Phage display technology is a common approach for discovery of therapeutic antibodies. Drug candidates are typically isolated in two steps: First, a pool of antibodies is enriched through consecutive rounds of selection on a target antigen, and then individual clones are characterized in a screening procedure. When whole cells are used as targets, as in phenotypic discovery, the output phage pool typically contains thousands of antibodies, binding, in theory, hundreds of different cell surface receptors. Clonal expansion throughout the phage display enrichment process is affected by multiple factors resulting in extremely complex output phage pools where a few antibodies are highly abundant and the majority is very rare. This is a huge challenge in the screening where only a fraction of the antibodies can be tested using a conventional binding analysis, identifying mainly the most abundant clones typically binding only one or a few targets. As the expected number of antibodies and specificities in the pool is much higher, complementing methods, to reach deeper into the pool, are required, called deep mining methods. In this study, four deep mining methods were evaluated: 1) isolation of rare sub-pools of specific antibodies through selection on recombinant proteins predicted to be expressed on the target cells, 2) isolation of a sub-pool enriched for antibodies of unknown specificities through depletion of the primary phage pool on recombinant proteins corresponding to receptors known to generate many binders, 3) isolation of a sub-pool enriched for antibodies through selection on cells blocked with antibodies dominating the primary phage pool, and 4) next-generation sequencing-based analysis of isolated antibody pools followed by antibody gene synthesis and production of rare but enriched clones. We demonstrate that antibodies binding new targets and epitopes, not discovered through screening alone, can be discovered using described deep mining methods. Overall, we demonstrate the complexity of phage pools generated through selection on cells and show that a combination of conventional screening and deep mining methods are needed to fully utilize such pools. Deep mining will be important in future phenotypic antibody drug discovery efforts to increase the diversity of identified antibodies and targets.

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-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

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