The aim of the study is to explore a new immunotherapy for celiac disease. Celiac disease is an autoimmune enteropathy driven by inflammatory gluten-specific CD4+ T cells. Our goal is to deplete the antigen-presenting cells that activate gluten-specific CD4+ T cells and thus are critical modulators of the inflammatory immune response in celiac disease. This will be achieved by using the BiTE (bi-specific T cell engager) platform. BiTE molecules are artificial bi-specific antibodies that have shown promising results as anti-cancer drugs, where they connect the patient’s own T cells to malignant cells. We have generated BiTE molecules that bind CD3 on T cells with one arm, and a gluten-peptide presented on HLA-DQ2.5 on antigen-presenting cells with the other arm. The BiTE will thus specifically guide cytolytic T cells to the disease-driving antigen-presenting cells. After in vitro testing the specificity and ability of the BiTE molecules to kill antigen-specific HLA-DQ2.5-expressing B cells, we are now planning to test the efficacy and safety of these BiTE molecules in vivo using our humanized mouse strains. We plan to test the efficacy of the BiTEs in our established mouse model of humanized HLA-DQ2.5 KI mice where celiac disease-specific autoantibodies targeting the enzyme transglutaminase-2 (TG2) are induced upon immunization with a recombinant TG2-gluten protein. In these pilot experiments we hypothesize that two injections with the BiTEs will inhibit the activation of TG2-specific B cells and development of TG2-specific autoantibodies. Testing the safety of our BiTE molecules for in vivo use is equally important in this application. It is known that T-cell immunotherapy can be accompanied by side effects caused by the cytokines that are produced upon T-cell activation. This phenomenon is referred to as “cytokine release syndrome” (CRS), and is induced by on-target effects following binding of the bispecific antibody to its antigen and subsequent activation of bystander immune cells. The incidence rate of such side effects varies with the type of immunotherapy, but for BiTE’s it is less frequently observed than with for instance chimeric antigen receptor (CAR) T-cell therapy (Cozenza et al, Int J Mol Sci 2021). It is thus unsure whether our BiTE will cause any side effects.

GlutenTCR HLA-DQ2 KI mice and 14E06 KI HLA-DQ2 KI mice (mice used as T and B cell donor mice for adoptive cell transfer) will be euthanized by cervical dislocation. HLA-DQ2 KI recipient mice will be earmarked with a hole puncher for identification purposes. (Some of the mice are already earmarked for genotyping purposes as described in FOTS 26426). Procedures outlined per day (where first immunization is set on day 0): Day -1: Adoptive transfer of gluten-specific T cells and TG2-specific B cells to HLA-DQ2KI recipient mice by intravenous (i.v.) injection. Mice will be restrained and isolated T cells and B cells will be injected in 200 µl PBS in the tail vein of mice that have been pre-warmed on warming-pads to dilate the veins. Day 0: Mice will receive the first BiTE (or control) treatment by i.v. injection in up to 200 µl PBS. I.v. injection will be done as above. Approximately 1 hour after i.v. BiTE injection, mice will be immunized with 100 µg TG2-gluten protein emulsified in Complete Freund's Adjuvant (CFA). 100 µl of a 1:1 PBS:CFA emulsion will be injected intra peritoneally (i.p.). Mice are restrained by holding the animal. Day 2: Mice will receive a second injection of BiTE molecules via the i.v. route, as described on day 0. Day 12: Mice will receive a booster immunization with 100 µg TG2-gluten protein emulsified in Incomplete Freund's Adjuvant (IFA). 100 µl of a 1:1 PBS:IFA emulsion will be injected intra peritoneally (i.p.). Day 19, 20 or 21: euthanasia by cervical dislocation. Throughout the experiment we will collect 4 number or blood samples from the mice. We will shave the hind leg and puncture the vein, and collect ~50 µl blood in eppendorf tubes. We will use alternate left and right leg. Blood samples will be collected on: - Day 0, approximately 4 hours after 1st BiTE injection - Day 2, approximately 4 hours after 2nd BiTE injection - Day 12 - Day 19-21

The experiments aim at evaluation the therapeutic effects and side effects of bispecific T cell engager (BiTE) molecules against gluten-presenting cells in celiac disease. No in vitro model can mimick the complex interactions between the many cell types involved in an immune response, nor predict side effects. Thus, pre-clinical testing can only be done using in vivo models. Prior to this application, we have done the in vitro experiments necessary to validate the BiTES and shown that these BiTEs indeed are able to induce killing of gluten-presenting B cells.