Il1rap antibodies
An antibody and receptor technology, applied in the field of IL1RAP antibody, can solve the problem of destroying LSC
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[0378] Applicants show that interleukin-1 receptor accessory protein (IL1RAP) is only expressed on human AML blasts, including a subset rich in leukemia stem cells (LSCs), but not on normal hematopoietic cells including hematopoietic stem cells (HSCs) expression, thus providing better targets for AML than the immunotherapeutic targets currently used in clinical trials (i.e. CD33 and CD123), which are also expressed on normal HSCs. Accordingly, Applicants designed an anti-IL1RAP / CD3 T cell-dependent bispecific antibody to target leukemia cells and LSCs, while sparing normal HSCs. In vitro functional analysis showed that the IL1RAP / CD3 bispecific antibody combined with T cells could effectively eliminate not only AML cell lines, but also leukemia cells, including LSCs, from samples from patients with different subtypes of AML. Compared with treatment with IgG or T cells alone, in vivo treatment with IL1RAP / CD3 bispecific antibody plus T cells significantly reduced leukemia burde...
example 2
[0379] Example 2: Antibody Generation and Selection
[0380] Human phage display libraries were constructed using purified peripheral blood mononuclear cells (PBMCs) from ten healthy donors as previously described. Twelve unique clones were identified as IL1RAP-binding clones. The gene sequences of these cloned single-chain fragment variants (scFv) were ligated into the scFv-FC expression vector (TEGX-SCblue, antibody design), respectively. Antibodies were expressed in transient expi293 cells and purified by protein A affinity chromatography. The IL1RAP EC50 (Table 2) of these antibodies was determined by ELISA and antibody-dependent T cell-mediated cytotoxicity (ADCC). Select the one with the highest binding affinity (ie, lowest EC 50 ) antibodies were used to construct bispecific antibodies.
[0381] Table 2. Half-maximal effective concentrations (EC) of exemplary IL1RAP antibodies and fragments thereof provided herein 50 )Research.
[0382] IL1RAP antibody...
example 3
[0383] Example 3: T Cell Dependent Cytotoxicity (TDCC) Long Term Killing Assay
[0384] To determine T cell-mediated killing of AML cell lines and AML primary cells, different concentrations of anti-IL1RAP antibody were combined with purified T cells from healthy donor or AML patient samples at 1x10 per well. 4 Target cells were incubated at different effector-target (E:T) ratios. After 48 hours of incubation, cells were stained with antibody, CD34 + CD45 dim or CD33 + CD45 dim CD14 - cells called leukemia cells, CD45 + CD4 + or CD45 + CD8 + for T cells, CD25 + or CD69 + for activated T cells. The numbers of leukemia cells and T cells were counted and cytotoxicity was calculated: cytotoxicity (%) = 100x (1 - treated target cells / control target cells).
[0385] Table 3. TDCC studies using recombinant proteins (IgG antibodies) provided herein.
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[0387]
PUM
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