Extended half-life target module for sustainable UniCAR T-cell treatment of STn-expressing cancers

Background: Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn). Methods: The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM. Results: The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM. Conclusion: In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.publishersversionpublishe

Cryogel-supported stem cell factory for customized sustained release of bispecific antibodies for cancer immunotherapy

Combining stem cells with biomaterial scaffolds provides a promising strategy for the development of drug delivery systems. Here we propose an innovative immunotherapeutic organoid by housing human mesenchymal stromal cells (MSCs), gene-modified for the secretion of an anti-CD33-anti-CD3 bispecific antibody (bsAb), in a small biocompatible star-shaped poly(ethylene glycol)-heparin cryogel scaffold as a transplantable and low invasive therapeutic machinery for the treatment of acute myeloid leukemia (AML). The macroporous biohybrid cryogel platform displays effectiveness in supporting proliferation and survival of bsAb-releasing-MSCs overtime in vitro and in vivo, avoiding cell loss and ensuring a constant release of sustained and detectable levels of bsAb capable of triggering T-cell-mediated anti-tumor responses and a rapid regression of CD33 + AML blasts. This therapeutic device results as a promising and safe alternative to the continuous administration of short-lived immunoagents and paves the way for effective bsAb-based therapeutic strategies for future tumor treatments

Retargeting of UniCAR T cells with an in vivo synthesized target module directed against CD19 positive tumor cells

Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells

Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL-2, TNF and interferon-γ and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells

Tumor cell elimination mediated by the novel scBsTaFv CD8-PSCA(MB1).

<p>To analyze killing properties of the novel CD8-engaging single-chain bsAb standard chromium release assays were performed. <i>A</i>, to compare the anti-tumor effect of the novel scBsTaFv CD8-PSCA(MB1) with the conventional bsAb CD3-PSCA(MB1), [⁵¹Cr]-labeled PC3-PSCA cells and either pre-activated PBMCs (<i>left panel</i>), isolated pre-activated CD8+ T cells (<i>middle panel</i>) or freshly isolated CD8+ T cells (<i>right panel</i>) were cultivated in the presence or absence of 30 pmol/ml of recombinant bsAbs for 22 h. Mean of specific lysis ± SD of one representative donor is shown. <i>BI</i>, summary of (<i>A, left panel</i>) seven different donors is shown. Statistical significance was determined with one-way ANOVA and Bonferroni Multiple Comparison test (***p<0.001 with respect to control: no Ab; *p<0.05 significant difference between the conventional anti-CD3 bsAb and the novel anti-CD8 bsAb). <i>BII,</i> In order to estimate the ratio of CD8+ to CD4+ T cells, the pre-activated PBMC preparations of all seven donors were stained for CD8+ and CD4+ T cells and analyzed by FACS.</p

Binding properties of the novel scBsTaFv CD8-PSCA(MB1).

<p><i>A</i>, in order to investigate binding properties of the novel single-chain bsAb, PC3-PSCA cells and isolated CD8+ T cells were stained with 10 ng/µl of recombinant Ab. Specific binding of the scBsTaFv CD8-PSCA(MB1) was detected with anti-myc/FITC. Histograms show percentage and mean fluorescence intensity (MFI) of stained antigen-positive cells (<i>black</i>) in comparison to the negative control incubated only with the secondary anti-myc/FITC mAb (white). <i>B</i>, to demonstrate simultaneous binding of the novel scBsTaFv CD8-PSCA(MB1) to PC3-PSCA cells and CD8+ T cells and, hence, to visualize a cross-linkage between the two cell types, microscopic images were taken. Therefore, PC3-PSCA cells and isolated CD8+ T cells were co-cultivated in the presence of scBsTaFv CD8-PSCA(MB1) for 22 h and fixed with 90% methanol. The scBsTaFv CD8-PSCA(MB1) was detected with anti-myc/FITC mAb (<i>green</i>) and cell nuclei were stained with DAPI (<i>blue</i>) containing sample cover medium. Microscopic image (<i>a</i>) shows DAPI-stained nuclei of one PC3-PSCA cell (<i>P</i>) surrounded by four T cells (<i>T</i>). A homogenous cell surface staining of the PC3-PSCA cell and T cells is shown in picture (<i>b</i>) after detection of the scBsTaFv CD8-PSCA(MB1) with anti-myc/FITC. Furthermore, a cross-linkage between the PC3-PSCA cell and a T cell is visible (<i>white triangle</i>). Image (<i>c</i>) is an overlay of (<i>a</i>) and (<i>b</i>).</p

Comparison of conventional CD3-engaging bsAbs with CD8-engaging bsAbs with regard to (A) principal idea, (B) schematic structure, (C) production and purification.

<p><i>A</i>, cross-linkage of T cells and tumor cells can be mediated by conventional bsAbs via simultaneous binding to the CD3 part of the TCR/CD3 complex and a tumor-associated surface antigen (TAA) (<i>AI</i>). A subtype-specific cross-linkage of CD8+ T cells can theoretically be achieved by a CD8-engaging bsAb (<i>AII</i>). <i>B</i>, schematic structure of recombinant single-chain bsAbs. For construction of the novel CD8-engaging single-chain bsAb, the anti-CD3 domain of scBsTaFv CD3-PSCA(MB1) was replaced by the scFv derived from the novel anti-CD8 mAb clone MB10. Recombinant Ab constructs were further equipped N-terminally with an Igκ leader as signal peptide (SP) for Ab secretion and C-terminally with a myc- and a his-tag for protein purification and detection. <i>C</i>, recombinant Abs were purified via Ni-NTA affinity chromatography from cell culture supernatant. Purified bsAbs were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue G250 to estimate protein purity and concentration (<i>CI</i>). Purified bsAbs were further analyzed by immunoblotting (<i>CII</i>). After transfer onto nitrocellulose membranes recombinant Abs were detected via their C-terminal his-tag.</p