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Smoldering multiple myeloma (SMM) is an indolent disease stage, considered to represent the transition phase from the premalignant MGUS (Monoclonal Gammopathy of Undetermined Significance) state towards symptomatic multiple myeloma (MM). Even though this diagnosis provides an opportunity for early intervention, few treatment studies have been done and the current standard of care is observation until progression. BI-505, a monoclonal antibody directed against intercellular adhesion molecule 1 (ICAM-1) with promising anti-myeloma activity in preclinical trials, is a possible treatment approach for this patient category with potential to eliminate tumor cells with minimal long-term side effects. BI-505 was well tolerated in an earlier phase 1 trial. In this phase 2 trial the effects of BI-505 in patients with SMM were studied. Four patients were enrolled and three of them completed the first cycle of treatment defined as 5 doses of BI-505, a total of 43 mg/kg BW, over a 7-week period. In the three evaluable patients, BI-505 showed a benign safety profile. None of the patients achieved a response as defined per protocol. EudraCT number: 2012-004884-29. The study was conducted to assess the efficacy, safety and pharmacodynamics of BI-505 in patients with SMM. BI-505 showed no clinically relevant efficacy on disease activity in these patients with SMM, even if well tolerated. ClinicalTrials.gov Identifier: NCT01838369.

Immunotherapy with therapeutic antibodies has increased survival for patients with hematologic and solid cancers. Still, a significant fraction of patients fails to respond to therapy or acquire resistance. Understanding and overcoming mechanisms of resistance to antibody drugs, and in particular those common to antibody drugs as a class, is therefore highly warranted and holds promise to improve response rates, duration of response and potentially overall survival. Activating and inhibitory Fc gamma receptors (FcγR) are known to coordinately regulate therapeutic activity of tumor direct-targeting antibodies. Similar, but also divergent, roles for FcγRs in controlling efficacy of immune modulatory antibodies e.g., checkpoint inhibitors have been indicated from mouse studies, and were recently implicated in contributing to efficacy in the human clinical setting. Here we discuss evidence and mechanisms by which Fc gamma receptors-the \"antibody checkpoints\"-regulate antibody-induced antitumor immunity. We further discuss how targeted blockade of the sole known inhibitory antibody checkpoint FcγRIIB may help overcome resistance and boost activity of clinically validated and emerging antibodies in cancer immunotherapy.

Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Yet, deconvoluting the targets of phenotypically discovered antibodies remains a bottleneck; efficient deconvolution methods are needed for phenotypic discovery to reach its full potential. Here, we report a comprehensive investigation of a target deconvolution approach based on pooled CRISPR/Cas9. Applying this approach within three real-world phenotypic discovery programs, we rapidly deconvolute the targets of 38 of 39 test antibodies (97%), a success rate far higher than with existing approaches. Moreover, the approach scales well, requires much less work, and robustly identifies antibodies against the major histocompatibility complex. Our data establish CRISPR/Cas9 as a highly efficient target deconvolution approach, with immediate implications for the development of antibody-based drugs. Efficient deconvolution of antibody targets is needed for phenotype-based discovery. Here, the authors investigate a deconvolution approach based on pooled CRISPR Cas9 to achieve 97% deconvolution success rate.

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

B cells are critical to the development of multiple sclerosis (MS), but the mechanisms by which they contribute to the disease are poorly defined. We hypothesised that the expression of CD32b (FcγRIIb), a receptor for the Fc region of IgG with inhibitory activities in B cells, is lower on B cell subsets from people with clinically isolated syndrome (CIS) or MS. CD32b expression was highest on post-naive IgM + B cell subsets in healthy controls. For females with MS or CIS, significantly lower CD32b expression was identified on IgM + B cell subsets, including naive and IgM hi MZ-like B cells, when compared with control females. Lower CD32b expression on these B cell subsets was associated with detectable anti-Epstein Barr Virus viral capsid antigen IgM antibodies, and higher serum levels of B cell activating factor. To investigate the effects of lower CD32b expression, B cells were polyclonally activated in the presence of IgG immune complexes, with or without a CD32b blocking antibody, and the expression of TNF and IL-10 in B cell subsets was assessed. The reduction of TNF but not IL-10 expression in controls mediated by IgG immune complexes was reversed by CD32b blockade in naive and IgM hi MZ-like B cells only. However, no consequence of lower CD32b expression on these cells from females with CIS or MS was detected. Our findings highlight a potential role for naive and marginal zone-like B cells in the immunopathogenesis of MS in females, which requires further investigation.

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated ‘cold’ tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VVGM-αCTLA-4).ResultsThe identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VVGM-αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VVGM-αCTLA-4 synergized with αPD-1 to reject cold tumors.ConclusionOur findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VVGM-αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced.

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

BackgroundPrevious data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit.MethodsThis study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb.ResultsBinding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype—with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope—with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms.ConclusionThese findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.

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