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.

The small intestinal lamina propria contains large numbers of IFNγ-producing T helper (Th1) cells that play important roles in intestinal homeostasis and host defense, but the mechanisms underlying their development remain poorly understood. Here, we demonstrate that Th1 cells accumulate in the SI-LP after weaning and are maintained there long term. While both Th17 and Th1 cell accumulation in the SI-LP was microbiota dependent, Th1 cell accumulation uniquely required IL-27 and MHCII expression by cDC1. This reflected a requirement for IL-27 signaling in the priming of Th1 cells rather than for their maintenance once in the mucosa. cDC1-derived IL-27 was essential for maintaining the Th1–Th17 balance within the SI-LP, and in its absence, remaining Th1 cells expressed enhanced levels of Th17 signature genes. In conclusion, we identify cDC1-derived IL-27 as a key regulator of SI-LP Th1–Th17 cell homeostasis.

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.

Bio-based 5-hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5-furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil-based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5-Hydroxymethylfurfural was converted to 5-formyl-2-furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30-40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3-fold higher k cat /K m and ~1.7-fold lower K m with HMF.

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of cancer patients including those with poorly immune infiltrated “cold” tumors are resistant to currently available ICB therapies. CTLA-4 is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved anti-CTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for anti-CTLA-4; immune checkpoint blockade and Treg depletion are both thought to contribute efficacy and toxicity in available, systemic, anti-CTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe, anti-CTLA-4 therapies have been lacking. Here, BioInvent and Transgene present and preclinically characterize a highly efficacious and potentially safe strategy to target CTLA-4 in the context of oncolytic virotherapy.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for 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, anti-CTLA-4 antibody and GM-CSF (VVGM-ahCTLA4, BT-001). Viruses encoding a matching Treg-depleting mouse surrogate antibody were additionally generated, enabling proof-of-concept studies in syngeneic immune competent mouse tumor models.ResultsOur studies demonstrate that intratumoral (i.t.) administration of VVGM-aCTLA4 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 anti-CTLA-4 antibody therapy. Efficacy correlated with FcgR-mediated intratumoral Treg-depletion and the reduction of exhausted CD8+ T cells. Remarkably, in a clinically relevant mouse model resistant to systemic immune checkpoint blockade, i.t. VVGM-aCTLA4 synergized with anti-PD-1 to reject “cold” tumors.ConclusionsOur findings demonstrate in vivo proof-of-concept for spatial restriction of strongly Treg-depleting, immune checkpoint blocking, vectorized anti-CTLA-4 as a highly effective and safe strategy to target CTLA-4 which is able to overcome current limitations of approved anti-CTLA-4 regimens. A clinical trial evaluating i.t. VVGM-ahCTLA4 (BT-001) alone and in combination with anti-PD-1 in metastatic or advanced solid tumors has commenced.Ethics ApprovalAll mouse experiments were approved by the local ethical committee for experimental animals (Malmö/Lunds djurförsöksetiska nämnd); at BioInvent under permit numbers 17196/2018 or 2934/2020; or at Transgene APAFIS Nr21622 project 2019072414343465 and performed in accordance with local ethical guidelines.