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Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4 and CD8 T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33 + and CD123 + AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment. Keypoints Modular platform enabling controlled targeting of AML by SAR-transduced T cells in combination with tandem scFv constructs. Efficient lysis of primary AML blasts in vitro and strong antitumoral effects and T cell persistence in xenograft models.

Background Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 “don’t eat me signal” is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. Methods SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. Results SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123 + CD47 + AML cells even in the presence of CD47 + healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. Conclusions SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.

BackgroundDespite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects.MethodsAntibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).ResultsThree different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model.ConclusionThis study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo .

Objectives Innovative post‐remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti‐leukaemic immune responses. Methods We conducted a first‐in‐human phase I study using TLR7/8‐matured DCs transfected with RNA encoding the two AML‐associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks. Results Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T‐cell infiltration. In peripheral blood, increased antigen‐specific CD8+ T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4+ T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen‐specific immune responses. Conclusions Administration of TLR7/8‐matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen‐specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses. Trial registration The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT‐Number 2010‐022446‐24) on 10 October 2013. Dendritic cell (DC) vaccination is a promising strategy to induce anti‐leukaemic immune responses. In this first‐in‐human phase I trial, TLR7/8‐matured DCs transfected with RNA encoding two leukaemia‐associated antigens (WT1 and PRAME) and CMVpp65 were used as post‐remission therapy for AML patients at high risk of relapse. DC generation was feasible, and administration was safe and resulted in local inflammatory responses and expanded antigen‐specific CD8+ and CD4+ T cells in peripheral blood; clinical benefit correlated with younger age and immune responders.

Background Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 \"don't eat me signal\" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRP[alpha]). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRP[alpha]-[alpha]CD123 fusion antibodies that localize the disruption of CD47/SIRP[alpha] signalling to AML while specifically enhancing LSC clearance. Methods SIRP[alpha]-[alpha]CD123 antibodies were generated by fusing the extracellular domain of SIRP[alpha] to an [alpha]CD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. Results SIRP[alpha]-[alpha]CD123 fusion antibodies exhibited increased binding and preferential targeting of CD123.sup.+ CD47.sup.+ AML cells even in the presence of CD47.sup.+ healthy cells. Furthermore, SIRP[alpha]-[alpha]CD123 fusion antibodies confined disruption of the CD47-SIRP[alpha] axis locally to AML cells. In vitro experiments demonstrated that SIRP[alpha]-[alpha]CD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRP[alpha]-[alpha]CD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. Conclusions SIRP[alpha]-[alpha]CD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRP[alpha]-[alpha]CD123 antibodies as promising therapeutic interventions for AML. Keywords: CD47, Acute myeloid leukaemia, CD123, Leukemic stem cells, Phagocytosis, Immunotherapy

BackgroundExtensive investigations into the tumor microenvironment (TME) have uncovered molecular mechanisms linking aberrant complement activation and cancer progression. Specifically, C5a, as a highly potent chemoattractant, recruits immune suppressive myeloid derived suppressive cells (MDSCs), neutrophils and M2 macrophages into the tumor site and accelerates tumor progression. Blockade of C5a/C5aR (CD88) pathway has been identified as a promising target to control MDSCs and restore tumor-killing ability of T and NK cells. TJ210, in licensed from MorphoSys as MOR210, is a differentiated anti-C5aR monoclonal antibody with a unique binding epitope.MethodsInteraction of TJ210 with C5aR was assessed through binding of the recombinant antigen, Flp-In CHO cells expressing C5aR and primary neutrophils. In vitro blockade of C5a/C5aR pathway was tested by inhibition of CD11b upregulation on granulocytes and monocytes induced by C5a, as well as neutrophil migration towards C5a. The in vitro synergistic effect of TJ210 with anti-PD-1 antibody was assessed in a T cell and differentiated MDSC co-culture system. The in vivo anti-tumor effect was tested in the MC38 syngeneic mouse model, in which mice were treated with a TJ210 mouse surrogate antibody either alone or in combination with an anti-PD-1 antibody.ResultsTJ210 bound to C5aR with high affinity and did not cross-react with other GPCR members including C5L2, ChemR23, FPR1 and C3aR. Unlike the reference antibody, TJ210 specifically interacted with the N-terminus of C5aR but not extracellular loops. TJ210 effectively inhibited CD11b upregulation on granulocytes and monocytes as well as neutrophil migration mediated by C5a. When compared with the reference antibody, TJ210 maintained potent antagonism at high ligand concentrations and over longer duration, properties that might translate into beneficial in vivo effects at pathophysiological conditions. In the in vitro co-culture system, presence of TJ210 and anti-PD-1 antibody enhanced IFN-γ release compared to either single agent, indicating a synergistic effect on T cells. In the in vivo syngeneic mouse model, combination treatment effectively inhibited tumor growth. Immune cell population analysis revealed significant elevation of CD8+ T cells and M1 macrophages compared to mono-treatment.ConclusionsThis series of in vitro and in vivo data demonstrate that TJ210 is a differentiated anti-C5aR antibody with unique binding epitope exhibiting superior anti-tumor potential especially in combination with an anti-PD-1 antibody. These data support further clinical studies of TJ210 in patients with solid tumors.