Glycoprotein D, a checkpoint inhibitor of early T-cell activation, to improve immunogenicity and efficacy of an HPV-16 vaccine in preclinical studies.

2020 ◽  
Vol 38 (5_suppl) ◽  
pp. 71-71
Author(s):  
Hildegund Ertl ◽  
Zhiquan Xiang ◽  
Yan Li ◽  
Andrew Luber ◽  
Colin Magowan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Checkpoint Inhibitor ◽  
Wild Type ◽  
Hpv 16 ◽  
Cell Responses

71 Background: CD8+ T cells can inhibit tumor progression, but their induction is hampered by the low immunogenicity of most tumor antigens. HSV-1 glycoprotein D (gD), when genetically expressed as a fusion protein with tumor antigens, serves as a checkpoint inhibitor of the B and T cell attenuator (BTLA)-herpes virus entry mediator (HVEM) pathway, which acts early during T cell activation. HSV-1 gD thereby augments antigen-driven CD8+ T cell responses. We describe the immunogenicity and efficacy of a chimpanzee adenoviral vector (AdC) vaccine containing a detoxified E7/E6/E5(AdC-gDE765dt) sequence of HPV-16 fused into gD. Methods: The frequency of HPV-16 E7-specific CD8+ T-cells was assessed by tetramer staining in C57/Bl6 mice 14 days after a single IM vaccination with AdC vectors encoding wild-type or mutant HPV-16 oncoproteins expressed within gD, a non-HVEM-binding form of gD or without gD. Efficacy was tested in a TC-1 tumor cell challenge model with mice receiving no treatment or a single IM vaccine injection 3 days after tumor cell transplantation. Mice were followed for 80 days. Results: The addition of gD increases HPV-16 E7-specific CD8+ T-cell frequencies approximately 10-fold. T cell responses are similar to AdC vaccines expressing wild-type or mutant oncoproteins within gD. All AdC-gDE765dt treated mice show delayed tumor progression after a single vaccination with 50% of animals remaining tumor-free at study completion. Conclusions: These results show that the addition of gD, an early checkpoint inhibitor, which acts locally at the site of T cell stimulation, to an HPV-16 vaccine markedly improves the vaccine’s immunogenicity and efficacy. AdC-gDE765dt is currently in GMP manufacture for Phase 1 investigation in HPV-16 infected patients.

scholarly journals Co-Stimulatory Bispecific Antibodies Induce Enhanced T Cell Activation and Tumor Cell Killing in Breast Cancer Models

2021 ◽  
Vol 12 ◽  
Author(s):  
Karsten M. Warwas ◽  
Marten Meyer ◽  
Márcia Gonçalves ◽  
Gerhard Moldenhauer ◽  
Nadja Bulbuc ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Fusion Proteins ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Bispecific Antibodies ◽  
Tumoricidal Activity

Although T cell-recruiting CD3-binding bispecific antibodies (BiMAb) have been proven to be clinically effective for hematologic malignancies, the success of BiMAb targeting solid tumor-associated antigens (TAA) in carcinomas so far remains poor. We reasoned that provision of co-stimulatory BiMAb in combination with αTAA–αCD3 BiMAb would boost T cell activation and proliferative capacity, and thereby facilitate the targeting of weakly or heterogeneously expressed tumor antigens. Various αTAA–αCD3 and αTAA–αCD28 BiMAb in a tetravalent IgG1-Fc based format have been analyzed, targeting multiple breast cancer antigens including HER2, EGFR, CEA, and EpCAM. Moreover, bifunctional fusion proteins of αTAA–tumor necrosis factor ligand (TNFL) superfamily members including 4-1BBL, OX40L, CD70 and TL1A have been tested. The functional activity of BiMAb was assessed using co-cultures of tumor cell lines and purified T cells in monolayer and tumor spheroid models. Only in the presence of tumor cells, αTAA–αCD3 BiMAb activated T cells and induced cytotoxicity in vitro, indicating a strict dependence on cross-linking. Combination treatment of αTAA–αCD3 BiMAb and co-stimulatory αTAA–αCD28 or αTAA–TNFL fusion proteins drastically enhanced T cell activation in terms of proliferation, activation marker expression, cytokine secretion and tumor cytotoxicity. Furthermore, BiMAb providing co-stimulation were shown to reduce the minimally required dose to achieve T cell activation by at least tenfold. Immuno-suppressive effects of TGF-β and IL-10 on T cell activation and memory cell formation could be overcome by co-stimulation. BiMAb-mediated co-stimulation was further augmented by immune checkpoint-inhibiting antibodies. Effective co-stimulation could be achieved by targeting a second breast cancer antigen, or by targeting fibroblast activation protein (FAP) expressed on another target cell. In tumor spheroids derived from pleural effusions of breast cancer patients, co-stimulatory BiMAb were essential for the activation tumor-infiltrating lymphocytes and cytotoxic anti-tumor responses against breast cancer cells. Taken together we showed that co-stimulation significantly potentiated the tumoricidal activity of T cell-activating BiMAb while preserving the dependence on TAA recognition. This approach could provide for a more localized activation of the immune system with higher efficacy and reduced peripheral toxicities.

scholarly journals A role for CD9 molecules in T cell activation.

1996 ◽  
Vol 184 (2) ◽  
pp. 753-758 ◽  
Author(s):  
X G Tai ◽  
Y Yashiro ◽  
R Abe ◽  
K Toyooka ◽  
C R Wood ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Expression Cloning ◽  
Wild Type ◽  
Almost All ◽  
Antigen Presenting ◽  
Deficient Mice

Costimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC). The molecule recognized by this mAb, 9D3, was found to be expressed on almost all mature T cells and to be a protein of approximately 24 kD molecular mass. By expression cloning, this molecule was identified as CD9, 9D3 (anti-CD9) synergized with suboptimal doses of anti-CD3 mAb in inducing proliferation by virgin T cells. Costimulation was induced by independent ligation of CD3 and CD9, suggesting that colocalization of these two molecules is not required for T cell activation. The costimulation by anti-CD9 was as potent as that by anti-CD28. Moreover, anti-CD9 costimulated in a CD28-independent way because anti-CD9 equally costimulated T cells from the CD28-deficient as well as wild-type mice. Thus, these results indicate that CD9 serves as a molecule on T cells that can deliver a potent CD28-independent costimulatory signal.

scholarly journals High Frequencies of Functional Virus-Specific CD4+ T Cells in SARS-CoV-2 Subjects With Olfactory and Taste Disorders

2021 ◽  
Vol 12 ◽  
Author(s):  
Dalila Mele ◽  
Anna Calastri ◽  
Eugenia Maiorano ◽  
Antonella Cerino ◽  
Michele Sachs ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Responses ◽  
Control Group ◽  
Presenting Symptoms ◽  
Cell Responses ◽  
Taste Disorders ◽  
Specific Igg

Olfactory and taste disorders (OTD) are commonly found as presenting symptoms of SARS-CoV-2 infection in patients with clinically mild COVID-19. Virus-specific T cells are thought to play an important role in the clearance of SARS-CoV-2; therefore the study of T cell specific immune responses in patients with mild symptoms may help to understand their possible role in protection from severe disease. We evaluated SARS-CoV-2-specific T cell responses to four different peptide megapools covering all SARS-CoV-2 proteins during the acute phase of the disease in 33 individuals with mild or no other symptom beside OTD and in 22 age-matched patients with severe infection. A control group of 15 outpatients with OTD and consistently negative nasopharyngeal SARS-CoV-2 RNA swabs and virus-specific IgG serology was included in the study. Increased frequencies of virus-specific CD4+ and CD8+ T cells were found in SARS-CoV-2 positive patients with OTD compared with those with severe COVID-19 and with SARS-CoV-2 negative OTD individuals. Moreover, enhanced CD4+ and CD8+ T-cell activation induced by SARS-CoV-2 peptides was associated with higher interferon (IFN)γ production. Increased frequencies of Spike (S1/S2)-specific CD4+ T cells showing enhanced IFNγ secretion and granzyme B content were associated with serum spike-specific IgG in the OTD group. In conclusion, patients with SARS-CoV-2 induced OTD develop highly functional virus-specific CD4+ and CD8+ T cells during the symptomatic phase of the disease, suggesting that robust and coordinated T-cell responses provide protection against extension of COVID-19 to the lower respiratory tract.

Abstract 449: Novel Role of Bim in the Regulation of Allogeneic T-Cell Activation and Development of Transplant Vasculopathy

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Anna von Rossum ◽  
Winnie Enns ◽  
Yu P Shi ◽  
Jonathan C Choy
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
T Cell Responses ◽  
Intimal Thickening ◽  
Cell Responses ◽  
Transplant Vasculopathy

Transplant vasculopathy (TV) is an arteriosclerotic disease characterized by intimal thickening of allograft arteries and is a leading cause of heart transplant rejection. T cell responses towards allograft arteries are responsible for the development of TV and understanding the regulatory pathways controlling T cell activation in allograft arteries provides opportunities for the therapeutic attenuation of TV as well as other arteriosclerotic diseases. Bim is a pro-apoptotic Bcl-2 protein known to down-regulate immune responses after viral infections by inducing cell death of effector T cells but its role in regulating allogeneic T cell responses is not known. We compared cell death and alloantigen-driven activation of T cells from Bim +/+ (wild-type), Bim +/- and Bim -/- mice as well as the development of TV in these mice. Bim was required for cell death of both CD4 and CD8 T cells in response to cytokine deprivation in vitro . Unexpectedly, Bim was also required for alloantigen-induced proliferation of both CD4 and CD8 T cells as well as for IL-2 production. When TV was examined in aortic interposition grafts implanted into complete major histocompatibility complex-mismatched mice, intimal thickening was significantly reduced in Bim +/- but not Bim -/- recipients as compared to Bim +/+ counterparts. There was signficantly less CD4 T cell accumulation in the intima of arteries from Bim +/- as compared to Bim +/+ recipients but this effect was not observed in Bim -/- recipients. The accumulation of CD8 T cells in allograft arteries was not affected by differences in Bim expression. Taken together, our data support a novel role for Bim in driving T cell activation in response to allogeneic stimuli and indicate that the effects of this Bcl-2 protein in the pathogenesis of TV likely depends on its dual role in supporting T cell activation and death.

scholarly journals HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India

2019 ◽  
Vol 16 (4) ◽  
pp. 302-314
Author(s):  
Chinnambedu Ravichandran Swathirajan ◽  
Ramachandran Vignesh ◽  
Greer Waldrop ◽  
Uma Shanmugasundaram ◽  
Pannerselvam Nandagopal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Activation Rates ◽  
Hiv 1

Background:Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations.Objective:This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP.Methods:HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-γ, TNF-α and MIP-1β against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38.Results:Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP.Conclusion:LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

scholarly journals MALT1 Controls Attenuated Rabies Virus by Inducing Early Inflammation and T Cell Activation in the Brain

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
E. Kip ◽  
J. Staal ◽  
L. Verstrepen ◽  
H. G. Tima ◽  
S. Terryn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Infection ◽  
Rabies Virus ◽  
T Cell Activation ◽  
Therapeutic Target ◽  
Cell Activation ◽  
Wild Type ◽  
The Impact ◽  
The Brain

ABSTRACTMALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1−/−mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1−/−mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1−/−mice at 10 dpi compared to MALT1+/+infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+mice. Moreover, MALT1−/−mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain.IMPORTANCERabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.

scholarly journals Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Ulrike Sauermann ◽  
Antonia Radaelli ◽  
Nicole Stolte-Leeb ◽  
Katharina Raue ◽  
Massimiliano Bissa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Responses ◽  
Target Cells ◽  
Aids Vaccine ◽  
Cell Responses ◽  
Antibody Titers

ABSTRACT An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen. IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

scholarly journals RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity

2020 ◽  
Author(s):  
Daniel Peltier ◽  
Molly Radosevich ◽  
Guoqing Hou ◽  
Cynthia Zajac ◽  
Katherine Oravecz-Wilson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Solid Organ Transplantation ◽  
Solid Organ ◽  
Hematopoietic Stem ◽  
Cell Responses ◽  
Human T Cells ◽  
Allogeneic Stimulation

ABSTRACTMechanisms governing allogeneic T-cell responses after allogeneic hematopoietic stem cell (HSC) and solid organ transplantation are incompletely understood. Long non-coding RNAs (lncRNA) do not code for, but control gene expression with tissue specificity. However, their role in T-cell alloimmunity is unknown. We performed RNA-seq on donor T-cells from HSCT patients and found that increasing strength of allogeneic stimulation caused greater differential expression of lncRNAs. The differential expression was validated in an independent patient cohort, and also following ex vivo allogeneic stimulation of healthy human T-cells. Linc00402, a novel, conserved lncRNA, was identified as the most differentially expressed and was enriched 88 fold in human T-cells. Mechanistically, it was mainly located in the cytoplasm, and its expression was rapidly reduced following T-cell activation. Consistent with this, tacrolimus preserved the expression of Linc00402 following T-cell activation, and lower levels of Linc00402 were found in patients who subsequently went on to develop acute graft versus host disease (GVHD). The dysregulated expression of Linc00402 was also validated in murine T-cells, both in vitro and in vivo. Functional studies using multiple modalities to deplete Linc00402 in both mouse and human T-cells, demonstrated a critical role for Linc00402 in the T-cell proliferative response to an allogeneic stimulus but not a non-specific anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a novel, conserved regulator of allogeneic T-cell function. Because of its T-cell specific expression and its impact on allogeneic T-cell responses, targeting Linc00402 may improve outcomes after allogeneic HSC and solid organ transplantation.One sentence summaryLncRNAs are differentially expressed by allogeneic antigen-stimulated T-cells, and the novel lncRNA, Linc00402, is a specific regulator of mouse and human allogeneic T-cells.

scholarly journals In Vitro Monitoring of Human T Cell Responses to Skin Sensitizing Chemicals—A Systematic Review

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 83
Author(s):  
Marina Aparicio-Soto ◽  
Caterina Curato ◽  
Franziska Riedel ◽  
Hermann-Josef Thierse ◽  
Andreas Luch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Cell Responses ◽  
Human T Cell ◽  
The Individual

Background: Chemical allergies are T cell-mediated diseases that often manifest in the skin as allergic contact dermatitis (ACD). To prevent ACD on a public health scale and avoid elicitation reactions at the individual patient level, predictive and diagnostic tests, respectively, are indispensable. Currently, there is no validated in vitro T cell assay available. The main bottlenecks concern the inefficient generation of T cell epitopes and the detection of rare antigen-specific T cells. Methods: Here, we systematically review original experimental research papers describing T cell activation to chemical skin sensitizers. We focus our search on studies published in the PubMed and Scopus databases on non-metallic allergens in the last 20 years. Results: We identified 37 papers, among them 32 (86%) describing antigen-specific human T cell activation to 31 different chemical allergens. The remaining studies measured the general effects of chemical allergens on T cell function (five studies, 14%). Most antigen-specific studies used peripheral blood mononuclear cells (PBMC) as antigen-presenting cells (APC, 75%) and interrogated the blood T cell pool (91%). Depending on the individual chemical properties, T cell epitopes were generated either by direct administration into the culture medium (72%), separate modification of autologous APC (29%) or by use of hapten-modified model proteins (13%). Read-outs were mainly based on proliferation (91%), often combined with cytokine secretion (53%). The analysis of T cell clones offers additional opportunities to elucidate the mechanisms of epitope formation and cross-reactivity (13%). The best researched allergen was p-phenylenediamine (PPD, 12 studies, 38%). For this and some other allergens, stronger immune responses were observed in some allergic patients (15/31 chemicals, 48%), illustrating the in vivo relevance of the identified T cells while detection limits remain challenging in many cases. Interpretation: Our results illustrate current hardships and possible solutions to monitoring T cell responses to individual chemical skin sensitizers. The provided data can guide the further development of T cell assays to unfold their full predictive and diagnostic potential, including cross-reactivity assessments.

scholarly journals Local Activator and T Cell Engager (αBCMA x αPD-L1 x αCD3) with Enhanced Tumor Killing and Minimal Cytokine Release for the Treatment of Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Melissa Vrohlings ◽  
Stephanie Jungmichel ◽  
Jan Müller ◽  
David Senn ◽  
Thomas Schleier ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cytokine Release ◽  
Private Company

BCMA-targeting bispecific T-cell engagers in clinical development have demonstrated encouraging preclinical efficacy. The most advanced of these is AMG 420, which showed significantly improved response rates in relapsed/refractory multiple myeloma (MM) patients. Nevertheless, median duration until relapse is currently limited to approximately 12 months, highlighting the need for new drugs with novel MoA. Recently, we reported on a Local Activator and T cell Engager (LocATE) antibody that targets BCMA and selectively blocks programmed death-ligand 1 (PD-L1) on malignant cells (ASCO, June 2019). LocATE induced superior T cell activation and cancer cell killing, in vitro and ex vivo, compared to a BCMAxCD3 BiTE alone or in combination with a PD-L1 inhibitor. Here, we sought to further characterize the novel MoA of our LocATE. To assess the therapeutic potential of the LocATE, we first investigated whether potent cytotoxicity is uncoupled from high levels of cytokine release. We evaluated three LocATE molecules with different PD-L1 affinities (low, medium, high). Using BCMA-expressing MM cell lines (U-266, MM.1S, RPMI-8226 and H929) with distinct PD-L1 surface expression levels (3 - 53%), we determined the cytokine profile (IL-2, IL-6, IFN-γ, TNF-α) and target cell lysis induced by each candidate in the presence of CD3-positive human T cells. All three candidates exhibited comparable killing potency, however, low-affinity PD-L1 LocATE antibodies induced significantly less cytokine release (up to 10-fold) than its higher PD-L1 affinity counterparts across all cell lines investigated. Notably, using the low-affinity PD-L1 LocATE, we observed a 2-fold increase in tumor cell killing compared to bispecific BCMAxCD3 targeting controls in cell lines expressing high PD-L1 levels (53%), underlining the contribution of PD-L1 inhibition. Accordingly, phenotypic profiling of effector cells showed that the LocATE more potently induced dose-dependent upregulation of the activation markers CD69, CD25 and HLA-DR compared to bispecific controls. Importantly, cytotoxic activity, T cell activation and cytokine release were not induced when BCMA-negative cells expressing high levels of PD-L1 were treated with LocATE, underlining the BCMA-selective killing mechanism. Since the superior efficacy of LocATE was found to correlate with the expression level of PD-L1 on MM cell lines and upregulation of PD-L1/PD-1 has been reported as one of the major myeloma cell escape mechanisms during treatment with BiTEs, we subsequently investigated the efficacy of LocATE using primary bone marrow samples and peripheral blood mononuclear cell (PBMCs) obtained from MM patients. Six bone marrow mononuclear cell (BMMC) and eight PBMC samples from MM donors of different disease stages were characterized for PD-1/PD-L1 expression levels; analysis of T cell frequency and level of activation/exhaustion was performed based on CD4, CD8, CD25, CD69, Tim-3, Lag-3 and PD-1 marker expression. Using patient samples with high frequencies of PD-1 expressing T cells prior to treatment, LocATE induced superior MM tumor cell lysis and T cell activation compared to BCMAxCD3 bispecific antibodies. No activity was observed on healthy cells, underlining the safe and selective killing mechanism through tumor-local reactivation of exhausted T cells. Collectively, these findings demonstrate that the simultaneous T cell redirection and tumor-specific checkpoint inhibition with the LocATE leads to an improved therapeutic index with robust tumor cell killing and low levels of cytokine release. Capable of counteracting adaptive immune resistance caused by increased PD-1/PD-L1 signaling, the LocATE antibody has the prospect to significantly improve survival for multiple myeloma patients. Disclosures Vrohlings: CDR-Life: Current Employment. Jungmichel:CDR-Life: Current Employment, Other: current option holder. Senn:CDR-Life: Current Employment. Schleier:CDR-Life: Current Employment, Current equity holder in private company. Scheifele:CDR-Life: Current Employment, Current equity holder in private company. Wendelspiess:CDR-Life: Current Employment. Leisner:CDR-Life: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Jaeger:CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria; Karyopharm: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding. Borras:CDR-Life: Current Employment, Current equity holder in private company.

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