Antigen-specific Signal Research Articles

Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens.

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

On antigen-specific signals, immune class regulation and energetics: Report III from the workshops on foundational concepts of immune regulation.

This is a report from a one-week workshop held in Athens, Greece in July of 2022. The workshop aimed to identify emerging concepts relevant to the fundamentals of immune regulation and areas for future research. Theories of immune regulation emphasize the role of T cell help or co-stimulation (signal 2). The workshop participants considered how new data on the characteristics of agonist antigens, the role of the antigen receptor signals (signal 1) in driving fate decisions, the effect of energetics on immunity and a better understanding of class-control in the immune response, may impact theories of immune regulation. These ideas were discussed in the context of tumour immunology, autoimmunity, pregnancy and transplantation. Here we present the discussions as a narrative of different viewpoints to allow the reader to join the conversation. These discussions highlight the evolving understanding of the nature of specific antigen recognition and how both antigen-specific and non-specific mechanisms impact immune responses.

Impaired tolerance to the autoantigen LL-37 in acute coronary syndrome.

LL-37 is the only member of the cathelicidin family of antimicrobial peptides in humans and is an autoantigen in several autoimmune diseases and in acute coronary syndrome (ACS). In this report, we profiled the specific T cell response to the autoimmune self-antigen LL-37 and investigated the factors modulating the response in peripheral blood mononuclear cells (PBMCs) of healthy subjects and ACS patients. The activation induced marker (AIM) assay demonstrated differential T cell profiles characterized by the persistence of CD134 and CD137, markers that impair tolerance and promote immune effector and memory response, in ACS compared to Controls. Specifically, CD8+CD69+CD137+ T cells were significantly increased by LL-37 stimulation in ACS PBMCs. T effector cell response to LL-37 were either HLA dependent or independent as determined by blocking with monoclonal antibody to either Class-I HLA or Class-II HLA. Blocking of immune checkpoints PD-1 and CTLA-4 demonstrated the control of self-reactive T cell response to LL-37 was modulated predominantly by CTLA-4. Platelets from healthy controls down-modulated CD8+CD69+CD137+ T cell response to LL-37 in autologous PBMCs. CD8+CD69+CD137+ T cell AIM profile negatively correlated with platelet count in ACS patients. Our report demonstrates that the immune response to the autoantigen LL-37 in ACS patients is characterized specifically by CD8+CD69+CD137+ T cell AIM profile with persistent T cell activation and the generation of immunologic memory. The results provide potentially novel insight into mechanistic pathways of antigen-specific immune signaling in ACS.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools.

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Engineered, fully human nanobody-based CAR T cells have enhanced antitumor activity against hepatocellular carcinoma in preclinical models.

e14512 Background: Glypican 3 (GPC3), a cell surface protein associated with early development, is an established biomarker of hepatocellular carcinoma (HCC). In a vast compendium of work, HN3, a human GPC3 nanobody (VH) isolated from a phage library, was shown to abrogate HCC tumors and block the distally located Wnt binding domain of GPC3. To date, it remains challenging how to precisely engineer CAR T cells for improved efficacy in solid tumors. In the context of hepatocellular carcinoma, we determined how hinges and transmembrane portions of varying structures and sizes affect CAR T cell function. Here, we show how rationally designed, engineered Chimeric Antigen Receptor (CAR) T cells containing HN3, demonstrate enhanced CAR T activity in HCC. Methods: We generated and compared multiple permutations of GPC3 targeted CAR T cells containing HN3, CD8, CD28, IgG4 and Fc domains. Incorporation of HN3, into multiple CAR formats yielded nine constructs, which were screened for antigen-specific and antigen-independent signaling using a luciferase reporter system in Hep3B, Huh7 and HepG2 cell lines. For in vivo assessment, immunodeficient NSG mice were intraperitonially injected with Hep3B or Huh7 cells and treated with a single dose (5 or 15 million) of engineered HN3 CAR T cells. Blood was collected at 2, 4 and 5 weeks to assess proliferation, T cell subtypes and exhaustion. Results: In vitro, the HN3-IgG4H-CD28TM CAR T cells induced high specificity and cytotoxic activity. HN3-IgG4H-CD28TM CAR T cells markedly improved HN3 cell killing activity by 30-40% in low (1.6:1) and high (25:1) effector to target ratios. Similarly, in vivo, in a high antigen environment, two determinative changes in the hinge region and transmembrane domains led to complete tumor eradication in immunodeficient mice bearing HCC tumors within 7-10 days. Moreover, HN3-IgG4H-CD28TM CAR T cells maintained specific tumor killing and averted exhaustion by producing a clear T cell response signature of enriched cytotoxic-memory (Temra) CD8+ along with a subset of naïve T cells. Conclusions: Engineered nanobody based CAR T cells containing the appropriate hinge and transmembrane domains can lead to determinative T cell signaling capable of inducing swift and durable eradication of HCC tumors. Altogether, we show that engineered HN3 CAR T cell therapy demonstrates strong potential for treatment of aggressive HCCs enabled by Wnt dysregulation.

The historical postulate is not the basis of self-nonself discrimination: A response to Bretscher's proposal.

Peter Bretscher was the first to envision that the problem of self-nonself discrimination in the adaptive immune system could be solved by positing that antigen inactivates single lymphocytes, whereas antigen-mediated lymphocyte cooperation is required to stimulate their activation. These ideas led to a two-signal model for lymphocyte activation: an antigen-specific signal that when generated alone results in tolerance and the combination of two (or more) antigen-specific signals resulting in lymphocyte activation and immunity. This 'quorum model' is consistent with the concept known as the historical postulate that posits that the early life timing of antigen exposure is the key to self-tolerance. Bretscher proposes that the historical postulate is 'the basis, at level of the system, for self-nonself discrimination' and contends that the Danger model violates this postulate. Herein I argue that the data do not support putting the historical postulate alone at the top of the hierarchy of concepts underlying self-nonself discrimination. The location of antigen is at least as important because it determines whether central tolerance will be engaged. Location of antigen together with timing of antigen exposure are major factors determining whether quorum, the basis for self-nonself discrimination, is achieved.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by “redirecting” polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Thyrotropin Receptor-Specific Lymphocytes in Adenovirus-TSHR-Immunized Native and Human Leukocyte Antigen-DR3-Transgenic Mice and in Graves' Disease Patient Blood.

Background: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Methods and Results: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IAd). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25high, CD127low). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Conclusions: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.

Aeromonas salmonicida activates rainbow trout IgM+ B cells signalling through Toll like receptors

As B cells are singularly equipped with a B cell receptor (BCR) and a range of innate receptors, they are able to integrate both antigen-specific and innate signals, with the latter being essential to reach an adequate level of activation. Whether teleost B cells sense pathogens through innate mechanisms has not yet been explored, despite the fact that fish B cells display a wider array of innate receptors than many mammalian B cell subsets. Hence, in the current study, we have investigated the effects of inactivated Aeromonas salmonicida, a Gram negative rainbow trout pathogen, on trout splenic IgM+ B cells in vitro in the presence or absence of different inhibitors of Toll-like receptor (TLR) signalling, to establish to what degree innate signals are contributing to the activation of B cells in teleosts. Our results demonstrate that most of the effects that A. salmonicida exerts on trout IgM+ B cells are significantly blocked in the presence of inhibitors of MyD88 and TRIF, important nodes in TLR signal pathways. Thus, the data presented demonstrates that, also in teleost, TLR signalling is essential for the activation of IgM+ B cells. These results will be useful for the future optimization of novel vaccines and adjuvants.

Single Domain Antibody-Mediated Blockade of Programmed Death-Ligand 1 on Dendritic Cells Enhances CD8 T-cell Activation and Cytokine Production.

Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1pos) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8pos) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.

Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding.

B lymphocytes use B cell receptors (BCRs) to recognize antigens. It is still not clear how BCR transduces antigen-specific physical signals upon binding across cell membrane for the conversion to chemical signals, triggering downstream signaling cascades. It is hypothesized that through a series of conformational changes within BCR, antigen engagement in the extracellular domain of BCR is transduced to its intracellular domain. By combining site-specific labeling methodology and FRET-based assay, we monitored conformational changes in the extracellular domains within BCR upon antigen engagement. Conformational changes within heavy chain of membrane-bound immunoglobulin (mIg), as well as conformational changes in the spatial relationship between mIg and Igβ were observed. These conformational changes were correlated with the strength of BCR activation and were distinct in IgM- and IgG-BCR. These findings provide molecular mechanisms to explain the fundamental aspects of BCR activation and a framework to investigate ligand-induced molecular events in immune receptors.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

Lack of T-cell-mediated IL-2 and TNFα production is linked to decreased CD58 expression in intestinal tissue during acute simian immunodeficiency virus infection.

For an effective T-cell activation and response, co-stimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the most important T-cell co-stimulatory pathways for T-cell activation and proliferation, and its role in regulating intestinal T-cell function in acute and chronic SIV -infected macaques is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell populations during acute SIV infection along with high plasma viral load and a loss of intestinal CD4+ T cells compared to SIV-uninfected control macaques. The reduction of CD58 expression in T cells was correlated with the reduced expression of T-cell-mediated IL-2 and TNFα production. Together, these results indicate that reduction in the CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.

IFNγ-Induced MHC Class II Expression on Islet Endothelial Cells Is an Early Marker of Insulitis but Is Not Required for Diabetogenic CD4+ T Cell Migration.

Diabetogenic T cells infiltrate the pancreatic islets by transmigrating across the microcapillaries residing close to, or within, the pancreatic islets. Deficiency in IFNγ signaling prevents efficient migration of T cells into the pancreatic islets, but the IFNγ-regulated molecules that mediate this are uncertain. Homing of autoreactive T cells into target tissues may require antigen specificity through presentation of cognate antigen by MHC expressed on the vascular endothelium. We investigated the hypothesis that IFNγ promotes the migration of islet antigen-specific CD4+ T cells by upregulating MHC class II on islet endothelial cells (IEC), thereby providing an antigen-specific signal for islet infiltration. Upon IFNγ stimulation, MHC class II, which is not constitutively expressed on IEC, was induced. IFNγ-dependent upregulation of MHC class II was detected in IEC isolated from prediabetic NOD mice at the earliest stages of insulitis, before other markers of inflammation were present. Using a CD4+ T cell-mediated adoptive transfer model of autoimmune diabetes we observed that even though diabetes does not develop in recipient mice lacking IFNγ receptors, mice with MHC class II-deficient IEC were not protected from disease. Thus, IFNγ-regulated molecules, but not MHC class II or antigen presentation by IECs is required for the early migration of antigen-specific CD4+ T cells into the pancreatic islets.

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in Tcells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumoractivity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survivalcompared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate invivo activity similar to that of T-CAR-expressing Tcells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy.

Lack of T-cell mediated IL2 and TNFa production is linked with the decreased CD58 expression in intestinal tissue during acute simian immunodeficiency virus infection

Abstract Human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection causes a progressive impairment of the immune system characterized by massive CD4+ T-cell loss, CD8+ T-cell expansion and sustained immune activation and inflammation. Our data also suggest that pathogenic SIVMAC251 infection in rhesus macaques (RMs) leads to decreased production of several T-helper 1 (TH1) and TH2 cytokines and increased production of IL-17, IFNg, CCL4 and GM-CSF by intestinal CD8+ T-cells 21 days after infection. For an effective T-cell activation and response, costimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the important T-cell costimulatory pathway for T-cell activation and proliferation. Intestinal lamina propria T cells were reported to be highly responsive to a stimulus delivered by CD2 pathway compared to the conventional CD3/TCR pathway. The role of CD2/CD58 in regulating intestinal T-cell function of acute and chronic SIV infected RMs is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell population during acute SIV infection along with the loss of intestinal CD4+ T-cells and high plasma viral load compared to SIV-uninfected normal RMs. The reduction of CD58 expression in T-cells was positively correlated with the reduced expression of T-cell mediated IL2 and TNFa production. Together, these results indicate that the reduction in CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.

CD8+ T cells with characteristic T cell receptor beta motif are detected in blood and expanded in synovial fluid of ankylosing spondylitis patients

The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.

Abstract P1-09-10: ICOSL anti-HER2 V-mAbs: Localizing engineered ICOSL costimulatory agonists to HER2+ tumors through trastuzumab

Abstract Background: The presence of tumor infiltrating lymphocytes (TILs) has been associated with improved prognosis in HER2+ breast cancer patients. Antigen specific TCR and costimulatory receptor signaling drive increases in TIL number, effector function, and tumor cytotoxicity. Improving the number and effector phenotype of tumor localized TILs has curative potential by enhancing the adaptive and memory immune response. Targeting HER2 with the monoclonal anti-HER2 antibody trastuzumabhas improved survival in HER2+ breast cancer patients and is known to increase peripheral type I immunity, which may be reflected by increased TILs. The Immunoglobulin Superfamily (IgSF) includes a large, diverse family of immunotherapy targets expressed on immune cells and tumors. Transmembrane IgSF receptors,CD28 and inducible T cell co-stimulator (ICOS), related costimulatory molecules expressed on T cells, interact with CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity.The Alpine Immune Science's VIgDTM platform uses directed evolution to derive novel, therapeutically-applicable IgSF extracellular domains with tailored specificity and affinity. The vIgD platform has generated human ICOSL vIgDs capable of binding both ICOS and CD28, activating both pathways. To promote anti-tumor activity of TILs in HER2+ tumors, we developed trastuzumab-ICOSL “V-mAbs” consisting of trastuzumab fused to activating ICOSL vIgDs. These V-mAbs are designed to localize to HER2+ tumors and activate antigen-specific, resident T-cells through costimulatory receptor agonism. Methods: V-mAbs were generated by fusing ICOSL vIgDs to either the N- or C- termini of the heavy and/or light chains of trastuzumab. V-mAb binding to CD28, ICOS or HER2 was measured by flow cytometric analysis of transfected cells or ForteBio analysis. V-mAb costimulatory activity was confirmed by immobilization in the presence of anti-CD3 in a primary human T cell assay. Finally, V-mAbs were co-cultured with HER2+ target cells and human T cells; T-cell activity was measured by proliferation, cytokine production, and target lysis. Results: V-mAbs were successfully produced and bound to CD28, ICOS and HER2. In a plate bound costimulation assay, the V-mAbs increased the amount of IFN-gamma produced by T-cells stimulated with anti-CD3. When incubated with HER2+ target cells, V-mAbs promoted T-cell proliferation, cytokine secretion, and target cell lysis. Data from in vivo studies, to determine the impact of trastuzumab V-mAbs on HER2+ cancers, will be presented when available. Conclusions: Trastuzumab-ICOSL V-mAbs are novel ICOS- and CD28-activating immunotherapies for HER2-positive tumors, promoting T-cell proliferation, cytokine secretion, and target cell lysis in a HER2 dependent fashion. The V-mAb platform has broad potential to enable tumor-localized immune modulation via the diverse array of IgSF members. Preclinical development of trastuzumab-ICOSL clinical therapeutics is in progress. Citation Format: Rickel E, Evans L, Swanson R, Levin SS, Rixon M, Wolfson M, Bhandari J, MacNeil S, Hoover J, Kornacker M, Capuano I, Peng SL. ICOSL anti-HER2 V-mAbs: Localizing engineered ICOSL costimulatory agonists to HER2+ tumors through trastuzumab [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-10.

Abstract B005: FPT155, a novel therapeutic CD80-Fc fusion protein with potent antitumor activity in preclinical models

Abstract Optimal activation of T cells requires both antigen-specific signaling and induction of costimulatory pathways. Costimulatory pathways are upregulated by foreign antigenic challenge but are not active in the normal immune state, thus maintaining peripheral tolerance to self-antigens. The absence of costimulatory signaling may likewise play a role in tolerance to cancer cells. Five Prime has developed a CD80 (B7.1) extracellular domain (ECD)-Fc fusion protein (FPT155) as a costimulatory molecule that can stimulate T-cell activation and break tumor immune tolerance. In vitro studies show that FPT155 induces T-cell activation and cytokine production via CD28. FPT155 activity requires T-cell receptor engagement, which indicates that FPT155 is not a CD28 “superagonist” that can drive T-cell activation independently of antigenic stimulation. To investigate the impact of CD80-Fc in preclinical models, we generated murine FPT155 (mFPT155), a surrogate fusion protein, composed of the mouse CD80 ECD fused with the murine Fc region of IgG2a. mFPT155 is well tolerated in mice and has potent efficacy in syngeneic tumor models, including the induction of complete tumor regression in the CT26 model. mFPT155 promotes the infiltration of T cells into the tumor core and increases the effector T:Treg ratio, thus inducing a favorable microenvironment for an effective antitumor immune response. Furthermore, combination of mFPT155 and anti-PD1 therapy leads to stronger antitumor efficacy compared to either therapy alone. Together these results indicate that mFPT155 is a potent T-cell costimulator with strong antitumor activity in mouse tumor models. We are currently performing IND-enabling studies and plan to initiate a clinical study with FPT155 in 2018. Citation Format: Susannah D. Barbee, Barbara Sennino, Jacqueline De La Torre, Monica Macal, Quinn Walker, Marc R. Jabon, Amanda Chen, David A. Busha, Margaret Best, Kristen L. Pierce, Luis Borges, Kevin P. Baker, Thomas Brennan. FPT155, a novel therapeutic CD80-Fc fusion protein with potent antitumor activity in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B005.