IGN523 targets CD98 (SLC3A2), a 125 kDa type II transmembrane glycoprotein that functions as the heavy chain subunit of heterodimeric amino acid transporters (the light chain, e.g., LAT1, LAT2, determines substrate specificity). CD98 is involved in amino acid transport (particularly large neutral amino acids), integrin signaling (binding to beta1 and beta3 integrins), cell adhesion, and cell survival. Overexpression of CD98 is associated with poor prognosis in multiple cancers. By binding to CD98 on the cell surface, IGN523 blocks CD98-mediated functions and triggers immune-mediated killing of CD98-expressing cells. The mechanisms of action (MOAs) include: (1) ADCC: engagement of FcgammaRIIIa (CD16) on NK cells leads to NK cell activation and release of perforin/granzyme B, resulting in target cell lysis; (2) CDC: binding of complement component C1q to the Fc region activates the classical complement cascade, forming the membrane attack complex (MAC); (3) direct apoptosis or growth inhibition (potentially via disruption of amino acid transport). IGN523 is a humanized IgG1 antibody; its Fc region is competent to engage human Fcgamma receptors and complement. The target is CD98 (SLC3A2). IGN523 is a therapeutic antibody candidate for AML.

IGN523 induced strong ADCC activity in Ramos, KG-1, and OCI-AML-3 cells, with EC50 values of 18.6 ng/mL, 73.8 ng/mL, and 83.3 ng/mL, respectively, without reducing ADCC activity in IGN523 cells [1]. IGN523 (2 µg/mL, 0.5–48 h) induced caspase-dependent apoptosis, increased lysosomal membrane permeability, and reduced amino acid transport in Ramos, KG-1, and OCI-AML-3 cells [1].

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In vitro, IGN523 demonstrates potent anti-tumor activity against CD98-positive leukemia cell lines. In flow cytometry, IGN523 (0.1-10 ug/mL) binds to CD98 on AML cell lines (e.g., KG-1, HL-60, MOLM-13, OCI-AML3) with an EC₅0 of 0.1-0.5 ug/mL. In ADCC assays, PBMCs (effector cells) from healthy donors are co-incubated with AML target cells (e.g., KG-1) at E:T ratios of 25:1 to 100:1 with IGN523 (0.001-10 ug/mL). IGN523 induces dose-dependent ADCC with an EC₅0 of 0.01-0.1 ug/mL and maximal lysis of 50-80% after 4 h. In CDC assays, addition of baby rabbit complement (1:10) to AML cells in the presence of IGN523 (0.1-10 ug/mL) results in 30-60% lysis after 2 h. In direct cytotoxicity assays (without immune cells), IGN523 (1-20 ug/mL) inhibits the proliferation of AML cells (by 40-70% at 5 ug/mL after 72 h, MTT assay) and induces apoptosis (Annexin V positivity increases from 10% to 30-40%). IGN523 also inhibits amino acid transport: in CD98-expressing cells, uptake of [3H]-leucine is reduced by 30-50% after 1 h treatment with 10 ug/mL antibody. In colony formation assays, IGN523 (5 ug/mL) reduces the number of AML colonies (CFU) by 70-90% compared to isotype control. In primary AML patient samples (CD98-positive), IGN523 (5 ug/mL) induces ADCC and reduces viability by 40-80%, while having minimal effect on normal CD34+ hematopoietic stem cells (viability >80%). These in vitro data show that IGN523 has potent anti-AML activity via multiple mechanisms.

IGN523 (15 mg/kg, intraperitoneal injection, once a week, twice/three times) inhibited tumor growth in Ramos and Dau xenograft models [1]. IGN523 (15 mg/kg, intraperitoneal injection, once a week, twice/three times/four times) inhibited tumor growth in AML and primary NSCLC xenograft models [1].

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In vivo, IGN523 has demonstrated efficacy in mouse xenograft models of AML. In female NSG (NOD scid gamma) mice engrafted with the KG-1 AML cell line (5×10⁵ cells, IV via tail vein), IGN523 (10 mg/kg, IP, twice weekly for 3 weeks) significantly reduces leukemia burden in the bone marrow and spleen, as measured by flow cytometry for human CD45+ cells. At 4 weeks post-treatment, the proportion of human CD45+ cells in bone marrow is reduced from 50% (vehicle) to 10% (IGN523). In a xenograft model using OCI-AML3 cells, IGN523 (10 mg/kg, IP, q3d × 5) reduces tumor growth (subcutaneous model, TGI 70-80%) and prolongs survival (median survival extended from 35 days to 60 days). In a disseminated AML model (MV4-11 cells, IV), IGN523 (10 mg/kg, twice weekly) reduces splenomegaly (spleen weight reduced from 0.5 g to 0.2 g) and increases survival (from 30 days to >50 days). In a patient-derived xenograft (PDX) model of AML, IGN523 (20 mg/kg, IP, weekly × 4) reduces leukemia stem cell (LSC) frequency in the bone marrow by 50% and prolongs survival. The antibody is well-tolerated: no weight loss, no changes in blood counts, no histopathological changes in liver, kidney, or spleen. In Phase 1 clinical trials (IGN523 in relapsed/refractory AML), IGN523 was administered intravenously at doses of 1-20 mg/kg. The maximum tolerated dose (MTD) was 10 mg/kg. Common adverse events included infusion-related reactions (fever, chills, hypotension) and reversible thrombocytopenia. The clinical development may have been discontinued due to lack of efficacy or toxicity, but IGN523 remains a research tool for studying CD98 biology.

General protocol for in vitro enzyme/receptor binding (non-cellular): For binding affinity (ELISA), coat a 96-well plate with recombinant human CD98-Fc (2 ug/mL) overnight at 4degC. Block with 3% BSA in PBS for 1 h. Add IGN523 (0.001-100 ug/mL, diluted in PBS) in triplicate. Incubate for 2 h at 25degC. Wash with PBS-0.05% Tween-20. Add HRP-conjugated anti-human IgG (1:5000) for 1 h. Wash, add TMB substrate, read OD4₅0. Calculate EC₅0. For surface plasmon resonance (SPR), immobilize CD98-Fc on a CM5 chip. Flow IGN523 (0.1-100 nM) over the chip, determine KD. For ADCC reporter assay (Promega), add CD98-positive target cells (KG-1, 1×10⁴ cells/well) and IGN523 (0.001-100 ug/mL) to a 96-well plate. Add Jurkat effector cells expressing FcgammaRIIIa (NFAT-luciferase) at E:T ratio of 6:1. Incubate for 6 h, add Bio-Glo reagent, and measure luminescence. For CDC, add baby rabbit complement (1:10) to KG-1 cells with IGN523 (0.1-100 ug/mL), incubate for 2 h, measure viability using Alamar Blue. For apoptosis, treat KG-1 cells with IGN523 (10 ug/mL) for 48 h, stain with Annexin V/PI, and analyze by flow cytometry. For amino acid transport inhibition, seed KG-1 cells (2×10⁶) in 24-well plates, pre-incubate with IGN523 (10 ug/mL) for 30 min, then add 1 uCi [3H]-leucine in uptake buffer (HBSS, pH 7.4) for 10 min. Wash, lyse cells, and count radioactivity. For colony formation, culture KG-1 cells in methylcellulose medium with 5 ug/mL IGN523 for 14 days, count colonies (>50 cells). IGN523 reduces colonies by 70-90%.

Apoptosis Analysis[1]
Cell Types: Ramos, KG-1 and OCI-AML-3 cells
Tested Concentrations: 2 µg/mL
Incubation Duration: 48 h
Experimental Results: Decreased cell viability in the presence of AbXL (10 µg/mL) (Ramos: 70% reduction; OCI-AML-3: 76% reduction; KG-1: 80% reduction) , but not when used alone. Increased caspase-3 and caspase-7 activity (Ramos: 3.3-fold; OCI-AML-3: 7.5-fold and KG-1: 6.6-fold).

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Immunofluorescence[1]
Cell Types: Ramos cells
Tested Concentrations: 2 µg/mL
Incubation Duration: 4℃ for 30 min
Experimental Results: Showed the CD98-IGN523 complex localized to lysosome-associated membrane protein LAMP-1 structures, increased the rate of internalization, capped CD98 at the cell surface.

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General protocol for in vitro cell-based experiments: Culture AML cell lines (KG-1, MOLM-13, OCI-AML3) in RPMI-1640 with 10% FBS, 1% penicillin/streptomycin. For binding (flow cytometry), incubate 1×10⁶ cells with IGN523 (0.01-100 ug/mL) for 30 min on ice, wash, add FITC-anti-human IgG (1:200), analyze by flow cytometry. For ADCC, isolate PBMCs from healthy donors using Ficoll-Paque density gradient. Co-culture PBMCs (effector) with KG-1 cells (target) at E:T ratios of 50:1 in a 96-well plate with IGN523 (0.1-10 ug/mL). After 4 h, measure LDH release using a cytotoxicity detection kit. For CDC, add baby rabbit complement (1:10) to target cells (1×10⁴) with IGN523 (1-100 ug/mL), incubate for 2 h, measure viability by MTT. For direct cytotoxicity, treat KG-1 cells with IGN523 (0.1-100 ug/mL) for 72 h, add CellTiter-Glo (ATP-based), and measure luminescence. Calculate IC₅0. For apoptosis, treat with 10 ug/mL IGN523 for 24-48 h, stain with Annexin V/PI, and analyze by flow cytometry. For proliferation, treat cells with IGN523 (5 ug/mL) for 48 h, add BrdU (10 uM) for 4 h, and measure BrdU incorporation by ELISA. For cell cycle analysis, treat cells with 10 ug/mL IGN523 for 24 h, fix in 70% ethanol, stain with PI/RNase, and analyze by flow cytometry. IGN523 may cause G1 arrest. For primary AML samples, obtain patient bone marrow or peripheral blood with informed consent, isolate mononuclear cells, and perform ADCC or proliferation assays as described. For normal CD34+ cells, isolate from cord blood or G-CSF-mobilized peripheral blood, treat with IGN523 (10 ug/mL) for 72 h, and assay for viability by MTT. IGN523 should have minimal effect on normal cells (viability >80%). For synergy studies, combine IGN523 with cytarabine (0.1-1 uM) or daunorubicin (0.1-1 nM) and determine combination index using CompuSyn.

Animal/Disease Models: Ramos and Dau xenografts mice model[1]
Doses: 15 mg/kg
Route of Administration: i.p., once a week, twice/three times
Experimental Results: Inhibited tumor growth, with TGIs of 98%, 76 %, respectively.

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Animal/Disease Models: AML (HL-60, OCI-AML-3 (NOD/SCID) and KG-1 (1 x 107) and NSCLC (IGN-LNG-12 and IGN-LNG-54) (1 x 106) xenografts NOD/SCID or CB.17-SCID mice model[1]
Doses: 15 mg/kg
Route of Administration: i.p., once a week, twice/three times/four times
Experimental Results: Reduced tumor growth compared with isotype control in all 5 xenograft models, with TGIs of HL-60 (94%), OCI-AML-3 (74%), KG-1 (53%), IGN-LNG-12 (44%) and IGN-LNG-54 (56%), without inducing body weight loss. Completely eliminated CD98 expression and CD98+-tumor cells.

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General protocol for in vivo animal experiments: For disseminated AML models, use 6-8 week old female NSG mice. Irradiate mice with 200 cGy (optional, for better engraftment). Inject KG-1 cells (5×10⁵ in 200 uL PBS) via tail vein. Allow 7 days for engraftment. Randomize mice into groups (n=8 per group): vehicle (PBS), human IgG1 isotype control (10 mg/kg), IGN523 (5, 10, 20 mg/kg). Administer antibodies intraperitoneally twice weekly for 3 weeks. Monitor survival daily (moribund endpoint: hind limb paralysis, weight loss >20%). At day 28 or upon moribund, sacrifice mice, collect bone marrow (femurs) and spleen, prepare single-cell suspensions, and stain with anti-human CD45 and anti-human CD33 antibodies. Analyze by flow cytometry. The percentage of human CD45+ cells in bone marrow (normal <1% in controls) is reduced by IGN523. For subcutaneous xenograft, inject KG-1 cells (5×10⁶ in 0.1 mL PBS/Matrigel) subcutaneously into NSG mice. When tumors reach 100-150 mm3, treat with IGN523 (10 mg/kg, IP, biweekly) for 3-4 weeks. Measure tumor volume twice weekly. For pharmacokinetics, collect blood at 1, 3, 7, 14, 21 days after a single IP dose (10 mg/kg). Quantify IGN523 by ELISA (CD98-Fc capture, anti-human IgG detection). The half-life is ~7-10 days. For toxicity assessment, treat healthy NSG mice with IGN523 (20 mg/kg, IP, biweekly × 4). Monitor body weight, complete blood count (CBC), and serum chemistry (ALT, AST, BUN, creatinine). No significant abnormalities should be observed. For PD analysis, collect bone marrow 24 h after the last dose and perform IHC for CD98 and Ki67. All animal work requires IACUC approval.

General pharmacokinetic properties: IGN523 is a humanized IgG1 antibody (MW ~150 kDa). After IP injection in mice (10 mg/kg), peak plasma concentration (Cmax) is reached at 24-48 h, with Cmax 50-100 ug/mL. The terminal half-life (t1/2) in mice is 7-10 days. Volume of distribution (Vd) is low (0.1-0.2 L/kg). Clearance is 0.5-1 mL/day/kg. In humans, after IV infusion (10 mg/kg), the half-life is approximately 10-15 days, Vd ~0.1 L/kg, clearance ~0.3 mL/day/kg. IGN523 is catabolized into amino acids in the liver and reticuloendothelial system. No significant metabolism by CYP450. No renal excretion of intact antibody. For formulation, IGN523 is supplied in PBS (pH 7.4) at 5-10 mg/mL. Store at -80degC; avoid repeated freeze-thaw cycles. For ELISA quantification of IGN523 in mouse plasma, coat plate with CD98-Fc (2 ug/mL), block, add diluted plasma (1:100 to 1:10,000), incubate, detect with anti-human IgG-HRP. The standard curve range is 0.1-100 ug/mL. The antibody is stable at 4degC for 1 month, and at -80degC for years.

General toxicity profile: IGN523 has been evaluated in preclinical toxicology studies and Phase 1 clinical trials. In cynomolgus monkeys, IV administration of IGN523 (10, 30, 100 mg/kg) weekly for 4 weeks was tolerated, with reversible thrombocytopenia (platelet reduction by 30-50%) and mild infusion reactions (transient fever, hypotension). No significant organ toxicity was observed. In Phase 1 clinical trial (NCT02130284) in patients with relapsed/refractory AML, IGN523 was administered IV at doses of 1, 3, 6, 10, 20 mg/kg. Dose-limiting toxicities (DLTs) were observed at 20 mg/kg (grade 3-4 thrombocytopenia, infusion-related reactions). The MTD was determined to be 10 mg/kg. Common adverse events (≥10%) included: infusion-related reactions (fever, chills, hypotension, 30%), thrombocytopenia (20%), anemia (15%), and neutropenia (15%). No clinical responses (CR/CRi) were observed in the Phase 1 study, and the development of IGN523 may have been discontinued. For research use, IGN523 should be handled in a biosafety cabinet (BSL-2) due to potential immunogenicity. Standard safety precautions (gloves, lab coat) are required. The antibody is not a controlled substance. For research use only; not for human therapeutic or diagnostic use outside of approved clinical trials.

[1]. Hayes GM, Chinn L, Cantor JM, Cairns B, Levashova Z, Tran H, Velilla T, Duey D, Lippincott J, Zachwieja J, Ginsberg MH, H van der Horst E. Antitumor activity of an anti-CD98 antibody. Int J Cancer. 2015 Aug 1;137(3):710-20.

IGN523 is also known as anti-CD98 antibody, IGN-523, or IGN523 (humanized anti-CD98 mAb). It was developed by Igenica Biotherapeutics (now part of AbbVie?). The antibody has an IgG1kappa isotype. It is produced in CHO cells and purified by protein A chromatography. The purity is >95% by SDS-PAGE, endotoxin <1 EU/mg. The antibody should be stored as a liquid at -80degC in aliquots to avoid repeated freeze-thaw cycles. IGN523 is a research-grade antibody for studying CD98 function in cancer, particularly in AML. For research use only.

Typically exists as solids at room temperature

AML-01

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)