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Antibody- and Peptide-based Immunotherapies: Proof-of-concept and safety considerations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of cancer immunotherapy is to eradicate tumours by inducing a tumour-specific immune response. This thesis focuses on how antibodies and peptides can improve antigen presentation and the subsequent tumour-specific T cell response. Tumour recognition by the immune system can be promoted through delivery of antigen in the form of a vaccine. One example is the development of a therapeutic peptide vaccine containing both CD4+ and CD8+ T cell epitopes. So far, peptide vaccinations have shown limited success in clinical trials and further improvements are needed, such as choice of adjuvant and T cell epitopes, as well as targeted delivery of peptides and adjuvants to the same DC.

In paper I, we describe the development of a peptide-peptide conjugate (with a tumour T cell epitope) that, via immune complex formation and FcγR binding, enhance antigen uptake and activation of DCs. The conjugate consists of three tetanus toxin-derived linear B cell epitopes (MTTE) that were identified based on specific IgG antibodies in human serum. Three MTTE peptide sequences were conjugated to a synthetic long peptide (SLP) that consists of a T cell epitope derived from the desired target tumour.

In paper II, the conjugate was evaluated in a modified Chandler loop model containing human blood, mimicking blood in circulation. The conjugate was internalised by human monocytes in an antibody-dependent manner. A conjugate containing the model CMV-derived T cell epitope pp65NLV generated recall T cell responses dependent on MTTE-specific antibodies and the covalent conjugation of the three MTTE with the SLP.

In paper III, a CD40-specific antibody was characterised for local treatment of solid tumours. The antibody eradicated bladder tumours in mice and induced T cell-mediated immunological memory against the tumour.

In paper IV, we characterised the Chandler loop model (used in paper II) for its potential use in predicting cytokine release syndrome (CRS) in response to monoclonal antibodies (mAbs). Superagonistic antibodies (e.g., OKT3) induced rapid cytokine release whereas no cytokine release was induced by antibodies (e.g., cetuximab) associated with low incidence of CRS in the clinic.

In conclusion, this thesis work demonstrates proof-of-concept of improved strategies for antibody- and peptides-based cancer immunotherapies and their potential use in multiple cancer indications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 73
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1370
Keyword [en]
Immune complex, conjugat, vaccine, CD40, whole blood, cytokine release syndrome
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-329038ISBN: 978-91-513-0064-1 (print)OAI: oai:DiVA.org:uu-329038DiVA, id: diva2:1139193
Public defence
2017-10-26, Rudbecksalen, Dag hammarskjöldsväg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-10-04 Created: 2017-09-07 Last updated: 2018-01-13
List of papers
1. Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses
Open this publication in new window or tab >>Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses
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2018 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 93, p. 115-124Article in journal (Refereed) Published
Abstract [en]

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8 + T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MITE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MITE antibodies with KM-containing conjugates are able to induce DC and T cell activation using model antigens.

Keyword
immune complex, FcgRs, dendritic cells, peptide conjugate
National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-329037 (URN)10.1016/j.molimm.2017.11.004 (DOI)000424181000014 ()29175591 (PubMedID)
Funder
Swedish Society for Medical Research (SSMF)Göran Gustafsson Foundation for Research in Natural Sciences and MedicineVINNOVA
Available from: 2017-09-06 Created: 2017-09-06 Last updated: 2018-04-03Bibliographically approved
2. Formation ofimmune-complexes by a defined linear tetanus toxin-derived B cell epitope boosts human T cell responses to long peptides: ICs boost specific-T cells
Open this publication in new window or tab >>Formation ofimmune-complexes by a defined linear tetanus toxin-derived B cell epitope boosts human T cell responses to long peptides: ICs boost specific-T cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Enhancing T cell responses against both viral and tumor antigens requires efficient co-stimulation and directed delivery of peptide antigens into APCs. As short peptides can lead to T cell tolerance and can only be used in a specific set of patients, long peptides are considered favourable vaccine moieties from a clinical perspective as they can harbor more than one immunogenic epitope enabling a broader target population. In addition, longer peptides are not bound unselectively to MHC class I on any cell, but rather require processing and will thereby be presented to T cells in secondary lymphoid organs. However, as peptides are not actively targeted to and taken up by APCs, and the standard non-conjugated adjuvant-peptide mixtures do not ensure co-targeting of the two to the same APC. We have identified a linear tetanus-toxin derived B cell epitope that can mediate the formation of immune-complexes by the presence of circulating antibodies. Herein, we show that these complexes, improve both antigen uptake by APCs (blood monocytes and CD1c+ DCs) and thereby CD8+ T cell recall responses in a human ex-vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on antibodies and the complement component C1q.  The defined linear peptide steers the immune-complex formation to a monoclonal-like complex and as a consequence we show that the number of linear tetanus sequences per T cell epitope determines the outcome of the response. We envision that this strategy can be used to facilitate active uptake of antigens into antigen-presenting cells to improve T cell responses against pathogens or cancer.

Keyword
peptide conjugate, therapeutic vaccine, whole blood, immune complexes
National Category
Other Health Sciences
Research subject
Immunology; Immunology; History of Sciences and Ideas
Identifiers
urn:nbn:se:uu:diva-328860 (URN)
Funder
Swedish Research Council
Available from: 2017-09-06 Created: 2017-09-06 Last updated: 2017-09-07
3. The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T cell dependent tumor immunity
Open this publication in new window or tab >>The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T cell dependent tumor immunity
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2015 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 21, no 5, p. 1115-1126Article in journal (Refereed) Published
Abstract [en]

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 has been investigated in human and murine in vitro models. The in vivo effect has been investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. Results: Activation of dendritic cells (DCs) by ADC-1013 results in up-regulation of the co-stimulatory molecules CD80 and CD86, and secretion of IL-12. ADC-1013 also activates dendritic cells from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated dendritic cells induce antigen-specific T cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant anti-tumor effects and long-term tumor-specific immunity. Further, ADC-1013 demonstrates significant anti-tumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. Conclusions: Our data demonstrate that ADC-1013 induces long-lasting anti-tumor responses and immunological memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-239514 (URN)10.1158/1078-0432.CCR-14-0913 (DOI)000351982800025 ()25316820 (PubMedID)
Available from: 2014-12-29 Created: 2014-12-29 Last updated: 2017-12-05Bibliographically approved
4. Extracorporeal human whole blood in motion, as a tool to predict first-infusion reactions and mechanisms-of-action of immunotherapeutics: CRS prediction in human whole blood
Open this publication in new window or tab >>Extracorporeal human whole blood in motion, as a tool to predict first-infusion reactions and mechanisms-of-action of immunotherapeutics: CRS prediction in human whole blood
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

First infusion reactions along with severeanaphylactic responses can occur as a result of systemic administration oftherapeutic antibodies. The underlying mechanisms by which monoclonal antibodiesinduce cytokine release syndrome (CRS) can involve direct agonistic effects viathe drug target, or a combination of target-engagement along with innatereceptor interactions. Despite the wide variety of pathways and cells that canplay a role in CRS, many currently used assays are devoid of one or morecomponents that must be present for these responses to occur. To date, oneassay that has not been used for studying CRS is the Chandler loop model. Thismodel is commonly used to study surface/blood interface interactions and has alsobeen used to study the instant blood-mediated inflammatory reaction (IBMIR). Herein we use a modified Chandler loopmodel with a heparin conjugate lining the inner surface of the loops to studyCRS. This allows for an assay harboring immune cells, intact cascade systemsalong with endogenous antibodies. Here, we evaluated a plethora of commerciallyavailable monoclonal antibodies to assess the capacity of the Chandler loopmodel for CRS prediction. We demonstrated that in a 4-hour loop assay both thesuperagonistic antibodies, anti-CD3 (OKT3) and anti-CD28 (ANC28.1), displayed aclear cytokine response with a mixed adaptive/innate cytokine source. OKT3 induced TNFα and IFN-g release in 20 out of23 donors tested, whereas ANC28.1 induced TNF-α, IL-2 and IFN-g release in all donors tested (n=18-22). On theother hand, non-agonistic antibodies associated with no or low infusionreactions in the clinic, namely cetuximab and natalizumab, neither induced cytokinerelease nor caused false positive responses. A TGN1412-like antibody alsodisplayed a clear cytokine release with an adaptive cytokine profile (IFN-g and IL-2) and all donors (n=9) inducing adistinct IL-2 response. Additionally, the value of an intact complement systemin the assay was highlighted by the possibility to dissect out themechanism-of-action (MOA) of alemtuzumab and rituximab. The loop assay can eithercomplement lymph node-like assays or stand-alone to investigate drug/bloodinteractions during preclinical development, or for individual safety screeningprior to a first-in-man clinical trial.

Keyword
Cytokine release syndrome, Chandler loop, TGN1412, alemtuzumab
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-328816 (URN)
Funder
Swedish Research Council
Available from: 2017-09-06 Created: 2017-09-06 Last updated: 2018-01-13

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