Malmö University Publications
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  • Public defence: 2025-02-21 09:15 Allmänna sjukhuset, aulan (AS:E002), Malmö
    Dao Nyesiga, Gillian
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV).
    Immune tolerance: induction and disruption for therapeutic immune modulation2025Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This study investigated the tolerance induction and disruption of immune tolerance as strategies for immune modulation.

    For induction of immune tolerance, novel monocyte-derived tolerogenic dendritic cells (ItolDCs) were generated, and their ability to modulate the immune system was assessed using in vitro assays in hemophilia A patients who had developed neutralizing antibodies against their factor VIII replacement therapy. The cells were characterized, their functionality was assessed, and their feasibility as a safe cell therapy was further evaluated using both in vitro and in vivo studies for the induction of immune tolerance against factor VIII.

    For research on disruption of immune tolerance, meningioma, the most common brain tumor, was studied. To map the immune cell composition in meningiomas, a protocol was optimized for shorter enzymatic digestion, which breaks down the tissue into single-cell suspensions of viable immune cells. Since CD8+ T cells are vital in tumor suppression, further studies were conducted to explore their characteristics and identify possible targetable processes for immunotherapy.

    To investigate both induction and disruption of immune tolerance, various techniques were employed, including flow cytometry, immunohistochemistry, and functional-cell-based assays.

    Our investigation demonstrated that ItolDCs are a feasible and safe option for cell therapy aimed at inducing immune tolerance. Thus, factor VIII-loaded ItolDCs are ready for clinical evaluation to reduce inhibitor levels in patients with hemophilia A.

    Several tolerance-associated markers (PD-1, TIM-3, TIGIT, and LAG-3) were identified in CD8+ T cells in meningioma. These findings highlight how tumor cells may evade immune defenses and suggest potential immunotherapeutic targets, including immune checkpoint inhibitors.

    Taken together, various approaches may be employed for immune modulations to either induce or disrupt immune tolerance.

    List of papers
    1. Tolerogenic dendritic cells generated in vitro using a novel protocol mimicking mucosal tolerance mechanisms represent a potential therapeutic cell platform for induction of immune tolerance.
    Open this publication in new window or tab >>Tolerogenic dendritic cells generated in vitro using a novel protocol mimicking mucosal tolerance mechanisms represent a potential therapeutic cell platform for induction of immune tolerance.
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    2023 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1045183Article in journal (Refereed) Published
    Abstract [en]

    Dendritic cells (DCs) are mediators between innate and adaptive immunity and vital in initiating and modulating antigen-specific immune responses. The most important site for induction of tolerance is the gut mucosa, where TGF-β, retinoic acid, and aryl hydrocarbon receptors collaborate in DCs to induce a tolerogenic phenotype. To mimic this, a novel combination of compounds – the synthetic aryl hydrocarbon receptor (AhR) agonist IGN-512 together with TGF-β and retinoic acid – was developed to create a platform technology for induction of tolerogenic DCs intended for treatment of several conditions caused by unwanted immune activation. These in vitro-generated cells, designated ItolDCs, are phenotypically characterized by their low expression of co-stimulatory and activating molecules along with high expression of tolerance-associated markers such as ILT3, CD103, and LAP, and a weak pro-inflammatory cytokine profile. When co-cultured with T cells and/or B cells, ItolDC-cultures contain higher frequencies of CD25+Foxp3+ regulatory T cells (Tregs), CD49b+LAG3+ ‘type 1 regulatory (Tr1) T cells, and IL-10-producing B cells and are less T cell stimulatory compared to cultures with matured DCs. Factor VIII (FVIII) and tetanus toxoid (TT) were used as model antigens to study ItolDC antigen-loading. ItolDCs can take up FVIII, process, and present FVIII peptides on HLA-DR. By loading both ItolDCs and mDCs with TT, antigen-specific T cell proliferation was observed. Cryo-preserved ItolDCs showed a stable tolerogenic phenotype that was maintained after stimulation with LPS, CD40L, or a pro-inflammatory cocktail. Moreover, exposure to other immune cells did not negatively impact ItolDCs’ expression of tolerogenic markers. In summary, a novel protocol was developed supporting the generation of a stable population of human DCs in vitro that exhibited a tolerogenic phenotype with an ability to increase proportions of induced regulatory T and B cells in mixed cultures. This protocol has the potential to constitute the base of a tolDC platform for inducing antigen-specific tolerance in disorders caused by undesired antigen-specific immune cell activation.

    Place, publisher, year, edition, pages
    Frontiers Media S.A., 2023
    Keywords
    antigen loading, antigen-specific response, cell therapy, immune tolerance, regulatory B cells (Bregs), regulatory T cells (Tregs), tolerogenic dendritic cells (tolDCs)
    National Category
    Immunology in the medical area
    Identifiers
    urn:nbn:se:mau:diva-63661 (URN)10.3389/fimmu.2023.1045183 (DOI)001092545900001 ()37901231 (PubMedID)2-s2.0-85175369630 (Scopus ID)
    Available from: 2023-11-13 Created: 2023-11-13 Last updated: 2025-01-28Bibliographically approved
    2. Pre-Clinical Safety Evaluation of an Autologous Tolerogenic DendriticCell Therapy for Patients with Hemophilia A with Inhibitory Antibodies
    Open this publication in new window or tab >>Pre-Clinical Safety Evaluation of an Autologous Tolerogenic DendriticCell Therapy for Patients with Hemophilia A with Inhibitory Antibodies
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biomedical Laboratory Science/Technology
    Identifiers
    urn:nbn:se:mau:diva-73385 (URN)
    Available from: 2025-01-28 Created: 2025-01-28 Last updated: 2025-01-28Bibliographically approved
    3. Flow Cytometry Analyses of Meningioma Immune Cell Composition Using a Short, Optimized Digestion Protocol
    Open this publication in new window or tab >>Flow Cytometry Analyses of Meningioma Immune Cell Composition Using a Short, Optimized Digestion Protocol
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    2024 (English)In: Cancers, ISSN 2072-6694, Vol. 16, no 23, article id 3942Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND: Current challenges in meningioma treatment, including post-surgical complications and cognitive impairments, highlight the need for new treatment alternatives. Immunological interventions have shown promise. However, there is a knowledge gap in characterizing infiltrating immune cells in meningioma and their interplay. Further studies on immune cells in single-cell suspensions from digested meningioma tissues could identify targetable mechanisms for non-surgical treatment options with fewer side effects. This study aimed to optimize a protocol for faster digestion of meningioma tissues into viable single-cell suspensions and to identify infiltrating immune cell populations.

    METHODS: We modified a commercial kit intended for whole skin dissociation to digest resected meningioma tissues into viable single-cell suspensions. Tumor-infiltrating immune cell populations were characterized using flow cytometry.

    RESULTS: , with a small proportion co-expressing CD83. Women were more likely to have a lower proportion of immune cells, B cells, and NK cells. Female patients with a high proportion of immune cells had a higher proportion of macrophages.

    CONCLUSION: We successfully optimized a protocol for generating single-cell suspensions with viable immune cells from meningioma tissues, revealing infiltrating antigen-presenting cells with an immunosuppressive phenotype, and lymphocytes. This short protocol allows advanced analyses of tumor-infiltrating cells using techniques such as single-cell RNA sequencing and flow cytometry, which require live, dissociated cells.

    Place, publisher, year, edition, pages
    MDPI, 2024
    Keywords
    T cells, TIM-3, brain tumor, immune cells, lymphocytes, macrophages, meningioma, single-cell suspension, tumor digestion, tumor-infiltrating cells
    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:mau:diva-72847 (URN)10.3390/cancers16233942 (DOI)001376117400001 ()39682129 (PubMedID)2-s2.0-85211949815 (Scopus ID)
    Available from: 2024-12-20 Created: 2024-12-20 Last updated: 2025-01-28Bibliographically approved
    4. Evaluating CD8+ T Cell Immune Dynamics in Meningioma: A Comprehensive Study of Inhibitory Molecules, Cytokines, and Proliferation
    Open this publication in new window or tab >>Evaluating CD8+ T Cell Immune Dynamics in Meningioma: A Comprehensive Study of Inhibitory Molecules, Cytokines, and Proliferation
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biomedical Laboratory Science/Technology
    Identifiers
    urn:nbn:se:mau:diva-73387 (URN)
    Available from: 2025-01-28 Created: 2025-01-28 Last updated: 2025-01-29Bibliographically approved
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  • Public defence: 2025-03-07 10:00 Auditorium (AS: E002), Faculty of Health and Society, Malmö
    Nyesiga, Barnabas
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV).
    Advancing cancer immunotherapy through novel CD40-focused antibody engineering platforms2025Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cancer poses a major global challenge due to its ability to evade the immune system. Conventional treatments, such as surgery, chemotherapy, and radiotherapy, often cause severe side effects. In contrast, immunotherapy offers a promising alternative by strengthening the immune system’s ability to generate strong anti-tumor responses. This thesis aimed at developing technologies that can enhance the efficacy of recombinant antibodies in cancer immunotherapy. Paper I focused on developing a bispecific antibody (bsAb) format called RUBY designed to address the bioprocessing challenges associated with bsAb production such as poor stability and chain mispairing. Findings demonstrate that the RUBY format allows for the mitigation of many of the development challenges associated with other bsAb formats and facilitates the generation of stable and functional bsAbs. In Paper II, the RUBY format was utilized to develop bsAbs capable of enabling enhanced priming and activation of tumor-specific T cells. RUBY bsAbs targeting CD40 and tumor-associated antigens (TAAs) EpCAM and CEACAM5 were evaluated. Results indicate that these molecules induced clustering of tumor debris and CD40-expressing cells, promoted effective cross-priming of T-cells, and induced anti-tumor responses superior to monospecific antibodies. In Paper III, a CD40-coiled coil affinity-based technology designed to deliver antigenic peptides to CD40-expressing cells was evaluated. Results suggest that antibody-peptide fusion constructs can be produced with favorable manufacturability and stability, and this technology effectively induces biological functions both in vitro and in vivo. Paper IV gives a comprehensive overview of the next generation CD40 targeting therapies; highlighting their potential to transform immuno-oncology and the challenges that lie ahead. In conclusion, this thesis highlights the potential of innovative antibody engineering in developing novel compounds that can improve cancer immunotherapy.

    List of papers
    1. RUBY® - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties
    Open this publication in new window or tab >>RUBY® - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties
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    2024 (English)In: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 16, no 1, article id 2330113Article in journal (Refereed) Published
    Abstract [en]

    Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.

    Place, publisher, year, edition, pages
    Taylor & Francis, 2024
    Keywords
    Antibody format, bispecific antibodies, IgG, immuno-oncology, RUBY (R), tetravalent
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:mau:diva-66835 (URN)10.1080/19420862.2024.2330113 (DOI)001190866000001 ()38527972 (PubMedID)2-s2.0-85188901748 (Scopus ID)
    Available from: 2024-04-22 Created: 2024-04-22 Last updated: 2025-02-13Bibliographically approved
    2. Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies
    Open this publication in new window or tab >>Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies
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    2022 (English)In: Journal for ImmunoTherapy of Cancer, E-ISSN 2051-1426, Vol. 10, no 11, article id e005018Article in journal (Refereed) Published
    Abstract [en]

    Background Indications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation, generate new tumor-specific T cells, and activate tumor-infiltrating myeloid cells to remodel the tumor microenvironment, represent a promising opportunity to meet this need. In this study, we present the first in vivo data supporting a role for tumor-associated antigen (TAA)-mediated uptake and cross-presentation of tumor antigens to enhance tumor-specific T-cell priming using CD40×TAA bispecific antibodies, a concept we named Neo-X-Prime.

    Methods Bispecific antibodies targeting CD40 and either of two cell-surface expressed TAA, carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) or epithelial cell adhesion molecule (EpCAM), were developed in a tetravalent format. TAA-conditional CD40 agonism, activation of tumor-infiltrating immune cells, antitumor efficacy and the role of delivery of tumor-derived material such as extracellular vesicles, tumor debris and exosomes by the CD40×TAA bispecific antibodies were demonstrated in vitro using primary human and murine cells and in vivo using human CD40 transgenic mice with different tumor models.

    Results The results showed that the CD40×TAA bispecific antibodies induced TAA-conditional CD40 activation both in vitro and in vivo. Further, it was demonstrated in vitro that they induced clustering of tumor debris and CD40-expressing cells in a dose-dependent manner and superior T-cell priming when added to dendritic cells (DC), ovalbumin (OVA)-specific T cells and OVA-containing tumor debris or exosomes. The antitumor activity of the Neo-X-Prime bispecific antibodies was demonstrated to be significantly superior to the monospecific CD40 antibody, and the resulting T-cell dependent antitumor immunity was directed to tumor antigens other than the TAA used for targeting (EpCAM).

    Conclusions The data presented herein support the hypothesis that CD40×TAA bispecific antibodies can engage tumor-derived vesicles containing tumor neoantigens to myeloid cells such as DCs resulting in an improved DC-mediated cross-priming of tumor-specific CD8+ T cells. Thus, this principle may offer therapeutics strategies to enhance tumor-specific T-cell immunity and associated clinical benefit in indications characterized by poor T-cell infiltration or deficiencies in T-cell priming.

    Place, publisher, year, edition, pages
    BMJ Publishing Group Ltd, 2022
    Keywords
    antigen presentation, antigens, neoplasm, dendritic cells, drug evaluation, preclinical, immunotherapy, active
    National Category
    Immunology in the medical area
    Identifiers
    urn:nbn:se:mau:diva-73762 (URN)10.1136/jitc-2022-005018 (DOI)
    Available from: 2025-02-13 Created: 2025-02-13 Last updated: 2025-02-13Bibliographically approved
    3. Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coilaffinity-based platform
    Open this publication in new window or tab >>Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coilaffinity-based platform
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Biomedical Laboratory Science/Technology
    Identifiers
    urn:nbn:se:mau:diva-73763 (URN)
    Available from: 2025-02-13 Created: 2025-02-13 Last updated: 2025-02-13Bibliographically approved
    4. Next-generation CD40 agonists for cancer immunotherapy
    Open this publication in new window or tab >>Next-generation CD40 agonists for cancer immunotherapy
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    2024 (English)In: Expert Opinion on Biological Therapy, ISSN 1471-2598, E-ISSN 1744-7682, Vol. 24, no 5, p. 351-363Article, review/survey (Refereed) Published
    Abstract [en]

    Introduction: There is a need for new therapies that can enhance response rates and broaden the number of cancer indications where immunotherapies provide clinical benefit. CD40 targeting therapies provide an opportunity to meet this need by promoting priming of tumor-specific T cells and reverting the suppressive tumor microenvironment. This is supported by emerging clinical evidence demonstrating the benefits of immunotherapy with CD40 antibodies in combination with standard of care chemotherapy.

    Areas covered: This review is focused on the coming wave of next-generation CD40 agonists aiming to improve efficacy and safety, using new approaches and formats beyond monospecific antibodies. Further, the current understanding of the role of different CD40 expressing immune cell populations in the tumor microenvironment is reviewed.

    Expert opinion: There are multiple promising next-generation approaches beyond monospecific antibodies targeting CD40 in immuno-oncology. Enhancing efficacy is the most important driver for this development, and approaches that maximize the ability of CD40 to both remodel the tumor microenvironment and boost the anti-tumor T cell response provide great opportunities to benefit cancer patients. Enhanced understanding of the role of different CD40 expressing immune cells in the tumor microenvironment may facilitate more efficient clinical development of these compounds.

    Place, publisher, year, edition, pages
    TAYLOR & FRANCIS LTD, 2024
    Keywords
    Antibodies, antigen-presenting cells, bispecific antibodies, CD40, immuno-oncology, tumor-associated antigens
    National Category
    Immunology in the medical area
    Identifiers
    urn:nbn:se:mau:diva-70013 (URN)10.1080/14712598.2024.2357714 (DOI)001229959500001 ()38764393 (PubMedID)2-s2.0-85193961344 (Scopus ID)
    Available from: 2024-08-01 Created: 2024-08-01 Last updated: 2025-02-13Bibliographically approved
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