B7-H3-mediated cis-inhibition of EGFR by a tumor-selective bispecific antibody enhances anti-tumor efficacy and minimizes toxicities

Domain: chemistry
Published: 2026-02-24
Editorial outcome: major_revision

Abstract
Therapeutic targeting of epidermal growth factor receptor (EGFR) in solid tumors faces significant limitations due to on-target/off-tumor toxicities, underscoring the urgent need for tumor-selective anti-EGFR therapies. Comprehensive bioinformatics and histopathological analyses identify marked upregulation of B7-H3 across EGFR-positive malignancies, contrasting with its minimal expression in healthy tissues. Leveraging an unbiased functional screen of bispecific antibodies (bsAbs) combining diverse B7-H3 and EGFR binders, we develop IBI334, a EGFR/B7-H3 bsAb exhibiting exceptional tumor selectivity. In preclinical models, IBI334 outperforms conventional EGFR antibodies by demonstrating superior EGFR occupancy, enhanced ligand-blocking efficacy, accelerated receptor degradation, and potent suppression of downstream EGFR signaling. Mechanistic studies demonstrate B7-H3-mediated cis-inhibition. The human B7-H3 extracellular domain (ECD) in complex with anti-B7-H3 Fab is resolved by cryo-EM, revealing critical residues for the antibody-B7-H3 interaction. IBI334 demonstrates robust anti-tumor activity in vitro and in vivo across EGFR-driven tumor models and synergized effectively with KRAS inhibitors. Toxicological evaluations in non-human primates reveal a favorable safety profile, with no EGFR-related adverse effects observed at doses up to 120 mg/kg over 4 weeks. Supported by these preclinical findings, IBI334 has advanced to a phase 1 clinical trial (NCT05774873) for advanced/metastatic solid tumors.

Introduction
Epidermal growth factor receptor (EGFR) activating mutations and amplifications are common oncogenic drivers in multiple cancers, including colorectal, gastric, breast, non-small cell lung, head and neck squamous cell and pancreatic cancers and diffuse intrinsic pontine glioma and cholangiocarcinoma1,2. Many EGFR-targeted therapies, such as monoclonal antibodies (e.g., Cetuximab3,4, Panitumumab5, Necitumumab6, Nimotuzumab7), bispecific antibody (e.g., Amivantamab8) and small molecule tyrosine kinase inhibitors (e.g., Gefitinib9, Afatinib10, Osimertinib11), have received approval for treating EGFR-positive tumors. Despite the clinical benefits of EGFR inhibitors, they are associated with various dose-limiting adverse events such as acneiform eruption, severe gastrointestinal (GI) toxicities, and rare but severe skin toxicities (e.g., toxic epidermal necrolysis), which not only significantly compromise patient quality of life but also limit the maximal therapeutic potential of the drugs12–14.

B7 Homolog 3 (B7-H3) is a single-pass transmembrane protein that is minimally expressed in normal tissues but is upregulated in several malignancies including lung, prostate, breast, pancreatic, colorectal, cutaneous squamous cell, and esophageal squamous cell carcinoma15,16. Elevated B7-H3 expression correlates with immune evasion, metastasis, disease progression and recurrence, poor prognosis, and resistance to therapy17–23. Human B7-H3 has two isoforms, 2Ig-B7-H3 (IgV-IgC domains similar to murine B7-H3) and 4Ig-B7-H3 (duplicated IgV-IgC domains)24, and the human 4Ig-B7-H3 isoform (referred to as “B7-H3” hereafter for simplicity) is the predominant form in human tumors25,26. B7-H3 is a promising therapeutic target, with various B7-H3-targeted therapies under clinical development, including monoclonal antibodies, bispecific/trispecific antibodies, antibody-drug conjugates (ADCs), CAR-T cell therapies and radionuclide-drug conjugates (RDCs)23,27.

In this work, we conduct bioinformatics analysis and immunohistochemistry staining to show strong co-expression of EGFR and B7-H3 in multiple solid tumours, but not in normal tissues. Then we design and screen B7-H3/EGFR bispecific antibodies to selectively augment EGFR signal suppression in B7-H3 positive tumors. The preclinical characterization of the lead bispecific antibody, IBI334, is comprehensively described in this report, encompassing its design rationale, mechanisms of action, structural analysis, anti-tumor efficacy, and toxicity profiles in animal studies.

Results
B7-H3 and EGFR are co-expressed in various tumors
To assess B7-H3 and EGFR co-expression across different cancer types, we analyzed RNA expression data of both targets from the TCGA database using linear regression. Statistically significant co-expression was observed in lung, head and neck, pancreatic, esophageal, thyroid, and kidney cancers (Fig. 1a). This co-expression pattern persisted when analyzing all cancer types collectively from TCGA, as well as in various cancer cell lines (Fig. 1b, c). Immunohistochemistry (IHC) staining confirmed the co-expression of both targets in lung squamous carcinoma, but not in normal lung or skin (Fig. 1d, e). Notably, B7-H3 was highly expressed in lung squamous carcinoma patient samples with minimal expression in normal tissues (Fig. 1d, and Supplementary Fig. 1a–c); whereas EGFR, but not B7-H3, was expressed in normal skin tissues (Fig. 1d, and Supplementary Fig. 1d). Immunofluorescent staining demonstrated co-localization of EGFR with B7-H3 on the same cancer cells (Fig. 1f). These data suggest that a B7-H3/EGFR bispecific antibody (bsAb) strategy could potentially reduce EGFR-related off-tumour toxicities and enhance efficacy in tumours co-expressing both antigens.

20G5/Zalu balances anti-tumor efficacy and safety
To enhance EGFR inhibition while minimising on-target toxicities, we assembled half-antibodies from five anti-EGFR clones with varying inhibition potencies (Supplementary Fig. 2a–d, and Supplementary Table 1) and 11 anti-B7-H3 clones differing in affinity, binding epitopes and ADCC activities (Supplementary Fig. 2e–h, Supplementary Table 2) into the “1+1” asymmetric IgG-like bsAbs using Innobody platform. These bsAbs were then extensively screened using unbiased functional bioassays across both cancerous and normal cell lines (Fig. 2a). The degree of optimization was assessed based on the efficacy and safety parameters. The efficacy was measured by the ratio of IC50 for growth inhibition between a null/EGFR bsAb control and a B7-H3/EGFR bsAb in B7-H3 positive (NCI-H358) or overexpressing (PC9-B7-H3) cancer cell lines. A B7-H3/EGFR clone demonstrating strong in vitro anti-tumor effects, indicated by a lower IC50 value, received a higher efficacy score. Safety was gauged by the ratio of IC50 for growth inhibition between a B7-H3/EGFR bsAb in A431 skin cell lines vs. PC9-B7-H3 cancer cell lines. Clones showing minimal toxicity in the skin cell line, reflected by higher IC50 values, achieved higher safety scores.

To identify the optimal EGFR binder, three distinct B7-H3 clones (20G5, 5C2, and 19A2) with varying epitopes on Ig domains, and different ADCC activities were evaluated against five EGFR binders in bsAb format (Supplementary Fig. 2i–k, and Supplementary Table 1–2). Among these, the bsAb combining anti-B7-H3 clone 20G5 and anti-EGFR clone Zalutumumab (20G5/Zalu) emerged as the most promising candidate based on efficacy and safety parameters (Fig. 2b, and Supplementary Fig. 2l), demonstrating the most potent anti-tumor efficacy (Supplementary Fig. 2m), reduced skin cytotoxicity compared to Zalutumumab (Fig. 2c, and Supplementary Fig. 2n, o) and superior ADCC activity (Supplementary Fig. 3a-d).

Mechanistically, 20G5/Zalu showed much stronger binding activity to EGFR than Zalutumumab on B7-H3-positive cancer cells as measured by both binding competition and receptor occupancy assays (Fig. 2d-e, Supplementary Fig. 3e-g). This resulted in better internalization of EGFR, and a more potent and sustained suppression of the downstream ERK phosphorylation than Zalutumumab (Fig. 2f–i, and Supplementary Fig. 3h–j), even in the presence of EGFR ligands, EGF and TNF-α (Fig. 2i, j). In addition, growth inhibition of IBI334 on SK-MES1 and PC-9 cells co-cultured with CHO-S cells overexpressing B7-H3 (B7-H3OE) (trans-inhibition) showed minimal increase in efficacy (Supplementary Fig. 3k, l), indicating that the anti-tumor effects of IBI334 are primarily cis-mediated. To further delineate the mechanisms of cis versus trans binding, we established a sparse plating model by culturing SK-MES-1 wt and SK-MES1-B7-H3OE cells at low density to minimize cell-cell contacts, thereby isolating cis-interaction effects (Fig. 2k; and Supplementary Fig. 3m). Western blot analysis showed that 20G5/Zalu exerted strong suppression of EGFR phosphorylation (pEGFR) in SK-MES-1-B7-H3OE cells, whereas only marginal inhibition was observed in SK-MES-1wt cells (Fig. 2l). Importantly, the control antibody null/Zalu (lacking the anti-B7-H3 arm) exhibited minimal pEGFR inhibition in B7-H3OE cells (Fig. 2m), demonstrating the essential role of B7-H3 targeting for effective EGFR signal blockade. Fluorescence microscopy analysis of pEGFR levels revealed stronger suppression in 20G5/Zalu-treated B7-H3OE single cells (cis-inhibition) compared to wild-type (WT) cells contacting B7-H3OE neighbors (trans-inhibition) (Fig. 2n). The untreated control groups showed comparable baseline pEGFR signals between B7-H3OE and WT cells. These data collectively suggest that 20G5/Zalu-mediated inhibition is primarily cis-mediated.

20G5/Zalu showed binding preference to the IgC1 of human B7-H3
To further elucidate the binding mechanism of 20G5/Zalu, we mapped the epitope(s) of 20G5 Fab (fragment-antigen binding) on the redundant IgC domains of human B7-H3. Sequence analysis of the IgC domain of human B7-H3 (Fig. 3a), which is a target domain for 20G5, revealed sequence differences at six and one amino acid positions from mouse B7-H3 and cynomolgus monkey B7-H3, respectively. Since 20G5 binds to human and monkey B7-H3s similarly but not to mouse B7-H3 (Supplementary Fig. 4a), the unique six amino acids in mouse likely contain the critical binding epitope. To confirm that 20G5 can bind to the two human B7H3 domains, we generated cells expressing B7H3 with 2 or 4 Ig domains (Figure S4b), and 20G5 can bind to both IgC1 and IgC2, as indicated by higher binding to 4-Ig B7H3 (Figure S4c). Structure prediction28 of human 2Ig-B7-H3 indicated that Q179, Q199, D159 and T160 are solvent-exposed residues (Supplementary Fig. 4d) that can be accessed by 20G5. We generated CHO cell lines overexpressing either wild-type or mutant 2Ig-B7-H3 containing mouse back mutations (Q179K, Q199R, or D159T+T160M). In contrast to Q199R or D159T+T160M, only Q179K mutation significantly reduced the binding activity of 20G5, indicating Q179 as the essential residue in the epitope on 2Ig-B7-H3 (IgC) (Fig. 3b). As human B7-H3 (4Ig-B7-H3) contains two redundant IgC domains (IgV1-IgC1-IgV2-IgC2), to investigate whether 20G5 binds equally to the two epitopes containing either Q179 (IgC1) or its counterpart Q397 (IgC2), we created NCI-H292 cell lines overexpressing human B7-H3 with Q179K, Q397K, or Q179K+Q397K mutations. Interestingly, Q179K single mutation only partially weakened 20G5 binding, indicating a second epitope on IgC2 which can compensate for the disruption of epitope on IgC1 (Fig. 3c, d). In contrast, Q397K single mutation had minimal impact on the binding of 20G5/null, suggesting the epitope containing Q397 on IgC2 is dispensable if 20G5 has access to the epitope on IgC1. Moreover, Q179K+Q397K double mutations synergized to nearly eliminate 20G5 binding. Consistently, the NCI-H292 cell line expressing B7-H3 Q179K+Q397K exhibited the weakest tumor growth inhibition by 20G5/Zalu compared to cells expressing B7-H3 Q179K, B7-H3 Q397K, or B7-H3 WT, while cytotoxicity was only marginally affected in B7-H3 Q397K expressing H292 cells (Fig. 3e). These results confirm that 20G5 can bind to either epitope on IgC1 and IgC2 of 4Ig-B7-H3, but with a binding preference to the Q179-containing epitope in the IgC1 domain.

Cryo-EM analysis of 20G5 (Fab’)2 and human B7-H3 complex
Human B7-H3 has four extracellular immunoglobulin domains arranged in an IgV1-IgC1-IgV2-IgC2 configuration (Supplementary Fig. 4e). The presence of a long, flexible loop between the IgC1 and IgV2 domains has made it difficult to resolve the complete structure of the B7-H3 ECD using cryo-electron microscopy (cryo-EM). Since 20G5 Fab recognizes both epitopes on the IgC1 and IgC2 domains, bivalent 20G5 (Fab’)2 binding to B7-H3 with high avidity can stabilize the 4Ig-B7-H3 ECD.

Given the possibility of different binding valences of 20G5 F(ab’)2 to 4Ig-B7-H3, the study identified two complexes: the bivalent 20G5 F(ab’)2 and human B7-H3 (4Ig-B7-H3) complex at a resolution of 3.59 Å (Supplementary Fig. 4f, g), and the monovalent 20G5 Fab and B7-H3 (IgC) complex at a resolution of 2.98 Å (Fig. 3f-g, Supplementary Fig. 4f-i). Unfortunately, the density of (Fab’)2 and the B7-H3 complex is insufficient for further interpretation.

The IgC domain of B7-H3 is characterized by a typical β-sandwich structure formed by ABED and CFG sheets. The 20G5 Fab engages all three CDRs to bind the IgC domain, primarily interacting with the CFG β-sheets through electrostatic interactions, hydrogen bonds, and hydrophobic interactions (Fig. 3g). The epitope residues from the CD loop (Q179-P185) are deeply embedded between the VH and VL domains of 20G5 Fab, where they establish hydrogen bonds and hydrophobic interactions with the side chains of HCDR1-3 and LCDR3. Notably, F177 aligns with aromatic residues W103 (HCDR3) and Y31 (LCDR1), while R223 engages in an electrostatic interaction with D49 in LCDR2. This structural insight prompted us to engineer single-point mutations in 2Ig-B7-H3 (Fig. 3h), pinpointing F177, Q179, D180, Q182, V184, P185, and R223 as crucial residues for the binding of 20G5 Fab. These interactions between the epitope and antigen are key to the high specificity of 20G5 Fab for the IgC domain of B7-H3.

Low fucosylation of 20G5/Zalu enhances ADCC effects
For therapeutic enhancement, the fragment crystallizable (Fc) region of the 20G5/Zalu was afucosylated using GlymaxX® technology, forming the lead molecule IBI334. This modification enhances its affinity to human RcγRIIIa (CD16A) and antibody-dependent cellular cytotoxicity (ADCC) effects, making IBI334 more effective than IBI334-IgG1, which has a standard IgG1 Fc (as shown in Supplementary Fig. 5a and Supplementary Table 4). Both versions of the molecule inhibit EGFR-mediated growth similarly, which is expected since the modification affects only the Fc region (Supplementary Fig. 5b). In an in vivo non-small cell lung cancer (NSCLC) model using NCI-H1975 cells (with EGFR L858R and T790M mutations) implanted in nude mice, IBI334 showed limited anti-tumor efficacy (Supplementary Fig. 5c). However, when these cells were implanted in NOG mice reconstituted with human NK cells, IBI334 demonstrated significant efficacy, highlighting its potent ADCC effects in vivo (Supplementary Fig. 5d).

IBI334 showed potent in vitro and in vivo anti-tumor efficacy across various cancer types
IBI334 showed potent in vitro efficacy against various cancer types, including colorectal cancer, non-small cell lung cancer, head and neck squamous cell carcinoma and pancreatic cancer, which varied in their mutational profiles and levels of B7-H3 and EGFR expression (Fig. 4a). In SK-MES-1 xenograft model, IBI334 showed better efficacy than Zalutumumab, 20G5/null, null/Zalu and combination of 20G5/null and null/Zalu (Fig. 4b, Supplementary Fig. 6a, b). In an in vivo PD study using the NCI-H358 xenograft model, Western blot analysis revealed that IBI334 significantly suppressed downstream EGFR signaling pathways compared to Zalutumumab, while immunohistochemistry results indicated a reduction in ERK phosphorylation and Ki67 marker (Fig. 4c, d,). Subsequently, IBI334 was compared with FDA-approved Amivantamab, a c-MET/EGFR bispecific antibody, and Cetuximab, an EGFR monoclonal antibody. IBI334 exhibited superior in vivo efficacy compared to both Amivantamab and Cetuximab in the NCI-H292-EGFR WT model and the NCI-H322-EGFR SVD model, which ectopically expressed an EGFR S786-D770 duplication, respectively (Fig. 4e-f, Supplementary Fig. 6c).

It was previously reported that EGFR mutated NSCLC patients treated with Osimertinib usually developed resistance due to the additional EGFR C797S mutation. Therefore, we generated SK-MES-1 LTC cell line harboring L858R, T790M and C797S (LTC) mutations, named as LTC (Supplementary Fig. 6d, e), and confirmed that Osimertinib was ineffective in this model. IBI334, in contrast, induced dose-dependent tumor regressions as monotherapy with 3/6 and 5/6 animals achieving complete responses at low and high doses, respectively, highlighting its potential in treating Osimertinib-resistant patients (Fig. 4g, and Supplementary Fig. 6f). In addition, IBI334 showed milder effect than Zalutumumab on A431 human xenograft model, a commonly used model to assess skin toxicity in vivo29, suggesting a potentially improved safety profile (Fig. 4h, and Supplementary Fig. 6g). Collectively, IBI334 displayed robust in vivo efficacy across various cancer models (Fig. 4i, and Supplementary Fig. 6h–k).

IBI334 enhanced Osimertinib activity and synergized with KRAS inhibitors
Combination therapy is often employed to enhance therapeutic effectiveness, and synergistic anti-tumor efficacy, rather than combinatorial effect, is highly desirable. We developed the NCI-H1975 LTC cell line, which harbors the Osimertinib-resistant EGFR LTC mutation, and found that IBI334 exhibited strong synergy with Osimertinib, achieving a synergy score of 21.74 (Fig. 5a–c). This combination was more effective at suppressing AKT phosphorylation than either drug alone (Fig. 5b). Consistent with in vitro results, the combination of IBI334 and Osimertinib demonstrated enhanced anti-tumor efficacy compared to individual treatments in the NCI-1975 LTC in vivo model as indicated by the number of animals achieving partial response (Fig. 5d, and Supplementary Fig. 7a). IHC staining of the extracted tumors showed reduced levels of pEGFR and the Ki67 marker (Fig. 5e).

Preclinical and early clinical studies have indicated synergistic effects between EGFR inhibitors and KRAS inhibitors30–33. This prompted us to investigate whether IBI334 could synergize with KRAS inhibitors. Indeed, IBI334 displayed strong synergy with AMG510, a KRAS G12C inhibitor, in NCI-H358 cells harboring the KRAS G12C mutation, both in vitro and in vivo (Fig. 5f–h, Supplementary Fig. 7b). The drug combination group exhibited more pronounced suppression of AKT and ERK phosphorylation than the individual drugs (Fig. 5g). Synergy was also observed with MRTX1133, a KRAS G12D inhibitor, and LY3009120, a pan-RAF inhibitor (Supplementary Fig. 7c–e).

IBI334 showed good pharmacokinetics and safety profiles in cynomolgus monkey
The pharmacokinetics (PK) of IBI334 was assessed in cynomolgus monkeys following a single intravenous administration at doses of 3, 10, or 30 mg/kg (Fig. 6a, Supplementary Table 5). Within this dose range, IBI334 exhibited a non-linear PK with slightly more than dose-proportional increase of drug exposure (mean AUC values were 3430±686, 17100±1840, and 66400±9810 h*µg/mL for 3, 10 and 30 mg/kg, respectively), and higher clearance at low dose, possibly due to target-mediated drug disposition (TMDD) effect34. The average terminal half-life (T1/2) of IBI334 was ~3 days, which support weekly or longer dosing interval. No significant differences in PK parameters were noted between male and female subjects within each dose group (Supplementary Table 5), providing valuable insights for establishing clinical dosing strategies.

To assess the toxicokinetics (TK) and safety profile of IBI334, cynomolgus monkeys of both sexes received intravenous doses of 30, 60, and 120 mg/kg of IBI334 weekly for a total of five doses, adhering to good laboratory practice (GLP) conditions (Fig. 6b). The mean serum exposure to IBI334 increased proportionally with the dose across the range from 30 to 120 mg/kg. After the first and fourth doses, there were no significant differences in serum IBI334 exposure between sexes, and no significant accumulation (accumulation factor < 1.56±0.29) was detected (Fig. 6c, Supplementary Table 6).

Throughout the dosing period, there were no deaths, moribund states, or other significant toxic symptoms observed in any of the monkey groups. At 2 hours post-first dose, serum IL-6 increase was noted in most monkeys that received IBI334, with highest average increase observed at 60 mg/kg group (7.2~242.9 pg/mL), but the increase was transient and returned to normal 24h after dosing (Fig. 6d). There were no significant changes in ALT and AST levels (Fig. 6e). Other blood biochemical parameters remained within normal ranges across all groups. Histopathological evaluations revealed mild increase in sinusoidal cells in the liver, moderate perivascular inflammation at the injection site, minimal to mild interstitial mononuclear cell infiltration in the kidneys, and minimal to mild mucosal mononuclear cell infiltration in the stomach for all IBI334-treated groups (Fig. 6f, Supplementary Table 7). Overall, IBI334 demonstrated a favorable TK and safety profile.

Methods
Study approval
This study complies with all relevant ethical regulations. All in vivo studies involving mice were performed according to Regulations for Care and Use of Laboratory Animals at Innovent Biologics and were approved by Innovent’s Institutional Animal Care and Use Committee (IACUC). Cynomolgus monkey pharmacokinetics (PK) study and GLP toxicity study were approved by the Institutional Animal Care and Use Committee of West China-Frontier PharmaTech Co., Ltd. (Approval Number: A2022147-T014-01) and were performed in strict accordance with the guidelines for the ethical use and care of animals established by the Chinese Council on Animal Care.

Cell lines and reagents
Cancer cell lines were sourced from various institutions: YBio (PC9), Cobioer (TE-1, SK-MES-1, NCI-H322, NCI-H1650, CCK-81, HT-55, NCI-H508, LS180, COLO680, NCI-H358, HCT116, COLO205, NCI-H2122, NCI-H1993, SW-48, AsPC-1, NCI-H820, SCC-9), Cell lines service (HaCat), Cell Bank/Stem Cell Bank of Chinese Academy of Sciences (NCI-H292, SW480), and the American Type Culture Collection (ATCC; A431, NCI-H1975, NCI-H1703, NCI-H2030, NCI-H520, SW-1573, NCI-H441, Calu-6, HCC4006, NCI-H1792, FaDu, LoVo), Meisen CTCC (HCC2279). These cell lines were maintained under manufacturers’ recommended conditions in growth media supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, SH30406.05) and Penicillin-Streptomycin antibiotics (100 U/ml penicillin and 100 ug/ml streptomycin) (Gibco, 15140-122) at 37 oC in a 5% CO2 humidified incubator.

Genetically modified stable cell lines, including PC9-B7-H3, SK-MES-1-B7-H3, SK-MES-1-B7-H3-GFP, NCI-H322SVD (EGFR exon 20 ins-S768_D770dup), SK-MES-1LTC (EGFR-L858R/T790M/C797S), NCI-H1975LTC, and NCI-H292-B7-H3 mutants, were generated using the LV-MAX™ Lentiviral Production System (Gibco, A35684CN). Following lentiviral transduction, these cells were selected for 2 weeks in media containing 1 μg/ml puromycin (Invitrogen, A11138-02). Human peripheral blood mononuclear cells (PBMCs) from healthy donors were procured from TPCS and All Cells. The small molecule inhibitors employed in this study included Osimertinib (MCE, HY-15772), AMG510 (MCE, HY-114277), MRTX133 (MCE, HY-134813), and LY3009120 (Macklin, L873098).

Protein expression and production
The sequences of the variable regions from the heavy and light chains of Panitumumab (CAS NO., 339177-26-3), Cetuximab (CAS NO., 205923-56-4), Necitumumab (CAS NO., 906805-06-9), Zalutumumab (Patent, WO2002100348A), Nimotuzumab (CAS NO., 828933-61-3), MGA271 (CAS NO., 1353485-38-7), M30 (INN, PL126), 8H9 (CAS NO., 1895083-75-6) and internally generated B7-H3 monoclonal antibodies (5C2, 20G5, 38G9, 19A2, 6C2, 1C5, 20E4, 7B6; PCT patent CN2021/140449) were cloned into the pcDNA3.1 vector (Invitrogen, V790-20). This vector includes the Fc region of wild-type human IgG1 for monoclonal antibodies or the Fc region with S364R+D399K or K370S+K409D+Y349T substitutions from the Innobody platform (WO Patent 2022143912A1) for bispecific antibodies. The plasmids were synthesized by Genewiz, and the antibodies were produced using the ExpiCHO Expression System (Invitrogen, A29133). GlymaxX technology is used to produce afucosylated antibodies44. Briefly, antibodies were expressed in Chinese hamster ovary (CHO) cells overexpressing GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, and the resulting antibodies are afucosylated.

Antibodies
For the FACS internalization assay, AF647-anti-human EGFR antibody (BD, 563577) was used. For IHC, multiplexed immunofluorescence (mIF), and western blotting (WB), the following antibodies were utilized: EGFR (CST, 4267), pEGFR (CST, 3777), B7-H3 (CST, 14508), AKT (CST, 4691), pAKT (CST, 4060), ERK (CST, 4695), pERK (CST, 4370), S6 (CST, 2217S), pS6 (CST, 2211S), GAPDH (CST, 2218), PanCK (GeneTech, GM351514), and Ki67 (CST, 9027S).

Affinity and epitope binning measurement
The binding affinity and kinetics were assessed using biolayer interferometry (BLI) on a Fortebio Octet RED96. The monoclonal antibodies for B7-H3, EGFR, and the bispecific antibody for B7-H3/EGFR were immobilized on AH sensors (Sartorius, 18-5060) at a concentration of 100 nM for 100 seconds. The analytes, 2Ig-B7-H3 (Sino Biological, 29849-H08H), 4Ig-B7-H3 (Sino Biological, 11188-H08H), and EGFR (Acro Biosystems, EGR-H5222) were prepared at 100 nM, associated with the antibody for 100 seconds, and then dissociated in buffer for 120 seconds. The affinity data was processed using Fortebio data analysis software (Version 10.0), employing a 1:1 binding model.

Epitope binning for the B7-H3 mAbs were conducted on the Octet Red96 using an in-tandem format. B7-H3 (Sino Biological, 29849-H08H) was immobilized onto HIS1K biosensors (Sartorius, 18-5120) at an approximate density of 0.5 nm. The biosensors were first saturated with the primary antibody (20G5), followed by the introduction of a secondary B7-H3 mAb. The detection of a significant binding signal from the secondary antibody indicated that its binding epitope is distinct from that of 20G5.

Cell growth inhibition assay
Growth inhibition assays were performed by seeding 1000–2000 cells per well into 96-well plates in 2D (Nest, 701001) or 3D (Corning, CLS7007-24EA) culture system. On the same day, the cells were treated with either proteins or small molecule inhibitors. After a 5-day treatment period, cell viability was assessed using the Cell Titer-Glo assay (Promega, G7572) as per the manufacturer’s instructions. The percentage of surviving cells was calculated using the formula: % of surviving cells = (Luminescence_test/Luminescence_blank) x 100%, where blank is IgG1 control.

Drug synergy between IBI334 and small-molecule inhibitors was evaluated using SynergyFinder+ algorithm according to the instruction45. Briefly, cell inhibition was quantified using the Cell Titer-Glo assay and experimental data were processed through a web-based SynergyFinder+ algorithm (http://www.synergyfinderplus.org) to obtain the synergy score. Synergy scores were interpreted as follow: <−10: Antagonistic interaction; −10 to 10: Additive interaction; >10: Synergistic interaction.

Cytotoxicity ADCC assay
For the ADCC reporter assay, Jurkat T cells engineered to express FcγRIIIa (reporter cells) were obtained from Promega (Catalog No. G7102). 1.5×105 reporter cells and 1.5 ×104 tumor cells were co-cultured in 96-well culture plates using RPMI1640 Medium (Gibco, Catalog No. 22400-071) supplemented with 4% low IgG FBS (Invitrogen, Catalog No. 1921005PJ). The co-culture was incubated for 20 h at 37°C in a 5% CO2 atmosphere. Subsequently, the signals were quantified using the Bio-Glo Luciferase Assay System (Promega, Catalog No. G7940) and measured with the Spectra Max I3 (Molecular Devices) according to the manufacturer’s instructions. The ADCC effect was calculated using the formula: Fold change in relative to IgG = (Luminescence_test/Luminescence_blank), where blank is IgG1 control.

For the ADCC assay involving human PBMCs (huPBMCs), 7.5×105 huPBMCs and 1.5×104 tumor cells were co-cultured in 96-well culture plates using CTS media (Gibco, Catalog No. 0870112DK). This setup was incubated for 6–8 h at 37°C in a 5% CO2 atmosphere. Lactate dehydrogenase (LDH) levels in the co-culture supernatants were quantified using the LDH kit (Roche, Catalog No. 4744926001). The percentage of cytotoxicity (LDH) was calculated following the guidelines provided in the manufacturer’s protocol. Cytotoxicity was quantified by measuring LDH release using the formula: % cytotoxicity = [(OD490_ test − OD490_ blank) / (OD490_ max tumor − OD490_ blank tumor)] × 100, in which “blank” represents PBMC and tumor cells co-culture with IgG1 control, “max tumor” is the tumor cells completely lysed and “blank tumor” is the tumor cell with IgG1 treatment.

FACS internalization assay
NCI-H358 cells were seeded in 6-well plates and allowed to adhere overnight. The following day, the cells were treated with PBS, null/Zalu, Zalutumumab, and 20G5/Zalu bsAb, and incubated at 37°C with 5% CO2 for various durations. Cells were harvested at specific time points (4h, 24h, 48h, and 72h) and stained with AF647-anti-human EGFR antibody (BD, 563577). The mean fluorescence intensity (MFI) of EGFR on the cell surface was measured using a FACSymphony™ A3 flow cytometer. The percentage of EGFR internalization (% EGFR internalization) was calculated using the following formula: EGFR internalization % = (1 - EGFR_test/EGFR_control) × 100%, where EGFR_control represents the mean fluorescence intensity (MFI) of NCI-H358 cells stained with AF647-anti-human antibodies following treatment with PBS alone.

EGFR occupancy assay
Zalutumumab was labeled using the AF647 antibody labeling kit (Invitrogen, A20186) to prepare AF647-labeled Zalutumumab (AF647-Zalu). Tumor cells were treated with either Zalutumumab or the 20G5/Zalu at 4°C for 1 h, washed with FACS buffer (PBS containing 2% FBS), and followed by an additional 1-h incubation with AF647-Zalu. Subsequently, the cells were washed with FACS buffer (PBS containing 2% FBS) and analyzed using the FACSymphony™ A3 (BD). Cells that were not treated and stained solely with the AF647-Zalu antibody served as the negative control (representing 0% EGFR occupancy and 100% AF647-Zalu staining). Receptor occupancy (RO%) is calculated using the formula: RO% = 100% - X, where 5 represents the percentage of cells gated within the population showing an AF647 signal below that of the negative control.

Western blotting
Cells were lysed using RIPA buffer (Thermo, 89900) supplemented with both phosphatase inhibitors (Abcam, ab201112) and a protease inhibitor (Roche, 11836170001). Following centrifugation at 13,523 xg for 15 minutes at 4°C, the supernatants were collected, and protein concentrations were determined using the BCA Protein Assay (Beyotime, P0012). Equal amounts of protein were resolved by SDS-PAGE (Thermo, NP0321Box) and subsequently transferred onto polyvinylidene difluoride membranes using the Invitrogen Trans-Blot system (Invitrogen, IBCOT2). Immunoblotting was conducted using primary antibodies and HRP-conjugated secondary antibodies. The membranes were then treated with Western HRP Substrate (Beyotime, P0018AM) and the signals were visualized with the Chemi-Doc MP Imaging System (BIO-RAD).

Fluorescence microscopy images of phosphorylated EGFR
SK-MES-1 cells (25,000 cells) and SK-MES-1-B7-H3OE-GFP cells (25,000 cells) were co-seeded onto chamber slides (Beyotime, FSL108) and cultured overnight. Cells were then treated with 20G5/Zalu bsAb or untreatment for 4 hours. Co-cultured cells were fixed and permeabilized according to the manufacturer’s protocol (Invitrogen, 00-5523-00). Phosphorylation of EGFR was detected using a phospho-EGF receptor antibody (CST, 3777S) followed by an anti-rabbit IgG secondary antibody (Biolegend, 406414). Nuclei were counterstained with DAPI before mounting with anti-fade mounting medium. Images were acquired using an EVOS M7000 high-resolution fluorescence imaging system (Thermo Fisher Scientific).

Cryo-EM complex and structure determination
Preparation of the 4Ig-B7-H3-20G5 F(ab’)2 complex. The 4Ig-B7-H3 and 20G5 F(ab’)2 proteins were mixed in a molar ratio of 1:1.2 and incubated at 4°C overnight. Subsequently, the complex underwent further purification via size exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare) with a running buffer composed of 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The fractions containing the complex were collected and concentrated to a final concentration of 0.92 mg/mL for cryo-EM sample preparation.

Cryo-EM sample preparation and data acquisition. A droplet of the protein sample was placed onto a glow-discharged Cu holey carbon film grid (Quantifoil), treated for 60 seconds at 15 mA current using a PELCO easiGlow™ Glow Discharge system. The grid was blotted for 4.0 seconds with a blot force of 0 at 4°C and 100% humidity, then plunge-frozen in liquid ethane using a Vitrobot Mark IV. Cryo-EM datasets were acquired using a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific, USA) equipped with a Falcon IV direct electron detector (Thermo Fisher Scientific, USA) in counting mode. Movies were automatically recorded using EPU software at a magnification of 165K, yielding a physical pixel size of 0.743 Å and a total dose of 55 e-/Å^2. A total of 5017 movies were collected with a defocus range from −1.0 μm to −2.4 μm, and the slit width of the Selectris X energy filter (Thermo Fisher Scientific, USA) was set at 10 eV.

Image Processing. Image processing was conducted using cryoSPARC v4.4.0, unless specified otherwise. The 5,017 dose-fractionated movies underwent patch motion correction using cryoSPARC’s native tools. Patch CTF estimation, 2D classification, heterogeneous refinement, and homogeneous refinements were also performed in cryoSPARC. A total of 1,520,193 particles were automatically picked using the blob picker and extracted with 2×2 binning (220-pixel box size, 1.486 Å per pixel). After two rounds of 2D classification to select high-quality particles, the remaining 538,193 particles underwent ab-initio reconstruction. These selected particles were then subjected to heterogeneous refinement across five volumes. The refined coordinates facilitated re-centering and re-extraction of unbinned particles (0.743 Å per pixel, 440-pixel box size). Particles of the B7-H3-20G5 Fab complex underwent further homogeneous refinements, resulting in a final density map at a resolution of 2.98 Å. Concurrently, particles of the 4Ig-B7-H3-20G5 F(ab’)2 complex were refined homogeneously, achieving a final density map at a resolution of 3.59 Å.

Immunohistochemistry and multiplex immunohistochemical staining
Tissue microarrays from both LUSC and normal tissues, as well as paraffin sections from xenograft models, were deparaffinized and stained using the Ventana Discovery Ultra automated slide stainer (Roche). Following the initial deparaffinization, heat-induced epitope retrieval (HIER) was conducted in DISCOVERY CC1 (Roche, 950-500) at 100°C for 40 minutes. This was succeeded by multiple rounds of staining, which included antigen retrieval, blocking, application of the primary antibody, HRP-coupled secondary antibody, and hematoxylin.

For multiplex immunohistochemical staining, immunofluorescent signals were captured using the DISCOVERY FAM Kit, DISCOVERY Cy5 Kit, DISCOVERY Rhodamine 6G Kit, and QD DAPI (Roche). Imaging was performed on the Leica Aperio Versa 8 Automated Imaging System, and the images were analyzed using the Aperio ImageScope Software Versa 12.3.3 (Leica Biosystems).

In vivo xenograft model efficacy
For in vivo efficacy and safety evaluations, female Balb/C nude mice and female NOG mice (PF grade, 16–20 g) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed under SPF conditions with a controlled temperature of 22±2°C, relative humidity of 50±5%, and a 12-h light/12-h dark cycle for efficacy and safety evaluations. Various cancer cell lines including SK-MES-1 (4 × 10^6 cells/mouse), NCI-H292 (4 × 10^6 cells/mouse), NCI-H1975 (5 × 10^6 cells/mouse), SK-MES-1LTC (3 × 10^6 cells/mouse), NCI-H508 (5 × 10^6 cells/mouse), NCI-H358 (5 × 10^6 cells/mouse), and NCI-H322SVD (5 × 10^6 cells/mouse) were implanted into female Balb/C nude mice or NOG mice to evaluate anti-tumor efficacy. The safety of Zalutumumab and IBI334 was assessed in the A431 model by implanting A431 cells (3 × 10^6 cells/mouse) into female Balb/C nude mice. Furthermore, the combined antitumor activity of IBI334 and small-molecule inhibitors was tested in vivo using NCI-H1975LTC (5 × 10^6 cells/mouse) and NCI-H358 (4 × 10^6 cells/mouse) implanted in either female Balb/C nude mice or NOG mice to evaluate the efficacy of IBI334 and small inhibitors combination therapy.

Tumor-bearing mice were monitored, and when tumor volumes reached ~120–220 mm3, the animals were randomly assigned to treatment groups to receive either IBI334 or benchmark. For all efficacy studies, body weight and tumor dimensions (length and width) were measured bi-weekly. Tumor volumes were calculated using the formula: (width)2 × length/2. Mice were humanely euthanized when tumor volumes exceeded 2000 mm3 or when body weight loss surpassed 20%.

Cynomolgus monkey pharmacokinetics (PK) study
Cynomolgus monkey were obtained from Guangxi Xiongsen Primate Laboratory Animal Breeding Development Co., Ltd., which is certified with a production license (No. SCXK(Gui) 2021-0004) and a quality certificate (No. 0002997). West China- Cynomolgus monkey were housed under animal house with a controlled temperature of 22±2°C, relative humidity of 60±20%, and a 12-h light/12-h dark cycle for PK and GLP toxicity study.

For the experimental procedure of PK study, the eighteen cynomolgus monkeys were randomly assigned into three groups, each consisting of six monkeys, balanced for gender. Each group received a single intravenous injection of IBI334 at dosages of 3, 10, and 30 mg/kg, respectively. Blood samples were collected at specified time points before and after the treatment of IBI334, specifically at 1, 2, 4, 8, 24, 72, 120, 168, 240, 336, 504, 672, 840, and 1008 hours post-treatment.

The serum concentration of IBI334 was quantified using an ELISA method. Human EGFR Protein (Acro, EGR-H522a) served as the coating antigen, samples containing IBI334 were placed on the ELISA plate and incubated. The detection involved a primary antibody specific to 20G5 of IBI334 (prepared in house), which bound to the antigen-antibody complexes on the plate. This was followed by the addition of a secondary antibody (Jackson Immuno Research, AB_10015289). After washing away unbound secondary antibodies, a substrate solution was added. Detection was performed using the Varioskan LUX (Thermo), and pharmacokinetic parameters were subsequently analyzed using Phoenix WinNonlin V8.2 software.

Cynomolgus monkey GLP toxicity study
In this study, forty cynomolgus monkeys were recruited and divided into four groups: a control group and three treatment groups receiving IBI334 at dosages of 30, 60, and 120 mg/kg, respectively. Each group comprised ten monkeys with an equal distribution of genders. The treatment of intravenous injections occurred weekly over a four-week period, totaling five doses, followed by a four-week recovery phase. The onset of dosing was defined as Day 1, and the commencement of the recovery phase was the day following the final dose.

The study encompassed various assessments including clinical observations, hematology, blood chemistry, cytokine profiling, immune response analysis, histopathology, and toxicokinetic (TK) analysis. Throughout the dosing and recovery phases, the general condition of the monkeys was monitored daily, with weekly measurements of body weight and food consumption. Urinalysis and ophthalmic examinations were conducted at the end of the dosing and recovery periods. Neurological assessments were conducted, electrocardiograms (ECGs) were obtained, and respiratory rate and blood pressure were measured at several time points: prior to treatment, and at 2, 24, 72, 120, and 168 h post-initial treatment, one day post the third and fifth treatments, and at the end of the recovery phase. Hematology using ADVIA 2120i Hematology System (Siemens), blood biochemistry using Cobas 8000 Modular Analyzer (Roche), and lymphocyte subsets using LSR Fortessa™ flow cytometer (BD) were analyzed one day after the third and fifth treatment and at the end of the recovery phase. Cytokine levels (IL-2, IL-6, IL-10, TNF-α, IFN-γ) were measured at 2 and 24 h following the initial treatment, 2 h post the fifth treatment, and at the end of the recovery phase.

Blood samples were drawn from the IBI334 treatment group at prior to treatment, the first and fourth treatments, and at 1 minute, 2, 8, 24, 48, 72, 96, and 168 h post IBI334 treatment to measure IBI334 concentrations and analyze toxicokinetic parameters. Following the dosing period, six monkeys from each group were selected for histopathology analysis; After the recovery phase, four more were chosen from each group for euthanasia and subsequent post-mortem and histopathological analysis.

Statistical and reproducibility
GraphPad Prism 10.0 software was utilized for graph generation and statistical analysis. The results from in vitro studies, pharmacokinetics (PK) study, and GLP toxicity study were presented as mean ± SD, while the in vivo xenograft efficacy results were expressed as mean ± SEM. Linear regression module was used for correlation analysis. Statistical significance was assessed using one-way or two-way ANOVA with Tukey’s post-hoc tests for continuous variables. P values ≥ 0.05 is denoted as non-significance. All immunoblots and core data shown are at least two independent experiments. For in vivo efficacy and monkey studies, animals were randomly assigned to different groups. The investigators who performed the experiments were blinded to the group assignments during data collection and/or analysis to reduce bias.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Given that current therapeutic targeting epidermal growth factor receptor (EGFR) in solid tumors have on-target/off-tumor toxicities, the authors functional screened and developed a leading bispecific antibodie IBI334 targeting both EGFR and B7-H3. IBI334 showed better tumor selectivity in tissues and tumors. In preclinical models, IBI334 outperformed other EGFR antibodies in occupancy, ligand-blocking efficacy, and suppression of downstream EGFR signaling. IBI334 also demonstrated robust antitumor activity in vitro and in vivo and synergized effectively with osimertinib and KRAS inhibitors. The authors further solved cryo-EM structure of B7-H3 extracellular domain in complex with anti-B7-H3 (Fab’). Finally, the authors revealed a favorable safety profile of IBI334i n non-human primates.\nOverall, they have performed a substantial range of experiments to supporting the notion that IBI334 outperformed conventional EGFR antibodies in both therapeutic efficacy and safety profile in preclinical studies. Importantly, IBI334 has advanced to a phase 1 clinical trial for advanced/metastatic solid tumors. The study has provided data that suggests more work in this area is warranted. I have requested for a revision. The data shown in this manuscript is compelling, but not yet definitive, which has led to some data over-interpretation.\nMajor questions and concerns are outlined below.\n1) Zalutumumab was originally developed by Denmab. Clinical trials of Zalutumumab yielded mixed results, so Denmab decided to discontinue the development of Zalutumumab. Whereas authors selected Zalutumumab for their bsAb 20G5/Zalu studies and clinical trial. Given the status of Zalutumumab, the authors should further justify their selection on using Zalutumumab as the mAb for their 20G5/Zalu. In addition, how about the patent status of Zalutumumab?\n2) in the Abstract and the Discussion, the authors stated “accelerated receptor degradation”, “Additionally, IBI334 prompts rapid and enduring degradation of EGFR in both cancer cells and xenograft tumors, likely initiated by antibody-induced receptor endocytosis.”, etc. However, it seemed the authors did not provide real experiment data to support this statement.\n3) Fig 1D, the quality of EGFR IHC staining at ++ and + is low.\n4) In Fig S2.j, high concentrations of 19A2/Nimo increased the survival of PC9-B7H3 tumor line, so did 19A2/Nimo induce EGFR signaling rather than inhibition? Please explain the result.", "reviewer_2": "The authors decsribe development of a bispecific antibody targeting EGFR and B7-H3, together with a series of studies of its efficacy in various cancer cell lines/models and in tumor xenograft studies. Cryo-EM structures of the B7-H3 targeting FAB with B7-H3 are also reported.\nWhile there is a tremendous amount of data here, and studies are appear to be carefully executed, I find the work exceptionally difficult to follow and interpret. In particular, the wide variety of cell lines studied, seemingly both with and without exogenous over-expression of B7-H3 or various oncogenic variants of EGFR, add to the confusion. THis is exacerbated by the lack of explanation for the underlying rationale of the choice of cell lines or an explanation of their genotype with respect to EGFR.\nA few specific comments:\n1. I do not find the correlation/co-expression analysis in Fig. 1 compelling.\n2. Along the same lines, in FIg. 4A it is not clear that there is any correlation of response to co-expression of the two target antigens.\n3. In the in vivo efficacy studies in Fig. 4 and elsewhere, why no control with the B7-H3 targeting antibody alone? or Two separate antibodies targeting EGFR an B7-H3 as compared with the bi-specific?\n4. Difficult to understand how these mice were dosed. For example, legend states treated with 4 doses of the specified antibodies at 10 mg/kg twice per week. Does this mean that there was an initial two week treatment period, followed by continued tumor measurement but w/o treatment? If so would be good to indicate dosing on the plots.\n5.With respect to the cryo-EM structures: The map and model for the single FAB structure (as illustrated in fig. 3h) looks fine and is of a reasonable resolution for the conclusions drawn.Very nice work.\n6. But there are issues with the structure with 2 FABs bound to B7-H3:\na. the density for the light chain of one FAB and the heavy chain of the other appear to be rather weak. This must be an imaging artifact of some sort...I do not have a good explanation but it does not make physical sense.\nb. While it is possible that the two FABs are bound the the duplicated domains of one B7-H3, it is not clear that this is the case - also possible that particles are self-associating via the FAB leading to this reconstruction.\nc. In any case, the PDB file for the 2-FAB structure models it as two copies of the IgC2 and IgV2 domains, each with a FAB bound, not as one FAB bound to IgC2-IgV2 and one to IgC1-IgV1.\nd. In fact the 2 version of the structure in the 2-FAB complex and the version in the 1-FAB complex are all almost identical (the antibody portions appear to be precisely the same, there are some very minor differences in the B7-H3 portion of the 1-FAB structure. Given that the 2-FAB structure has problems and that it does not add anything to the work, I would simply remove it. The interesting part is recognition of the epitope on B7-H3 by the FAb, and that is best described with the 1-FAB structure.", "reviewer_3": "In this study, the authors present the development of a novel bispecific antibody, IBI334, targeting EGFR-associated tumors. This antibody combines binding epitopes for EGFR and B7-H3 — a single-pass transmembrane protein that is upregulated in various tumors, including lung cancer, but shows minimal expression in normal tissue, making it a promising target for selective cancer therapy and several examples targeting B7-H3 are currently under clinical investigation. The clinical relevance of the presented study for EGFR-related cancer types makes it particularly compelling and should justify publication in NC. The design rationale and methodology are thoroughly described and clearly presented. The anti-tumor efficacy (in-vitro and in-vivo) and toxicity profiles observed in animal models are promising and have prompted the authors to advance to an ongoing phase I clinical trial.\nA few things should be considered:\nCould the authors elucidate the calculated synergy score from (Synergy Finder+) and discuss the presented values and meaningfulness?\nFigure 5b: combination of Osimertinib (50 nM) and IBI334. What might be the reason why only pAKT is suppressed by the combination (and not ERK)? And is the conc. of Osi high enough for the observed effect? 5a suggests an EC50 of OSI around 1 micromolar at LRTMCS (appears consistent with other literature). How should this be understood in context with the differences to the H358 xenograft model in Fig 4c?\nTable S6 – by nature of potential variability in pk studies. Are all Tmax values correctly stated?\nThe selectivity of the described bsAb is exceptional and raises interest in the approach. Since EGFR-driven cancers are often associated with prominent and relatively quickly appearing resistance/escape pathways – did the authors conduct experiments to assess such potential outcomes for their bsAb (e.g., downregulation of B7-H3)? This should help to predict the long-term potential of their approach.", "reviewer_4": "This manuscript by Jian Guan and colleagues describes the development of a novel bispecific antibody (bsAb), IBI334, targeting B7-H3 and EGFR, with the goal of achieving enhanced anti-tumor efficacy while minimizing on-target toxicity. The authors propose that the therapeutic benefit of IBI334 is based on its higher affinity for B7-H3 relative to EGFR, leading to preferential binding to tumor cells and reduced systemic toxicity. While the concept is promising and the generation of a functional bsAb with dual specificity is noteworthy, the manuscript suffers from several conceptual and methodological weaknesses that must be addressed to support the authors’ claims.\nMajor Comments\n1. Lack of Evidence for Sequential Cis-Binding Mechanism\nThe central mechanistic claim of the manuscript is that IBI334 first binds B7-H3 and subsequently engages EGFR on the same cell surface via a cis-acting mechanism. However, this hypothesis is not directly supported by the experimental data. The only related evidence (Figure S3h) is indirect and lacks sufficient clarity. Without robust biochemical or biophysical data demonstrating this sequential binding, this mechanistic assertion remains speculative.\n2. Discrepancies in Binding Affinity Reporting\nThe manuscript reports that converting Zalutumumab to a monovalent format increases EGFR affinity from a KD of 3.89 × 10⁻⁹ M to 4.69 × 10⁻¹⁰ M (Table S3). However, Table S3 contains an apparent error, listing the bsAb KD as 4.69 × 10⁻⁹ , As the KD is calculated from observed Kon and Kdis values. This inconsistency must be corrected and clarified.\n3. Safety Claims Based on Incomplete Context\nThe bsAb is reported to be safe in mouse models while demonstrating strong efficacy in tumor volume reduction thereby suggesting a broadended therapeutic window. However, this safety profile is likely attributable to its lack of cross-reactivity with mouse EGFR and B7-H3, as noted in Table S7 and the EMA report for Rybrevant. Therefore, murine studies cannot reliably inform on-target toxicity, and this critical limitation should be explicitly acknowledged.\n4. Inconsistent Claims Regarding Receptor Degradation\nThe authors state that IBI334 induces accelerated EGFR degradation; however, Figures 2g and 2h show no significant change in EGFR levels over five days. These contradictory data undermine the claim and require resolution.\nMinor Comments\n1. Lack of Methodological Detail for Antibody Generation\nThe bsAb was reportedly generated using the Innobody platform as a 1+1 asymmetric IgG-like format. However, no information or references are provided regarding the Fc heterodimer design or bsAb assembly. Given that ADCC is proposed as a mechanism of action, Fc structure and function must be described to allow reproducibility and proper interpretation.\n2. Redundancy in Antibody Selection Description (Lines 94–128)\nThe manuscript devotes excessive space to describing antibodies that were ultimately discarded. This content could be moved to the supplementary materials or summarized briefly.\n3. Inadequate Figure Legends and Method Descriptions\nSeveral figures, particularly in the supplementary section, lack sufficient explanation. For example, in Figure S3h, it is unclear whether the X-axis refers to labeled Zalutumumab or competitors. Overall, the figure legends and Methods section are too abbreviated to allow reproducibility.\n4. Mismatch Between Methods and Results Sections\nA FACS-based internalization assay is described in the Methods section but is not shown or discussed in the Results. Yet the Discussion (line 345) claims that IBI334 facilitates significantly greater EGFR internalization and degradation. This disconnect must be addressed.\nRecommendation\nWhile the development of IBI334 is a potentially valuable contribution to the field of bispecific antibody therapeutics targeting EGFR, the current version of the manuscript lacks sufficient mechanistic evidence and contains several inconsistencies that limit its impact. The authors should:\n• Provide direct evidence (or remove claims) related to the proposed cis-binding mechanism;\n• Clarify discrepancies in affinity data;\n• Substantiate or retract claims about receptor degradation;\n• Expand methodological detail, especially regarding antibody generation and Fc design;\n• Improve figure legends and ensure consistency between Methods and Results.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: Thank you for your question. Zalutumumab is an EGFR blocking antibody with moderate ligand blocking activity (weaker than Cetuximab and Panitumumab), and therefore the monovalent form of Zalutumumab showed more balanced efficacy and safety profiles in preclinical studies (Cancer Res; 76(13); 3942–53). The exact sequence of Zalutumumab was used in Amivantamab, the FDA-approved c-MET/EGFR bispecific antibody for non-small cell lung cancer with EGFR activating mutations. Amivantamab also showed positive results (objective response rate of 13% to 47% in different cohorts) in phase 1 study for colorectal cancer (ASCO Gastrointestinal Cancers Symposium 2024 Abstract #135). These data collectively show that Zalutumumab, at least in the form of bispecific antibody, is clinically validated and justify its use in IBI334 design.\nThe PCT for Zalutumumab sequence was filed by Genmab in 2002 (WO02/100348 A2) and the associated molecular entity patents were expired in 2022.\n2) in the Abstract and the Discussion, the authors stated “accelerated receptor degradation”, “Additionally, IBI334 prompts rapid and enduring degradation of EGFR in both cancer cells and xenograft tumors, likely initiated by antibody-induced receptor endocytosis.”, etc. However, it seemed the authors did not provide real experiment data to support this statement.\nResponse: We appreciate the reviewer’s feedback. As demonstrated in Fig2h of the manuscript and below, the Western Blot assay revealed a time-dependent reduction in EGFR levels in the 20G5/Zalu group. Additionally, we performed further EGFR internalization experiments, which demonstrated that 20G5/Zalu induced stronger time-dependent EGFR internalization compared to Zalutumumab (bivalent) and null/Zalu (monovalent) (Fig 2f and below). Supporting this observation, flow cytometry data showed that 20G5/Zalu had the most pronounced reduction in surface EGFR levels (Fig S3h and below).\nFig 2h in the updated manuscript:\nAdditional figures:\nFigure legend: EGFR internalization after antibody treatment in NCI-H358 cells. Cells were treated with 5 nM IBI334, Zalutumumab or Null/Zalu for the indicated durations, and cell surface EGFR levels were detected using a detecting antibody against epitope different from Zalutumumab (left figure). Data are shown as mean ± SEM. Representative flow cytometry analysis of EGFR expression levels at 72 hours from two independent experiments (N=3) (right figure). PC, positive control (NCI-H358 cells stained with the detecting EGFR antibody only); NC, negative control (unstained NCI-H358 cells).\nIn H358 xenograft tumors, we observed clear EGFR down-regulation after 24 hrs treatment of IBI334 in both western blot and IHC analyses (Fig 4c-d). In H1975LTC model, IBI334 treatment induced EGFR down-regulation in vivo, and this effect was further amplified when combining with Osimertinib treatment (Fig 5e).\nFig 4c-d in the original manuscript:\nFig 5e in the original manuscript:\n3) Fig 1D, the quality of EGFR IHC staining at ++ and + is low.\nResponse: We appreciate the reviewer’s feedback. The scoring was performed independently by two trained technicians, considering both staining intensity and the proportion of positive cells to account for tumor heterogeneity. Figure 1D displays only representative EGFR staining sections, and we regret any confusion this may have caused. As shown in the figure below, the EGFR+ sample shows clear positivity compared to EGFR- samples, and EGFR++ samples demonstrated greater staining intensity and higher proportion of positivity.\nEntire tumor section and magnified view of EGFR + & ++ tumor section in original Fig 1D:\nNote: Rectangle indicates the tumor section shown in Fig 1D of the original manuscript. Circles show magnified views of different sections on the same tumor.\nMore examples of EGFR++, + & - staining:\n4) In Fig S2.j, high concentrations of 19A2/Nimo increased the survival of PC9-B7-H3 tumor line, so did 19A2/Nimo induce EGFR signaling rather than inhibition? Please explain the result.\nResponse: Thank you for your insightful observation. We found it intriguing and therefore repeated the experiments three times (figure below). However, we did not observe a consistent increase in survival and concluded that the initial result was likely due to technical variability, based on the following observation: (1) the increase was marginal, with the last data point showing high variability; (2) in the absence of killing activity, baseline fluctuations are common; and (3) we did not detect a similar effect in repeated experiments (figure below) or in other cell lines (eg. A431 in Fig S2k of the original manuscript), which would be expected if 19A2/Nemo truly enhances EGFR signaling.\nFig S2j in the original manuscript:\nFig S2k in the original manuscript:\nRepeated experiments:\nFigure legend: Growth inhibition of 19A2/Nimo bsAb in PC9-B7-H3 cells. PC9-B7-H3 cells were treated for 5 days in 3D culture; Experiment performed in triplicate (N=3). Data are shown as mean ± SEM.\nReviewer #2 (Remarks to the Author):\nThe authors describe development of a bispecific antibody targeting EGFR and B7-H3, together with a series of studies of its efficacy in various cancer cell lines/models and in tumor xenograft studies. Cryo-EM structures of the B7-H3 targeting FAB with B7-H3 are also reported.\nWhile there is a tremendous amount of data here, and studies appear to be carefully executed, I find the work exceptionally difficult to follow and interpret. In particular, the wide variety of cell lines studied, seemingly both with and without exogenous over-expression of B7-H3 or various oncogenic variants of EGFR, add to the confusion. This is exacerbated by the lack of explanation for the underlying rationale of the choice of cell lines or an explanation of their genotype with respect to EGFR.\nA few specific comments:\n1. I do not find the correlation/co-expression analysis in Fig. 1 compelling.\nResponse: Thank you for your feedback. Fig 1 depicts the procedure of B7-H3/EGFR pair discovery, starting from bioinformatics analysis of marker expressions, evaluating the RNA expression as well as performing flow cytometry on different cell lines. All the data showed positive correlation with p value less than 0.0001, R2 > 0.2 for RNA data (Fig 1b,c) and R2 >0.3 for protein data (Fig 1c). We agree that R2 values of 0.2 – 0.3 may suggest weak correlation despite very low p value scores. Therefore, we did immunohistochemistry staining and showed that both markers are positive in >80% patient samples (Fig S1b). These results align with previous reports showing EGFR overexpression in 40%-89% of non-small cell lung cancer (NSCLC) cases (Transl Lung Cancer Res. 2015;4:110) and B7-H3 in 80% NSCLC (J Hematol Oncol. 2022;15:153).\nFig S1b:\n2. Along the same lines, in FIg. 4A it is not clear that there is any correlation of response to co-expression of the two target antigens.\nResponse: Thank you for your comment. Tumor sensitivity to EGFR inhibition does not directly correlate to the expression level of EGFR because tumor cells often harbor EGFR downstream mutations, such as KRAS G12 mutations, that make them less sensitive to EGFR antibody therapy. For example, in the phase III trial of Cetuximab (an FDA approved anti-EGFR antibody), neither the proportion of EGFR-positive cells nor staining intensity correlated with patient response rates (p = 0.87 and p = 0.64, respectively) (N Engl J Med. 2004;351:337). So based our observation and previous clinical data, we hypothesize that the response is largely determined by tumor cell’s intrinsic sensitivity to EGFR pathway inhibition, instead of EGFR expression. For example, cancer cells independent of EGFR signaling are less likely to respond to our bispecific Ab regardless of antigen expression levels. However, for cancer cells relying on EGFR oncogenic drivers, we do see a trend that higher B7-H3 expression may facilitate EGFR signal blockade by our bispecific antibody (eg. PC9-B7-H3 vs. PC9 cells).\nFig 4a:\n3. In the in vivo efficacy studies in Fig. 4 and elsewhere, why no control with the B7-H3 targeting antibody alone? or Two separate antibodies targeting EGFR an B7-H3 as compared with the bi-specific?\nResponse: Thank you for your question. We did not include a B7-H3-targeting antibody alone in this study because prior data demonstrated that none of the B7-H3 monoclonal antibodies tested could induce tumor growth inhibition (Fig S2f). Instead, we evaluated both B7-H3 and combination controls in our SK-MES-1 model (Fig S6a). In this model, IBI334 exhibited superior anti-tumor activity compared to B7-H3/null monotherapy and the combination of B7-H3/null bsAb and EGFR/null bsAb.\nFig S2f\nFig S6a\n4. Difficult to understand how these mice were dosed. For example, legend states treated with 4 doses of the specified antibodies at 10 mg/kg twice per week. Does this mean that there was an initial two week treatment period, followed by continued tumor measurement but w/o treatment? If so would be good to indicate dosing on the plots.\nResponse: We apologize for the confusion. Yes, “treated with 4 doses of the specified antibodies at 10 mg/kg twice per week” means an initial 2-week treatment period followed by continued tumor measurement without treatment. We included a dosing plots in our updated manuscript for clarity and we deeply appreciate your suggestion.\nExample of updated in vivo data figure with dosing labelled (Fig 4e): black arrowhead indicates the dosing of antibodies.\n5. With respect to the cryo-EM structures: The map and model for the single FAB structure (as illustrated in fig. 3h) looks fine and is of a reasonable resolution for the conclusions drawn. Very nice work.\nResponse: We appreciate that. Thank you.\n6. But there are issues with the structure with 2 FABs bound to B7-H3:\na. the density for the light chain of one FAB and the heavy chain of the other appear to be rather weak. This must be an imaging artifact of some sort...I do not have a good explanation but it does not make physical sense.\nResponse: Thank you for your feedback. The issue you raised is indeed present. As shown in the 2D classification (Figure S4f), the density of the two chains located near the adjacent regions of the two Fabs appears weaker compared to the other two chains. Furthermore, the 20G5 (Fab’)2/hB7-H3 complex exhibits a clear orientation preference due to its overall flat structure (Figure S4h). These factors likely contribute to the relatively weaker density of these two chains in comparison to the others.\nb. While it is possible that the two FABs are bound the duplicated domains of one B7-H3, it is not clear that this is the case - also possible that particles are self-associating via the FAB leading to this reconstruction.\nResponse: Thank you for your feedback and we agree with your point that the alternative case likely exists under our experimental conditions. During cryo-EM sample screening, we tested both 20G5 Fab and 20G5 (Fab’)2 in complex with 4Ig-B7-H3. We observed that samples containing 20G5 (Fab’)2 exhibited better homogeneity in micrographs, with a higher proportion of particles showing two Fabs bound to a single B7-H3 molecule. We attribute this stabilization to the disulfide bonds between the heavy chains in 20G5 (Fab’)2, which help maintain complex integrity. Additionally, Fab-Fab interactions—a well-documented phenomenon (Yin et al., 2022, Comput Struct Biotechnol J)—likely contribute to the formation of a stable triangular structure between 4Ig-B7-H3 and 20G5 (Fab’)2. This configuration stabilizes the B7-H3’s flexible extracellular domains, ultimately enabling us to resolve the structure of the 20G5 (Fab’)2 and hB7-H3 complex. In contrast, 20G5 Fab + 4Ig-B7-H3 failed to yield high resolution complex structures in our Cryo-EM experiment.\nc. In any case, the PDB file for the 2-FAB structure models it as two copies of the IgC2 and IgV2 domains, each with a FAB bound, not as one FAB bound to IgC2-IgV2 and one to IgC1-IgV1.\nResponse: We sincerely apologize for providing the incorrect PDB file. We have corrected it and uploaded the revised file in the submitting system. The figure S4h in the manuscript has been updated as shown below.\nd. In fact the 2 version of the structure in the 2-FAB complex and the version in the 1-FAB complex are all almost identical (the antibody portions appear to be precisely the same, there are some very minor differences in the B7-H3 portion of the 1-FAB structure. Given that the 2-FAB structure has problems and that it does not add anything to the work, I would simply remove it. The interesting part is recognition of the epitope on B7-H3 by the FAb, and that is best described with the 1-FAB structure.\nResponse: Thank you for the feedback. We have removed the 2-Fab structure from the main figure. While the 20G5 (Fab’)2/hB7-H3 complex structure lacks high-resolution details due to limited map density, it suggests the possibility of simultaneous binding of (Fab’)2 to B7-H3's IgC1 and IgC2 domains. We think this is important for interpreting our experimental results and have kept this information in the supplementary Figure 4. Because only (Fab’)2, but not Fab’, can theoretically stabilize the highly flexible domains of 4Ig-B7-H3, and thereby enabling us to resolve the structure of this complex (Fab’ data collection on Krios shown below). This may also explain why there was only murine 2Ig-B7-H3 structure resolved (Structure. 2013 May 7;21(5):707-17), but no human 4Ig-B7-H3 structure available before the current study.\nReviewer #3 (Remarks to the Author):\nIn this study, the authors present the development of a novel bispecific antibody, IBI334, targeting EGFR-associated tumors. This antibody combines binding epitopes for EGFR and B7-H3 — a single-pass transmembrane protein that is upregulated in various tumors, including lung cancer, but shows minimal expression in normal tissue, making it a promising target for selective cancer therapy and several examples targeting B7-H3 are currently under clinical investigation. The clinical relevance of the presented study for EGFR-related cancer types makes it particularly compelling and should justify publication in NC. The design rationale and methodology are thoroughly described and clearly presented. The anti-tumor efficacy (in-vitro and in-vivo) and toxicity profiles observed in animal models are promising and have prompted the authors to advance to an ongoing phase I clinical trial.\nA few things should be considered:\nCould the authors elucidate the calculated synergy score from (Synergy Finder+) and discuss the presented values and meaningfulness?\nResponse: Thank you for your question. To evaluate the potential synergy of combined drugs, the observed drug combination responses are usually compared with expected combination responses calculated by means of synergy scoring models. Consequently, based on deviation of observed and expected responses one can classify the drug combination either as synergistic or antagonistic (Genomics Proteomics Bioinformatics. 2022; 20: 587, https://synergyfinder.aittokallio.group/synfin_docs/).\nWhen the synergy score is:\n• Less than -10: the interaction between two drugs is likely to be antagonistic;\n• From -10 to 10: the interaction between two drugs is likely to be additive;\n• Larger than 10: the interaction between two drugs is likely to be synergistic.\nSynergy scores reported in Figure 5 and Supplementary Figure S7 are all above 10, suggesting synergistic effects of IBI334 and KRAS inhibitors/Osimertinib.\nFigure 5b: combination of Osimertinib (50 nM) and IBI334. What might be the reason why only pAKT is suppressed by the combination (and not ERK)? And is the conc. of Osi high enough for the observed effect? 5a suggests an EC50 of OSI around 1 micromolar at LRTMCS (appears consistent with other literature). How should this be understood in context with the differences to the H358 xenograft model in Fig 4c?\nResponse: Thank you for the questions. In Figure 5b, bispecific Ab or combination therapy could effectively suppress pAKT, as well as pERK (highlighted in the red rectangle below). However, Osimertinib itself has no effect in suppressing either pAKT or pERK in this EGFR LTC triple mutation cell line (NCI-1975LTC). To examine whether this is due to insufficient dose of Osimertinib, we have performed additional experiments using high concentration Osimertinib (1000nM), and similar trend was observed (Figure below).\nFigure 5b\nAdditional experiments:\nFigure legend: Western blot analysis of IBI334 and Osimertinib in NCI-H1975LTC cells. Cells were treated with 100nM IBI334, 50 nM or 1000 nM Osimertinib, or combination for 4 hours. Left and right figures are from two independent experiments.\nThis finding is consistent with Uchibori K. et al study (Nat Commun. 2017: 8: 14768) whereby 1000 nM Osmertinib showed minimal suppression of pERK & pAKT on PC9 triple mutant (C797S/T790M/del19) cells (figure below), as Osimertinib is ineffective to cells harboring C797S mutation. Unlike NCI-1975LTC cells, H358 is a different lung cancer model (with KRAS G12C mutation) that does not contain LTC mutations. Both cellular models exhibited sensitivity to IBI334 inhibition, because this agent functions not only as an EGFR ligand blocker but also disrupts the scaffold function of EGFR in downstream signal transduction. Specifically, IBI334 interferes with EGFR dimerization and promotes EGFR degradation. This effect was particularly pronounced in the H358 model, where we observed more substantial EGFR degradation compared to the H1975LTC model (which ectopically overexpresses the EGFR L858R/T790M mutant). The differential degradation patterns may consequently affect EGFR-mediated adaptor protein recruitment and downstream signaling pathways, including PI3K/Akt and KRAS/MAPK cascades.\nFigure adapted from Nat Commun. 2017: 8: 14768\n(https://www.nature.com/articles/ncomms14768)\n(f) Western blotting of PC9 triple mutant (C797S/T790M/del19) cells indicated that brigatinib and AP26113 analog, but not afatinib or osimertinib, suppressed phosphorylation of EGFR and its downstream signaling\nTable S6 – by nature of potential variability in pk studies. Are all Tmax values correctly stated?\nResponse: All reported Tmax values were accurate. Blood sampling occurred at 0.017 hr (first timepoint) and 2 hr (second timepoint). Inter-animal variability led to some subjects reaching Tmax at the initial timepoint while others peaked at the second, accounting for the observed variation in mean Tmax.\nThe selectivity of the described bsAb is exceptional and raises interest in the approach. Since EGFR-driven cancers are often associated with prominent and relatively quickly appearing resistance/escape pathways – did the authors conduct experiments to assess such potential outcomes for their bsAb (e.g., downregulation of B7-H3)? This should help to predict the long-term potential of their approach.\nResponse: Thank you for raising this very interesting point here. As part of our research, we conjugated IBI334 with DAR4 Exatecan to generate an antibody drug conjugate that showed better efficacy than IBI334 in vivo (left figure below). Notably, repeated administration of IBI334 led to the downregulation of both B7-H3 and EGFR, which is consistent with your speculation, and the tumor showed reduced sensitivity to subsequent IBI334-ADC treatment (right figure below).\nHowever, it is worth mentioning that the observed downregulation of B7-H3 and EGFR was assessed just four days after IBI334 treatment. Residual bispecific antibody in circulation or on the tumor could potentially influence these measurements. Nevertheless, we speculated that a longer time interval before re-challenge may restore some B7-H3 or EGFR expression given their oncogenic roles and potential growth advantages for tumor cells. For instance, it was reported that after anti-EGFR withdrawal, RAS and EGFR mutant clones lack growth advantage relative to other clones and decay over time, and relapsed patients may again benefit from anti-EGFR treatment (Ann Oncol. 2019; 30: 243). Indeed, J&J presented data at ASCO GI 2024 (Abstract #135) indicating that longer intervals between amivantamab (a c-MET/EGFR bispecific antibody) treatment and prior EGFR mAb progression correlate with better outcomes. We appreciate the reviewer for highlighting this intriguing topic. While drug resistance falls outside the scope of this article, it certainly merits further investigation in future studies.\nFigure legend: In vivo efficacy results of IBI334 and IBI334-ADC in NCI-H358 models. (Left) NOG mice (n=6/group) bearing NCI-H358 tumor were treated with a single dose of the indicated antibodies or ADC. (Right) NOG mice (n=6/group) bearing NCI-H358 tumor were first treated with IBI334, followed by IBI334-ADC. Two mice in each group were sacrificed on the same day as ADC was administrated and the tumor expression of B7-H3 and EGFR were measured by flow cytometry.\nReviewer #4 (Remarks to the Author):\nThis manuscript by Jian Guan and colleagues describes the development of a novel bispecific antibody (bsAb), IBI334, targeting B7-H3 and EGFR, with the goal of achieving enhanced anti-tumor efficacy while minimizing on-target toxicity. The authors propose that the therapeutic benefit of IBI334 is based on its higher affinity for B7-H3 relative to EGFR, leading to preferential binding to tumor cells and reduced systemic toxicity. While the concept is promising and the generation of a functional bsAb with dual specificity is noteworthy, the manuscript suffers from several conceptual and methodological weaknesses that must be addressed to support the authors’ claims.\nMajor Comments\n1. Lack of Evidence for Sequential Cis-Binding Mechanism\nThe central mechanistic claim of the manuscript is that IBI334 first binds B7-H3 and subsequently engages EGFR on the same cell surface via a cis-acting mechanism. However, this hypothesis is not directly supported by the experimental data. The only related evidence (Figure S3h) is indirect and lacks sufficient clarity. Without robust biochemical or biophysical data demonstrating this sequential binding, this mechanistic assertion remains speculative.\nResponse: We thank the reviewer for this critical feedback and agree that additional data would strengthen the evidence for the proposed cis-acting mechanism.\nTherefore, we have conducted additional experiments to support the claim.\nFirst, to examine the cis-binding effect of IBI334 (20G5/Zalu), we used flow cytometry-based cell binding assays (gated on single cells while excluding doublets) to demonstrate that 20G5/Zalu binds to NCI-H358 cells (EGFR and B7-H3 double positive) at a much higher rate than Zalu alone, as evidenced by stronger binding at low concentrations (Figure S3e and below). This data suggests that the high affinity anti-B7-H3 arm (20G5) helps IBI334 to more efficiently engage tumor antigens and anchor onto cell surface in comparison to bivalent Zalutumumab (two anti-EGFR Fabs).\nFigure legend: Representative gating strategy for single cells (top) used in cell binding assay of 20G5/Zalu and zalutumumab in NCI-H358 (bottom).\nFurthermore, to evaluate how B7-H3-mediated faster and more potent binding impacts EGFR engagement on the same cell (in cis), we compared 20G5/Zalu and zalutumumab in EGFR occupancy assays (experimental details described in method section). EGFR occupancy was assessed by flow cytometry gated for single cells. 20G5/Zalu exhibited superior EGFR receptor occupancy compared to Zalu even at lower concentrations, and 20G5/Zalu achieved maximal occupancy (plateau) but not zalutumumab (Figure 2e and below (bottom left)). And the difference was even more dramatic after antibody washout and incubation on ice for 4 hr (Figure S3f and below (bottom right)). These data suggest that the high-affinity B7-H3 arm of IBI334 firmly anchors the molecule to the cell surface (due to slow dissociation rate of 20G5), allowing the EGFR arm to efficiently locate and bind to nearby EGFRs (avid binding) on the same cell surface in cis position.\nFigure legend: Representative gating strategy for single cells (top) used in EGFR occupancy assay of 20G5/Zalu bsAb and zalutumumab (bottom). NCI-H358 cells were incubated with 20G5/Zalu bsAb or zalutumumab for 1 hr, thoroughly washed, and then further incubated on ice for 0 h (bottom left) or 4 h (bottom right). EGFR occupancy was assessed using AF647-labeled zalutumumab by flow cytometry.\nTo examine how cis-inhibition mechanism of IBI334 affects its biological activities, we performed cell growth inhibition assays, signaling transduction assay and immunofluorescence microscopy using cancer cell models in low density cell culture or co-culture assays to mimic in-cis or in-trans conditions, respectively.\nFirst, IBI334 exhibited only a slight increase in growth inhibition against SK-MES-1 and PC-9 cells co-cultured with B7-H3-overexpressing CHO-S cells (trans-inhibition), whereas SK-MES-1 and PC-9 cells overexpressing B7-H3 showed stronger growth suppression. This suggests that IBI334’s anti-tumor activity is mainly mediated through cis-inhibition. (Fig. S3k-l).\nFig S3k\nFig S3l\nFigure legend: k-l，Growth inhibition of 20G5/Zalu bsAb on SK-MES-1 (k) or PC-9 cells (l) co-cultured with CHO-S cells with or without B7-H3 overexpression (Left), and on SK-MES-1 or PC9 cells with or without B7-H3 overexpression co-cultured with CHO-S cells (Right).\nSecond, to minimize the interference from cell-cell contact (trans-inhibition), we plated SK-MES-1 (wt) or SK-MES-1 overexpressing B7-H3 (B7-H3OE) cells at low density to isolate single cells for subsequent analyses, representing only cis-inhibition effect. Western blot analysis revealed that 20G5/Zalu significantly suppressed EGFR phosphorylation (pEGFR) in B7-H3OE cells, while less effective in WT cells (Figure 2l). In contrast, Null/Zalu (no anti-B7-H3 help) had only minimal inhibition of pEGFR in comparison to 20G5/Zalu in B7-H3OE cells (Fig 2m).\nFig 2k-m\nFigure Legend:\nk, A schematic illustrating the approach for assessing cis-inhibition. Cells were seeded at a low density of 3×105 cells per 10 cm² dish to prevent cell-cell contact for subsequent (l) and (m) western blot analyses. l, Western blot analysis of 20G5/Zalu in SK-MES-1 cells (Abbreviated as WT) or B7-H3-overexpressing SK-MES-1 cells (SK-MES-1-B7-H3OE, abbreviated as B7-H3OE). Cells were treatment with 20G5/Zalu for 4 hours before analysis. m, Western blot of 20G5/Zalu or null/Zalu in SK-MES-1-B7-H3OE cells after treated with indicated antibody for 4 h.\nThird, we assessed both cis- and trans-inhibition by analyzing pEGFR levels through fluorescence microscopy. Treatment with Zalu/20G5 led to a greater suppression of pEGFR in B7-H3-overexpressing (B7-H3OE) SK-MES-1 cells compared to wild-type SK-MES-1 cells (Figure 2n). In co-culture assays, SK-MES-1-B7-H3OE cells (GFP positive) exhibited largely reduced pEGFR levels compared to neighboring wild-type SK-MES-1 cells (GFP negative), indicating that cis-inhibition predominates over trans-inhibition.\nFig 2n\nFigure legend: Phosphorylated EGFR levels (red) were analyzed by fluorescence microscopy in SK-MES1 (GFP negative), SK-MES1-B7-H3OE (expressing GFP for visualization), and co-cultured cells, with or without 1 nM 20G5/Zalu (4 h treatment). SK-MES1-B7-H3OE and SK-MES1 cells are indicated by green and white arrows, respectively.\nThese new data were included in the revised manuscript (main text and Figure 2k-n).\n2. Discrepancies in Binding Affinity Reporting\nThe manuscript reports that converting Zalutumumab to a monovalent format increases EGFR affinity from a KD of 3.89 × 10⁻⁹ M to 4.69 × 10⁻¹⁰ M (Table S3). However, Table S3 contains an apparent error, listing the bsAb KD as 4.69 × 10⁻⁹ , As the KD is calculated from observed Kon and Kdis values. This inconsistency must be corrected and clarified.\nResponse: We thank the reviewer for pointing this out and apologize for the apparent mistake when copying the values from the datasheet. We corrected the values of Table S3 in the revised manuscript as shown below.\nTable S3 (updated). Binding affinity (KD) of 20G5/Zalu based on BLI\nRaw data (EGFR affinity):\n3. The bsAb is reported to be safe in mouse models while demonstrating strong efficacy in tumor volume reduction thereby suggesting a broadended therapeutic window. However, this safety profile is likely attributable to its lack of cross-reactivity with mouse EGFR and B7-H3, as noted in Table S7 and the EMA report for Rybrevant. Therefore, murine studies cannot reliably inform on-target toxicity, and this critical limitation should be explicitly acknowledged.\nResponse: Thank you for the comment. Indeed, bsAb does not cross react with mouse antigens, and the on-target toxicities in mice can not be directly measured. Therefore, we used A431 xenograft as a surrogate model to preliminarily evaluate the EGFR on-target toxicities (such as skin side effects) as shown in Fig 4h. A431 is a human epidermoid carcinoma cell line that was commonly used as an in vivo to evaluate human epithelium-related toxicity (J Med Chem. 2014 Oct 23;57(20):8249-67; Sci Transl Med. 2023; 15: eabo0684). Since this model is of human origin, it eliminates the need for the molecule to cross react with mouse EGFR or B7-H3.\nWe recognize that our description in the original manuscript may be unclear and have updated the manuscript to clarify this:\n“In addition, IBI334 showed milder effect than Zalutumumab on A431 human xenograft model, a commonly used surrogate model to assess skin toxicity in vivo29, suggesting a potentially improved safety profile (Fig.4h, Supplementary Fig.6g).”\n4. Inconsistent Claims Regarding Receptor Degradation\nThe authors state that IBI334 induces accelerated EGFR degradation; however, Figures 2g and 2h show no significant change in EGFR levels over five days. These contradictory data undermine the claim and require resolution.\nResponse: We thank the reviewer for the feedback. We agree that Figure 2g did not show significant downregulation of EGFR by Western blot assay but would like to note that these cells were only treated for 4 hrs, which may not be optimal to evaluate EGFR degradation, as the assay is primarily for evaluating EGFR downstream signaling (phosphorylation). Nonetheless, the Western Blot assay in Figure 2h showed decrease of EGFR in 20G/Zalu group on Day 5.\nFig 2g-h:\nHowever, since the Western Blot assay can detect both intracellular and transmembrane EGFR, it may not be the most suitable approach for measuring EGFR internalization. Hence, we performed additional experiments to support our hypothesis. As shown below, 20G5/Zalu induced a significant internalization (p<0.0001) of EGFR than Zalutumumab or Null/Zalu bispecific control antibody as early as 4hr after treatment (Fig 2f and Fig S3g).\nFig 2f:\nFigure legend: EGFR internalization of 20G5/Zalu bsAb in NCI-H358 cells measured by flow cytometry. Cells were treated with 5 nM IBI334, Zalutumumab or Null/Zalu for indicated durations, and cell surface EGFR levels were detected using an anti-EGFR antibody targeting an different epitope from Zalu‘s binding site.\nFig S3g:\nFigure legend: A representative flow cytometry analysis of EGFR expression in NCI-H358 cells. NCI-H358 cells were treated with 5 nM 20G5/Zalu, Zalutumumab or Null/Zalu for 72h, and cell surface EGFR levels were detected using an antibody targeting a different epitope from Zalu‘s binding site. Experiments were performed in triplicate.\nMinor Comments\n1. Lack of Methodological Detail for Antibody Generation\nThe bsAb was reportedly generated using the Innobody platform as a 1+1 asymmetric IgG-like format. However, no information or references are provided regarding the Fc heterodimer design or bsAb assembly. Given that ADCC is proposed as a mechanism of action, Fc structure and function must be described to allow reproducibility and proper interpretation.\nResponse: Thank you for the feedback. The Innobody platform can increase Fc-containing formation of the heavy chain of an Fc heterodimer protein by mutations in the CH3 region. The mutations, expression, purification and in vitro assembly of Innobody Fc heterodimers are described in the patent WO2022143912A1，entitled “Protein containing heterodimer antibody fc, and preparation method therefore”. The patent is cited in the method section under the subtitle “Protein expression and production”:\n“This vector includes the Fc region of wild-type human IgG1 for monoclonal antibodies or the Fc region with S364R+D399K or K370S+K409D+Y349T substitutions from the Innobody platform (WO Patent2022143912A1) for bispecific antibodies.”\nMeanwhile, ADCC of IBI334 is enhanced by using the GlymaxX low-fucose technology (Gargi Roy, et al. MAbs. 2018; 10: 416). The following description was added to the method section under the subtitle “Protein expression and production” for further clarity:\n“GlymaxX Technology is used to produce afucosylated antibodies44. Briefly, antibodies were expressed in Chinese hamster ovary (CHO) cells overexpressing GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, and the resulting antibodies are afucosylated.”\n2. Redundancy in Antibody Selection Description (Lines 94–128)\nThe manuscript devotes excessive space to describing antibodies that were ultimately discarded. This content could be moved to the supplementary materials or summarized briefly.\nResponse: We appreciate the reviewer’s feedback. These paragraphs outline the selection system and process for our lead antibody. Including non-selected candidates helps readers to understand the rationale behind our choices, with detailed characteristics of all antibodies provided in the supplementary data.\nWe agree that the text could be more succinct and have revised the manuscript accordingly (added text highlighted in red, deleted text in green):\n“Among these, the bsAb combining anti-B7-H3 clone 20G5 and anti-EGFR clone Zalutumumab (20G5/Zalu) emerged as the most promising candidate based on efficacy and safety parameters (Fig. 2b, Supplementary Fig. 2l), demonstrating the most potent anti-tumor efficacy (Supplementary Fig. 2m), reduced skin cytotoxicity compared to Zalutumumab (Fig. 2c, Supplementary Fig. 2n-o), superior ADCC activity (Supplementary Fig. 3a-d), ”\nDeleted text:\n“Further validation involved pairing the 11 anti-B7-H3 clones with Zalutumumab, and 20G5/Zalu consistently demonstrated the best anti-tumor potency (Supplementary Fig. 2m). In addition, clone 20G5 was combined with various EGFR binders, and 20G5/Zalu exhibited minimal toxicity in skin cell lines (Supplementary Fig. 2n). Notably, 20G5/Zalu not only showed enhanced efficacy and reduced skin cytotoxicity compared to Zalutumumab (Fig. 2c, Supplementary Fig. 2o) but also displayed superior ADCC activity among all other tested bsAbs (Supplementary Fig. 3a-d).”\n3. Inadequate Figure Legends and Method Descriptions\nSeveral figures, particularly in the supplementary section, lack sufficient explanation. For example, in Figure S3h, it is unclear whether the X-axis refers to labeled Zalutumumab or competitors. Overall, the figure legends and Methods section are too abbreviated to allow reproducibility.\nResponse: We thank the reviewer for the feedback and have updated the figure legends and methods for clarity (highlighted in red in the updated manuscript), including innobody mutations at the Fc and cited the relevant patent, description on the generation of afucosylated Fc and the reference, further description on the ADCC and growth inhibition assays, and method on the internalization assay.\nWe also updated the x-axis and figure legends of Fig S3h of the original manuscript (Fig S3l in the updated manuscript) to avoid confusion. The x-axis refers to the 20G5/Zalu concentration and the assay contains only 20G5/Zalu but not Zalutumumab or competitors. We apologize for the confusion.\nFig S3k-I in updated manuscript:\nFigure legend: k-l，Growth inhibition of 20G5/Zalu bsAb on SK-MES-1 (k) or PC-9 (l) cells co-cultured with CHO-S cells with or without B7-H3 overexpression (left), and on SK-MES-1 (k) or PC-9 (l) cells with or without B7-H3 overexpression co-cultured with CHO-S cells 9 (right). Cells were treated with 20G5/Zalu for 5 days.\n4. Mismatch Between Methods and Results Sections\nA FACS-based internalization assay is described in the Methods section but is not shown or discussed in the Results. Yet the Discussion (line 345) claims that IBI334 facilitates significantly greater EGFR internalization and degradation. This disconnect must be addressed.\nResponse: We thank the reviewer for pointing this out and apologized for our mistake. The FACS-based internalization assay results are included in Fig 2f of the revised manuscript.\nFig 2f:\nRecommendation\nWhile the development of IBI334 is a potentially valuable contribution to the field of bispecific antibody therapeutics targeting EGFR, the current version of the manuscript lacks sufficient mechanistic evidence and contains several inconsistencies that limit its impact. The authors should:\n• Provide direct evidence (or remove claims) related to the proposed cis-binding mechanism;\n• Clarify discrepancies in affinity data;\n• Substantiate or retract claims about receptor degradation;\n• Expand methodological detail, especially regarding antibody generation and Fc design;\n• Improve figure legends and ensure consistency between Methods and Results.\nResponse: We sincerely accept all the kind recommendation from the reviewer, and made the following changes to the revised manuscript:\n• We included additional data supporting the proposed cis-binding mechanism in Fig 2k-n & Fig S3j-m\n• We updated the affinity data at Table S3\n• We included additional data on receptor degradation in Fig 2F and Fig S2g\n• We updated the method section with more clarity and details, including the innobody mutations at the Fc and cited the relevant patent, description on the generation of afucosylated Fc and the reference, further description on the ADCC and growth inhibition methods, and method on the internalization assay.\n• We expanded the figure legend description, added the internalization data on Fig 2f and double checked the Methods & Results section.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all of my questions.", "reviewer_2": "While the authors have satisfactorily addressed some of my the issues I raised in my review, problems remain. The problems with the single particle reconstruction for the (Fab)2 structure have not been addressed. Not acceptable to just move low quality data to a supplemental figure. While I think the authors are likely correct in their interpretation of this structure as a (Fab)2 bound to one 4-domain B7-H3, their response is not reassuring in that they seem to be acknowledging the possibility that it could instead be one(Fab)2 bound to two 4-domain B7-H3s, where the non-bound domains of B7-H3 are disordered and not present. If this is the case, one might expect to see \"clouds\" of faint extr: density in the 2D class averages. Unfortunately the 2D class averages provided are two low resolution to evaluate for this possibility. I feel the authors either need to do additional experimental and/or computational work as to improve the quality of this reconstruction, remove it from the manuscript, or at a bare minimum clearly note in the text the issues with the quality of the map and the resulting uncertainty in their interpretation.\nA few additional things that I might or might not have mentioned in my initial review:\n1. The superlatives in the abstract are off-putting.\n2. This sentence in the abstract incorrectly implies that the cryoEM work in the paper supports their cis-inhibition model.\n\"Mechanistic studies revealed B7-H3-mediated cis inhibition of EGFR through structural elucidation of the full-length human B7-H3 extracellular domain (ECD) in complex with anti-B7-H3 (Fab’)₂resolved via cryo-EM for the first time\"\n3. The degree of structural detail in the model in FIg. 3j goes beyond what is supported in the paper. I worry that when presented together with cryoEM structures it will lead some readers to presume that it is supported by structural data. Better to show models like this in more schematic form. (I will leave it to the other reviewers to comment on whether they are now convinced that there is sufficient direct evidence in the paper to support this cis-inhibition model).", "reviewer_3": "Reviewer #3 (Remarks to the Author):\nThank you for your careful and extensive replies!\nPlease adjust the following prior publication:\n1) Thank you for your detailed explanation of the Synergy Finder+.\nI’m convinced that the reader would benefit from a short explanation in your manuscript or the SI, too.\n2) Thanks for the additional experiments with Osi. I honestly can’t decipher a clear difference in the blots between the two dosages of 50 vs 1000 nM (without the antibody). And cannot see a difference in the gel without and with antibody. One the one hand: could the stated synergistic effect of osi and IBI334 also stem from another target, which osi is inhibiting? Other than that, Figure 5d states a p-value of 0.116 between IBI334 and the combo. Please elaborate and reconsider the synergistic effects of antibody with 3rd gen inhibitor.\n3) Thanks for your clarifying statement and the presented data in context with pk studies\n4) Thank you for the interesting insights into your latest ADC.", "reviewer_4": "The authors have carefully and comprehensively addressed the concerns raised In my review. The revised manuscriipt shows substantial improvements in clarity, methodological rigor, and overall presentation. The new data and analyses provided convincingly support the central conclusions, and the authors have responded thoughtfully to each reviewer comment.\nIn my assessment, the work now meets the high standards of novelty, technical quality, and broad interest expected for publication in Nature Communications. The findings will be of significant relevance to researchers in the field and will likely stimulate further work and discussion.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely accept the comments and have removed 4-domain B7-H3 figures from the supplementary figure 4. In addition, we also noted the issue in the revised manuscript:\nAdded the sentence:\n“Unfortunately, the density of (Fab’)2 and the B7-H3 complex is insufficient for further interpretation.”\nRemoved the sentence:\n“we were able to solve the complete cryo-EM structure of human B7-H3 in complex with 20G5 (Fab’)2 (Supplementary Fig. 4f-m).”\nChanges were highlighted in red in the revised manuscript.\nA few additional things that I might or might not have mentioned in my initial review:\n1. The superlatives in the abstract are off-putting.\n2. This sentence in the abstract incorrectly implies that the cryoEM work in the paper supports their cis-inhibition model.\n\"Mechanistic studies revealed B7-H3-mediated cis inhibition of EGFR through structural elucidation of the full-length human B7-H3 extracellular domain (ECD) in complex with anti-B7-H3 (Fab’)₂resolved via cryo-EM for the first time\"\nWe appreciate the reviewer’s comments. In the revised abstract, we eliminated superlative terms such as “for the first time” and restructured sentences to more accurately describe the cryo-EM work. Our mechanistic studies (fluorescence microscopy, co-culture assays etc.) support B7-H3-mediated cis-inhibition, while cryo-EM analysis identified critical residues for the antibody-B7-H3 interaction, informing our mutagenesis studies and revealing IBI334’s binding preference for the IgC1 domain of B7-H3.\nThe revised abstract section is shown below:\n“Mechanistic studies demonstrated B7-H3-mediated cis-inhibition. The full-length human B7-H3 extracellular domain (ECD) in complex with anti-B7-H3 (Fab’)₂ was resolved by cryo-EM, revealing critical residues for the antibody-B7-H3 interaction”\n3. The degree of structural detail in the model in FIg. 3j goes beyond what is supported in the paper. I worry that when presented together with cryoEM structures it will lead some readers to presume that it is supported by structural data. Better to show models like this in more schematic form. (I will leave it to the other reviewers to comment on whether they are now convinced that there is sufficient direct evidence in the paper to support this cis-inhibition model).\nWe thank the review for the comment and have updated Fig 3j to schematic diagram instead as shown below.\nReviewer #3 (Remarks to the Author):\nReviewer #3 (Remarks to the Author):\nThank you for your careful and extensive replies!\nPlease adjust the following prior publication:\n1) Thank you for your detailed explanation of the Synergy Finder+.\nI’m convinced that the reader would benefit from a short explanation in your manuscript or the SI, too.\nWe thank the review for the comment and have included a short explanation in the method section and reference.\nAdded a paragraph:\nDrug synergy between IBI334 and small-molecule inhibitors was evaluated using SynergyFinder+ algorithm according to the instruction.45 Briefly, cell inhibition was quantified using the CellTiter-Glo assay and experimental data were processed through a web-based SynergyFinder+ algorithm (http://www.synergyfinderplus.org) to obtain the synergy score. Synergy scores were interpreted as follow:\n< -10: Antagonistic interaction\n-10 to 10: Additive interaction\n> 10: Synergistic interaction\n2) Thanks for the additional experiments with Osi. I honestly can’t decipher a clear difference in the blots between the two dosages of 50 vs 1000 nM (without the antibody). And cannot see a difference in the gel without and with antibody. One the one hand: could the stated synergistic effect of osi and IBI334 also stem from another target, which osi is inhibiting? Other than that, Figure 5d states a p-value of 0.116 between IBI334 and the combo. Please elaborate and reconsider the synergistic effects of antibody with 3rd gen inhibitor.\nThank you for the reviewer’s question. As shown below, parental NCI-H1975 cells harboring EGFR L858R/T790M mutation (or EGFRLT) responded to osimertinib, whereas NCI-H1975 cells overexpressing EGFR L858R/T790M/C797S mutation (or EGFRLTC, used in our synergy experiments) did not, indicating that the mutant EGFR is the primary target of osimertinib in our model, and the observed synergistic effects in vitro are unlikely due to off-target of Osimertinib. In addition, Osimertinib is FDA-approved for non-small cell lung cancer (NSCLC) with activating EGFR mutations (exon 19 deletions or L858R) or T790M resistance mutations, and its selectivity for mutant EGFR is well-established (Biochemistry. 2020;59(14):1428-1437; Cancer Discov. 2014;4(9):1046-1059).\nRegarding Fig. 5d, IBI334 treatment resulted in progressive disease in four mice and partial response in two, whereas all animals in the combination group exhibited tumor shrinkage, with four achieving partial response. We acknowledged that we observed a promising trend for the combo group than individual treatments but it is not statistically significant in this in vivo study (p=0.116), despite strong synergy in vitro (synergy score > 10). Therefore, we revised the manuscript to exclude any reference to synergistic effects in vivo.\nThe revision includes:\nAbstract:\nIBI334 demonstrated robust antitumor activity in vitro and in vivo across EGFR-driven tumor models and synergized effectively with osimertinib and KRAS inhibitors.\nMain text:\nIBI334 enhanced Osimertinib activity and synergized with KRAS inhibitors\nCombination therapy is often employed to enhance therapeutic effectiveness, and synergistic antitumor efficacy, rather than combinatorial effect, is highly desirable. We developed the NCI-H1975LTC cell line, which harbors the Osimertinib-resistant EGFRLTC mutation, and found that IBI334 exhibited strong synergy with Osimertinib, achieving a synergy score of 21.74 (Fig. 5a-c). This combination was more effective at suppressing AKT phosphorylation than either drug alone (Fig. 5b). Consistent with in vitro results In addition, the combination of IBI334 and Osimertinib demonstrated enhanced antitumor efficacy compared to individual treatments in the NCI-1975LTC in vivo model as indicated by the number of animals achieving partial response (Fig.5d, Supplementary Fig.7a). IHC staining of the extracted tumors showed reduced levels of pEGFR and the Ki67 marker (Fig. 5e)."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have now adequately addressed my concerns.\nBut I do notice that their edit below created a sentence fragment:\nSince 20G5 Fab recognizes both epitopes on the IgC1 and IgC2 domains, by utilizing\nbivalent 20G5 (Fab’)2 to avidly bind and stabilize the 4Ig-B7-H3 ECD.", "reviewer_3": "The R1 covers my issues well.\ntks", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response:\nThank you for pointing it out. It is revised in the updated manuscript as follow:\n“Since 20G5 Fab recognizes both epitopes on the IgC1 and IgC2 domains, bivalent 20G5 (Fab’)2 binding to B7-H3 with high avidity can stabilize the 4Ig-B7-H3 ECD”"}]

Decision letter