Genetic alteration of a bispecific ligand-directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy

Abstract

A bispecific ligand-directed toxin (BLT) called DT2219ARL consisting of two scFv ligands recognizing CD19 and CD22 and catalytic DT390 was genetically enhanced for superior in vivo anti-leukemia activity. Genetic alterations included reverse orienting VH-VL domains and adding aggregation reducing/stabilizing linkers. In vivo, these improvements resulted in previously unseen long-term tumor-free survivors measured in a bioluminescent xenograft imaging model in which the progression of human Raji Burkitt's lymphoma could be tracked in real time and in a Daudi model as well. Studies showed DT2219ARL was potent (IC50s 0.06-0.2 nM range) and selectively blockable. Imaging studies indicated the highly invasive nature of this B cell malignancy model and showed it likely induced pre-terminal hind limb paralysis because of metastasis to spinal regions prevented by DT2219ARL. DT2219ARL represents a new class of bispecific biological that can be continually improved by genetic mutation.

Figure 1

A) Construction of DT2219ARL. 1) The original DT2219EA construct consisting of the first 389 amino acids of the DT (DT₃₉₀), the V_H and V_L regions of anti- CD22 (sFv) and anti-CD19 (31) linked by a 20 amino acid segment of human muscle aldolase (hma). 2) To construct DT2219ARL the V_H-V_L orientation was reversed and the V_L and V_H genes of each sFv were conjoined by a fragment encoding the ARL linker. 3) The final target gene was spliced into pET21d vector. B) SDS-PAGE gel containing all 3 DT2219 variants used in these studies. Lane 1 and 8 -Molecular weight standards, Lane 2–95 kDa DT2219ARL, Lane 3-DT2219EA, Lane 4-DT2219EB1, Lane 5-RFB4 monoclonal antibody, Lane 6-HD37 monoclonal antibody, Lane 7-DT₃₉₀ (partially purified), The gel was stained using Coomassie blue and shows size and purity of the agents.

Figure 2

The in vitro effect of the DT2219 mutant proteins. A) Daudi cells were cultured with fusion proteins and proliferation was measured by uptake of tritiated thymidine. Data are percentage of control response where control response is untreated cells. Data are expressed as mean ± standard deviation (SD). The mean values of untreated Daudi cells were 121,001 ± 8,276 cpm/20,000 cells. B.) Selectivity was determined on the CD19⁻CD22⁻ HPBMLT cell line in a separate experiement. The mean cpm of untreated HPBMLT in this experiment was 61,993 ± 7,178 cpm/20,000 cells. DT2219ARL differed significantly from the control BIC3 group at 0.01 – 100 nM by Student t test (p<0.0001). C) In a third experiment, the ability of the ligands themselves to mediate cytotoxicity was tested by inactivating the diphtheria toxin with the DT2219GE mutation that disrupts toxin activity and leaves the ligands intact. The mean cpm of untreated Daudi in this experiment was 112,164 ± 10,379 cpm/20,000 cells. D) The anti-proliferative effect of DT2219ARL, DT22, DTIL19, and a mixture of DT22/DT19 on Daudi cells was tested by measuring 3H-thymidine uptake 72 hours following IT exposure. Points on each graph represent mean of triplicate samples ± SD. Control counts= 61,993 ± 7,178 cpm/20,000 cells.

Figure 3

The activity of mutated DT2219ARL is mediated by both anti-CD19 sFv and the anti-CD22 sFv ligands. Proliferation studies were performed in which Daudi cells were treated with a constant concentration of 10 nM DT2219ARL and then blocked with increasing concentrations of HD37 monoclonal antibody, RFB4 monoclonal antibody, or non-reactive control Ly5.2 antibody. Thymidine uptake was then measured. Each line represents the mean of triplicate determinations ± standard deviation (SD). Percent blocking was calculated in comparison to the unblocked control and then graphed. Counts for untreated Daudi cells were 59,301 ± 2,804 cpm/20,000 cells.

Figure 4

Binding of DT2219ARL-FITC to monkey PBMC by direct immunofluorescence. Monkey PBMC, normal human PBMC, or normal human CD22+ magnetic bead enriched PBMC cells were incubated with DT2219ARL-FITC, or negative control DTEpCam23. Positive controls included FITC labeled conventional monoclonal antibodies RFB4-FITC or HD37-FITC. Flow cytometry was performed and data expressed as a contour plot showing cells versus increasing fluorescent intensity. The top 3 panels show human CD22+ magnetic bead enriched PBMC, the lowest 3 panels show normal monkey PBMC, while the middle 3 panels show normal human PBMC. The box in each panel outlines the gated area which shows binding that exceeds the values obtained for the negative control. The number in the box is the percentage of positive cells. NE- not evaluated.

Figure 5

Groups of scid mice were given 10⁶ Daudi cells iv to induce systemic disease. A) Three days following Daudi injection, mice were given the exact same injection schedule of multiple IP injections of DT2219ARL and DT2219EB1 in order to compare them to no treatment controls. Data was graphed as proportion surviving versus time. Statistical analysis was performed using the LogRank test and the DT2219ARL group significantly differed from the no treatment group (p<0.001). B) Three days following Daudi injection, mice were given ip treatment with DT2219EA and DT2219ARL in comparison to no treatment controls and to Bic3 immunotoxin control treated mice. The DT2219ARL group significantly differed from the DT2219EA group, the Bic3 group, and the no treatment group (p<0.001). C) Three days following Daudi injection, mice were given a single ip injection of DT2219EA and DT2219ARL in comparison to no treatment controls. Only the DT2219ARL group significantly differed from the no treatment group.

Figure 6

Effect of IP administration of DT2219ARL on mice given systemic B cell cancer by IV injection of Raji-luc. Raji-luc cells stably expressing the luciferase gene were administered IV to scid mice. A) Mice were either treated with DT2219ARL (M1, M2, M3, and M4) on days 3, 5, 11, 16, and 18 or untreated (M5, M6, M7, and M8). Luciferase bioluminescence was measured as photons/sec/cm²/sr. B) The same data as shown in A is graphed in B. Data are expressed as total activity graphed over time for each individual animal (M1–M8). C) Digital Images of illustrating tumor progression in untreated Raji-luc mice. Bioluminescent imaging is shown for 3 untreated mice M9–M11 on day 21. Because the Raji-luc line has a GFP reporter gene as well as a luciferase reporter gene, fluorescent imaging is also shown for animals M9, M10, and M11. Lymphoma can be seen in lung, bone marrow, lymph node and compressing the spinal chord which likely causes hind limb paralysis (HLP). GFP imaging correlates with luciferase imaging.

Figure 7

Toxicity of DT2219ARL in rabbits. Rabbits were given IV injection of DT2219ARL on days 1, 3, 5, 7. A) Average weight of two rabbits B) ALT enzyme levels from the same rabbits C) Frozen liver section from a rabbit treated with 500 ug/kg DT2219ARL. The section was stained with H and E and is shown at 100X magnification.