Tbx21−/− CD4+ T cells from C57BL/6 mice instead of BALB/c mice. An important difference between Tbx21−/− CD4+ T cells from these genetic backgrounds appears to be their differential susceptibility to suppression by IL-4/STAT6 signals. We found that transfer of Tbx21−/− T cells induced IL-17A-dependent colitis despite increased frequencies of IL-4-expressing cells in the intestine. This discrepancy may be due to higher amounts of IL-4 produced by activated CD4+ T cells from BALB/c versus C57BL/6 mice45, leading to the well-described Th2-bias of the BALB/c strain45. Second, differences in the composition of the intestinal microbiota between animal facilities can have a substantial effect on skewing CD4+ T cells responses. In particular, the Clostridium-related segmented filamentous bacteria (SFB) have been shown to drive the emergence of IL-17 and IL-22 producing CD4+ T cells in the intestine46. Importantly, the ability of naïve CD4+ T cells to induce colitis is dependent on the presence of intestinal bacteria, as germ-free mice do not develop pathology upon T cell transfer47. In line with this, we previously described that colonization of germ-free mice with intestinal microbiota containing SFB was necessary to restore the development of colitis47. Since our Rag1−/− colony is SFB+ and the presence of SFB was not reported in the previous studies, it is possible that differences in SFB colonization status contributed to the observed differences in pathogenicity ofTbx21−/− T cells.
It is important to note that T-bet-deficient T cells did not induce more severe colitis than WT T cells but rather promoted a distinct mucosal inflammatory response. Colitis induced by WT T cells is characterized by a multifunctional response with high amounts of IFN-γ and GM-CSF and a lower IL-17A and IL-22 response. Consistent with this, we have shown that blockade of GM-CSF abrogates T cell transfer colitis48 as well as bacteria-driven intestinal inflammation49 in T-bet sufficiency whereas blockade of IL-17A or IL-22 fails to do so. By contrast T-bet deficiency leads to production of high amounts of IL-17A and IL-22 in the colon and neutralization of either was sufficient to reduce intestinal pathology. Our in vitro experiments suggest that IL-17A and IL-22 synergise to promote intestinal epithelial cell responses, which may in part explain the efficacy of blocking IL-17A or IL-22 in colitis induced by T-bet-deficient T cells. A similar synergistic interplay has been described in the lung where IL-22 served a tissue protective function in homeostasis but induced airway inflammation in the presence of IL-17A (ref. 50). This highlights the complexity of the system in health and disease, and the need for a controlled production of both cytokines. We describe here only one mechanism of how IL-17A/IL-22 induce a context-specific epithelial cell response that potentially impacts on the order or composition of immune cell infiltration. Overall, these results provide a new perspective on T-bet, revealing its role in shaping the qualitative nature of the IL-23-driven colitogenic T cell response.
We also describe here the unexpected finding that a substantial proportion of T-bet-deficient intestinal T cells retain the ability to express IFN-γ. To investigate the potential mechanisms responsible for T-bet-independent IFN-γ production by intestinal CD4+ T cells we focused on two transcription factors, Runx3 and Eomes. Runx3 has been shown to promote IFN-γ expression directly through binding to the Ifng promoter38 and Eomes is known to compensate for IFN-γproduction in T-bet-deficient Th1 cells37. We found IL-23-mediated induction of Runx3 protein in WT and Tbx21−/− T cells isolated from the intestine, thus identifying Runx3 downstream of IL-23R signalling. By contrast, we could only detect Eomes protein and its induction by IL-23 in T-bet-deficient but not WT T cells. Thus, Runx3 and Eomes are activated in response to IL-23 in T-bet-deficient cells and are likely to be drivers of T-bet-independent IFN-γ production. In support of this we found that the majority of T-bet-deficient IL-17A−IFN-γ+ T cells expressed Eomes. However, only a minor population of IL-17A+IFN-γ+ T cells stained positive for Eomes, suggesting the existence of alternative pathways for IFN-γ production by Th17 cells. Intriguingly, a recent study identified Runx3 and Runx1 as the transcriptional regulators critical for the differentiation of IFN-γ-producing Th17 cells51. The author’s demonstrated that ectopic expression of Runx transcription factors was sufficient to induce IFN-γ production by Th17 cells even in the absence of T-bet. These findings, combined with our data on Runx3 activation downstream of IL-23R signalling strongly suggest that Runx3 rather than Eomes is driving IFN-γ expression by intestinal Th17 cells.
We have not formally addressed the role of IFN-γ in colitis driven by T-bet-deficient T cells. A recent report by Zimmermann et al.52 found that antibody-mediated blockade of IFN-γ ameliorates colitis induced by WT or T-bet-deficient T cells suggesting IFN-γ also contributes to the colitogneic response mediated by T-bet-deficient T cells as originally described for WT T cells53, 54. By contrast with our results the Zimmerman study found that IL-17A blockade exacerbated colitis following transfer of Tbx21−/− T cells. The reason for the differential role of IL-17A in the two studies is not clear but it is notable that the Zimmerman study was performed in the presence of co-infection with SFB and Hh, and this strong inflammatory drive may alter the pathophysiological role of particular cytokines. Together the data indicate that T-bet deficiency in T cells does not impede their colitogenic activity but that the downstream effector cytokines of the response are context dependent.
In conclusion, our data further underline the essential role for IL-23 in intestinal inflammation and demonstrate that T-bet is an important modulator of the IL–23-driven effector T cell response. The colitogenic T cell response in a T-bet sufficient environment is multifunctional with a dominant GM-CSF and IFN-γ response. By contrast T-bet-deficient colitogenic responses are dominated by IL-17A and IL-22-mediated immune pathology. These results may have significant bearing on human IBD where it is now recognized that differential responsiveness to treatment may reflect considerable disease heterogeneity. As such, identification of suitable biomarkers such as immunological parameters, that allow stratification of patient groups, is becoming increasingly important55. Genome-wide association studies have identified polymorphisms in loci related to innate and adaptive immune arms that confer increased susceptibility to IBD. Among these are Th1 (STAT4, IFNG and STAT1) as well as Th17-related genes (RORC, IL23R and STAT3) (refs56, 57). Thus, detailed profiling of the T cell response in IBD patients may help identify appropriate patient groups that are most likely to benefit from therapeutic blockade of certain effector cytokines. Finally, our studies highlight the importance of IL-23 in the intestinal inflammatory hierarchy and suggest that IL-23 could be an effective therapeutic target across a variety of patient groups.
Yale study: How antibodies access neurons to fight infection
Yale scientists have solved a puzzle of the immune system: how antibodies enter the nervous system to control viral infections. Their finding may have implications for the prevention and treatment of a range of conditions, including herpes and Guillain-Barre syndrome, which has been linked to the Zika virus.
Many viruses — such as West Nile, Zika, and the herpes simplex virus — enter the nervous system, where they were thought to be beyond the reach of antibodies. Yale immunobiologists Akiko Iwasaki and Norifumi Iijima used mice models to investigate how antibodies could gain access to nerve tissue in order to control infection.
In mice infected with herpes, they observed a previously under-recognized role of CD4 T cells, a type of white blood cell that guards against infection by sending signals to activate the immune system. In response to herpes infection, CD4 T cells entered the nerve tissue, secreted signaling proteins, and allowed antibody access to infected sites. Combined, CD4 T cells and antibodies limited viral spread.
“This is a very elegant design of the immune system to allow antibodies to go to the sites of infection,” said Iwasaki. “The CD4 T cells will only go to the site where there is a virus. It’s a targeted delivery system for antibodies.”
Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help
Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood–brain barrier1 and blood–nerve barrier2, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.
T Cells Help Reverse Ovarian Cancer Drug Resistance
T cells (red) attack ovarian cancer cells (green). [University of Michigan Health System]
Researchers at the University of Michigan have recently published the results from a new study that they believe underscores why so many ovarian tumors develop resistance to chemotherapy. The tumor microenvironment is made up of an array of cell types, yet effector T cells and fibroblasts constitute the bulk of the tissue. The investigators believe that understanding the interplay between these two cell types holds the key to how ovarian cancer cells develop resistance.
The new study suggests that the fibroblasts surrounding the tumor work to block chemotherapy, which is why nearly every woman with ovarian cancer becomes resistant to treatment. Conversely, the scientists published evidence that T cells in the microenvironment can reverse the resistance phenotype—suggesting a whole different way of thinking about chemotherapy resistance and the potential to harness immunotherapy drugs to treat ovarian cancer.
“Ovarian cancer is often diagnosed at late stages, so chemotherapy is a key part of treatment,” explained co-senior study author J. Rebecca Liu, M.D., associate professor of obstetrics and gynecology at the University of Michigan. “Most patients will respond to it at first, but everybody develops chemoresistance. And that’s when ovarian cancer becomes deadly.”
Dr. Liu continued, stating that “in the past, we’ve thought the resistance was caused by genetic changes in tumor cells. But we found that’s not the whole story.”
The University of Michigan team looked at tissue samples from ovarian cancer patients and separated the cells by type to study the tumor microenvironment in vitro and in mice. More importantly, the scientists linked their findings back to actual patient outcomes.
The results of this study were published recently in Cell through an article entitled “Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.”
Ovarian cancer is typically treated with cisplatin, a platinum-based chemotherapy. The researchers found that fibroblasts blocked platinum. These cells prevented platinum from accumulating in the tumor and protected tumor cells from being killed off by cisplatin.
Diagram depicting how T cells can reverse chemotherapeutic resistance. [Cell, Volume 165, Issue 5, May 19, 2016]
“We show that fibroblasts diminish the nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy,” the authors wrote. “We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance.”
T cells, on the other hand, overruled the protection of the fibroblasts. When researchers added the T cells to the fibroblast population, the tumor cells began to die off.
“CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts,” the authors explained. “CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc−cystine and glutamate antiporter via the JAK/STAT1 pathway.”
By boosting the effector T cell numbers, the researchers were able to overcome the chemotherapy resistance in mouse models. Moreover, the team used interferon, an immune cell-secreted cytokine, to manipulate the pathways involved in cisplatin.
“T cells are the soldiers of the immune system,” noted co-senior study author Weiping Zou, M.D., Ph.D., professor of surgery, immunology, and biology at the University of Michigan. “We already know that if you have a lot of T cells in a tumor, you have better outcomes. Now we see that the immune system can also impact chemotherapy resistance.”
The researchers suggest that combining chemotherapy with immunotherapy may be effective against ovarian cancer. Programmed death ligand 1 (PD-L1) and PD-1 pathway blockers are currently FDA-approved treatments for some cancers, although not ovarian cancer.
“We can imagine re-educating the fibroblasts and tumor cells with immune T cells after chemoresistance develops,” Dr. Zou remarked.
“Then we could potentially go back to the same chemotherapy drug that we thought the patient was resistant to. Only now we have reversed that, and it’s effective again,” Dr. Liu concluded.
Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer
•Fibroblasts diminish platinum content in cancer cells, resulting in drug resistance
•GSH and cysteine released by fibroblasts contribute to platinum resistance
•T cells alter fibroblast GSH and cystine metabolism and abolish the resistance
•Fibroblasts and CD8+ T cells associate with patient chemotherapy response
Summary
Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc− cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8+ T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation. Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α.. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation. CD28 co-stimulation further activates the PI3K/Akt/mTOR pathway in particular, and provides a signal for Glut1 expression and cell surface localization. Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway. The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions
UPDATE 6/11/2021
Bispecific Antibodies Emerging as Effective Cancer Therapeutics
Science 28 May 2021: Vol. 372, Issue 6545, pp. 916-917 DOI: 10.1126/science.abg1209
Bispecific antibodies (bsAbs) bind two different epitopes on the same or different antigens. Through this dual specificity for soluble or cell-surface antigens, bsAbs exert activities beyond those of natural antibodies, offering numerous opportunities for therapeutic applications. Although initially developed for retargeting T cells to tumors, with a first bsAb approved in 2009 (catumaxomab, withdrawn in 2017), exploring new modes of action opened the door to many additional applications beyond those of simply combining the activity of two different antibodies within one molecule. Examples include agonistic “assembly activities” that mimic the activity of natural ligands and cofactors (for example, factor VIII replacement in hemophilia A), inactivation of receptors or ligands, and delivery of payloads to cells or tissues or across biological barriers. Over the past years, the bsAb field transformed from early research to clinical applications and drugs. New developments offer a glimpse into the future promise of this exciting and rapidly progressing field.
Monoclonal antibodies (mAbs) comprise antigen-binding sites formed by the variable domains of the heavy and light chain and an Fc region that mediates immune responses. BsAbs, produced through genetic engineering, combine the antigen-binding sites of two different antibodies within one molecule, with a plethora of formats available (1). Conceptually, one can discriminate between bsAbs with combinatorial modes of action where the antigen-binding sites act independently from each other, and bsAbs with obligate modes of action where activity needs binding of both, either in a sequential (temporal) way or dependent on the physical (spatial) linkage of both (see the figure) (2). BsAbs approved as drugs are so far in the obligate dual-binding category: A T cell recruiter (blinatumomab) against cancer and a factor VIIIa mimetic to treat hemophilia A (emicizumab). Most but not all of the more than 100 bsAbs in clinical development address cancers. Some are in late stage (such as amivantamab, epcoritamab, faricimab, and KNO46), but most are still in early stages (2). Most of these entities enable effector cell retargeting to induce target cell destruction.
An increasing number of programs also explore alternative modes of action. This includes bsAbs that target pathways involved in tumor proliferation (such as amivantamab), invasion, ocular angiogenesis (such as faricimab), or immune regulation by blocking receptors and/or ligands, mainly in a combinatorial manner. Challenges for all of these entities are potential adverse effects, toxicity in normal tissues, and overshooting and systemic immune responses, especially with T cell retargeting or immune-modulating or activating entities. Such issues need to be carefully addressed.
Most of the bispecific T cell engagers comprise a binding site for a tumor-associated antigen and CD3 [a component of the T cell receptor (TCR) activation complex] as trigger molecule on T cells. To prevent or ameliorate “on-target, off-tumor” effects of T cell recruiters, approaches currently investigated include the modulation of target affinities and mechanisms to allow conditional activation upon target cell binding. Thus, a reduced affinity for CD3 increased tolerability by reducing peripheral cytokine concentrations that are associated with nonspecific or overshooting immune reactions (3). Similarly, reduced affinity for the target antigen was shown to ameliorate cytokine release and damage of target-expressing tissues (4). Tumor selectivity can be further increased by implementing avidity effects—for example, by using 2+1 bsAb formats with two low-affinity binding sites for target antigens and monovalent binding to CD3 (4).
In further approaches, binders to CD3 were identified that efficiently trigger target cell destruction without inducing undesired release of cytokines, demonstrating the importance of epitope specificity to potentially uncouple efficacy from cytokine release (5). Complementing these T cell–recruiting principles, the nonclassical T cell subset of γ9d2 T cells with strong cytotoxic activity emerged as potent effectors, which can be retargeted with bsAbs binding to the γ9d2 TCR. Thereby, global activation of all T cells, including inhibitory regulatory T cells (Treg cells), through CD3 binding, may be avoided (6). However, even these approaches might result in a narrow therapeutic window to treat solid tumors because of T cell activation in normal tissues.
Consequently, there are several approaches to conditionally activate T cells within tumors, including a local liberation of the CD3-binding sites or triggering local assembly of CD3-binding sites from two half-molecules. For example, CD3-binding sites have been masked by fusing antigen binding or blocking moieties—such as peptides, aptamers, or anti-idiotypic antibody fragments—to one or both variable domains. These moieties are released within the tumor by tumor-associated proteases, or through biochemical responses to hypoxia or low pH (7). This approach can also be applied to confer specific binding of antibody therapeutics, including bsAbs, to antigens on tumor cells (8).
An on-target restoration of CD3-binding sites requires application of two target-binding entities, each comprising parts of the CD3-binding site, which assemble into functional binding sites upon close binding of both half-antibodies. The feasibility of this approach was recently shown, for example, for a split T cell–engaging antibody derivative (Hemibody) that targets a cell surface antigen (9). Such approaches can also be applied to half-antibodies that recognize two different targets expressed on the same cell, further increasing tumor selectivity.
Regarding T cell engagers, increasing efforts are made to target not only cell-surface antigens expressed on tumor cells but also human leukocyte antigen (HLA)–presented tumor-specific peptides. This expands the target space of bsAbs toward tumor-specific intracellular antigens and can be achieved by using either recombinant TCRs or antibodies with TCR-like specificities combined with, for example, CD3-binding arms to engage T cell responses. A first TCR–anti-CD3 bispecific molecule is in phase I and II trials to treat metastatic melanoma (10). A challenge of this approach is the identification of TCRs or TCR-like antibodies that bind the peptide in the context of HLA with high affinity and specificity, without cross-reacting with related peptides to reduce or avoid off-target activities. Comprehensive screening tools and implementation of computational approaches are being developed to achieve this task.
A rapidly growing area of bsAbs in cancer therapy is their use to foster antitumor immune responses. Here, they are especially applied for dual inhibition of checkpoints that prevent immune responses—for example, programmed cell death protein 1 (PD-1) and its ligand (PD-L1), cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), or lymphocyte activation gene 3 (LAG-3; for example, KNO46). Tumor-targeted bsAbs can also target costimulatory factors such as CD28 or 4-1BB ligand (4-1BBL) to enhance T cell responses when combined with PD-1 blockade or to provide an activity-enhancing costimulatory signal in combination with CD3-based bsAbs (11). Furthermore, bsAbs are being developed for local effects by targeting one arm to antigens that are expressed by tumor cells or cells of the tumor microenvironment (2).
Clinical application of bsAbs now expands to other therapeutic areas, including chronic inflammatory, autoimmune, and neurodegenerative diseases; vascular, ocular, and hematologic disorders; and infections. In contrast to mAbs, bsAbs can inactivate the signaling of different cytokines with one molecule to treat inflammatory diseases (12). Simultaneous dual-target binding is not essential to elicit activity for bsAbs against combinations of proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1α (IL-1α), IL-1β, IL-4, IL-13, IL-17, inducible T cell costimulator ligand (ICOSL), or B cell–activating factor (BAFF). This presumably also applies to blockade of immune cell receptors, although dual targeting might confer increased efficacy due to avidity effects and increased selectivity through simultaneous binding of two different receptors.
A further application of combinatorial dual targeting is in ophthalmology. Loss of vision in wet age-related macular degeneration (AMD) results from abnormal proliferation and leakiness of blood vessels in the macula. This can be treated with antibodies that bind and inactivate factors that stimulate their proliferation (13). In contrast to mAbs or fragments that recognize individual factors, bsAbs bind two such factors. For example, faricimab that binds vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (ANG2), demonstrated dual efficacy in preclinical studies, and is currently in phase 3 trials.
BsAbs with obligate modes of action that mandate simultaneous dual-target binding are “assemblers” that replace the function of factors necessary to form functional protein complexes. One of these bsAbs with an assembly role (emicizumab, approved in 2018) replaces factor VIIIa in the clotting cascade. Deficiency of factor VIII causes hemophilia A, which can be overcome by substitution with recombinant factor VIII. However, a proportion of patients develop factor VIII–neutralizing immune responses and no longer respond to therapy. To overcome this, a bsAb was developed with binding sites that recognize and physically connect factors IXa and X, a process normally mediated by factor VIIIa. Extensive screening of a large set of bsAbs was required to identify those that combine suitable epitopes with optimized affinities and geometry to serve as functional factor VIIIa mimetics (14). This exemplifies the complexity of identifying the best bsAb for therapeutic applications.
A mode of action requiring sequential binding of two targets is the transport of bsAbs across the blood-brain barrier (BBB). This is a tight barrier of brain capillary endothelial cells that controls the transport of substances between the blood and the cerebrospinal fluid—the brain parenchyma. Passage of large molecules, including antibodies, across the BBB is thereby restricted. Some proteins, such as transferrin or insulin, pass through the BBB by way of transporters on endothelial cells. Antibodies that bind these shuttle molecules, such as the transferrin receptor (TfR), can hitchhike across the BBB. BsAbs that recognize brain targets (such as β-amyloid for Alzheimer’s disease) and TfR with optimized affinities, epitopes, and formats can thereby enter the brain. Such bsAbs are currently in clinical evaluation to treat neurodegenerative diseases (15).
In the past years, there has been a transition from a technology-driven phase, solving hurdles to generate bsAbs with defined composition, toward exploring and extending the modes of action for new therapeutic options. The challenge of generating bsAbs is not only to identify suitable antigen pairs to be targeted in a combined manner. It is now recognized that the molecular composition has a profound impact on bsAb functionality (13). That more than 30 different bsAb formats are in clinical trials proves that development is now driven by a “fit for purpose” or “format defines function” rationale. Many candidates differ in their composition, affecting valency, geometry, flexibility, size, and half-life (1). Not all members of this “zoo of bsAb formats” qualify to become drugs. Strong emphasis is therefore on identifying candidates that exhibit drug-like properties and fulfill safety, developability, and manufacturability criteria. There is likely to be an exciting new wave of bsAb therapeutics available in the coming years.
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