Formation Of Immunological Memory Research Articles

A-263 Single-cell multiomics identifies formation of immunologic memory in CTCL - transition from blood to skin

A-263 Single-cell multiomics identifies formation of immunologic memory in CTCL - transition from blood to skin

Longitudinal tracking of T-cell repertoire reveals long-lasting CD4⁺ yellow fever specific clone cluster

Infection is inconceivable without T cells. T cells not only eliminate virus-infected cells and participate in the formation of immunological memory, but also indirectly modulate the humoral response through the selection and maintenance of specific B cells. The T-cell receptor (TCR) recognizes processed antigen presented on the surface of cells in the MHC of one of two classes. Thus, the formed TCR repertoire reflects the history of encountered antigens through the prism of the specific organism with a particular set of MHC. To investigate changes in the TCR repertoire in response to acute viral infection, we utilized a yellow fever vaccination model. The yellow fever vaccine has been a benchmark for both safety and efficacy for over half a century. The vaccine is based on a live attenuated virus, allowing the study of the immune response under conditions closely to the viral infection. The yellow fever-specific T-cell response to immunodominant peptides presented on HLA-A02 is well studied, but experiments with HLA-A02-negative donors are still lacking. The aim of this study was to examine the dynamics of changes in the T-cell repertoire structure that occur in response to yellow fever vaccination in a donor without the HLA-A02 allele. We found that the overall T-cell response dynamics were similar to that in HLA-A02-positive donors: vaccination led to rapid expansion of yellow fever-reactive clones by day 14. Despite the absence of a known immunodominant epitope for HLA I alleles in this donor, the immune response also shifted towards CD8⁺ T cells, with increasing of the CD8⁺ clones fraction by day 53. The amino acid sequences of CDR3 TCRb yellow fever specific clones formed a stable cluster by CD4⁺ T cells, further confirming the presence of novel immunogenic epitopes.

Formation of immunological memory of salmonella antigens in cows using different phenotypes of T-lymphocyte populations

The study aimed to investigate the formation of immunological memory to Salmonella antigens in cows using different phenotypes of T-lymphocyte populations. The study was conducted on farms in Kazakhstan and Latvia. As part of the study, blood samples were collected from cows vaccinated against Salmonella to analyse T-lymphocyte phenotypes. A comparative study of different phenotypes of T-cell populations, including CD4+, CD8+, γδ T-cells and effector T-cells (CD45RA+ and CD45RA-), was conducted to assess their role in the formation of immunological memory. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to quantify T-cell populations and levels of specific antibodies. The main results showed that vaccination causes a significant increase in the population of CD4+ and CD8+ T-lymphocytes, which correlates with an increase in the level of specific antibodies to Salmonella antigens. The γδ T-cell population also showed significant activation, indicating their potential role in the formation of an immune response to Salmonella. Particular attention was devoted to effector cells of T-lymphocytes: CD45RA+ cells were actively involved in the primary immune response to Salmonella antigens, while CD45RA cells showed high activity during repeated exposure to the antigen, indicating their key role in maintaining longterm immunological memory. Comparative analysis of data from Kazakhstan and Latvia showed similar trends, indicating the universality of the observed immunological mechanisms. These findings highlight the importance of including different T-cell phenotypes in vaccination programmes to ensure more effective immunological memory in cows. Thus, this study provides valuable data on the role of different T-lymphocyte populations in the formation of immunological memory to Salmonella antigens in cows, which may contribute to the development of more effective vaccine programmes and improve the health and productivity of livestock in the regions of Kazakhstan and Latvia

Plasmid DNA ionisable lipid nanoparticles as non-inert carriers and potent immune activators for cancer immunotherapy

Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo.In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.

Abstract 7577: A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation

Abstract Cancer cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Uncontrolled cancer cell proliferation, as well as immune suppressive macrophage proliferation, contribute to tumor growth and spread. Effort to suppress tumor cell growth using Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth, but resistance develops due to upregulation of the alternative pathway. We identified a novel series of allosteric tyrosine kinase receptor inhibitors that induce synthetic lethality in AML, lung carcinoma, pancreatic carcinoma, and other tumor cells and in proliferating macrophages in vitro and in vivo by simultaneously inhibiting cMyc and Tor and activating the stress activated kinase JNK, leading to p53 mediated cell cycle arrest. Select kinase inhibitors with a unique positioning of a single side chain inhibited CDK8/19 and thereby Myc while stimulating phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting both tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Jason Rivera, Marc Paradise, Zihan Xu, Giuliana Mognol, Judith Varner. A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation [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 7577.

Recent advances in sialic acid-based active targeting chemoimmunotherapy promoting tumor shedding: a systematic review.

Tumors have always been a major public health concern worldwide, and attempts to look for effective treatments have never ceased. Sialic acid is known to be a crucial element for tumor development and its receptors are highly expressed on tumor-associated immune cells, which perform significant roles in establishing the immunosuppressive tumor microenvironment and further boosting tumorigenesis, progression, and metastasis. Obviously, it is essential to consider sophisticated crosstalk between tumors, the immune system, and preparations, and understand the links between pharmaceutics and immunology. Sialic acid-based chemoimmunotherapy enables active targeting drug delivery via mediating the recognition between the sialic acid-modified nano-drug delivery system represented by liposomes and sialic acid-binding receptors on tumor-associated immune cells, which inhibit their activity and utilize their homing ability to deliver drugs. Such a "Trojan horse" strategy has remarkably improved the shortcomings of traditional passive targeting treatments, unexpectedly promoted tumor shedding, and persistently induced robust immunological memory, thus highlighting its prospective application potential for targeting various tumors. Herein, we review recent advances in sialic acid-based active targeting chemoimmunotherapy to promote tumor shedding, summarize the current viewpoints on the tumor shedding mechanism, especially the formation of durable immunological memory, and analyze the challenges and opportunities of this attractive approach.

Dendritic Cells Pulsed with Tumor Lysates Induced by Tetracyanotetra(aryl)porphyrazines-Based Photodynamic Therapy Effectively Trigger Anti-Tumor Immunity in an Orthotopic Mouse Glioma Model.

Research in the past decade on immunogenic cell death (ICD) has shown that the immunogenicity of dying tumor cells is crucial for effective anticancer therapy. ICD induction leads to the emission of specific damage-associated molecular patterns (DAMPs), which act as danger signals and as adjuvants to activate specific anti-tumor immune responses, leading to the elimination of tumor cells and the formation of long-term immunological memory. ICD can be triggered by many anticancer treatment modalities, including photodynamic therapy (PDT). However, due to the variety of photosensitizers used and the lack of a universally adopted PDT protocol, there is a need to develop novel PDT with a proven ICD capability. In the present study, we characterized the abilities of two photoactive dyes to induce ICD in experimental glioma in vitro and in vivo. One dye was from the tetracyanotetra(aryl)porphyrazine group with 9-phenanthrenyl (pz I), and the other was from the 4-(4-fluorobenzyoxy)phenyl (pz III) group in the aryl frame of the macrocycle. We showed that after the photosensitizers penetrated into murine glioma GL261 cells, they localized predominantly in the Golgi apparatus and partially in the endoplasmic reticulum, providing efficient phototoxic activity against glioma GL261 cells upon light irradiation at a dose of 20 J/cm2 (λex 630 nm; 20 mW/cm2). We demonstrated that pz I-PDT and pz III-PDT can act as efficient ICD inducers when applied to glioma GL261 cells, facilitating the release of two crucial DAMPs (ATP and HMGB1). Moreover, glioma GL261 cells stimulated with pz I-PDT or pz III-PDT provided strong protection against tumor growth in a prophylactic subcutaneous glioma vaccination model. Finally, we showed that dendritic cell (DC) vaccines pulsed with the lysates of glioma GL261 cells pre-treated with pz-I-PDT or pz-III-PDT could act as effective inducers of adaptive anti-tumor immunity in an intracranial orthotopic glioma mouse model.

Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.

Abstract 1669: In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor

Abstract Tumor cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Unchecked tumor cell proliferation, as well as macrophage proliferation, contributes to tumor growth and spread. Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth until resistance due to upregulation of the alternative pathway develops. We identified a novel allosteric cKIT/FLT3/CSF1R inhibitor that induces synthetic lethality in AML, lung carcinoma, pancreatic carcinoma and other tumor cells and in proliferating macrophages in vitro and in tumors in vivo by simultaneously inhibiting the MEK-ERK and PI3K-AKT pathways and activating stress activated kinase-mediated cell cycle arrest. A series of related cKIT/FLT3/CSF1R/PDGFR allosteric inhibitors suppressed receptor tyrosine phosphorylation as well as downstream MEK-ERK and PI3K-AKT signaling and mTorc1 and cMyc gene expression pathways that control cell growth. Select cKIT/FLT3/CSF1R/PDGFR inhibitors with a unique positioning of a single side chain also inhibited CDK8/19 and stimulated phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Marc Paradise, Ryan Shepard, Judith Varner. In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1669.

Sonodynamic-immunomodulatory nanostimulators activate pyroptosis and remodel tumor microenvironment for enhanced tumor immunotherapy.

Rationale: Spatiotemporal control of pyroptosis has a profound impact on cancer immunotherapy. Owing to the precise spatiotemporal control and reduction in the side effects of ultrasound (US), sonodynamic therapy (SDT) is expected to be a promising mean to activate pyroptosis. Furthermore, the pyroptosis-initiated immune response can be amplified by enhanced lymphocyte infiltration occurring due to extracellular matrix (ECM) depletion. Therefore, it is highly desirable to develop a sonodynamic-immunomodulatory strategy to amplify pyroptosis-mediated tumor immunotherapy by remodeling of the tumor microenvironment, thereby enhancing tumor immunotherapy. Methods: We reported a potent strategy based on a sonosensitizer, which is composed of LY364947-loaded porous coordination network (PCN-224) camouflaged with a red blood cell (RBC) membrane and evaluated pyroptosis activation, collagen depletion, immunocyte infiltration, and adaptive immune response during the pyroptosis-initiated immune response in vitro and in vivo. Results: The sonosensitizer generated reactive oxygen species (ROS) under US irradiation and initiated the caspase-3 apoptotic signaling pathway, which is regarded as the key upstream activator of gasdermin E (GSDME)-mediated pyroptosis. During the subsequent anti-tumor immune response mediated by pyroptosis, LY364947 loosened the ECM structure via collagen depletion, resulting in enhanced T-lymphocyte infiltration and nearly complete eradication of tumors in a mouse model with the formation of immunological memory. Conclusion: Our findings indicate that sonodynamic-immunomodulatory pyroptotic strategy exhibits robust anti-tumor immune efficacy as well as provides novel insights into the role of pyroptosis in cancer immunology.

Immunomodulatory effects of microbiota-derived metabolites at the crossroad of neurodegenerative diseases and viral infection: network-based bioinformatics insights.

Bidirectional cross-talk between commensal microbiota and the immune system is essential for the regulation of immune responses and the formation of immunological memory. Perturbations of microbiome-immune system interactions can lead to dysregulated immune responses against invading pathogens and/or to the loss of self-tolerance, leading to systemic inflammation and genesis of several immune-mediated pathologies, including neurodegeneration. In this paper, we first investigated the contribution of the immunomodulatory effects of microbiota (bacteria and fungi) in shaping immune responses and influencing the formation of immunological memory cells using a network-based bioinformatics approach. In addition, we investigated the possible role of microbiota-host-immune system interactions and of microbiota-virus interactions in a group of neurodegenerative diseases (NDs): Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). Our analysis highlighted various aspects of the innate and adaptive immune response systems that can be modulated by microbiota, including the activation and maturation of microglia which are implicated in the development of NDs. It also led to the identification of specific microbiota components which might be able to influence immune system processes (ISPs) involved in the pathogenesis of NDs. In addition, it indicated that the impact of microbiota-derived metabolites in influencing disease-associated ISPs, is higher in MS disease, than in AD, PD and ALS suggesting a more important role of microbiota mediated-immune effects in MS.

Mechanisms of formation of hybrid immunity in people who recovered from COVID-19 and were vaccinated with SARS-CoV-2 peptide antigens

The relevance of the current epidemic situation of a new coronavirus infection is determined by new strains of the virus and the registration of cases of re-infection in COVID-19 survivors earlier. In this regard, the questions about the expediency and nature of vaccination of those who have been ill attract close attention, moreover it has affected the formation of the concept of “hybrid immunity”. The aim of this study was to analyze changes in the parameters of the immune system, reflecting their regulatory and functional potential, in response to the introduction of the peptide vaccine EpiVacCorona to persons who have suffered from the new coronavirus infection. To study the features of the formation of hybrid immunity, a retrospective analysis of the observation of 43 study participants was carried out. The inclusion criteria were data confirming COVID-19 in mild and moderate forms of the course in the period from six months to a year ago, a low level or absence of antibodies to the nucleocapsid protein SARS-CoV-2, a negative PCR result for the presence of the SARS-CoV-2 virus, the absence of comorbid pathology. The subpopulation composition, regulatory and functional potential of the immune system were determined by flow cytofluorimetry using a set of monoclonal antibodies corresponding to the goals. 21 days after the administration of a single dose of EpiVacCorona, antibodies to the vaccine peptide antigens were registered in all study participants at the highest coefficient of positivity values for the SARS-CoV-2-IgG-Vector test system used. In addition, there was a fourfold increase in the number of specific IgG to the N protein. A specific immune response to recombinant SARS-CoV-2 antigens was accompanied by a decrease in the circulation of the number of monocytes expressing TLR4, T helper cells expressing the interaction coreceptor with antigen-presenting cells, unconnected B memory with an increase in the number of B lymphocytes expressing the CD40 T-B coreceptor interaction molecule. The remaining differences in the functioning of the immune system identified in patients with COVID-19 before the vaccination in comparison with the control data have not changed. The differences consist in a decrease in the proportion of monocytes expressing HLA-DR, an increase in the expression of interaction molecules on T and B lymphocytes, an increase in the number of Treg, B1 cells, activated B lymphocytes with a decrease in the proportion of suppressor Breg and B memory. The totality of the presented data demonstrates that the COVID-19 infection that preceded vaccination in mild and moderate clinical course contributes to the formation of immunological memory, which made it possible to form a secondary immune response even to a single injection of peptide antigens of the virus.

Abstract 5616: Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate

Abstract Traditional antibody-drug conjugates (ADCs) selectively deliver cytotoxic payloads to tumor-associated antigen (TAA)-expressing tumor cells, thereby limiting healthy cell damage and toxicities associated with systemic administration. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to achieve tumor-targeted activation of anti-tumor immune responses, avoiding dose-limiting immunotoxicities seen with the systemic immune agonist delivery. Most of the ADC or ISAC to date utilize random payload conjugation to native surface-exposed lysines or cysteines, and relatively labile linkage chemistries, leading to conjugation site and drug-to-antibody ratio (DAR) heterogeneity, and ADC/ISAC instability in systemic circulation. In particular, the presence of high-DAR species and linker instability can lead to off-tumor toxicity, thus limiting the therapeutic window. Here we describe the generation and preclinical characterization of a site-specific TLR7-agonist (TLR7a) ISAC targeting an undisclosed TAA1. To specify the conjugation site and DAR, a non-natural amino acid (nnAA), para-acetyl-L-phenylalanine (pAF) was genetically incorporated at defined sites within the anti-TAA1 antibody, providing an orthogonal chemical handle for covalent TLR7 agonist payload conjugation through a highly stable oxime bond. Using this site-specific, homogenous ISAC platform, we systematically screened different conjugate sites and TLR7 agonist payloads to obtain an optimized TAA1-TLR7a ISAC drug candidate. ISAC platform stability, homogeneity, and flexibility were critical, because conjugation site and payload structure dramatically influenced ISAC in vitro activity, PK profile and in vivo efficacy. Using in vitro co-culture assays with human immune cells and tumor cell lines, we showed that the optimized TAA1 ISAC can induce multiple anti-tumor immune mechanisms, including proinflammatory cytokine production, the myeloid cell activation markers induction and enhanced ADCC mediated tumor cell killing. The TAA1 ISAC is 100-fold more active than the free, unconjugated TLR7 agonist, and all activity is conditional on the presence of TAA1-expressing tumor cells. Finally, we demonstrated in vivo efficacy in both xenograft and syngeneic tumor models. In a syngeneic MC38-tumor model expressing human TAA1, ISAC treatment led to complete tumor regression and formation of immunologic memory. These results provide a strong rationale for site-specific TLR7 agonist ISAC as a next generation platform for tumor-targeted, innate immune agonist immunotherapy. Citation Format: Mingchao Kang, Sung-Ju Moon, Ji Young Kim, Andy Beck, Molly Allen, Jay Nelson, Keith Tatsukawa, Hon Tran, Manoj Pal, Michael Gray, Nick Knudsen, Lillian Skidmore, David Mills, Yingchun Lu, Ying Buechler, Sukumar Sakamuri, Shawn Zhang, Feng Tian. Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5616.

Abstract 5591: Anti-FolRα ADC STRO-002 induces immunogenic cell death (ICD) to enhance anti-tumor activity

Abstract STRO-002 is an antibody-drug conjugate (ADC) composed of an anti-folate receptor alpha (FolRα) antibody conjugated to a tubulin-targeting hemiasterlin (SC209) warhead via a cleavable linker currently in clinical studies for ovarian and endometrial cancer. FolRα exhibits minimal expression in normal tissues but is overexpressed in several cancers such as ovarian, endometrial, and NSCLC. We previously showed that STRO-002 induces in vitro hallmarks of immunogenic cell death (ICD), such as increased calreticulin exposure and HMGB1 and ATP release in a FolRα dependent manner. Follow-up vaccination studies with murine MC38 tumor cells engineered to express human FolRα (MC38-hFolRa) were performed to further explore the significance of STRO-002-induced ICD and protective immunity in vivo. A single vaccination with STRO-002 or SC209 in vitro-treated MC38-hFolRa cells in immunocompetent mice resulted in 60% of animals rejecting a subsequent rechallenge with living MC38-hFolRa cells, while relapse was observed in most control mice. Including an additional booster vaccination further increased the proportion ( 90%) of animals remaining tumor free after rechallenge. These results demonstrate that tumor cells pre-treated with STRO-002 or SC209 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo. The ability of STRO-002 to induce ICD that elicits an effective and robust anti-tumor immune response may translate into several advantages in the clinic. Induction of ICD is likely to offer a complementary anti-tumor mechanism in combination therapy regimens such as with check point inhibitors or VEGF blockade. We have demonstrated that STRO-002 in combination with avelumab in MC38-hFolRα-bearing mice significantly enhanced efficacy and durable anti-tumor immunity. More recent rechallenge studies reveal that this combination treatment suppresses growth of not only MC38-hFolRα but also MC38 wild-type tumor cells, indicating formation of immunological memory with signs of epitope spreading. Additionally, co-administration of STRO-002 with anti-VEGF therapy demonstrates improved TGI compared to monotherapy (p < 0.0001) in human ovarian OV-90 tumors. The potent cytotoxic and immunostimulatory properties of STRO-002 may also contribute to enhancing efficacy in indications with low FolRα expression. Such tumor types include endometrial and NSCLC, in which STRO-002 has displayed potent monotherapy activity in preclinical studies. In a panel of endometrial PDX models, STRO-002 was significantly efficacious in 9/17 (53%) FolRα-positive tumors, including models with moderate and weak FolRα levels. In a NSCLC PDX model, a single dose of STRO-002 induced tumor regression and suppressed tumor growth for up to three months post-dose. In conclusion, the induction of ICD by STRO-002 has potential to increase anti-tumor activity in cancers with low FolRα expression. Citation Format: Cristina L. Abrahams, Andrew McGeehan, Jennifer Smith, Robert Yuan, Kshama A. Doshi, Sihong Zhou, Xiaofan Li, Cuong Tran, Gang Yin, Arturo Molina, Kristin Bedard, Trevor Hallam. Anti-FolRα ADC STRO-002 induces immunogenic cell death (ICD) to enhance anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5591.

Targeting intrinsic regulators of antibody and B cell memory to control chronic viral infection

Abstract The production of antibody and formation of immunological memory underpins vaccine success. Yet, chronic viral infections result in an inadequate humoral response, and little is known about the mechanisms underpinning this dysregulation. We therefore utilised acute or chronic strains of lymphocytic choriomeningitis virus (LCMV) to investigate B cell behaviour in vivo during chronic infection. Our work revealed that chronic infection altered epigenetic regulation of B cell fate, and a key epigenetic target could be therapeutically modulated to correct antibody responses. Deep-sequencing revealed key differences in chromatin accessibility of B cells responding to acute vs chronic infection, leading us to investigate the Polycomb repressive complex 1 component BMI-1. BMI-1 was upregulated in germinal centre B cells of mice infected with chronic LCMV, compared to acute LCMV. B cell-intrinsic deletion of BMI-1 resulted in a restoration of splenic architecture, and an enhanced and rapid ability to clear virus. Deletion of BMI-1 re-established appropriate c-Myc expression in B cells, concomitant with improved quality of antibody with enhanced antibody-dependent effector function and increased neutralising capacity. Together, our data defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection. Further, single-cell RNA-seq and genetic models revealed that excess immune complexes, in concert with dysregulated complement-mediated functions, contribute to ineffective memory formation during chronic viral infection. Understanding the molecular events that may sustain dysfunction lays the foundation for improving therapeutic interventions in chronic infectious diseases. Supported by Bellberry-Viertel Senior Medical Research Fellowship and an AAI Careers in Immunology Fellowship.

Gut microbial influences on the adaptive immune system and the development of cow milk allergy.

Allergic diseases constitute significant health and economic issues in both developed and developing nations, with epidemiological studies demonstrating a rapid increase in the global prevalence of food allergy among the pediatric population. Cow milk protein allergy (CMPA), one of the most common forms of food allergies observed in early childhood, affects between 2%–6% of infants and children under 3 years of age. CMPA can present as either an IgE-mediated atopic allergy or a non-IgE mediated allergic response. Antigen-specific T cells play a pivotal role in directing the type of inflammatory immune response that occurs as well as in the formation of immunological memory. IgE-mediated CMPA is thought to develop because of an abnormal expansion of allergen-specific type-2 helper T (Th2) cells and a corresponding deficiency in immune regulation by regulatory T cells (Tregs), thereby altering the Th2/Treg balance. The gut microbiota, established very early during childhood through host-microbe interactions, can influence the incidence of allergic diseases. In this study, we aimed to analyze both the microbiome composition and CD4+T cell differentiation patterns in pediatric patients with and without cow milk allergy to establish the association between these factors. Using 16S rRNA sequencing, we analyzed the microbiome composition in stool samples of allergic and non-allergic pediatric patients aged between 1–4 years and identified the microbial species abundant in IgE and non-IgE mediated cow milk allergies. To assess the CD4+T cell differentiation patterns, peripheral blood mononuclear cells (PBMCs) from these patients were re-stimulated with cow milk antigen, and T cell subsets were assessed using flow cytometry. Antigen-specific CD4+T cells were identified and sorted for high throughput sequencing and subsequent gene expression analysis. The CD4+T cell differentiation patterns of the total and antigen-specific T cells were analyzed and statistically compared with controls. The identification of the correlation between the CD4+T cell differentiation patterns and species-specific microbial abundance in IgE and non-IgE mediated cow milk allergies can help in determining how the gut microbiome influences the CD4+T cell immune compartment development, ultimately leading to the development of cow milk allergy in pediatric patients.

Local therapy with an engineered oncolytic adenovirus enables antitumor response in non-injected melanoma tumors in mice treated with aPD-1

ABSTRACT Intratumoral immunotherapies are entering clinical use but concerns remain regarding their effects on non-injected tumors. Here, we studied the impact of local treatment with an adenovirus coding for TNFa and IL-2 on systemic antitumor response in animals receiving aPD-1 (anti-programmed cell death protein 1) therapy. Using bilateral murine melanoma models, we tested systemic tumor response to combined therapy with anti-PD-1 and an adenovirus coding for TNFa and IL-2 (“virus”). Virus was given intratumorally (to one of the two tumors only) and aPD-1 monoclonal antibody systemically. We evaluated both tumors’ response to treatment, overall survival, metastasis development, and immunological mechanisms involved with response. Consistent tumor control was observed in both injected and non-injected tumors, including complete response in all treated animals receiving aPD-1+ virus therapy. Mechanistically, virus injections enabled potent effector lymphocyte response locally, with systemic effects in non-injected tumors facilitated by aPD-1 treatment. Moreover, adenovirus therapy demonstrated immunological memory formation. Virus therapy was effective in preventing metastasis development. Local treatment with TNFa and IL-2 coding adenovirus enhanced systemic response to aPD-1 therapy, by re-shaping the microenvironment of both injected and non-injected tumors. Therefore, our pre-clinical data support the rationale for a trial utilizing a combination of aPD-1 plus virus for the treatment of human cancer.

Immune Metabolism of IL-4-Activated B Cells and Th2 Cells in the Context of Allergic Diseases.

Over the last decades, the frequency of allergic disorders has steadily increased. Immunologically, allergies are caused by abnormal immune responses directed against otherwise harmless antigens derived from our environment. Two of the main cell types driving allergic sensitization and inflammation are IgE-producing plasma cells and Th2 cells. The acute activation of T and B cells, their differentiation into effector cells, as well as the formation of immunological memory are paralleled by distinct changes in cellular metabolism. Understanding the functional consequences of these metabolic changes is the focus of a new research field termed “immune metabolism”. Currently, the contribution of metabolic changes in T and B cells to either the development or maintenance of allergies is not completely understood. Therefore, this mini review will introduce the fundamentals of energy metabolism, its connection to immune metabolism, and subsequently focus on the metabolic phenotypes of IL-4-activated B cells and Th2 cells.

Evolution of anti-modified protein antibody responses can be driven by consecutive exposure to different post-translational modifications

BackgroundBesides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is “fixed” in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM.MethodsLongitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses.ResultsHuman data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the “booster” PTM.ConclusionsThe relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

Abstract P258: CBX-12 (alphalexTM-exatecan) sensitizes tumors to immune checkpoint blockade in an antigen agnostic manner by immune activation

Abstract Immune checkpoint blockade (ICB) in combination with chemotherapy is standard of care for several solid tumors. However, potent chemotherapies such as topoisomerase inhibitors can result in severe dose-limiting toxicities requiring dose reduction, limiting their efficacy. Moreover, leukocytopenia from chemotherapy induces immunosuppression that further limits the full potential of combinations with ICB. The use of targeted chemotherapies such as antibody-drug conjugates (ADCs) demonstrate potential synergy with ICB, however are limited to a defined subset of patients with tumors expressing the target antigen. Cybrexa has reported the development of CBX-12, a peptide-drug conjugate consisting of alphalexTM-exatecan (1). Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide (pHLIP®; 2-4) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. The alphalexTM component of CBX-12 forms an alpha helix only in low pH conditions, allowing for directional insertion of the peptide within the cancer cell membrane, delivery of C-terminally linked exatecan across the membrane, and subsequent intracellular release of active exatecan via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. Here we evaluated the potential for CBX-12 to effectively synergize with PD1 and CTLA4 blockade in multiple syngeneic mouse models without requirement for tumor specific antigens. We found that CBX-12/ICB combination treatment significantly delayed tumor growth, improved survival and led to complete tumor regressions. Mice cured with combined CBX-12/ICB therapy demonstrated formation of long-term immunological memory after in vivo and ex vivo tumor rechallenge. The ability of CBX-12 to induce immunogenic cell death was confirmed by vaccinating syngeneic mice with CBX-12 treated tumor cells and subsequent tumor rechallenge, which demonstrated near total anti-tumor immunity induced by CBX-12. Together, these preclinical data demonstrate the potential for CBX-12 to enhance tumor immunogenicity and potentiate the efficacy of ICB in patients with solid tumors affording a superior, universal tumor targeting mechanism that bypasses the limitations of ADCs. 1. Gayle S et al. 2021.Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity. NAR Cancer. 2. Rather than targeting a specific antigen, alphalexTM includes a pHLIP® peptide. pHLIP® peptides are a family of pH-Low Insertion Peptides that target acidic cell surfaces. pHLIP® was developed at Yale University and the University of Rhode Island, and is exclusively licensed to pHLIP, Inc. 3. Wyatt LC et al. 2017. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotech. 4. Wyatt LC et al. 2018. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. PNAS. Citation Format: Sophia Gayle, Juan Vasquez, Timothy Paradis, Kelli Jones, Ranjini Sundaram, Jinny van Doorn, Viswanathan Muthusamy, Ranjit S. Bindra, Robert J. Aiello, Vishwas Paralkar. CBX-12 (alphalexTM-exatecan) sensitizes tumors to immune checkpoint blockade in an antigen agnostic manner by immune activation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P258.