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Immune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance. The protein tyrosine phosphatase PTPN2 is a central regulator of inflammation, and genetic deletion of PTPN2 on either tumour cells or host immune cells promotes anti-tumour immunity. However, inhibitors of PTPN2 with suitable pharmacokinetic properties for oral administration have not been described. Here, we present the characterization of ABBV-CLS-484 (A484), a potent active site inhibitor of PTPN2 and the closely related phosphatase PTPN1. A484 treatment in vitro amplifies the response to interferon gamma, and monotherapy A484 treatment generates robust anti-tumour immunity in several murine cancer models. Through in vivo studies and single cell transcriptional profiling of tumour-infiltrating lymphocytes (TIL) from A484-treated mice, we show that A484 inflames the tumour microenvironment and promotes CD8+ T cell function by enhancing cytokine signaling and decreasing T cell exhaustion and dysfunction. Our results demonstrate that oral administration of small molecule inhibitors of PTPN2/N1 can induce potent anti-tumour immunity in mouse models. PTPN2/N1 inhibitors offer a promising new strategy for cancer immunotherapy and are currently being evaluated clinically in patients with advanced solid tumours (NCT04777994). More broadly, our study shows that small molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to current antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge A484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics targeting this important class of enzymes. Citation Format: Hakimeh Ebrahimi-Nik, Arvin Iracheta-Vellve, Kira E. Olander, Thomas R.G. Davis, Sarah Y. Kim, Mitchell D. Yeary, James C. Patti, Tyler M. Balon, Omar Ismail Avila, Cun Lan Chuong, Meng-Ju Wu, Christina K. Baumgartner, Keith M. Hamel, Kathleen A. McGuire, Rebecca Mathew, Carey Backus, Ian C. Kohnle, Zhaoming Xiong, Elliot P. Farney, Jennifer M. Frost, Geoff T. Halvorsen, Matthew Rees, Andrew Boghossian, Melissa Ronan, Jennifer A. Roth, Todd R. Golub, Gabriel K. Griffin, Nabeel El-Bardeesy, Clay C. Beauregard, Philip R. Kym, Kathleen B. Yates, Robert T. Manguso. Small molecule inhibition of PTPN2/1 inflames the tumour microenvironment and unleashes potent CD8+ T cell immunity [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A41.

Background: Pharmacologic inhibition of PTPN2 and PTPN1 (PTPN2/N1) represents a novel therapeutic approach in immuno-oncology that augments innate and adaptive immune responses in addition to enhancing tumor cell sensitivity to immune-mediated killing. PTPN2/N1 emerged as top hits in an in vivo CRISPR screen to identify tumor-intrinsic targets that enhance sensitivity and overcome resistance to anti-PD-1 treatment. PTPN2/N1 are phosphatases that act as negative regulators in numerous pathways including immune activation. While phosphatases have long been of interest, they are challenging drug targets, and the active site had been considered undruggable. Results: Here we report the discovery of the highly selective, active site PTPN2/N1 small molecule inhibitor, ABBV-CLS-484. Highly optimized ligand-protein interactions have led to the design of sub-nanomolar PTPN2/N1 inhibitors, confirmed through x-ray crystallography. PTPN2/N1 inhibitors increase the activation and function of cytotoxic T cells as well as increase the pro-inflammatory properties of CD103+ dendritic cells and macrophages in vitro. However, they do not cause non-specific activation in the absence of stimulation; rather, they augment signaling in cells that are already activated. PTPN2/N1 inhibition also has effects directly on tumor cells, where it amplifies sensitivity to immune-mediated killing by enhancing the interferon response. ABBV-CLS-484 promotes anti-tumor immunity as monotherapy and in combination with anti-PD-1 leading to dramatic tumor regression, even in models resistant to anti-PD-1 treatment such as 4T1, or those with minimal inflammation such as EMT6. Single-cell RNAseq analyses of tumor-infiltrating immune cells confirmed activation of T cells and demonstrated switching of myeloid-derived suppressor cells towards a proinflammatory phenotype, thereby revealing a distinct mechanism of action of ABBV-CLS-484 compared with PD-1 blockade. Our results show that PTPN2/N1 inhibitors have complementary effects on the immune system and tumor microenvironment that act to promote effective tumor killing. Based on these robust preclinical data, phase I clinical trials of ABBV-CLS-484 alone and in combination with an anti-PD-1 agent have been initiated to establish the safety, tolerability, and efficacy in diverse solid tumor indications. Conclusions: We have discovered a first-in-class PTPN2/N1 inhibitor, which represents a promising novel immunotherapy that both enhances the immune response and increases tumor sensitivity to immune-mediated killing. ABBV-CLS-484 is currently being evaluated in phase I clinical trials in patients with advanced solid tumors, as a monotherapy or in combination with a PD-1 targeting agent (NCT04777994). Citation Format: Christina K. Baumgartner, Marcia N. Paddock, Jennifer M. Frost, Keith M. Hamel, Kathleen A. McGuire, Kyle Halliwill, Zhaoming Xiong, Liang Mu, Kelly Klinge, Prasanthi Geda, Jaqueline Aguado, Marinka Bulic, Elliot P. Farney, Kathleen B. Yates, Robert T. Manguso, Clay Beauregard, Philip R. Kym. ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing [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 ND06.

Immune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance. The protein tyrosine phosphatase PTPN2 is a central regulator of inflammation, and genetic deletion of PTPN2 on either tumor cells or host immune cells promotes anti-tumor immunity. However, inhibitors of PTPN2 have not been described. Here, we present the validation of ABBV-CLS-484, a potent catalytic inhibitor of PTPN2 and the closely related phosphatase PTPN1. ABBV-CLS-484 treatment of tumor cells in vitro phenocopies the genetic deletion of PTPN2/N1, causing both amplified transcriptional responses to IFNg and reduced cell viability across human cancer cell lines. Monotherapy ABBV-CLS-484 treatment generates robust anti-tumor immunity in several murine cancer models with efficacy comparable to anti-PD-1 treatment. Through genetic studies, we show that while ABBV-CLS-484 can act on both tumor cells and the host immune system, IFN sensing and PTPN2/N1 expression on tumor cells are not always required for efficacy, suggesting that PTPN2/N1 inhibition on host immune cells may be sufficient for activity of the drug. Through scRNAseq profiling of TILs from both ABBV-CLS-484-treated and anti-PD-1-treated tumors, we show that ABBV-CLS-484 induces unique transcriptional changes to both myeloid and lymphoid populations in the tumor microenvironment which are dominated by enhanced IFN sensing and a shift from suppressive to pro-inflammatory phenotypes. ABBV-CLS-484 treatment enhances the activation and effector functions of CD8+ T cells while decreasing the expression of genes classically associated with T cell exhaustion and dysfunction such as Tox. The efficacy of ABBV-CLS-484 is critically dependent on CD8+ T cells and treatment with ABBV-CLS-484 results in greater levels of T cell infiltration into tumors and a more diverse repertoire of expanded T cell clones relative to anti-PD-1. Thus, the PTPN2/N1 inhibitor ABBV-CLS-484 is a highly effective immunotherapy with monotherapy efficacy across mouse tumor models. Small molecule inhibitors of PTPN2 offer a promising new strategy for cancer immunotherapy by targeting an IFN signaling checkpoint and are currently being evaluated clinically in patients with advanced solid tumors (NCT04777994). Citation Format: Arvin Iracheta-Vellve, Hakimeh Ebrahimi-Nik, Thomas R. Davis, Kira E. Olander, Sarah Y. Kim, Mitchell D. Yeary, James C. Patti, Ian C. Kohnle, Christina K. Baumgartner, Keith M. Hamel, Kathleen A. McGuire, Cun Lan Chuong, Zhaoming Xiong, Elliot P. Farney, Jennifer M. Frost, Matthew Rees, Andrew Boghossian, Melissa Ronan, Jennifer A. Roth, Todd R. Golub, Gabriel K. Griffin, Clay Beauregard, Philip R. Kym, Kathleen B. Yates, Robert T. Manguso. Targeting the immune checkpoint PTPN2 with ABBV-CLS-484 inflames the tumor microenvironment and unleashes potent CD8+ T cell immunity [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 606.

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

Background Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIIInull mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were use to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T cell proliferation assay, and memory B cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIIInull mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4+ T cells from treated animals failed to proliferate in response to rhF8 restimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of manipulated cells can introduce immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. This article is protected by copyright. All rights reserved.

Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. SummaryBackground Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin–antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet–leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.

Recent studies from our group and others have demonstrated that FVIII ectopically targeted to platelets under control of the platelet-specific αIIb promoter (2bF8) can efficiently restore hemostasis in hemophilia A mice even in the presence of high-titer inhibitory antibodies directed against FVIII (inhibitors). Our studies have demonstrated that platelet-targeted FVIII gene therapy can not only correct the hemophilic phenotype, but also induce FVIII-specific immune tolerance. In the platelet gene therapy model, hematopoietic stem cells (HSCs) are ex vivo transduced with lentivirus carrying 2bF8 and transplanted into the recipient. Sufficient preconditioning has to be employed to create space for therapeutic engraftment of the transduced HSCs. It is not clear whether preconditioning affects the potential for an immune response in the context of platelet-derived FVIII. Furthermore, if current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically, as potential transfusion alternative, in hemophilia A patients even with inhibitors. One important question that has not been explored, however, is the immunogenicity of platelet-derived FVIII. To investigate whether platelet-derived FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets into naïve FVIIInull mice without any preconditioning weekly for 8 weeks. These platelets were transfused to a level between 20 to 57% of total platelets upon infusion, and all animals survived the tail-clip survival test 13-hours after platelet infusion. The level of platelet-FVIII in the infused animals was 0.11 ± 0.01 mU/108 platelets (n = 6) even one week after infusion. Neither inhibitory nor non-inhibitory anti-FVIII antibodies were detected in the infused mice during the study course (n = 9). All animals developed inhibitors following further challenge with recombinant human FVIII (rhF8) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger an immune response in hemophilia A mice. We then explored whether platelets containing FVIII can act as an immunogen in FVIIInull mice with pre-existing anti-FVIII immunity. FVIIInull mice were immunized with rhF8 to induce anti-FVIII antibodies. Four week after the last immunization, 2bF8 transgenic platelets were transfused into rhF8-primed FVIIInull mice (n = 4) weekly for 4 weeks and anti-FVIII antibody titers were monitored. There was not significant augmentation of FVIII-specific antibodies as determined by Bethesda assay for inhibitory antibodies and ELISA assay for total anti-FVIII IgG, indicating that infusion of platelets containing FVIII does not stimulate an anti-FVIII memory response in the inhibitor model. To investigate whether preconditioning affects the anti-FVIII immune response, animals were pre-conditioned with a sub-lethal 660 cGy total body irradiation (TBI) followed by 2bF8 transgenic platelet infusion weekly for 8 weeks. No anti-FVIII antibodies were detected in recipients (n = 6) after 2bF8 transgenic platelet infusion. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower (75 ± 42 BU/ml) than in the naïve FVIIInull mice without preconditioning when the same infusion protocol was employed (270 ± 76 BU/ml), indicating that 660 cGy TBI plus 2bF8 transgenic platelet infusion may suppress anti-FVIII immune response. In conclusion, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in hemophilia A mice. Disclosures No relevant conflicts of interest to declare.

Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4+ T cells. However, CD11c+ dendritic cells facilitate the T cell–independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell–dependent restimulation, the antibody response after T cell–independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor–mediated effector functions, both are required for effective immune responses against pathogens.

Protective immunity against a variety of infections depends on the amplification and differentiation of rare naive antigen-specific CD4 and CD8 T cells. Recent evidence indicates that the clonotypic composition of the responding T-cell compartment has a critical role in the immune defense against pathogens. The present review compares and contrasts how naive CD4 and CD8 T cells recognize their cognate antigen, and discusses the factors that regulate the genesis and maintenance of the CD4 and CD8 T-cell receptor repertoire diversity.

Factor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

New vaccines based on soluble recombinant antigens (Ags) require adjuvants to elicit long-lasting protective humoral and cellular immunity. Despite the importance of CD4 T helper cells for the generation of long-lived memory B and CD8 T cells, the impact of adjuvants on CD4 T-cell responses is still poorly understood. Adjuvants are known to promote dendritic cell (DC) maturation and migration to secondary lymphoid organs where they present foreign peptides bound to class II major histocompatibility complex molecules (pMHCII) to naive CD4 T cells. Random and imprecise rearrangements of genetic elements during thymic development ensure that a vast amount of T-cell receptors (TCRs) are present in the naive CD4 T-cell repertoire. Ag-specific CD4 T cells are selected from this vast pre-immune repertoire based on the affinity of their TCR for pMHCII. Here, we review the evidence demonstrating a link between the adjuvant and the specificity and clonotypic diversity of the CD4 T-cell response, and consider the potential mechanisms at play.

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity, but the mechanisms regulating the responding TCR repertoire remain poorly defined. In this study, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that, although all peptides elicited similar T cell response size on immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high-affinity clonotypes expressing restricted public TCR, whereas peptides with longer half-lives broadened CD4 T cell response by recruiting lower-affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high-affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher-affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.

Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Our group has previously developed a gene therapy approach for hemophilia A in which FVIII expression is targeted to platelets. Platelet expressed FVIII successfully restores hemostasis in hemophilic mice even in the presence of high titer inhibitory anti-FVIII antibodies and induces immune tolerance to FVIII. Therapeutic efficacy was achieved in our original transgenic mouse line at a level of only 0.75mU FVIII per 108 platelets, which corresponded to an equivalent of 1.25% FVIII in plasma of wild-type (WT) mice. FVIII is not normally expressed in platelets but with platelet-FVIII gene therapy FVIII levels at the site of injury might dramatically increase due to platelet aggregation and activation, and consequent local FVIII release from platelets together with VWF. Furthermore, a higher embolism rate has been suggested in transgenic mice expressing FVIII in platelets on an otherwise FVIII deficient background. Thus, although this approach is very successful in restoring hemostasis, evaluating potential pathological consequences in conjunction with platelet-FVIII is of great importance. Here we explored whether a pro-thrombotic state was induced by platelet expressed FVIII in an attempt to define the breadth of the therapeutic window. To examine this, we analyzed high platelet-FVIII expressing mice using five techniques including 1) a native whole blood thrombin generation assay, 2) ex vivo clot formation using thomboelastometry (ROTEM), 3) assessment of plasma parameters linked with an increased thrombosis risk (D-Dimer, thrombin anti-thrombin complexes (TAT), fibrinogen), 4) in vivo clot formation using a ferric chloride carotid artery injury model, and 5) tissue fibrin deposition. In addition to steady state, mice were subjected to an inflammatory challenge to induce pro-thrombotic conditions. We generated transgenic mice, LV17/18tg, that expressed 18mU FVIII per 108 platelets on a FVIII deficient background. This platelet-FVIII level was 24-fold higher than in our originally described mouse line. While FVIII deficient mice had negligible thrombin generation, comparing WT control to LV17/18tg mice, neither thrombin generation nor ex vivo clot formation was increased above WT levels in the transgenic mice. In WT and LV17/18tg, respectively, peak thrombin was 216 ± 16 and 195 ± 13 nM and endogenous thrombin potential was 1718 ± 41 and 1642 ± 56 nM. Clotting time determined by ROTEM in WT and LV17/18tg, respectively, was 424 ± 20 and 705 ± 37 seconds and maximum clot firmness was 51.3 ± 1.4 and 54.0 ± 1.7 mm. Fibrinogen and D-Dimer levels were similar in WT and LV17/18tg mice. While TAT levels were significantly higher in LV17/18tg (11.6 ± 1.1 ng/ml) than in WT mice (6.0 ± 0.5 ng/ml), interestingly this increase was also observed in FVIIInull mice (11.0 ± 1.2 ng/ml). Therefore, we attributed the elevation of TAT levels to the FVIII deficient background. Why the FVIII deficient mice have elevated TAT levels is not clear and may reflect an abnormality in the FVIII deficient line that we are further investigating. When we monitored time to occlusion in an in vivo thrombus formation model using ferric chloride induced carotid artery injury, vessels in LV17/18tg mice did not occlude faster than in WT mice (time to occlusion: 14.5 ± 0.5 versus 5.9 ± 0.5 min, respectively). Thus, under steady state, platelet-FVIII did not seem to be pro-thrombotic. This led us to evaluate the thrombosis risk under pro-thrombotic conditions. Because inflammation induces a pro-thrombotic state, we challenged mice with 40mg/kg LPS and analyzed the animals 16 hours later. WT and LV17/18tg mice treated with LPS showed a similar significant increase in fibrinogen, D-Dimer and TAT levels over PBS treated control mice. While no fibrin deposition was observed in the liver of WT or LV17/18tg mice, LPS challenge did induce fibrin deposition, but there was no difference between WT and LV17/18tg mice. In conclusion, 24-fold higher platelet-FVIII levels than in our originally described mouse line in which hemostasis was fully restored, did not show any signs of thrombosis in steady state or under inflammatory conditions. Therefore, in this regard, platelet targeted FVIII gene therapy can be considered a safe therapy with a relatively wide therapeutic window that is in excess of 24-fold. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETED GENE EXPRESSION. . Montgomery:Biogen: Consultancy; Bayer: Consultancy; CSL Behring: Consultancy; Baxter: Consultancy; Octapharma: Consultancy; Grifols: Consultancy.

Our previous studies using hemophilia A and B mouse models have demonstrated that targeting FVIII or FIX expression to platelets under control of the aIIb promoter through lentivirus-mediated delivery to hematopoietic stem cells (HSCs) results in transgene protein expression and storage in platelet a-granules and that platelet-derived FVIII or FIX not only restores hemostasis but also induces immune tolerance in transduced recipients. In the current studies, we explored how immune tolerance is induced after platelet-specific gene therapy and whether this approach can be applied to induce immune tolerance to a non-coagulant protein. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). Since VWF propeptide (Vp) can reroute secreting proteins to a storage pathway, we designed another vector, 2bVpOVA, which contains Vp to secure OVA storage in platelet granules. We first confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to HSCs can induce anti-OVA immune tolerance in wild-type (WT) C57BL6 mice. 2bOVA or 2bVpOVA-transduced HSCs (CD45.2/B6) were transplanted into CD45.1/B6 recipients pre-conditioned with 6.6Gy total body irradiation (TBI). We found that 95% and 98% of OVA protein in whole blood was stored in platelets with an OVA protein level of 24.22±8.72 ng/108 platelets (n=10) and 1.41±0.73 ng/108 platelets (n=10) in 2bOVA and 2bVpOVA transduced recipients, respectively. Electronic microscope analysis demonstrated that the OVA transgene protein using both vectors was stored in transduced platelet a-granules. When the transduced recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA group (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in untransduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG titer in 2bF8 (an unrelated control vector) transduced FVIIInull/B6 recipients without OVA immunization was 413±61 (n=12), which was not significantly different compared to the 2bOVA or 2bVpOVA group even after OVA immunization. In another unrelated control group, 2bGFP, anti-OVA titer was 84±17 (n=9), which was significantly higher than the data obtained from untransduced WT animals without immunization (33±7, n=24). Why there were various levels of anti-OVA antibody titers in unrelated vectors transduced recipients is still unclear and needed further investigation. To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP (a control vector) and transplanted into CD45.1/B6 recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (86.4±2.3%, 86.2±2.2%, and 87.4±2.0%, respectively), but donor-derived CD45.2+ CD4+ T cells in the 2bOVA (0.2±0.1%, n=5) and 2bVPOVA groups (0.9±0.4%, n=6) were consistently significantly lower than in the 2bGFP group (3.1±0.9%, n=6) in peripheral blood during the entire study course. Similarly, donor-derived CD45.2+ CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in either percentage or total cell number of CD45.2+ CD4+ T cells in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy. Notably, the frequency and total number of endogenous CD4 T cells were similar in the 3 groups. Annexin-V staining revealed that the percentage of apoptotic CD45.2+ CD4+ T cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in both spleen and lymph nodes, but not in the thymus. The frequency of donor-derived regulatory T cells cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in peripheral blood, spleen, and lymph nodes, but not in the thymus. Taken together, our studies demonstrate that platelet-specific gene therapy induces immune tolerance through peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. Thus, platelet gene therapy can be a promising approach for immune tolerance induction. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Induction of antigen-specific immune tolerance is desirable in autoimmune diseases, transplantation, and gene therapy. Our previous studies have demonstrated that FVIII or FIX expression ectopically targeted to platelets under control of the platelet-specific αIIb promoter results in transgene protein storage in platelet α-granules. Further studies have demonstrated that lentivirus-mediated platelet-specific gene delivery to hematopoietic stem cells (HSCs) not only restores hemostasis but also induces antigen-specific immune tolerance in hemophilic mice. We wanted to explore whether platelet-specific gene transfer can be used as a means of immune tolerance induction. In the current study, we used ovalbumin (OVA) as a non-coagulant protein to further examine the potential of a platelet gene therapy-based immune tolerance induction approach. We constructed a lentiviral vector (LV) in which OVA is driven by the αIIb promoter (2bOVA). Evidence suggests that VWF propeptide can reroute unrelated secreting proteins to a storage pathway. Thus, we designed another vector, 2bVpOVA, which contains VWF propeptide to secure OVA storage in platelet granules. HSCs from wild type B6/CD45.2 mice were transduced with 2bOVA or 2bVpOVA LV and transplanted into B6/CD45.1 recipients preconditioned with 660 cGy total body irradiation. We found that 96% of OVA expression in whole blood was stored in platelets with a level of 51.3 ± 22.5 ng/108 platelets (n = 5) while 4% was detectable in plasma in 2bOVA-transduced recipients at 12-week after transplantation. This distribution is very similar to the results we obtained from the FIX study. In contrast, 98% of OVA was stored in platelets with a level of 3.9 ± 3.3 ng/108 platelets (n = 5) in 2bVpOVA-transduced recipients. The lower total OVA expression level in the 2bVpOVA group could be due to the size effect of transgene expression cassette as the 2bVpOVA cassette is 3-fold larger than the 2bOVA cassette. To investigate whether anti-OVA immune tolerance was established in recipients after platelet-specific OVA gene transfer, 16-weeks post-transplantation, animals were challenged with OVA. The titer of anti-OVA total IgG determined by ELISA assay was 640 ± 101 in the 2bOVA group and 320 ± 0 in the 2bVpOVA group. These titers were significantly lower than that obtained from the untransduced control group (10210 ± 3636), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG in the 2bVpOVA group was significantly lower than in the 2bOVA group although the total OVA expression levels in the 2bOVA group is 13-fold higher than in the 2bVpOVA group. The percentage of regulatory T cells in peripheral blood in 2bOVA and 2bVpOVA-transduced recipients was significantly higher than in untransduced control animals. In summary, our data demonstrate that targeting transgene expression and storage in platelet a-granules is a potentially promising approach for inducing immune tolerance. Disclosures No relevant conflicts of interest to declare.

Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%. Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (>6 half-lives, calculated FVIII levels In conclusion, the nWB-TGA provides a useful tool to monitor efficacy of FVIII replacement therapy and might assist in tailoring individual FVIII treatment regimens. This close to physiological whole blood assay allows distinguishing blood samples with FVIII levels below 1% in vivo, and might help to explain the heterogeneity in bleeding phenotypes observed in severe hemophilia A patients. This assay may also be useful in assessing therapeutic benefit of “long acting” FVIII or FIX products. Disclosures No relevant conflicts of interest to declare.

Factor VIII (FVIII) gene therapy is a promising approach to potentially permanently and cost-effectively correct the bleeding phenotype of hemophilia A patients and improve patients quality of life. Our group has developed a successful gene therapy approach in which FVIII expression is targeted to platelets. Platelet expressed FVIII protects hemophilic mice from lethal blood loss after vessel injury. Most importantly this therapy does not induce FVIII inhibitory antibodies and is even successful in the treatment of mice with pre-existing high titer inhibitors. Therefore this approach is among the first to hold promise for patients who develop inhibitory antibodies against FVIII that render FVIII replacement therapy ineffective. Levels of platelet expressed FVIII achieved by gene therapy may vary between individuals due to differences in ex vivotransduction and gene expression efficiency. We determined hemostatic efficacy over a wide therapeutic dose range with a novel native whole blood thrombin generation assay. Tracking the correction of abnormal bleeding phenotypes during the treatment of patients with hemostatic disorders is crucial to evaluate success of therapy. Global coagulation assays in contrast to single clotting factor assays are desirable to better understand the overall hemostatic condition of patients. Here we evaluated thrombin generation using a modified protocol of a recently described whole blood assay. In our native assay we initiated coagulation without the addition of tissue factor. Sole recalcification of whole blood resulted in thrombin generation with high reproducibility. Lag time (LT) determined in blood from C57BL/6 WT mice was 6 ± 0.2 min (Mean ± SEM) , thrombin generation rate was 58 ± 6 nM/min and thrombin peak was 188 ± 7 nM. In contrast, FVIII deficient blood had negligible thrombin generation with 39 ± 7 min LT, 1.4 ± 0.3 nM/min thrombin generation rate and 12 ± 3 nM thrombin peak. Spiking hemophilic blood with increasing concentrations of recombinant FVIII ex vivo resulted in a dose dependent increase in thrombin generation. Reconstitution of hemophilic blood with FVIII to a 1%, 10% and 100% level shortened LT to 19 ± 1, 12 ± 0.3 and 9 ± 0.5 min, respectively. To evaluate efficacy of platelet-derived FVIII we utilized a newly developed transgenic mouse model that expresses high levels of FVIII in platelets. Homozygous mice express platelet FVIII levels corresponding to 20% endogenous FVIII in whole blood. We combined different ratios of FVIII deficient blood with blood from platelet FVIII expressing transgenic mice. At low ratios of transgenic blood, similar to ex vivospiking with recombinant FVIII, thrombin generation parameters were dose-dependent. Remarkably, a corresponding dose of as low as 0.2% platelet-derived FVIII significantly elevated thrombin generation above FVIII deficient blood and had comparable therapeutic efficacy as a 5-fold higher dose of recombinant FVIII (LT, 18 ± 2 vs 19 ± 1). Similarly, efficacy of 1.5% of platelet-derived FVIII compared with the 6.7-fold higher, 10% dose of recombinant FVIII (LT, 13 ± 1 vs 12 ± 0.3). Further increase of thrombin generation was noticed with platelet FVIII expressing transgenic blood ratios corresponding to 2% and 5% FVIII levels (LT, 11 ± 0.3 and 8.7 ± 0.3 min, respectively). Interestingly, our native assay showed that the platelet FVIII expressing transgenic blood ratio corresponding to a FVIII level of only 5% was sufficient to induce maximal thrombin generation, similar to that obtained with undiluted transgenic blood (LT, 8.7 ± 0.6 min). A similar FVIII dose-dependency was identified for additional thrombin generation parameters including endogenous thrombin potential, thrombin peak, peak time and thrombin generation rate. We conclude that this native whole blood thrombin generation assay could be used to track therapeutic efficacy of hemophilia A treatment. Using this assay, our data indicate that similar to FVIII replacement therapy our previously established platelet targeted FVIII gene therapy approach enhances hemostasis over a wide therapeutic dose level. This is of great importance because levels of platelet expressed FVIII achieved upon gene therapy in mice vary. In agreement with our previous reports our data from native whole blood thrombin generation assay confirm that at lower FVIII dose levels platelet targeted FVIII gene therapy might be more efficient than factor replacement therapy. Disclosures: No relevant conflicts of interest to declare.