Robust Memory Responses Research Articles

A Near-Infrared II Photo-Triggered Multifunctional Plasmonic Hyperthermia Immunomodulator for SERS-Guided Combination Cancer Immunotherapy.

Immunotherapy represents a promising therapeutic strategy for cancer treatment, but its clinical applications are currently hindered by insufficient therapeutic potency, nonspecific delivery, and adverse side effects. Herein, a novel near-infrared II (NIR-II) photo-triggered plasmonic hyperthermia immunomodulator (RP@IR-pcNS@HA nanoparticles (NPs)) for anticancer treatment of both primary and distant cancers is reported. This immunomodulator comprises an IR-1061 dye-encoded NIR-II porous cubic AuAg nanoshell (pcNS) loaded with a Toll-like receptor 7 agonist - R837 in phase change materials (PCMs), further modified with hyaluronic acid (HA). In response to NIR-II photoirradiation, the RP@IR-pcNS@HA NPs controllably deliver and release R837 to tumor sites, subsequently perform plasmonic hyperthermia therapy for direct ablation of primary tumors, and elicit robust anticancer immune responses. It is demonstrated that upon NIR-II irradiation, such a plasmonic hyperthermia immunomodulator combined with anti-programmed death 1 antibody (αPD-1) completely eradicates both primary and distant cancers. In addition, this combination treatment successfully elicits robust immune memory responses for effective suppression of recurrence and distant metastasis of cancer. With the excellent NIR-II surface-enhanced Raman scattering (SERS) detection ability, the RP@IR-pcNS@HA NPs combined with αPD-1 represent an efficient way to develop high-performance theranostic agents for SERS-guided combination cancer photoimmunotherapy.

Intranasal Immunization for Zika in a Pre-Clinical Model.

Humans continue to be at risk from the Zika virus. Although there have been significant research advancements regarding Zika, the absence of a vaccine or approved treatment poses further challenges for healthcare providers. In this study, we developed a microparticulate Zika vaccine using an inactivated whole Zika virus as the antigen that can be administered pain-free via intranasal (IN) immunization. These microparticles (MP) were formulated using a double emulsion method developed by our lab. We explored a prime dose and two-booster-dose vaccination strategy using MPL-A® and Alhydrogel® as adjuvants to further stimulate the immune response. MPL-A® induces a Th1-mediated immune response and Alhydrogel® (alum) induces a Th2-mediated immune response. There was a high recovery yield of MPs, less than 5 µm in size, and particle charge of -19.42 ± 0.66 mV. IN immunization of Zika MP vaccine and the adjuvanted Zika MP vaccine showed a robust humoral response as indicated by several antibodies (IgA, IgM, and IgG) and several IgG subtypes (IgG1, IgG2a, and IgG3). Vaccine MP elicited a balance Th1- and Th2-mediated immune response. Immune organs, such as the spleen and lymph nodes, exhibited a significant increase in CD4+ helper and CD8+ cytotoxic T-cell cellular response in both vaccine groups. Zika MP vaccine and adjuvanted Zika MP vaccine displayed a robust memory response (CD27 and CD45R) in the spleen and lymph nodes. Adjuvanted vaccine-induced higher Zika-specific intracellular cytokines than the unadjuvanted vaccine. Our results suggest that more than one dose or multiple doses may be necessary to achieve necessary immunological responses. Compared to unvaccinated mice, the Zika vaccine MP and adjuvanted MP vaccine when administered via intranasal route demonstrated robust humoral, cellular, and memory responses. In this pre-clinical study, we established a pain-free microparticulate Zika vaccine that produced a significant immune response when administered intranasally.

Abstract 2652: NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade

Abstract Introduction HPK1 is a member of the MAP4K family of protein serine/threonine kinases that negatively regulates activation signals in multiple immune cells and is an attractive therapeutic target for many cancers. Using structure-based drug design, we developed a highly selective HPK1 inhibitor, NDI-101150, with nanomolar potency and physiochemical properties suitable for once daily oral administration. Methods & Results HPK1 inhibition with NDI-101150 has a differentiated pharmacology profile when compared directly to PD1 blockade. NDI-101150 was able to overcome the immunosuppressive effects of prostaglandin E2, adenosine, and TGF-β on human T cell activation. HPK1 inhibition also directly limited the suppressive capacity of T regulatory cells. In contrast, anti-PD1 treatment was not able to overcome the immunosuppressive activity associated with any of these mechanisms. In vivo, HPK1 inhibition significantly enhanced the production of antigen-specific antibodies in response to immunization with both T-dependent and T-independent antigens, while anti-PD1 treatment had minimal to no effects on antibody production. In the EMT-6 syngeneic model, 7/10 mice receiving NDI-101150 exhibited complete tumor regressions, compared to only 1/10 from an anti-PD1 cohort. Furthermore, NDI-101150 established a robust and durable immune memory response as 100% of treated mice showed complete rejection of tumor growth upon subsequent re-challenge. HPK1 inhibition with NDI-101150 also shows robust synergy with PD1 blockade in vitro and in vivo. Both NDI-101150 and anti-PD1 were able to reinvigorate exhausted human T cells, restoring cytokine production and proliferative effects in mixed lymphocyte reactions. Further, in combination HPK1 inhibition and PD1 blockade synergized to enhance the activity of exhausted human T cells to an extent exceeding that observed with naïve human T cells in similar experiments. In vivo, while NDI-101150 or anti-PD1 treatments alone induced tumor growth inhibition in the murine CT-26 syngeneic tumor model, NDI-101150 and anti-PD1 in combination mediated complete tumor regressions in several mice. Furthermore, NDI-101150 seemed to induce a durable immune memory response as evidenced by the combination-treated animals showing complete rejection of tumor growth upon subsequent re-challenge, without any further dosing of NDI-101150. Conclusions Pharmacological inhibition of HPK1 with NDI-101150 represents a powerful system-wide immunomodulatory approach with the potential to enhance anti-tumor immunity in patients failing to respond to currently approved immune checkpoint therapies or in combination with immune checkpoint blockade therapies. NDI-101150 is currently being tested in a Phase 1/2 trial (NCT05128487) in patients with advanced solid tumors. Citation Format: David Ciccone, Fu-Shan Kuo, Scott Daigle, Neelu Kaila, Gene Yau, Mark Carlson, Denise Levasseur, Bhaskar Srivastava, Frank G. Basile, Christine Loh. NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2652.

Notch2 controls developmental fate choices between germinal center and marginal zone B cells upon immunization

Sustained Notch2 signals induce trans-differentiation of Follicular B (FoB) cells into Marginal Zone B (MZB) cells in mice, but the physiology underlying this differentiation pathway is still elusive. Here, we demonstrate that most B cells receive a basal Notch signal, which is intensified in pre-MZB and MZB cells. Ablation or constitutive activation of Notch2 upon T-cell-dependent immunization reveals an interplay between antigen-induced activation and Notch2 signaling, in which FoB cells that turn off Notch2 signaling enter germinal centers (GC), while high Notch2 signaling leads to generation of MZB cells or to initiation of plasmablast differentiation. Notch2 signaling is dispensable for GC dynamics but appears to be re-induced in some centrocytes to govern expansion of IgG1+ GCB cells. Mathematical modelling suggests that antigen-activated FoB cells make a Notch2 dependent binary fate-decision to differentiate into either GCB or MZB cells. This bifurcation might serve as a mechanism to archive antigen-specific clones into functionally and spatially diverse B cell states to generate robust antibody and memory responses.

Characterization, functional validation, and analysis of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 6 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors over multiple infections may create a narrowed repertoire consisting of a few clonotypes, covering a narrow range of antigenic space representing the mutant epitopes encountered. Our data shows that cross-reactive TCRs are more similar to each other than single epitope specific cells and that cross-reactivity can be replicated during peptide stimulation when cross-reactive TCRs are transduced into TCR null cell lines invitro. Given the narrowing nature of cross-reactive responses elicited over different infections, selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools. Supported by NIH Grant R01112404040 and the St. Jude Graduate School of Biomedical Sciences

Shape memory mechanical metamaterials

Shape memory materials can maintain temporary shapes without external constraints and revert to their permanent shape upon exposure to an external stimulus, such as heat, light, or moisture. This behavior, often named the shape memory effect, has led to the use of shape memory materials in a variety of applications including deployable aerospace structures, biomedical devices, flexible electronics, and untethered soft robots. Most thermally triggered reconfigurable metamaterials using shape memory polymers require a laborious process of thermomechanical programming at high temperature, above their transition value, to maintain a temporary shape. In this paper, we utilize two 3D-printable polymeric materials that do not rely upon their shape memory effect to generate robust shape memory response in a set of mechanical metamaterials. The enabling characteristic is the mismatch of the temperature-dependent moduli of the constitutive materials leveraged in rationally interconnected reconfigurable units, and their hallmark is the freedom to forego the complex programming process of typical shape memory polymers. Their shape reconfiguration and rapid recovery are solely governed by mechanical loading and temperature change, leading to sequentially programmable multistability, hyperelasticity, giant thermal deformations, and shape memory capacity. Theoretical models, numerical simulations, and thermomechanical experiments are performed to demonstrate their functionality, stability transition mechanism, and potential applications.

Upper respiratory tract mucosal immunity for SARS-CoV-2 vaccines.

SARS-CoV-2 vaccination significantly reduces morbidity and mortality, but has less impact on viral transmission rates, thus aiding viral evolution, and the longevity of vaccine-induced immunity rapidly declines. Immune responses in respiratory tract mucosal tissues are crucial for early control of infection, and can generate long-term antigen-specific protection with prompt recall responses. However, currently approved SARS-CoV-2 vaccines are not amenable to adequate respiratory mucosal delivery, particularly in the upper airways, which could account for the high vaccine breakthrough infection rates and limited duration of vaccine-mediated protection. In view of these drawbacks, we outline a strategy that has the potential to enhance both the efficacy and durability of existing SARS-CoV-2 vaccines, by inducing robust memory responses in the upper respiratory tract (URT) mucosa.

Boosting compromised SARS-CoV-2-specific immunity with mRNA vaccination in liver transplant recipients

Liver transplant recipients (LTRs) demonstrate a reduced response to COVID-19 mRNA vaccination; however, a detailed understanding of the interplay between humoral and cellular immunity, especially after a third (and fourth) vaccine dose, is lacking. We longitudinally compared the humoral, as well as CD4+ and CD8+ T-cell, responses between LTRs (n= 24) and healthy controls (n= 19) after three (LTRs: n= 9 to 16; healthy controls: n= 9 to 14 per experiment) to four (LTRs: n= 4; healthy controls: n= 4) vaccine doses, including in-depth phenotypical and functional characterization. Compared to healthy controls, development of high antibody titers required a third vaccine dose in most LTRs, while spike-specific CD8+ T cells with robust recall capacity plateaued after the second vaccine dose, albeit with a reduced frequency and epitope repertoire compared to healthy controls. This overall attenuated vaccine response was linked to a reduced frequency of spike-reactive follicular T helper cells in LTRs. Three doses of a COVID-19 mRNA vaccine induce an overall robust humoral and cellular memory response in most LTRs. Decisions regarding additional booster doses may thus be based on individual vaccine responses as well as evolution of novel variants of concern. Due to immunosuppressive medication, liver transplant recipients (LTR) display reduced antibody titers upon COVID-19 mRNA vaccination, but the impact on long-term immune memory is not clear. Herein, we demonstrate that after three vaccine doses, the majority of LTRs not only exhibit substantial antibody titers, but also a robust memory T-cell response. Additional booster vaccine doses may be of special benefit for a small subset of LTRs with inferior vaccine response and may provide superior protection against evolving novel viral variants. These findings will help physicians to guide LTRs regarding the benefit of booster vaccinations.

Longitudinal Analysis of the Phenotype, Transcriptional Profile, and Anatomic Location of Memory CD8 T Cell Subsets after Acute Viral Infection.

Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1+IL-7Rα- terminal effectors (TEs) and KLRG1-IL-7Rα+ memory precursors (MPs). But we also characterized a third KLRG1+IL-7Rα+ subset which we refer to as KLRG1+ MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1+ MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1+ MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.

Gambogic Acid Induces Pyroptosis of Colorectal Cancer Cells through the GSDME-Dependent Pathway and Elicits an Antitumor Immune Response.

Pyroptosis is a recently identified form of programmed cell death (PCD) that exerts a vital influence on the antitumor immune response. GA, a xanthone structure isolated from gamboge resin, is a naturally occurring bioactive ingredient with several anticancer activities, such as activities that affect cell cycle arrest, apoptosis, and autophagy. Here, we found that GA decreased the viability of the CRC cell lines, HCT116 and CT26, in a dose- and time-dependent manner, and multiple pores and large bubbles in the membranes, which are morphological characteristics of pyroptosis, were observed by light microscopy and transmission electron microscopy (TEM). Furthermore, the cleavage of gasdermin E (GSDME) was observed after exposure to GA, along with concomitant activation of caspase-3. Additionally, GSDME-dependent pyroptosis triggered by GA could be attenuated by siRNA-mediated knockdown of GSDME and treatment with the caspase-3 inhibitor. Moreover, we found that GA induced pyroptosis and significantly inhibited tumor growth in CT26 tumor-bearing mice. Strikingly, significantly increased proportions of CD3+ T cells, cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) were observed in the tumor microenvironment in the GA-treated groups. Moreover, significantly increased proportions of CTLs and effector memory T cells (TEM) (CD8+ CD44+ CD62L-) were also detected in the spleens of the GA-treated groups, suggesting that the pyroptosis-induced immune response generated a robust memory response that mediated protective immunity. In this study, we revealed a previously unrecognized mechanism by which GA induces GSDME-dependent pyroptosis and enhances the anticancer immune response. Based on this mechanism, GA is a promising antitumor drug for CRC treatment that induces caspase-3-GSDME-dependent pyroptosis. This study provides novel insight into cancer chemoimmunotherapy.

Early and Long-Term HIV-1 Immunogenicity Induced in Macaques by the Combined Administration of DNA, NYVAC and Env Protein-Based Vaccine Candidates: The AUP512 Study.

To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection.

How do plants remember drought?

Plants develop both short-term and transgenerational memory of drought stress through epigenetic regulation of transcription for a better response to subsequent exposure. Recurrent spells of droughts are more common than a single drought, with intermittent moist recovery intervals. While the detrimental effects of the first drought on plant structure and physiology are unavoidable, if survived, plants can memorize the first drought to present a more robust response to the following droughts. This includes a partial stomatal opening in the watered recovery interval, higher levels of osmoprotectants and ABA, and attenuation of photosynthesis in the subsequent exposure. Short-term drought memory is regulated by ABA and other phytohormone signaling with transcriptional memory behavior in various genes. High levels of methylated histones are deposited at the drought-tolerance genes. During the recovery interval, the RNA polymerase is stalled to be activated by a pause-breaking factor in the subsequent drought. Drought leads to DNA demethylation near drought-response genes, with genetic control of the process. Progenies of the drought-exposed plants can better adapt to drought owing to the inheritance of particular methylation patterns. However, a prolonged watered recovery interval leads to loss of drought memory, mediated by certain demethylases and chromatin accessibility factors. Small RNAs act as critical regulators of drought memory by altering transcript levels of drought-responsive target genes. Further studies in the future will throw more light on the genetic control of drought memory and the interplay of genetic and epigenetic factors in its inheritance. Plants from extreme environments can give queues to understanding robust memory responses at the ecosystem level.

Characterization of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. The number of possible peptides presented to the immune system exceeds 1015, and this number is greater than the number of possible T cells the immune system can generate and sustain. Thus, a certain portion of the T cells in an immune system must be cross-reactive. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools. Supported by grants from NIH (R01 112404040) and the St. Jude Children's Research Hospital Graduate School

LncRNA-targeting bio-scaffold mediates triple immune effects for postoperative colorectal cancer immunotherapy

Colorectal cancer (CRC) recurrence after surgical resection results in poor clinical outcomes. Long noncoding RNAs (lncRNAs) are emerging new targets for mediating tumorigenesis and immunosuppression within tumor microenvironment. We develop a bio-scaffold encapsulating lncRNA-targeting biomimetic nanosystems for mediating triple immune effects against postoperative CRC recurrence. Liposome (termed as “D”)-decorated CRC cells’ membrane (CM) envelops a plasmid encoding a short hair-pinned RNA (shRNA) against plasmacytoma variant translocation 1 (Pvt1), forming the shPvt1-CM-D nanosystem. This nanosystem and the chemodrug Oxaliplatin (Oxa) are embedded in a hyaluronic acid and alginate-based bio-scaffold for postoperative implantation. (1) ShPvt1-CM-D-mediated Pvt1 knockdown strengthens Oxa-induced immunogenic cell death (ICD). (2) Such tumor antigens released from enhanced ICD and the CM from shPvt1-CM-D act as dual vaccines of dendritic cells. (3) Pvt1 knockdown by shPvt1-CM-D within granulocytic myeloid-derived suppressor cells (G-MDSCs) ameliorates G-MDSC-mediated immunosuppression. The nanosystem-carrying bio-scaffold significantly suppresses perioperative CRC local recurrence by 97.8% with survival rate (SR) of 62.5%. The bio-scaffold generates robust immune memory responses for completely suppressing tumor ectopic rechallenging and metachronous metastasis (SR: 100%). Additionally, the bio-scaffold reduces synchronously distant metastasis by 70.8%. This work presents a potent nanotechnology-facilitated lncRNA-targeting immunotherapy for postoperative CRC treatments.

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.

MDNA11 is a long-acting IL-2 superkine that potentiates durable immune response in mouse tumor models and non-human primates.

e14546 Background: The efficacy and safety of recombinant human IL-2 (rhIL-2; Proleukin) to treat certain cancers is limited by a short half-life, marked toxicity and selective high affinity binding to IL2Ra over IL2Rb, resulting in preferential activation of suppressive Tregs. In contrast, MDNA11 has been engineered as a long-acting IL-2 superkine with high affinity IL2Rb receptor selectivity, resulting in preferential anti-cancer effector immune cell activation. Methods: MDNA11 was characterized in both in vitro and in vivo studies including assessment of receptor binding kinetics using BLI/Octet, receptor-mediated signaling in human PMBCs, efficacy in syngeneic mouse tumor models including memory response, as well as safety and PK/PD assessments in non-human primates (NHP). Results: Unlike rhIL-2, MDNA11 does not bind to human IL2Ra but demonstrates a 30-fold higher affinity binding to human IL2Rb. This selectivity resulted in enhanced in vitro STAT5 signaling in human NK and resting CD8 T cells with diminished signaling in Tregs; validation studies in humanized mice are ongoing. In CT26 and MC38 syngeneic tumor models, MDNA11 demonstrates potent and durable efficacy as monotherapy following a Q1W dose schedule for 2 weeks. Synergy with anti-PD1 and anti-CTLA4 immune checkpoint inhibitors (ICIs) was observed and a robust immune memory response developed in all mice with complete tumor clearance. These mice were protected against relapse and tumor re-challenges for up to 8 months without any further treatment, and showed the presence of antigen-specific CD8 T cells. In binding studies with IL-2 receptors of different species, MDNA11 showed highly similar affinity towards human and cynomolgus IL2Rb, confirming the latter as a highly relevant model for toxicology study. MDNA11 was well tolerated in cynomolgus monkeys up to 0.6 mg/kg, while inducing durable (≥10 days) proliferation and expansion of NK and CD8 T cells. Effects on Tregs were minimal and there was no eosinophilia and hypotension (associated with vascular leak syndrome). At high doses of MDNA11, the most common clinical observations were transient loss of appetite and diarrhea. There was modest increase in levels of IFNg and TNFa, but no sign of cytokine release syndrome. Dosing did not trigger development of anti-drug antibodies or histopathologic evidence of pulmonary edema (a major IL-2 induced toxicity). Conclusions: MDNA11 is a long-acting IL-2 superkine that exhibits robust efficacy in mouse tumor models as a single agent and was synergistic in combination with ICIs (anti-CTLA4 and anti-PD1). In NHP, MDNA11 demonstrates selective immune effector cell activation and a favorable safety profile. These data constitute a strong framework for the design of a pivotal GLP toxicology study to further support the planned clinical study of MDNA11 either as a single agent or in combination with ICIs.

Characterization of the cross reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract Influenza A viruses (IAVs) are a major respiratory pathogen with the capacity to rapidly mutate and escape an individual’s existing immunity to a specific strain generated by vaccination or naturally acquired infection. CD8 T cells however, target conserved proteins that could be shared across multiple subtypes. Heterosubtypic immunity to IAVs, where memory T cells generated to a previous IAV are reactivated during re-challenge with an antigenically distinct virus, may be important in generating an effective immune response to different and/or novel strains of IAV. Specifically, cross reactive T cells, where a T cell receptor (TCR) can recognize more than one epitope displayed on an MHC-I molecule, could facilitate rapid recognition of conserved or nominally divergent epitopes, may have the potential to alter the TCR repertoire over the course of heterosubtypic infections, and might be selected for by the immune system. Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, even if the epitope has slightly mutated. However, selection for cross reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape and are shaped by these selected cross-reactive T cell pools.

Abstract B21: Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent

Abstract Background: Immune checkpoint blockade, specifically targeting PD-1/PD-L1, is a promising approach for cancer therapy. However, for some common cancers, such as epithelial ovarian cancer (EOC), PD1/PDL1 blockade as monotherapy has a low response rate. In these less immunogenic cancer types, focus has turned to combining checkpoint inhibitors with cancer therapies that elicit immunologic cell death (ICD). Antibody-drug conjugates (ADCs), comprising a tumor-targeting antibody armed with highly potent small-molecule payloads, are a clinically validated anticancer therapy. Mirvetuximab soravtansine is an ADC comprised of a folate receptor alpha (FRα)-binding antibody linked to the potent tubulin-disrupting maytansinoid DM4 via a cleavable disulfide sulfo-SPDB linker and is currently being evaluated in combination with pembrolizumab (anti-PD1 antibody) in a phase 1b study (FORWARDII, NCT02606305). Here we report preclinical studies that provide a mechanistic understanding for the antitumor activity of combination therapy consisting of PD-1/PD-L1 blockade and an FRα targeting disulfide linked DM4 ADC. Methods: A chimeric anti-mouse FRα antibody (rmFR1-12) was generated and its specificity for murine FRα was assessed by ELISA and FACS. rmFR1-12 was conjugated to DM4 via a s-SPDB linker to generate rmFR-1-12-s-SPDB-DM4 ADC, which was quality checked using standard analytical methods. In vitro assays were used to assess cytotoxicity against FRα-positive cell lines, as well as the ability of rmFR1-12-s-SPDB-DM4 to induce ICD and engage cells of the immune system. In vivo studies performed in albino C57/B6 mice evaluated the antitumor activity of rmFR1-12-s-SPDB-DM4, anti-PD-1 or the combination therapy against ID8 tumors, a translationally relevant murine model of EOC. Results: rmFR1-12 binding was specific for FRα. The rmFR1-12-s-SPDB-DM4 ADC was cytotoxic against FRα-positive cell lines and induced ICD as measured by upregulation of ICD markers. In vivo studies using syngeneic ID8 tumors demonstrated that rmFR1-12-s-SPDB-DM4 or anti-PD-1 monotherapy treatment resulted in modest antitumor activity. In contrast, combination treatment demonstrated synergistic antitumor activity, inducing complete tumor regressions and increased survival over control or monotherapy regimens. Depletion of CD8+ cells abrogated the antitumor activity of the combination, whereas depletion of CD4+ cells had no impact. Finally, combination-treated mice generated a robust memory response when rechallenged with ID8 cells. Conclusion: Anti-FRα-s-SPDB-DM4 induces ICD in FRα-positive cell lines and demonstrates synergistic antitumor activity when combined with anti-PD1 in a murine EOC model. The antitumor immunity is dependent upon CD8+ cells. These findings support the clinical assessment of mirvetuximab soravtansine and pembrolizumab. Citation Format: L. Cristina Gavrilescu, Yulius Setiady, Ling Dong, Sharon Chicklas, Luke Harris, Jan Pinkas, Richard Gregory, Joe Ponte. Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B21.

Icaritin Exacerbates Mitophagy and Synergizes with Doxorubicin to Induce Immunogenic Cell Death in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) resistant to both chemotherapy and immunotherapy is among the deadliest malignancies. Doxorubicin widely used in transarterial chemotherapy in HCC can induce immunogenic cell death (ICD), but the resulting immunogenicity is still weak. We aim to seek a strategy for improving the efficacy of ICD in HCC based on an immunoregulatory drug called icaritin. Icaritin induced mitophagy and apoptosis to provoke ICD both in mouse Hepa1-6 and human Huh7 HCC cells. A combination of icaritin and doxorubicin with a molar ratio of 1:2 played a synergistic role in ICD induction. The poly lactic-co-glycolic acid (PLGA)-polyethylene glycol (PEG)-aminoethyl anisamide (AEAA) nanoparticle (NP) targeted codelivery of icaritin and doxorubicin remodeled the immunosuppressive tumor microenvironment and triggered a robust immune memory response, which efficiently improved anti-HCC effect at an early stage in mouse HCC model. In addition, the combo PLGA-PEG-AEAA NP together with lenvatinib significantly prolonged survival time of mice at the advanced stage of HCC. Collectively, our findings reveal an anti-HCC mechanism of icaritin on mitophagy and provide an effective immune-based therapeutic strategy for HCC.

Effect of HIV exposure and timing of antiretroviral therapy initiation on immune memory responses to diphtheria, tetanus, whole cell pertussis and hepatitis B vaccines

ABSTRACT Objectives: We evaluated memory responses and antibody persistence to diphtheria-toxoid, tetanus-toxoid, whole-cell-pertussis (DTwP), and Hepatitis-B vaccines in HIV-unexposed, HIV-exposed-uninfected and HIV-infected children previously randomized to initiate time-limited ART at 6–10 weeks (ART-Immed) or when clinically/immunologically indicated (ART-Def). Methods: All children received DTwP booster at 15–18 months. Antibodies were measured for pertussis-toxoid, filamentous haemagglutinin (FHA), diphtheria-toxoid, tetanus-toxoid, and hepatitis-B prior to booster, 1–2 weeks post-booster and at 24 months of age. Results: Pre-booster antibody GMC were lower in HIV-infected groups than HIV-unexposed children for all epitopes. Post-booster and at 24 months of age, the ART-Def group had lower GMCs and antibody proportion ≥0.1 IU/ml for tetanus-toxoid and diphtheria-toxoid compared to HIV-unexposed children. At 24 months of age, the ART-Immed group had higher GMCs, and more likely to maintain antibody titres ≥1.0 IU/ml to tetanus-toxoid and diphtheria-toxoid compared to HIV-unexposed children. Compared to HIV-unexposed children, at 15 and 24 months of age, persistence of antibody to HBsAg of ≥10 mIU/ml was similar in the ART-Immed group but lower among the ART-Def group. Antibody kinetics indicated more robust memory responses in HIV-exposed-uninfected than HIV-unexposed children to diphtheria-toxoid and wP. Conclusion: HIV-infected children not on ART at primary vaccination had poorer memory responses, whereas HIV-exposed-uninfected children mounted robust memory responses.