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Inevitable relapses remain as the major therapeutic challenge in patients with mantle cell lymphoma (MCL) despite FDA approval of multiple targeted therapies and immunotherapies. Fc gamma receptors (FcγRs) play important roles in regulating antibody-mediated immunity. FcγRIIB, the unique immune-checkpoint inhibitory member of the FcγR family, has been implicated in immune cell desensitization and tumor cell resistance to the anti-CD20 antibody rituximab and other antibody-mediated immunotherapies; however, little is known about its expression and its immune-modulatory function in patients with aggressive MCL, especially those with multi-resistance. In this study, we found that FcγRIIB was ubiquitously expressed in both MCL cell lines and primary patient samples. FcγRIIB expression is significantly higher in CAR T-relapsed patient samples ( p  < 0.0001) compared to ibrutinib/rituximab-naïve, sensitive or resistant samples. Rituximab-induced CD20 internalization in JeKo-1 cells was completely blocked by concurrent treatment with BI-1206, a recombinant human monoclonal antibody targeting FcγRIIB. Combinational therapies with rituximab-ibrutinib, rituximab-venetoclax and rituximab-CHOP also induced CD20 internalization which was again effectively blocked by BI-1206. BI-1206 significantly enhanced the in vivo anti-MCL efficacy of rituximab-ibrutinib ( p  = 0.05) and rituximab-venetoclax ( p  = 0.02), but not the rituximab-CHOP combination in JeKo-1 cell line-derived xenograft models. In patient-derived xenograft (PDX) models, BI-1206, as a single agent, showed high potency ( p  < 0.0001, compared to vehicle control) in one aggressive PDX model that is resistant to both ibrutinib and venetoclax but sensitive to the combination of rituximab and lenalidomide (the preclinical mimetic of R 2 therapy). BI-1206 sensitized the efficacy of rituximab monotherapy in a PDX model with triple resistance to rituximab, ibrutinib and CAR T-therapies ( p  = 0.030). Moreover, BI-1206 significantly enhanced the efficacy of the rituximab-venetoclax combination ( p  < 0.05), which led to long-term tumor remission in 25% of mice. Altogether, these data support that targeting this new immune-checkpoint blockade enhances the therapeutic activity of rituximab-based regimens in aggressive MCL models with multi-resistance. Graphical Abstract

Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

CD8+ T cell-restricted immunity is important in the control of HIV-1 infection, but continued immune activation results in CD8+ T cell dysfunction. Early initiation of antiretroviral treatment (ART) and the duration of ART have been associated with immune reconstitution. Here, we evaluated whether restoration of CD8+ T cell function in HIV-1-infected individuals was dependent on early initiation of ART. HIV-specific CD107a, IFNγ, IL-2, TNFα and MIP-1β expression by CD8+ T cells and the frequency of CD8+ T cells expressing PD-1, 2B4 and CD160 were measured by flow cytometry. The frequency of CD8+ T cells expressing the inhibitory markers PD-1, 2B4 and CD160 was lower in ART-treated individuals compared with ART-naïve individuals and similar to the frequency in HIV-uninfected controls. The expression of the three markers was similarly independent of when therapy was initiated. Individuals treated before seroconversion displayed an HIV-specific CD8+ T cell response that included all five functional markers; this was not observed in individuals treated after seroconversion or in ART-naïve individuals. In summary, ART appears to restore the total CD8+ T cell population to a less exhausted phenotype, independent of the time point of initiation. However, to preserve multifunctional, HIV-1-specific CD8+ T cells, ART might have to be initiated before seroconversion.

BackgroundThe pleiotropic TNF-α:TNFR axis plays a central role in the immune system. TNFR2 has been proposed to be both essential for the survival of T regs, as well as providing important co-stimulatory signals for T cell activation and memory generation. In addition, the therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated. To gain further insight, we characterized the biophysical properties and in vitro and in vivo activities of human and mouse α-TNFR2-specific antibodies designed to agonize the receptor.MethodsA human lead candidate (BI-1910) and a mouse surrogate (mBI-1910) α-TNFR2 were identified. Agonistic activity on T cells were demonstrated for both antibodies in vitro. mBI-1910 showed potent anti-tumor activity both as a single agent and in combination with anti-PD1 in multiple immunocompetent tumor models. The antibody showed co-stimulation through TNFR2, which enhanced T cell activation and induced CD8+ T cell-dependent anti-tumor effects. These findings were confirmed using BI-1910 in human TNFR2 transgenic mice.To address safety, a GLP toxicological study was performed in cynomolgus macaques. Three doses (1, 5, and 25 mg/kg) were given weekly for four consecutive weeks followed by a recovery period of eight weeks. In addition, cytokine release was studied in T cell stimulation assays and in a humanized mouse model. In parallel, multiple immune stimulation assays were studied in vitro using human cells to establish EC50 values and a clear relationship with dose, receptor occupancy and immune cell activation.ResultsFour administrations of BI-1910 to cynomolgus macaques were well tolerated at all doses, with no associated clinical signs and no signs of cytokine release. Pharmacokinetic studies demonstrated an expected human IgG half-life at receptor-saturating doses. Interestingly, there was a clear dose-dependent T cell activation, evidenced by an increase in several T cell activation markers and a shift from naïve to effector memory T cells supporting the proposed mode-of action. Importantly, the nature of BI-1910-induced T cell activation in cynomolgus macaques closely mirrored that in TNFR2 humanized mice, in which clear anti-tumor effects were also demonstrated.ConclusionsThe strong similarities in BI-1910 induced immune response between mice and cynomolgus macaques shows promise that similar T cell activation and following anti-tumor effects will occur also in humans. Collectively, these studies support the upcoming phase I/II study in solid cancer patients planned to start in H2 2023.Ethics ApprovalAll data utilizing human blood or animals was approved by an ethic committé before the experiments were started.Experiments using human blood were approved by the Regional committé for ethichs approval in Lund, Sweden ID numbers 2018/37 and 2010/356The murine experiments were approved by the Ethical committé of animal experiment in Lund and Malmö, Sweden ID numbers 5.8.18–19686/2022; 5.8.18–03333-2020; 5.8.18–02934-2020; 5.8.18–17196-2018The non-human primate experiments were approved by Charles River Laboratories Evreux Ethics Committee (CEC), France, ID number 2850398

Specific cellular cytotoxic immune responses (CTL) are important in combating viral diseases and a highly desirable feature in the development of targeted HIV vaccines. Adjuvants are key components in vaccines and may assist the HIV immunogens in inducing the desired CTL responses. In search for appropriate adjuvants for CD8(+) T cells it is important to measure the necessary immunological features e.g. functional cell killing/lysis in addition to immunological markers that can be monitored by simple immunological laboratory methods. We tested the ability of a novel two component adjuvant, CAF01, consisting of the immune stimulating synthetic glycolipid TDB (Trehalose-Dibehenate) incorporated into cationic DDA (Dimethyldioctadecylammonium bromide) liposomes to induce CD8(+) T-cell restricted cellular immune responses towards subdominant minimal HLA-A0201-restricted CTL epitopes from HIV-1 proteins in HLA-A*0201 transgenic HHD mice. CAF01 has an acceptable safety profile and is used in preclinical development of vaccines against HIV-1, malaria and tuberculosis. We found that CAF01 induced cellular immune responses against HIV-1 minimal CTL epitopes in HLA-A*0201 transgenic mice to levels comparable with that of incomplete Freund's adjuvant.

BackgroundBI-1808 is a human IgG1 monoclonal antibody targeting TNFR2 by blocking the interaction of TNFR2 with its ligand TNF-α, confering FcγR-dependent depletion of intratumoral Tregs and mediating expansion of intratumoral CD8+ T cells. Upon co-administration of BI-1808 and anti-PD-1 surrogate antibodies to immunocompetent tumor-bearing mice, with partial sensitivity to checkpoint blockade, complete cures were observed in all treated mice, indicating a potentially synergistic activity.MethodsSafety and tolerability of BI-1808 as a single agent and in combination with pembrolizumab is currently investigated in the Phase 1/2a trial 19-BI-1808–01 in patients with advanced malignancies or cutaneous T-cell lymphoma (CTCL). The trial consists of Phase 1 Parts A and B (dose escalation with single agent and combination with pembrolizumab, respectively), and Phase 2a Parts A and B (dose expansion with single agent and combination therapy, respectively). Dose escalation uses a modified toxicity probability interval-2 protocol (mTPI-2), investigating ascending dose levels of 25–1000 mg every three weeks (Q3W). Dose escalation aims to select both single agent RP2D and combination RP2D of BI-1808 for Phase 2a.Patients are sampled for pharmacokinetics (PK) of BI-1808, antidrug-antibodies and pharmacodynamics including lymphocyte subsets, regulatory T cells, memory T-cells, soluble TNFR2 serum concentration (sTNFR2) and BI-1808 receptor occupancy (RO).ResultsAs of June 19th, 2023, 24 subjects with various advanced solid malignancies received doses of up to 1000 mg BI-1808 as single-agent treatment, and 7 subject received 225 mg doses of BI-1808 with pembrolizumab.Across the completed monotherapy arm, no Grade 3/4 AEs, AEs related to BI-1808 and no DLTs were observed. No MTD was defined. The number of potentially related AEs of Gr 1/2 are evenly distributed across the dose range, with no target system organ class of special notice identified. Best clinical response recorded are stable disease (SD) in 7/19 evaluable patients in the monotherapy arm. The first dose cohort for BI-1808 at 225 mg in combination with pembrolizumab is currently ongoing.BI-1808 exhibits a non-linear PK. At doses > 675 mg Q3W, t½ was approximately 1 week resulting in accumulation of drug, with complete RO throughout the dosing interval.ConclusionsPreliminary data from the BI-1808 monotherapy arm from the clinical trial 19-BI-1808–01 is promising. BI-1808 has a favorable safety profile, with no DLTs observed. SD was observed in 7/19 evaluable patients. Doses of 675 mg and higher are expected to provide complete RO throughout the dose interval, and will be further explored in Ph2a.

BackgroundThe pleiotropic TNF-alpha:TNFR axis plays a central role in the immune system. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated and to gain further insight, we characterized a wide panel antibodies, generated from the n-CoDeR F.I.R.S.T™ target and antibody discovery platform. We identified parallel human and mouse TNFR2 specific, complete ligand (TNF-alpha) blocking antibodies and could show potent anti-tumor activity in several immune-competent models, both as single agent and in combination with anti-PD1 using a BI-1808 murine surrogate. The mechanism-of-action was shown to be FcgR dependent and likely mediated through a combination of intra-tumor T reg depletion, CD8+ T cell expansion and modulation of tumor-associated myeloid cells. These findings were confirmed using BI-1808 in a humanized mouse model.MethodsTo address safety of the human lead-candidate BI-1808 two toxicological studies were performed in cynomolgus monkeys. The first study was a dose-range-finding study and the second a GLP study where three doses (2, 20 and 200 mg/kg) were given weekly for four consecutive weeks followed by a recovery period of eight weeks. In addition, cytokine release was further studied in T cell stimulation assays and in a humanized mouse model. Moreover, the BI-1808 murine surrogate was used to study the relationship between dose, receptor occupancy (RO) and efficacy in immune competent mouse cancer experimental models.ResultsFour weekly administrations of BI-1808 to cynomolgus monkeys were well tolerated at all doses, with no associated clinical signs, and no histopathological changes. Non-adverse and reversible increases in neutrophil counts and decreases in T cells were observed at all dose levels. No drug-related adverse events were observed and consequently the NOAEL for BI-1808 was determined to be 200 mg/kg. Pharmacokinetic studies demonstrated an expected half-life of two weeks at receptor saturation. There were no indications of cytokine release in any of the systems tested. Finally, we could show that to achieve max therapeutic effect, sustained RO was needed for approximately two weeks, covering the time it takes to generate a full adaptive Immune response.ConclusionsThere is a clear association between RO and therapeutic effect and BI-1808 is well tolerated at doses associated with high and sustained RO. Collectively, these studies were used to determine the starting dose in upcoming phase I/II study in solid cancer aiming for first-patient in during December 2020.Ethics ApprovalThe study on cynomolgous monkeys was conducted by Citox/Charles River Laboratories in compliance with animal health regulations, in particular: Council Directive No. 2010/63/EU of 22 September 2010 and French decree No. 2013-118 of 01 February 2013 on the protection of animals used for scientific purposes. Studies in mice were approved by the Swedish Animal Experiment Ethics Board, ethical permit/ethical license numbers 5.2.18-17196/2018 and 5.8.18-03333/2020

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

Background Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. Results The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. Conclusion We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad) viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression. Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow) phenotype (n = 20), but R5(broad) and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad) and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad) phenotype, however, the presence of the R5(broad) virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad) viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad) phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. Our results show that R5(broad) viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow) phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.