IOMX-0675 demonstrates strong binding to primary immune cells IOMX-0675 binds to primary cells of the myeloid and lymphoid immune lineage with single digit nM affinity FIGURE II A) Whole blood from healthy donors (n=3) was analyzed by flow cytometry regarding LILRB1 & LILRB2 target expression on relevant immune cell populations (NK = natural killer, CM = central memory; EM = effector memory, EMRA = effector memory cells re-expressing CD45RA). Data is depicted as mean ± SEM and is representative for 3 donors. B) IOMX-0675, competitor 1 and isotype-control antibodies were labelled with Alexa Fluor 647 and titrated on primary CD8+ T cells from healthy PBMC donors or in vitro differentiated M2-like macrophages according to the protocol depicted in Figure III A. Median fluorescence intensity was analyzed by flow cytometry and is depicted as mean ± SD. Data are representative of 3 donors. IOMX-0675 exhibits in vivo anti-tumor efficacy in a highly aggressive melanoma model In the highly aggressive A375 melanoma xenograft model, IOMX-0675 shows significant single-agent activity Inhibition of the LILRB1/2 pathway by IOMX-0675 or competitor 1 enhances T cell activation in vivo IOMX-0675 demonstrates its best-in-class potential by repolarizing the tumor microenvironment in contrast to a clinical competitor IOMX-0675 reprograms macrophages to a pro-inflammatory M1-like phenotype Across multiple donors, IOMX-0675 consistently demonstrates robust reprogramming of immunosuppressive macrophages Macrophage repolarization correlates with IOMX-0675 receptor occupancy FIGURE I A) Single-cell RNA sequencing (scRNAseq) datasets from Barkley et. al. 2022 Nat Genet, Lin et al 2020 Genome Med and Kim et al 2020 Nat Com. were analyzed using the Seurat R package. The extracted and quality-filtered RNAseq data from breast cancer (n=4), colorectal cancer (1), lung adenocarcinoma (4), ovarian cancer (3) and pancreatic cancer (9) were used to analyze LILRB1, LILRB2, LILRA1 and HLA-G expression in the annotated cell types. LILRA3 was not annotated in the analyzed data set. Cell annotations shown in the UMAP plot are taken from the original publication. B) Differential binding profile of IOMX-0675 against LILRB1/2 and LILRA1/3 compared to clinical competitor antibody (Competitor 1). C) Binding kinetics of IOMX-0675 or competitor 1 antibody at 10 nM to LILRB2 in the presence of 20 nM or 100 nM recombinant LILRA3 (rLILRA3). Interactions were measured by BLI on an Octet Red96e. Myeloid checkpoints in the tumor microenvironment have gained increased attention in the context of tumor immunity with a key role of these receptors in tumor resistance mechanisms. LILRB1 (ILT2) and LILRB2 (ILT4) are immunosuppressive receptors of the leukocyte immunoglobulin-like receptor (LILR) family that recognize both classical and non-classical MHC-I molecules (e.g., HLA-G). While tumor-infiltrating myeloid cells express both LILRB1 and LILRB2, lymphoid cells are restricted to LILRB1 expression. LILRB1 and LILRB2 are frequently upregulated in patients non-responsive to T cell checkpoint blockade, implicating a key role of these receptors in immune evasion. IOMX-0675 is a fully human, Fc-silenced monoclonal immunoglobulin G1 (IgG1) antibody, identified from iOmx’ proprietary phage display library. It displays a highly differentiated binding profile, with selective, high-affinity binding to the inhibitory receptors LILRB1 and LILRB2, while binding with only negligible affinity to the closely related immune-activating LILR family members LILRA1 and LILRA3. IOMX-0675 efficiently leads to macrophage repolarization and enhanced phagocytosis correlating with IOMX-0675 receptor occupancy in a flow-based assay. In autologous immune cell co-culture assays, IOMX-0675 demonstrates high potency in reprogramming immunosuppressive macrophages and restoring cytotoxic T cell activity. Additionally, in a CD34+ stem cell-engrafted humanized mouse xenograft melanoma model, IOMX-0675 demonstrates potent anti- tumor activity and pharmacodynamic modulation of tumor-associated macrophages, mirroring our in vitro findings. In summary, we discovered IOMX-0675, a cross-specific antibody antagonizing both LILRB1 and LILRB2 with high selectivity. Due to its highly differentiated binding profile which translates into superior potency, IOMX-0675 shows best-in-class potential for a dual-targeting myeloid checkpoint inhibitor that may maximize efficacy in patients across a variety of solid tumor indications. IOMX-0675, a cross-specific antibody selectively inhibiting LILRB1 and LILRB2, effectively repolarizes immunosuppressive myeloid cells and activates cytotoxic T cells leading to potent tumor cell killing IOMX-0675 selectively modulates the immunomodulatory LILRB1/2 receptors Single-cell RNAseq analysis reveals co-expression of LILRB1, LILRB2 and LILRA1 on individual immune cells in tumor biopsy samples HLA-G expression demonstrated on myeloid, lymphoid as well as cancer cells IOMX-0675 demonstrates high affinity to the immunosuppressive LILRB1 & LILRB2 receptors IOMX-0675 shows a superior binding profile over a clinical competitor targeting LILRB1/2 by avoiding binding to immuno-activating LILRA1/3 FIGURE VI A375 melanoma tumor cells were implanted subcutaneously into NOG-EXL mice, fully humanized with CD34+ stem cells from two donors. Randomized animals were treated twice weekly with 20 mg/kg IOMX-0675, competitor 1, or corresponding isotype control antibody. Tumor infiltrating immune cells were analyzed by flow cytometry. Activated CD69+ CD3+ T cells, CD68+CD11c-HLA-DR- M2-like macrophages and the ratio of CD3+ T cells versus M2-like macrophages are shown as mean ± SEM. Cell type LILRB1 expression LILRB2 expression LILRA1 expression HLA-G expression B) Differential binding profile of IOMX-0675 IOMX-0675 is a fully human, Fc-silenced cross-specific antibody binding with high affinity to the immunosuppressive receptors LILRB1 and LILRB2, while sparing their closely related immuno-activating LILR family members LILRA1/3. The highly differentiated binding profile of IOMX-0675 translates into remarkably superior potency in various binding and in vitro functional assays of macrophage repolarization, T cell suppression and tumor cell phagocytosis as compared to mono- or dual-targeting antibodies currently in clinical development. IOMX-0675 demonstrates its best-in-class potential by repolarizing the immunosuppressive tumor microenvironment in vivo and thereby inhibiting tumor growth in a humanized, myeloid engrafted melanoma model. CTA/IND-enabling studies for IOMX-0675 completed with CTA submission planned for Q4 2024. A) Co-expression of LILR family members and HLA-G in the tumor microenvironment of cancer patients C) Binding of IOMX-0675 to LILRB2 in the presence of LILRA3 C) Consistent repolarization of M2-like macrophages across multiple donors A) Macrophage generation B) LILRB1/2 RO correlates with phenotypic changes in M2-like macrophages A) Repolarization of M2-like macrophages and activation of effector T cells in an in vitro co-culture system B) Enhancement of tumor cell phagocytosis through inhibition of the LILRB1/2 pathway Christina A. Hartl, Kristina Heinig *, Ilona-Petra Maser, Marisa Stebegg-Wagner, Simone Friedrich, Jonas Schilz, Stefan Kaden, Thomas J. Jaquin, Eugenia Korotkova, Michail Maraslis, Carmen Ginzel, Bettina Langer, Michal Swiat, Alina Huth, Christine Rothe, Hannes Loferer, Maximilian Aigner, Alexander N. Marziale and Stefan Bissinger EORTC-NCI-AACR- 2024 Authors and affiliations iOmx Therapeutics AG, Martinsried/Munich, Germany; * …Presenting Author Introduction Conclusion IOMX-0675 reactivates immunosuppressed cytotoxic T cells and enhances tumor cell phagocytosis Superior repolarization of M2-like macrophages by IOMX-0675 translates into stronger activation of immunosuppressed cytotoxic T cells compared to clinical competitors on the LILRB1/LILRB2 pathways Dual targeting of LILRB1 and LILRB2 by IOMX-0675 enhances tumor cell phagocytosis and correlates with receptor occupancy FIGURE IV Differentiation of monocytes from healthy PBMC donors and polarization towards M1-like macrophages according to the depicted protocol. Treatment with IOMX-0675, competitor 1 or corresponding isotype control antibody in the presence of 37.5 nM or 150 nM recombinant LILRA3 (rLILRA3). Surface receptor CD163 were analyzed by flow cytometry on day 8 and GM-CSF was analyzed by bead-based multiplex immunoassays from the supernatants on day 8. Results are shown as the average for a concentration of 1nM (IOMX-0675 and Competitor 1) and 5nM (Isotype-ctrl.), respectively. FIGURE V A) M2-like macrophages were co-cultured with autologous T cells for 3 days. Treatment with IOMX-0675, competitor 1/2 or appropriate isotype control antibody was performed throughout the assay. Flow cytometry (CD163, CD69) and supernatant analysis by bead-based multiplex immunoassays were done on day 8 (Macrophages) and day 11 (T cells). B) M0-like macrophages were generated by differentiation of monocytes for 6 days with M-CSF. Cell Trace Far Red (CTFR) labelled A375 tumor cells were incubated with M0-like macrophages for 2h and treated with IOMX-0675, competitor molecules and corresponding isotype ctrl. Phagocytic activity was measured by flow cytometry. Phagocytosis induction upon treatment with IOMX-0675 and competitor molecules (10 nM) is shown across 13 donors as mean ± SD (left graph). Dose-dependent phagocytic activity (middle graph) as well as LILRB1/ LILRB2 receptor occupancy (right graph) was analyzed for n= 6 donors by flow cytometry. Results are shown as mean ± SD of n=6 donors (blue / red line) and each donor individually (grey lines). 0 25 50 75 100% LILRB1+ cells LILRB1 0 25 50 75 100% LILRB2+ cells LILRB2 NK cells T naive T CM T EM T EMRA B cells classical monocytes non- classical monocytes neutrophils 0.0010.01 0.1 1 10 1001000 0 1×106 2×106 3×106 4×106 AB conc. [nM] Fluorescence Intensity [Median] M2- Macrophages 0.1 1 10 100 1000 0 1×104 2×104 3×104 4×104 5×104 6×104 AB conc. [nM] CD8+ T cells IOMX0675 Competitor 1 (αLILRB1/2)Isotype-ctrl. B) Binding of IOMX-0675 to primary immune cellsA) LILRB1 / LILRB2 positive immune cell populations 0.001 0.01 0.1 1 10 0 20 40 60 80 100 120 IOMX-0675 conc. [nM] % Receptor Occupancy Iso-ctrl. RO mean ± SEM 0.001 0.01 0.1 1 10 0 100 200 300 400 500 600 IOMX-0675 conc. [nM] % Median FI relative to isotype-ctrl. CD86 EC50RO mean ± SEM M1-like 0.001 0.01 0.1 1 10 0 50 100 150 IOMX-0675 conc. [nM] CD163EC50RO mean ± SEM 0.001 0.01 0.1 1 10 0 50 100 150 IOMX-0675 conc. [nM] % Chemokine relative to isotype-ctrl. CCL13EC50RO mean ± SEM M2-like IOMX-0675 reprograms macrophages in a LILRA3 dominated environment Even in LILRA3-dominated environments, IOMX-0675 enhances the pro-inflammatory phenotype of M1-like macrophages FIGURE III A) Differentiation of monocytes from healthy PBMC donors and polarization towards M2-like macrophages according to the depicted protocol. B / C) Treatment with IOMX-0675 or corresponding isotype ctrl. antibody throughout the differentiation/polarization period. LILRB1/2 receptor occupancy as well as surface receptors CD86 and CD163 were analyzed by flow cytometry on day 8. CCL-13 was analyzed by a bead-based multiplex immunoassays from the supernatants on day 8. Results are shown as mean ± SEM of all donors (red / blue line) and each donor individually (grey lines) (B+C). 0.001 0.01 0.1 1 10 100 90 100 110 120 130 140 150 Antibody concentration [nM] % Macrophage phagocytosis rel. to isotype ctrl EC50 RO 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 120 Antibody concentration [nM] % Receptor occupancy Phagocytosis Receptor Occupancy 0% 100 150 200 250 % Macrophage Phagocytosis relative to isotype ctrl. Competitor 1 (αLILRB1/2) IOMX-0675 Competitor 2 (αLILRB2) Competitor 3 (αLILRB1) Phagocytosis 0 5 10 15 20 0 200 400 600 Days after start of therapy Tumour volume [mm3] mean ± SEM p= 0.0003 0 10 20 30 40 50 % CD69+ CD3+ Tcells of CD3+ Tcells 0 2000 4000 6000 8000 M2-like TAMs [count/g tumor] 0.002 0.002 0.0 1.0 2.0 3.0 4.0 T cell / M2-like TAMs ratio 0.03 0.004 Isotype-ctrl IOMX-0675Competitor 1 (αLILRB1/2) 0.01 0.1 1 10 0 2×103 4×103 6×103 8×103 1×104 1.2×104 AB conc. [nM] Fluorescence Intensity [Median] CD163mean ± SEM 0.01 0.1 1 10 0 2,500 5,000 7,500 10,000 AB conc. [nM] Concentration [pg/ml] IL-8mean ± SEM 0.01 0.1 1 10 0 100 200 300 400 500 AB conc. [nM] Fluorescence Intensity [Median] CD69mean ± SEM 0.01 0.1 1 10 0 5,000 10,000 15,000 AB conc. [nM] Concentration [pg/ml] IFNymean ± SEM Competitor 2 (αLILRB2) IOMX0675 Competitor 1 (αLILRB1/2) 150 nM 37.5 nM 0 10 20 30 40 50 60 70 rLILRA3 %CD163+ Macrophages <0.0001<0.0001 CD163 mean ± SD 150 nM 37.5 nM 0 100 200 300 400 rLILRA3 % Cytokine relative to Isotype-ctrl. 0.0003 0.0003 GM-CSF mean ± SD Isotype-ctrl. (5nM) Competitor 1 (αLILRB1/2) (1nM) IOMX-0675 (1nM) 0 300 600 900 1200 1500 0.0 0.5 1.0 Time [s] -10% -62% -100% -3% IOMX-0675 w/o rLILRA3 IOMX-0675 + 20nM rLILRA3 IOMX-0675 + 100nM rLILRA3 Competitor 1 w/o rLILRA3 Competitor 1 + 20nM rLILRA3 Competitor 1 + 100nM rLILRA3 LILRB2 + rLILRA3 Competitor 1 (αLILRB1/2) IOMX-0675 10-9 10-8 10-7 10-6 KD (M) LILRB2 LILRB1 LILRA1 LILRA3 non-binding < 10 nM 10 - 100 nM > 100 nM >200-fold 962 nM 575 nM 2.8 nM 2.3 nM
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