T cell-dependent lysis of CD19-positive leukemia cells mediated by single-chain triplebodies with dual-targeting

Targeted tumor therapy with multispecific antibody formats bears great potential to improve the efficacy of cancer immunotherapy: The simultaneous interaction of antibody derivatives with immune effector cells and multiple tumor-associated antigens is expected to increase cancer cell selectivity, to block cancer cell survival mechanisms and to hamper immune escape. For this purpose a large number of bi- and multispecific molecular platforms have been developed including the single-chain triplebody format. Triplebodies are composed of three antibody-derived single-chain variable fragments interconnected by flexible glycine-serine peptide linkers. They are used for re-targeting of cytotoxic immune effector cells towards cancer cells, which are bound bivalently by the triplebody. In the present work the triplebody-mediated engagement of T cells for the lysis of B lymphoid leukemia cells was established. A prototype with specificity for B lymphoid differentiation antigen CD19 and T cell trigger antigen CD3-epsilon – triplebody 19-3-19 – was shown to activate T lymphocytes at picomolar concentrations and to engage them for the efficient, serial lysis of target antigen-positive cancer cells. The triplebody 19-3-19 also induced T cell proliferation, which can lead to the partial regeneration of a patient’s immune effector cell pool. In these capacities the triplebody 19-3-19 was comparable to the bispecific T cell engager (BiTE®) blinatumomab, which is approved for the treatment of relapsed or refractory acute precursor B lymphoid leukemia in the USA and in the European Union since late 2014/2015. Furthermore, it was shown with the trispecific triplebody 33-3-19 that dual targeting of CD19 and myeloid surface marker CD33 on biphenotypic leukemia blasts results in selective lysis of these target cells. The CD19 and CD33 double-positive blasts were 145-fold more sensitive to treatment with the triplebody 33-3-19 than CD19 single-positive cells. Parts of the author’s work also contributed to the functional characterization of two previously developed NK cell-recruiting triplebodies – SPM-1 (19-16-19) and SPM-2 (33-16-123) – which are candidates for clinical development. The results of this thesis project have established the triplebody format as a molecular platform, which can be employed for the recruitment of any cytotoxic effector cell population as required in a particular therapeutic setting. Furthermore, the improved target cell selectivity that was achieved in vitro with the dual-targeting triplebody 33-3-19 adds weight to the concept of improved therapeutic efficacy of multispecific antibodies

T cell-dependent lysis of CD19-positive leukemia cells mediated by single-chain triplebodies with dual-targeting

Targeted tumor therapy with multispecific antibody formats bears great potential to improve the efficacy of cancer immunotherapy: The simultaneous interaction of antibody derivatives with immune effector cells and multiple tumor-associated antigens is expected to increase cancer cell selectivity, to block cancer cell survival mechanisms and to hamper immune escape. For this purpose a large number of bi- and multispecific molecular platforms have been developed including the single-chain triplebody format. Triplebodies are composed of three antibody-derived single-chain variable fragments interconnected by flexible glycine-serine peptide linkers. They are used for re-targeting of cytotoxic immune effector cells towards cancer cells, which are bound bivalently by the triplebody. In the present work the triplebody-mediated engagement of T cells for the lysis of B lymphoid leukemia cells was established. A prototype with specificity for B lymphoid differentiation antigen CD19 and T cell trigger antigen CD3-epsilon – triplebody 19-3-19 – was shown to activate T lymphocytes at picomolar concentrations and to engage them for the efficient, serial lysis of target antigen-positive cancer cells. The triplebody 19-3-19 also induced T cell proliferation, which can lead to the partial regeneration of a patient’s immune effector cell pool. In these capacities the triplebody 19-3-19 was comparable to the bispecific T cell engager (BiTE®) blinatumomab, which is approved for the treatment of relapsed or refractory acute precursor B lymphoid leukemia in the USA and in the European Union since late 2014/2015. Furthermore, it was shown with the trispecific triplebody 33-3-19 that dual targeting of CD19 and myeloid surface marker CD33 on biphenotypic leukemia blasts results in selective lysis of these target cells. The CD19 and CD33 double-positive blasts were 145-fold more sensitive to treatment with the triplebody 33-3-19 than CD19 single-positive cells. Parts of the author’s work also contributed to the functional characterization of two previously developed NK cell-recruiting triplebodies – SPM-1 (19-16-19) and SPM-2 (33-16-123) – which are candidates for clinical development. The results of this thesis project have established the triplebody format as a molecular platform, which can be employed for the recruitment of any cytotoxic effector cell population as required in a particular therapeutic setting. Furthermore, the improved target cell selectivity that was achieved in vitro with the dual-targeting triplebody 33-3-19 adds weight to the concept of improved therapeutic efficacy of multispecific antibodies

Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression

In vitro cell-based models of lung cancer are frequently employed to study invasion and the mechanisms behind metastasis. However, these models often study only one cell type with two-dimensional (2D) monolayer cell cultures, which do not accurately reflect the complexity of inflammation in vivo. Here, a three-dimensional (3D) cell co-culture collagen gel model was employed, containing human lung adenocarcinoma cells (HCC), human lung fibroblast cells (MRC-5), and macrophages. Cell culture media and cell images were collected, and matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor (VEGF) production was monitored under different cell culture conditions. We found that simulating hypoxia and/or serum starvation conditions induced elevated secretion of VEGF in the 3D co-culture model in vitro, but not MMP-1;the morphology of HCC in the 2D versus the 3D co-culture system was extremely different. MMP-1 and VEGF were secreted at higher levels in mixed cell groups rather than mono-culture groups. Therefore, incorporating lung cancer cells, fibroblasts, and macrophages may better reflect physiological metastasis mechanisms compared to mono-culture systems. Tumour stromal cells, macrophages, and fibroblast cells may promote invasion and metastasis, which also provides a new direction for the design of therapies targeted at destroying the stroma of tumor tissues

NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody Rituximab (TM) were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML

Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression.

In vitro cell-based models of lung cancer are frequently employed to study invasion and the mechanisms behind metastasis. However, these models often study only one cell type with two-dimensional (2D) monolayer cell cultures, which do not accurately reflect the complexity of inflammation in vivo. Here, a three-dimensional (3D) cell co-culture collagen gel model was employed, containing human lung adenocarcinoma cells (HCC), human lung fibroblast cells (MRC-5), and macrophages. Cell culture media and cell images were collected, and matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor (VEGF) production was monitored under different cell culture conditions. We found that simulating hypoxia and/or serum starvation conditions induced elevated secretion of VEGF in the 3D co-culture model in vitro, but not MMP-1; the morphology of HCC in the 2D versus the 3D co-culture system was extremely different. MMP-1 and VEGF were secreted at higher levels in mixed cell groups rather than mono-culture groups. Therefore, incorporating lung cancer cells, fibroblasts, and macrophages may better reflect physiological metastasis mechanisms compared to mono-culture systems. Tumour stromal cells, macrophages, and fibroblast cells may promote invasion and metastasis, which also provides a new direction for the design of therapies targeted at destroying the stroma of tumor tissues

The expression of MMP1.

<p><b>(A) Expression of MMP-1 in 3D mono- and co-culture lung cancer models at 48 h detected by ELISA.</b> The expression of MMP1 in HCC & MRC-5 & macrophage co-culture group was higher than that in HCC & MRC-5 co-culture group, or MRC-5/HCC/macrophage mono-culture groups. There was almost no expression of MMP1 in the HCC/macrophage mono-culture group. <b>(B) Expression of MMP-1 in 3D mono- and co-culture lung cancer model at 48 h detected by Western blotting.</b> In Fig 1B, a, the molecular weight of MMP-1 is 52 kD. From the left to right, the lanes are: HCC mono-culture group (2 x 10⁵ cells); MRC-5 mono-culture group (2 x 10⁵ cells); MRC-5 and HCC co-culture group (2 x10⁵ cells); HCC mono-culture group (1 x 10⁶ cells); MRC-5 and HCC co-culture group (1 x 10⁶ cells); MRC-5 mono-culture group (1 x 10⁶ cells). Expression of MMP-1 in co-culture groups was higher than in mono-culture groups (both 2 x 10⁵ cells and 1 x 10⁶ cells). Expression of MMP-1 in the 1 x 10⁶ cell group was higher than the 2 x 10⁵ cell group, regardless of mono-culture or co-culture group designations. In Fig 1B, b, the mean IOD values of the Western blot are shown. <b>(C) Expression of MMP-1 under different co-culture conditions.</b> Expression of MMP1 under 10% FBS and O₂ (10% FBS cell culture medium with O₂) was higher than that under w/o FBS and w/o O₂ (without FBS and without O₂) at 7 different time points. Furthermore, the expression trend of MMP1 under the condition of w/o FBS and w/o O₂ continued to decline from 120 h.</p

The expression of VEGF.

<p><b>(A) Expression of VEGF in HCC, MRC-5, and macrophage mono-cultures groups.</b> Expression of VEGF in the HCC mono-culture group was significantly higher than expression in the MRC-5/macrophage mono-culture group under 10% FBS and O₂ culture conditions. <b>(B) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups compared with the HCC mono-culture group.</b> Expression of VEGF in the HCC & MRC-5 & Macrophage co-culture group was higher than in the HCC & MRC-5 co-culture group and the HCC mono-culture group cultured with 10% FBS and O₂ for 48 h. <b>(C) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under different co-culture conditions.</b> The expression of VEGF in cells cultured w/o FBS (starved of FBS but with O₂), w/o FBS and w/o O₂ (without FBS and without O₂) was higher than that in 10% FBS or O₂ (10% FBS cell culture medium with O₂), while the expression of VEGF in the three different conditions first increased and then decreased.</p

Statistical analysis of the expression of MMP-1 by Western Blot (IOD value).

<p>Statistical analysis of the expression of MMP-1 by Western Blot (IOD value).</p

Statistical analysis of the expression of MMP-1 by ELISA assay (pg/ml).

<p>Statistical analysis of the expression of MMP-1 by ELISA assay (pg/ml).</p

Statistical analysis of the expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under three different co-culture conditions (pg/ml).

<p>Statistical analysis of the expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under three different co-culture conditions (pg/ml).</p