Immune Antibodies Research Articles

Evolution of antibody immunity following Omicron BA.1 breakthrough infection

Understanding the longitudinal dynamics of antibody immunity following heterologous SAR-CoV-2 breakthrough infection will inform the development of next-generation vaccines. Here, we track SARS-CoV-2 receptor binding domain (RBD)-specific antibody responses up to six months following Omicron BA.1 breakthrough infection in six mRNA-vaccinated individuals. Cross-reactive serum neutralizing antibody and memory B cell (MBC) responses decline by two- to four-fold through the study period. Breakthrough infection elicits minimal de novo Omicron BA.1-specific B cell responses but drives affinity maturation of pre-existing cross-reactive MBCs toward BA.1, which translates into enhanced breadth of activity across other variants. Public clones dominate the neutralizing antibody response at both early and late time points following breakthough infection, and their escape mutation profiles predict newly emergent Omicron sublineages, suggesting that convergent antibody responses continue to shape SARS-CoV-2 evolution. While the study is limited by our relatively small cohort size, these results suggest that heterologous SARS-CoV-2 variant exposure drives the evolution of B cell memory, supporting the continued development of next-generation variant-based vaccines.

Recruiting T-Cells toward the Brain for Enhanced Glioblastoma Immunotherapeutic Efficacy by Co-Delivery of Cytokines and Immune Checkpoint Antibodies with Macrophage-Membrane-Camouflaged Nanovesicles.

Immunotherapy with immune checkpoint inhibitors (CPIs) shows promising prospects for glioblastoma multiforme (GBM) but with restricted results, mainly attributed to the immunosuppressive tumor microenvironment (TME) and the limited antibody permeability of the blood-tumor barrier (BTB) in GBM. Here, nanovesicles with amacrophage-mimicking membrane are described, that co-deliverchemotactic CXC chemokine ligand 10 (CXCL10), to pre-activate the immune microenvironment, and anti-programmed death ligand 1 antibody (aPD-L1), to interrupt the immune checkpoint, aiming to enhance the effect of GBM immunotherapy. Consequently, the tumor tropism of the macrophage membrane and the receptor-mediated transcytosis of the angiopep-2 peptide allowthe nanovesicle to effectively cross the BTB and target the GBM region, with 19.75-fold higher accumulation of antibodies compared to the free aPD-L1 group. The CPI therapeutic efficacy is greatly enhanced by CXCL10-induced T-cells recruitment with significant expansion of CD8+ T-cells and effector memory T-cells, leading to the elimination of tumor, prolonged survival time, and long-term immune memory in orthotopic GBM mice. The nanovesicles, that relieve the tumor immunosuppressive microenvironment by CXCL10 to enhance aPD-L1 efficacy, may present a promising strategy for brain-tumor immunotherapy.

Antibodies for a bespoke plant immunity

Plants do not have antibodies. However, in a recent Science article, Kourelis and Marchal etal. have demonstrated that plant immune receptors can be retrofitted with animal antibodies to provide plants potentially with hundreds and thousands of options to perceive attacking microbes. This is the dawn of bespoke plant immunity.

Impact of Lymphatic Pump Treatment and Methotrexate on Inflammation, Cytokine Production, and Leukocyte Infiltration in a Rat Model of Rheumatoid Arthritis

Abstract Rheumatoid Arthritis in autoimmune disease afflicting synovial joints leading to chronic joint damage. While the specific mechanisms have not been fully identified, the immune system and inflammatory factors are actively involved. T cells release cytokines and chemokines, such as TNF-alpha, IL-1 and IL-6, CINC-1 and CINC-2, and B cells produce self-reactive antibodies including rheumatoid factor and anti-citrullinated protein antibodies. All these factors result in a pronounced and aggressive inflammatory response within the joints. Lymphatic pump treatment (LPT) promotes lymphatic flow within the body, causing a flushing out of the immune-complexes and pro-inflammatory cells. Using a rat AIA model, the goal was to determine if LPT in conjunction with methotrexate had a greater impact on rheumatoid arthritis symptoms than methotrexate alone. A 21-day AIA rat study was performed using LPT and methotrexate treatments on days 14–21. Ankle circumference and articular index scores were recorded daily as a measurement of inflammation. ELISAs of arthritic ankles homogenates and serum were used to analyze cytokine, chemokine, and autoantibody concentrations. Immune cell infiltration into the ankles was determined using immunohistochemistry. We found that LPT with methotrexate treatment did not significantly alter ankle circumference, articular index scores, immune cell infiltration, cytokine, chemokine, or antibody concentrations compared to methotrexate alone. Overall, methotrexate combined with LPT treatments on days 14–21 did not significantly alter disease progression, however, it is possible that earlier LPT treatment may have a more significant impact on disease progression. College of Graduate Studies Biomedical Sciences program at Midwestern University, Downers Grove, IL.

The anaphylatoxin C5a: Structure, function, signaling, physiology, disease, and therapeutics.

The complement system is one of the oldest known tightly regulated host defense systems evolved for efficiently functioning cell-based immune systems and antibodies. Essentially, the complement system acts as a pivot between the innate and adaptive arms of the immune system. The complement system collectively represents a cocktail of ∼50 cell-bound/soluble glycoproteins directly involved in controlling infection and inflammation. Activation of the complement cascade generates complement fragments like C3a, C4a, and C5a as anaphylatoxins. C5a is the most potent proinflammatory anaphylatoxin, which is involved in inflammatory signaling in a myriad of tissues. This review provides a comprehensive overview of human C5a in the context of its structure and signaling under several pathophysiological conditions, including the current and future therapeutic applications targeting C5a.

Intramuscular mRNA prime plus intranasal adenoviral vector booster elicit robust respiratory mucosal IgA responses against SARS-CoV-2

Abstract Current SARS-CoV-2 mRNA vaccines induce robust humoral and cellular immunity in the circulation, but its ability in eliciting respiratory mucosal immunity is less characterized. Here, we demonstrated that systemic mRNA expressing ancestral spike (mRNA-S) vaccination alone induced weak respiratory mucosal neutralizing antibody and cellular immunity, particularly against SARS-CoV-2 Omicron strain. In contrast, an immunization strategy combining systemic mRNA-S administration plus mucosal adenoviral vector expressing ancestral spike (Ad5-S) booster induced strong T cell, B cell, and antibody responses in the respiratory tract, which can last for a long time. Furthermore, we found that Ad5-S mucosal booster promoted robust IgA-producing B cells in the respiratory tract, which were correlated with the levels of mucosal S-specific IgA levels. Strikingly, these local IgA-producing B cells were more cross-reactive to the Delta and Omicron Spike proteins compared to those IgG-producing B cells. We further showed that CD4 T cell help was required for the development of IgA-producing B cell in the respiratory mucosa. Taken together, our study identified a vaccination strategy and its associated mechanisms for the induction of strong cross-reactive IgA responses, which were shown to correlate with optimal protection against breakthrough infection, especially by Omicron sub-lineages. Hence, our data provide insights into the rational design of next-generation SARS-CoV-2 mucosal vaccines required for the protection against infection by SARS-CoV-2 Omicron sub-lineages or future variants.

A case for teaching basic immunology through scientific journalism-lesson taught by a Coronavirus went viral!

Abstract Sudden switch to online mode of learning in the March of 2020 for a Basic Immunology course lead to a cascade of experimenting teaching strategies. We at College of Pharmacy Chicago State University experimented such innovative teaching techniques for two consecutive spring semesters. One such teaching method was to deliver course content material through use of mainstream media (MSM) via scientific journalism. Basic immunology course content was divided into media-inspired topics of public concern and the students were made to research and review selected topics before we discussed those in virtual classrooms. The most common topics included immunology terms (antigen, antibody, immune reaction, innate and adaptive immunity, immunology of infection, ARDS, non-pharmacological interventions (NPI) like lockdown, masking and herd immunity, development of vaccines and therapeutics of infectious diseases. The students researched those topics in daily news outlets, i.e., TV, Radio, Newspaper, Social media (YouTube, Facebook, Twitter, Instagram), and presented to the class the underlying and foundational immunology concepts making the breaking news. Subjective data was collected through student opinion surveys and objective data was analyzed through students’ performance, assessments, grades, and Course and Faculty evaluations. The study found a positive correlation between the use of scientific journalism as a teaching tool and students’ engagement, comprehension, retention and academic outcomes. The publications authored by Scientific Journalists presented the complex and complicated immunology material in a very simple and understandable way for the public, which complemented immunology students’ learning as well.

Sustained release hydrogel for durable locoregional chemoimmunotherapy for BRAF-mutated melanoma

The wide prevalence of BRAF mutations in diagnosed melanomas drove the clinical advancement of BRAF inhibitors in combination with immune checkpoint blockade for treatment of advanced disease. However, deficits in therapeutic potencies and safety profiles motivate the development of more effective strategies that improve the combination therapy's therapeutic index. Herein, we demonstrate the benefits of a locoregional chemoimmunotherapy delivery system, a novel thermosensitive hydrogel comprised of gelatin and Pluronic® F127 components already widely used in humans in both commercial and clinical products, for the co-delivery of a small molecule BRAF inhibitor with immune checkpoint blockade antibody for the treatment of BRAF-mutated melanoma. In vivo evaluation of administration route and immune checkpoint target effects revealed intratumoral administration of antagonistic programmed cell death protein 1 antibody (aPD-1) lead to potent antitumor therapy in combination with BRAF inhibitor vemurafenib. The thermosensitive F127-g-Gelatin hydrogel that was evaluated in multiple murine models of BRAF-mutated melanoma that facilitated prolonged local drug release within the tumor (>1 week) substantially improved local immunomodulation, tumor control, rates of tumor response, and animal survival. Thermosensitive F127-g-Gelatin hydrogels thus improve upon the clinical benefits of vemurafenib and aPD-1 in a locoregional chemoimmunotherapy approach for the treatment of BRAF-mutated melanoma.

Vaccination and Omicron BA.1/BA.2 Convalescence Enhance Systemic but Not Mucosal Immunity against BA.4/5.

Rising breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4/5 led to the performance of various studies investigating systemic immunity and neutralizing antibodies in sera, but mucosal immunity remains understudied. In this cohort study, the humoral immune responses, including immunoglobulin levels and the presence of virus-neutralizing antibodies, of 92 vaccinated and/or BA.1/BA.2 convalescent individuals were investigated. Cohorts received two doses of ChAdOx1, BNT162b2, or mRNA-1273 and subsequent booster vaccination with either BNT162b2 or mRNA-1273, following BA.1/BA.2 infection. In addition, vaccinated and nonconvalescent or unvaccinated and BA.1 convalescent individuals were studied. Serum and saliva samples were used to determine SARS-CoV-2 spike-specific IgG and IgA titers and neutralizing activity against replication-competent SARS-CoV-2 wild-type virus and the Omicron BA.4/5 variant. Vaccinated/convalescent cohorts demonstrated strongest neutralization against BA.4/5, with 50% neutralization titer (NT50) values reaching 174.2; however, neutralization was reduced up to 11-fold, compared to wild-type virus. Both BA.1 convalescent and vaccinated nonconvalescent cohorts displayed the weakest neutralization against BA.4/5, with NT50 values being reduced to 4.6, accompanied by lower numbers of positive neutralizers. Additionally, salivary neutralization against wild-type virus was strongest in vaccinated and BA.2 convalescent subjects, but this elevated neutralization efficiency was lost when challenged with BA.4/5. Our data support the contention that current coronavirus disease 2019 (COVID-19) vaccines efficiently induce humoral immunity. However, antiviral effectiveness in serum and saliva is greatly reduced against novel variants of concern. These results suggest an adjustment of current vaccine strategies to an adapted or alternative vaccine delivery, such as mucosal booster vaccinations, which might establish enhanced or even sterilizing immunity against novel SARS-CoV-2 variants. IMPORTANCE Rising incidences of breakthrough infections caused by SARS-CoV-2 Omicron BA.4/5 have been observed. Although various studies were conducted investigating neutralizing antibodies in sera, mucosal immunity was barely evaluated. Here, we investigated mucosal immunity, since the presence of neutralizing antibodies at mucosal entry sites plays a fundamental role in disease limitation. We found strong induction of serum IgG/IgA, salivary IgA, and neutralization against SARS-CoV-2 wild-type virus in vaccinated/convalescent subjects but detected 10-fold reduced (albeit positive) serum neutralization against BA.4/5. Interestingly, vaccinated and BA.2 convalescent patients demonstrated the greatest serum neutralization against BA.4/5, but this advantageous neutralizing effect was not observed in the saliva. Our data support the contention that current COVID-19 vaccines are very efficient against severe/critical disease progression. Moreover, these results suggest an adjustment of the current vaccine strategy to adapted and alternative vaccine delivery, such as mucosal booster vaccinations, to establish robust sterilizing immunity against novel SARS-CoV-2 variants.

Abstract LB102: Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model

Abstract Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p<0.001) and for HCB at 6.25 uM (p<0.001). Tregs decreased with HCB at a threshold of 12 uM (p <0.001) and with HBB at 25 uM (P < 0.001) while 14,15-EET increased Tregs (1.2 fold; P=0.02) and suppressed the CD8+:Treg ratio (0.79; P=0.02). In the SSM2ucd mammary allograft model, although HCB 12mg/kg daily did not inhibit tumor growth, it reduced intratumoral hypoxia by 20% (P=0.01), increased CD8+ TIL by 4.4-fold (P=0.04), decreased Treg:CD4+ TIL ratio by 76% (P=0.02), and decreased Treg:CD8+ TIL ratio by 88% (P=0.01) relative to control. Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.

Formulation Studies to Develop Low-Cost, Orally-Delivered Secretory IgA Monoclonal Antibodies for Passive Immunization Against Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common cause for diarrheal infections in children in low- and middle-income countries (LMICs). To date, no ETEC vaccine candidates have been approved. Passive immunization with low-cost, oral formulations of secretory IgA (sIgA) against ETEC is an alternative approach to protect high-risk populations in LMICs. Using a model sIgA monoclonal antibody (anti-LT sIgA2-mAb), the stability profiles of different formulations were assessed during storage and in in vitro digestion models (mimicking in vivo oral delivery). First, by employing various physicochemical techniques and a LT-antigen binding assay, three formulations with varying acid-neutralizing capacity (ANC) were evaluated to stabilize sIgA2-mAb during stress studies (freeze-thaw, agitation, elevated temperature) and during exposure to gastric phase digestion. Next, a low-volume, in vitro intestinal digestion model was developed to screen various additives to stabilize sIgA2-mAb in the intestinal phase. Finally, combinations of high ANC buffers and decoy proteins were assessed to collectively protect sIgA2-mAb during in vitro sequential (stomach to intestine) digestion. Based on the results, we demonstrate the feasibility of low-cost, ‘single-vial’, liquid formulations of sIgA-mAbs delivered orally after infant feeding for passive immunization, and we suggest future work based on a combination of in vitro and in vivo stability considerations.

Design and optimization of mRNAs encoding an Anti-TIGIT antibody with therapeutic potential for cancer in TIGIT-humanized BALB/c Mice

mRNA drugs are synthesized using cell-free systems without complex and stringent manufacturing processes, which makes their preparation simple, efficient, and economical. Over the past few years, mRNAs encoding antibodies have been one of the research frontiers of antibody drug development. In cancer immunotherapy, mRNAs encoding immune checkpoint antibodies may be advantageous regarding antibody persistence and durability of the anti-tumor immune response of patients. In our previous study, a candidate antibody—AET2010—targeting the novel immune checkpoint TIGIT was reported. Its anti-tumor activity was also investigated using adoptive transfer of NK-92MI cells in a xenograft mouse model, but the limitations of the model did not facilitate precise evaluation. In the present study, we further investigated the therapeutic potential of AET2010 for cancer in TIGIT-humanized BALB/c mice. Next, we explored the design, synthesis, and optimization of mRNAs encoding AET2010 and ultimately obtained a candidate mRNA (mRNA-BU) with favorable in vitro and in vivo expression levels of active AET2010. Particularly, lipid-nanoparticle-encapsulated mRNA-BU delivered to mice produced AET2010 with significantly higher peak concentration and expression duration than an equivalent dose of original AET2010. This study provides a sound basis for developing novel drugs targeting TIGIT.

Guanidine-modified nanoparticles as robust BTZ delivery carriers and activators of immune responses

Dendritic cells (DCs), the primary antigen-presenting cells in the immune system, play a critical role in regulating tumor immune responses. However, the tumor immunosuppressive microenvironment severely impedes the process of antigen-presenting and DC maturation, thereby limiting the efficacy of cancer immunotherapy. In this work, a pH-responsive polymer nanocarrier (PAG) modified with aminoguanidine (AG) was constructed for the efficient delivery of bortezomib (BTZ) through bidentate hydrogen bonds and electrostatic adsorption formed between guanidine groups of PAG and boronic acid groups of BTZ. The obtained PAG/BTZ nanoparticles exhibited pH-responsive release of BTZ and AG in the acidic tumor microenvironment. On the one hand, BTZ induced potent immune activation by eliciting immunogenic cell death (ICD) and releasing damage-associated molecular patterns. On the other hand, the cationic AG significantly promoted antigen uptake by DCs and activated DC maturation. As a result, PAG/BTZ significantly stimulated tumoral infiltration of cytotoxic T lymphocytes (CTLs) and triggered robust antitumor immune responses. Thus, it showed potent antitumor efficacy when synergizing with an immune checkpoint-blocking antibody.

Abstract 709: AST-301, a pDNA-based therapeutic cancer vaccine encoding HER2 ICD, improves the efficacy of checkpoint blockade therapy in CD34+ humanized gastric cancer mouse model

Abstract PD-1/PD-L1 play a crucial role in modulating immune response and promoting self-tolerance through activating apoptosis of antigen-specific T cells and suppressing apoptosis of regulatory T cells. Although immunotherapy to block this may provide a greater survival benefit for some patients, the therapeutic effect shown in clinical trials is limited by shortage of pre-existing CD8+ T cells infiltrating the tumor. AST-301 is a pDNA-based therapeutic cancer vaccine encoding HER2 ICD sequence. It was effective in eliciting HER2-specific T cells and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen and has proven safety as well as long-term immunological memory efficacy of T-cell immune by the phase 1 study (PN 109, NCT00436254). Our previous study has shown that AST-301 has anti-tumor effects by mediating NK cells in human HER2+ gastric cancer cell (NCI-N87) xenograft athymic mice. Here, we aimed to determine whether combined administration of AST-301 can enhance the anti-tumor effect of immune checkpoint inhibitor (ICI) using NCI-N87 xenograft humanized mouse. To confirm the tumor suppressive effect of pembrolizumab (anti-PD-1) treatment combined with AST-301 in the hu-CD34+ hematopoietic stem cell-engrafted NSG mice that formed tumors with NCI-N87, AST-301 (100 μg) and pembrolizumab (5 mg/kg) were administered into mice once a week for a total of 3 times and one booster doses of AST-301 was injected at week 4. Pembrolizumab low dose (5 mg/kg) test group administered with AST-301 had a relatively higher tumor growth inhibition (TGI) effect than the same concentration of Pembrolizumab alone group. TGI effect (%) of G2 (a combination of AST-301 and Pembrolizumab 5 mg/kg), G3 (Pembrolizumab 5 mg/kg), and G4 (Pembrolizumab 10 mg/kg) were 54%, 47%, and 78%, respectively. To evaluate the mechanism underlying the enhanced anti-tumor effect due to the combined administration of AST-301 and pembrolizumab, we measured the ratio of immune cells isolated from spleen and tumor in mice. in G2, the ratio of helper T cells and cytotoxic T cells in splenocytes higher than the vehicle-treated control group (G1), and the ratio of cytotoxic T cells infiltrated the tumor was also detected high in some subjects compared to those in all the other three groups. These results suggest that AST-301 can improve HER2+ gastric cancer treatment outcomes of immune checkpoint blockade therapy by inducing a robust cytotoxic CD8+ T cell response to tumor cells. Currently, we are ongoing study to prove the anti-tumor synergistic effects of the combination of AST-301 and various FDA-approved Antibody Drug Conjugates (ADCs). This effort is focused on narrowing the administration doses to low levels to resolve the safety issue of ADC or ICI, and it will strongly support the diverse applicability of the cancer therapeutic vaccine. Citation Format: Hyo-Hyun Park, Hye-Ran Kim, Jin Kyeong Choi, Jee Hyun Choi, JinHo Kang, Jinback Lim, Hyo Jung Lim, Jong Yeong Lee, Eunkyo Joung, Hun Jung. AST-301, a pDNA-based therapeutic cancer vaccine encoding HER2 ICD, improves the efficacy of checkpoint blockade therapy in CD34+ humanized gastric cancer mouse model [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 709.

Abstract 3159: Next generation immuno-oncology tumor profiling using a biophysics-based biomarker to predict immunotherapy response in early-stage breast cancer

Abstract Background: Immuno-oncology (IO) therapies, such as immune checkpoint inhibitor antibodies, have emerged as promising treatments for early-stage breast cancer (ESBC). Although immunotherapy is standard-of-care for ESBC, the number of benefiting patients remains small, and the therapy itself can prompt severe immune-related adverse events. To date, efforts to identify biomarkers of IO response, such as those targeting the PD-1/PD-L1 signaling axis, have failed to robustly demarcate predictive features that translate across cohorts. Here, we applied an integrative computational approach to develop the IOScope biomarker, which predicts IO response in ESBC. Our method overcomes many of the limitations of invasive, costly, and time-consuming IO response analyses derived from transcriptomics data, and advances beyond the inability of single-site biopsies to account for tumor heterogeneity. Methods: We analyzed single-cell and whole-tissue RNA-seq data from non-IO treated ESBC patients in the I-SPY trials, as well as 23 manually-curated gene expression signatures representing the “hallmarks of cancer”, in order to associate PD-L1 levels with local tumor biology. We then correlated PD-L1 expression to biophysical features derived from patient DCE-MRIs to generate the IOScope biomarker. We quantified IOScope within patient tumors using integrative computational modeling and developed a corresponding IOScope Score. The IOScope biomarker and Score were then applied to data from IO-treated patients in the I-SPY2 clinical trial to assess their performance in predicting pCR in response to IO therapy. We additionally validated IOScope in a virtual clinical trial, comparing pCR rates to empirical data. Results: We validated the IOScope biomarker and the IOScope Score in a small, independent cohort of IO-treated patients (n=17) and correctly predicted pCR response in 15/17 individuals (88.2% accuracy, comprising 10/12 in TNBC and 5/5 in HR+/HER2-). We further applied the IOScope Score in a virtual clinical trial (N = 292) simulating IO administration in IO-naïve cohort (Virtual Tumor Bank, SimBioSys, Inc.). Using this approach, we predicted an increase in pCR rates of 28.5% (TNBC) and 8.9% (HR+/HER2-) with the addition of IO therapy to a standard chemotherapy background. These predicted increases in pCR rate compare favorably to published rates increases produced by IO in both cancer subtypes. Conclusion: The IOScope biomarker and IOScope Score represent a next generation approach using integrative computational and biophysical analysis to assess cancer responsiveness to immunotherapy. This biomarker, predicated on MRI-derived tumor microenvironmental features, allows for immuno-oncology profiling of tumors in a rapid, non-invasive manner and may confer high clinical decision impact to further enable personalized oncologic care. Citation Format: Daniel Cook, John Pfeiffer, Joseph Peterson, Nicole Liadis, Matthew Biancalana, Dorys A. Lopez-Ramos, Tushar Pandey, John Cole, Anuja K. Antony. Next generation immuno-oncology tumor profiling using a biophysics-based biomarker to predict immunotherapy response in early-stage breast cancer [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 3159.

Abstract 3242: Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy

Abstract Antibody-drug conjugates (ADCs) are effective anti-tumor therapies with high specificity, with little toxicity and side effects. Xenograft tumor models based on immunodeficient mice have been used for evaluating the human anti-tumor efficacy of ADCs. However, the antibody-mediated immune response of ADCs is not well evaluated in immunodeficient mice. To this end, we have developed BALB/c-hPD1 humanized mouse model to evaluate the efficacy of ADCs in combination with immunotherapy. Trastuzumab deruxtecan (T-DXd, DS-8201a) is a HER2-targeting ADC. In vitro efficacy studies showed that T-DXd effectively bound to the HER2-positive cell line SK-BR-3, resulting in strong endocytosis and induction of apoptosis. It has also been shown to kill HER2-negative MDA-MB-468 cells through a bystander effect both in vitro and in vivo. In vivo efficacy studies have demonstrated that ADCs can effectively inhibit human tumor cell growth in immunodeficient mice. In addition to human tumor cell lines, the efficacy of ADCs was also studied in BALB/c-hPD1mice engrafted with murine colon cancer cells model, and the results showed that T-DXd and Keytruda could significantly inhibit CT26-hHER2 tumor growth, with the tumor growth inhibition rates being 57% and 66%, respectively, Tumor growth inhibition rate of the combined treatment group was about 91%, which was superior compared to the efficacy of single drug treatment. The evaluation of Fc segment-mediated effect (such as ADCC, CDC and ADCP) of ADCs antibodies was carried out in humanized immune system mouse models, such as NCG-hIL15 and NCG-hSGM3. In summary, we have established a drug evaluation platform using various humanized mouse models to study the synergistic anti-tumor effects of ADCs combined with immune checkpoint blockade antibodies. Citation Format: Fang Zhu, Hongyan Sun, Qian Lu, Yuan Fang, Jianming Xu, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy [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 3242.

Abstract 4701: Spatial resolution of tumor and immune cell lineages in the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC)

Abstract Understanding how the tumor microenvironment (TME) evolves during tumorigenesis and therapeutic response is crucial to developing personalized treatments with the goal of improving cancer therapy. With robust and comprehensive multiplexed imaging technologies, immune biomarker antibodies can be used to interrogate immune cell lineages and structures. When combined with specific oncology biomarker antibodies, this approach can capture the immune response within the TME in a variety of neoplasms. The Cell DIVETM Multiplex Imaging Solution allows probing and imaging of dozens of biomarkers on a whole single tissue section using an iterative staining and dye inactivation workflow. The broad portfolio of robust IHC-validated antibodies from Cell Signaling Technology (CST) enables the detection of key proteins in the TME, allowing immune cell detection and phenotyping in tissue. Here, we demonstrate multiplexed Cell DIVE imaging using a novel CST panel to probe pancreatic ductal adenocarcinoma (PDAC). These biomarker antibodies define the immune cell landscape in the hypoxic tumor. Development of the antibody panel required minimal optimization, enabled the identification of complex cell types and revealed their cell-to-cell interactions within the tumor microenvironment. The availability of cell type specific biomarkers, combined with the ability to interrogate using multiplexed tissue imaging, provides unprecedented and novel insights and spatial resolution of immune cell populations with many cell types in the TME. Citation Format: Lisa Arvidson, Reginaldo Prioli, Samuel Jensen, Michael J. Smith, Katie O. White, Richard A. Heil-Chapdelaine, Chi-Chou Huang, Tuan H. Phan, Hideki Sasaki, Melinda L. Angus-Hill. Spatial resolution of tumor and immune cell lineages in the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC). [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 4701.

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [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 1546.

Abstract 5105: TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis

Abstract Brain metastasis is an unmet clinical need, affecting between 10-30% of cancer patients with 200000 to 400000 newly diagnosis per annum in the US. Recently, several clinical trials have reported benefits using immunotherapy to treat brain metastasis. However, variability of the responses is broad and high benefit is found mainly in asymptomatic brain metastasis, while the benefit is dramatically reduced in the clinically relevant stage. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes in these advanced stages of the disease. Our current study further exploited the heterogeneity within the metastasis-associated microenvironment as a resource to identify novel therapeutic vulnerabilities to improve the benefits of immunotherapies based on immune checkpoint blocking antibodies (ICB) in symptomatic brain metastasis. Our results demonstrate that reactive astrocytes are strong immunomodulatory cells in brain tumors. We have identified the molecular profile of disease-associated glial cells and defined its connection to modulatory activities on specific lymphocyte populations in experimental brain metastasis as well as human-derived samples. scRNASeq and high content multiplex immunofluorescence allowed us to report a novel local immunomodulatory axis dependent on TIMP1 (astrocytes)/CD63 (CD8 T cells), which is present in brain metastasis patients with high immunoscore and would imply an additional immunosuppressive signal for potential ICB responders in brain metastasis. Genetic and pharmacologic approaches targeting this STAT3-dependent local immunomodulatory axis have allowed us to define the rationale to combine immune checkpoint blockade with a STAT3 inhibitor, which we previously used in patients. We proved that such combined immunotherapy boost the systemic activation of T cells while also preventing the local blockade. Additionally, our comprehensive strategy includes the possibility to stratify patients that are best qualified to benefit from this therapy by measuring TIMP1 in liquid biopsies from CSF. Even more, our data using Patient Derived Organotypic Cultures (PDOC) from fresh brain metastasis neurosurgeries confirms that our therapeutic strategy might benefit brain metastases generated from any primary source. In conclusion, we describe an immunosuppressive mechanism in the brain microenvironment that could explain the lack of response to ICB in patients with advanced brain metastasis. Our finding provides the rationale to implement complementary approaches targeting local immunosuppression to increase the benefit of immunotherapy in symptomatic brain metastasis. Citation Format: Neibla Priego, Pedro García-Gómez, Ana de Pablos-Aragoneses, María Perea-García, Laura Álvaro-Espinosa, Carolina Hernández-Oliver, Elena Martínez-Saez, Ángel Pérez-Núñez, Aurelio Hernández-Laín, Rebeca Sanz-Pamplona, Marc Schmitz, Stephen J. Crocker, Diego Serrano, Asís Palazón, RENACER Red Nacional de Metástasis Cerebral, Manuel Valiente. TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis. [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 5105.

Abstract 4296: Fast de novo antibody structure prediction with atomic accuracy

Abstract Background: In the field of antibody engineering, an essential task is to design a novel antibody whose paratopes bind to a specific antigen with correct epitopes. Understanding antibody structure and its paratope can facilitate a mechanistic understanding of its function. Therefore, antibody structure prediction from its sequence alone has always been a highly valuable problem for de novo antibody design. AlphaFold2 (AF2), a breakthrough in the field of structural biology, provides a solution to this protein structure prediction problem by learning a deep learning model. However, the computational efficiency and undesirable prediction accuracy on antibody, especially on the complementarity-determining regions limit its applications in de novo antibody design. Methods: To learn informative representation of antibodies, we trained a deep antibody language model (ALM) on curated sequences from observed antibody space database via a well-designed transformer model. We also developed a novel model named xTrimoABFold++ to predict antibody structure from antibody sequence only based on the pretrained ALM as well as efficient evoformers and structural modules. The model was trained end-to-end on the antibody structures in PDB by minimizing the ensemble loss of domain-specific focal loss on CDR and the frame aligned point loss. Results: xTrimoABFold++ outperforms AF2 and OmegaFold, HelixFold-Single with 30+% improvement on RMSD. Also, it is 151 times faster than AF2 and predicts antibody structure in atomic accuracy within 20 seconds. In recently released antibodies, for example, cemiplimab of PD-1 (PDB: 7WVM) and cross-neutralizing antibody 6D6 of SARS-CoV-2 (PDB: 7EAN), the RMSD of xTrimoABFold++ are 0.344 and 0.389 respectively. Conclusion: To the best of our knowledge, xTrimoABFold++ achieved the state-of-the-art in antibody structure prediction. Its improvement on both accuracy and efficiency makes it a valuable tool for de novo antibody design, and could make further improvement in immuno-theory. Experimental results on immune antibody dataset with 95% confidence interval. Method RMSD TMScore GDTTS GDTHA AlphaFold2 3.1254 ± 0.1410 0.8385 ± 0.0055 0.7948 ± 0.0057 0.6548 ± 0.0063 OmegaFold 3.2610 ± 0.1463 0.8384 ± 0.0057 0.7925 ± 0.0059 0.6586 ± 0.0063 HelixFold-Single 2.9648 ± 0.0997 0.8328 ± 0.0055 0.7805 ± 0.0057 0.6225 ± 0.0060 ESMFold 3.1549 ± 0.0067 0.8390 ± 0.0003 0.7952 ± 0.0003 0.6551 ± 0.0003 xTrimoABFold++ 1.9594 ± 0.0805 0.8986 ± 0.0052 0.8694 ± 0.0057 0.7456 ± 0.0066 Citation Format: Yining Wang, Xumeng Gong, Shaochuan Li, Bing Yang, Yiwu Sun, Yujie Luo, Hui Li, Le Song. Fast de novo antibody structure prediction with atomic accuracy. [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 4296.