On-target restoration of a split T cell-engaging antibody for precision immunotherapy

Abstract

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (V_L) or variable heavy (V_H) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

Fig. 1

Combinatorial immunotherapy by hemibodies. a Binding of two hemibodies to their respective antigens on a target cell, each consisting of a single-chain variable fragment (scFv) fused to the variable light (V_L) or variable heavy chain domain (V_H) of a CD3-specific antibody, enables V_H/V_L association and the reconstitution of a functional CD3-binding site to engage T cells. b Allogeneic mismatch transplantation model: The hematopoietic system of a HLA-A2-positive leukemia patient is replaced by blood stem cells of an HLA-A2-negative donor. Hemibodies directed against HLA-A2 and the pan-hematopoietic marker CD45 tag the patient’s dual antigen-positive diseased hematopoiesis for lysis by T cells, while sparing single antigen-positive healthy tissues and donor-derived blood cells.

Fig. 2

Dual antigen-restricted reconstitution of a functional CD3-binding site. a V_LαCD3-scFvαHLA-A2 (green curve) and V_HαCD3-scFvαCD45 (black curve) hemibodies were isolated by immobilized metal ion affinity chromatography (IMAC) and size-exclusion chromatography (SEC) and analyzed by FPLC at 1 mg/ml. V0 void volume/aggregates. Typical elution profiles of the hemibodies, the bispecific BiTE construct (scFvαCD3-FvαHLA-A2, red curve on top), and a mixture of the two hemibodies (blue curve, bottom) are shown for comparison. b HLA-A2/CD45 dual-positive THP-1 acute myeloid leukemia cells were co-incubated with HLA-A2-negative donor peripheral blood mononuclear cells (PBMCs) and constructs as indicated. T cell engagement was assessed by interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretion and target cell lysis. The bispecific HLA-A2 × CD3 BiTE was used as a positive control. Data represent the mean values with standard deviations (±SD) from triplicate wells from three independent experiments; the effector to target cell ratio (E:T) was 5:1. c Binding of hemibodies (6 nM each) to THP-1 cells was competitively blocked by molar excess of scFv specific for CD45 (left) or HLA-A2 (right). Stimulation of donor PBMCs was assessed by IL-2 secretion at E:T = 2:1. Data represent the mean from two replicate wells from three experiments that yielded similar results. d Dual antigen-positive THP-1 leukemia, single-positve RAJI Burkitt lymphoma, or HT1080 fibrosarcoma cells were loaded with the hemibody pair (blue bars) or BiTEs (red bars), washed twice, and incubated with donor PBMCs as indicated at a E:T of 2:1. Secretion of IL-2 was assessed by ELISA. Data represent the mean (±SD) from two replicate wells from three experiments. e HLA-A2-positive (red bars) and HLA-A2-negative (blue bars) PBMNC were incubated with constructs as indicated (10 nM each). % apoptosis was assessed after annexinV staining by flow cytometry techniques as a measure of fratricide reaction. Data are representative for three independent experiments yielding comparable results.

Fig. 3

Biochemical basis for hemibody complementation. a A plasmon resonance chip was coated with V_LαCD3-scFvαHLA-A2. V_HαCD3-scFvαCD45 was used as an analyte at 100, 200, 400, 800, 1600 (nM) as indicated. Association contact time was 300 s, dissociation contact time 3.600 s at a flow rate of 30 μl/min, data represent one out of three experiments. b FCS autocorrelation functions, normalized to the number of molecules, recorded for 1 nM V_LαCD3-scFvαHLA-A2 alone (dark red) and for the same sample in presence of 1 µM V_HαCD3-scFvαCD45 (light red) or 100 nM HLA-A2 antigen (blue). Curves are fitted using data derived from three independent experiments c Top: Equilibrium binding study of scFv-GpL (Gaussia princeps luciferase) fusion proteins specific for CD45, HLA-A2, or CD3 to the respective antigens on 10⁵ Jurkat (CD3⁺, CD45⁺) and U266 cells (HLA-A2⁺, CD45⁺). Specific binding (triangles, solid line) was calculated as the difference of total (circles, dashed line) and non-specific binding (squares, dashed line) determined by using an irrelevant scFv-GpL fusion protein, HLA-A2-negative KMS-12-BM cells or CD3-negative U266 cells as indicated. Bottom: For heterologous competition analysis, cells were incubated with scFvCD45-GpL (2 nM), scFvHLA-A2-GpL (2 nM), or scFvCD3-GpL (4 nM) and the indicated concentrations of the hemibodies or the bispecific BiTE construct. IC₅₀ values were determined and used to calculate the K_i of CD45-, HLA-A2-, and CD3-specific scFv domains by help of the previously determined K_D-values of the scFv-GpL fusion proteins. Data show one out of three experiments yielding comparable results. d scFvCD3 or isolated V_HCD3 and V_LCD3 domains were fused to GpL. No specific binding of individual or combined constructs to CD3-positive Jurkat cells were detected as assessed by cell-bound luciferase activity. Data show one out of three experiments yielding comparable results. e Preservation of T cell triggering activity after incubation of hemibodies and BiTEs in human serum for 72 h at 37 °C. Read out was specific lysis at E:T of 5:1. Data represent the mean (±SD) from three replicate wells from one out of three experiments yielding comparable results. f In vivo serum half-lives after intravenous injection of 8 µg of a hemibody or BiTE construct into Balb/c mice. Values are deduced from >40 mice as determined by ELISA.

Fig. 4

High precision cancer cell targeting in vivo. Immune deficient mice (6 per group) were challenged with 1 × 10⁶ luciferase-positive THP-1 (CD45 and HLA-A2 positive) tumor cells intravenously (i.v.) on day 1. HLA-A2 negative memory CD4 and CD8 donor T lymphocytes were added i.v. on day 1, 22, and 28. After tumor engraftment on day 7, mice were treated subcutaneously with either saline (PBS), individual hemibodies addressing CD45 or HLA-A2 antigens, the combination of both hemibodies, or the HLA-A2 targeting BiTE control with a starting dose of 2 µg/mouse per day for 1 week, followed by 8 µg/mouse per day at distant sites until day 39. a Tumor burden of luciferase-positive THP-1 cells were assessed on a weekly basis by IVIS Lumina XR Real-Time Bioluminescence Imaging and b survival was monitored daily until day 110. Significance was determined by the Kaplan–Meier estimator; p = 0.0017 for the hemibody combination versus V_LαCD3-scFvαHLA-A2, p = 0.0005 for the hemibody combination versus V_HαCD3-scFvαCD45. No significant difference for the hemibody combination versus the bispecific BiTE control, p = 0.6670. The data represent one experiment, statistical considerations on effect and group size are provided in Supplementary Table 1.

Fig. 5

Absence of bystander cytotoxicity and translation into solid tumor model. a Intracellular caspase-3 activation as a measure of apoptosis in HLA-A2/CD45 dual-positive THP-1 (upper panels) and CD45-positive but HLA-A2-negative bystander cells (bottom panels) was assessed by flow cytometry in vitro after co-culture with donor PBMCs and CD45 or HLA-A2 targeting hemibodies as indicated (3 nM). The HLA-A2 specific BiTE was used as a positive control (left panel); data represent one out of two experiments that yielded similar results. b Luciferase-expressing dual antigen-positive THP-1 and single antigen-positive RAJI cells were injected subcutaneously in the left and right thigh, respectively. Donor T cells were injected i.v. at day 1 and saline (PBS), paired hemibodies and the BiTE control (8 µg/mouse) were administered s.c. daily until day 7. Data are representative for two independent experiments. c Immune deficient mice were challenged i.v. with luciferase-expressing human breast cancer cells MDA-MB-231, which co-express EGFR and Her2/neu antigens. After engraftment of lung metastases on day 3, PBMCs from a healthy donor were administered i.v. followed by s.c. injection of a buffer control (saline), a CD3 × EGFR BiTE or the combination of EGFR and Her2/neu-specific hemibodies. Tumor burden was visualized by IVIS Lumina XR Real-Time Bioluminescence Imaging on days 1 and 8. One in vivo experiment out of two is shown. For in vitro analyses, data represent means (±SD) of triplicate wells from at least two independent experiments, E:T = 10:1.