Supplementary Figure S8: In vitro human RBC agglutination assay of TJH2201 using samples from three different PBMC donors.

Supplementary Figure S3: Binding ability of Hu5F9/B2B, and their respective parent antibodies.

Supplementary Figure S2: Binding ability and in vitro human RBC agglutination assay of Hu5F9/gp120.

Supplementary Figure S12: In vivo PK profiles of TJH2201 in Balb/c mice.

Supplementary Figure S10: HE staining of spleens from hCD47/SIRPα KI mice 4 days after treatment.

Supplementary Figure S5: Microscopic images of in vitro RBC agglutination induced by Hu5F9/gp120 at concentrations from 0.046 µg/ml to 100 µg/ml. Scale bar = 100µm

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 antitumor activity in vitro and in vivo across EGFR-driven tumor models and synergized effectively with KRAS inhibitors. Toxicological evaluations in non-human primates reveals 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.

Supplementary Figure S11: In vitro phagocytosis, in vivo anti-tumor efficacy, and body weight change data of TJH2201.

Supplementary Figure S1: Hematological effects of Hu5F9 on hCD47 KI mice and hCD47/SIRPα DKI mice.

Supplementary Figure S4: Hematological effects of biparatopic CD47 antibody, Hu5F9/B2B, in vitro and in vivo.

Supplementary Figure S7: Epitope binning of indicated CD47 antibodies by biolayer interferometry.

Supplementary Figure S9: Microscopic images of in vitro RBC agglutination induced by TJH2201 at concentrations from 0.046 µg/ml to 100 µg/ml. Scale bar = 100 µm

Antibody-drug conjugates (ADCs) have emerged as a distinguished class of anticancer therapeutics. ADCs targeting Trophoblast Cell Surface Antigen 2 (TROP2) have shown efficacy in treating various epithelial tumors, such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). Programmed Death Ligand 1 (PD-L1) serves as both an immune checkpoint and a tumor-associated antigen (TAA), commonly overexpressed in diverse tumor types. Growing evidence showed that ADCs can increase the efficacy of immunotherapeutic agents by multiple mechanisms, such as induction of immunogenic cell death, dendritic cell maturation, increase of T cell infiltration and expression of immune-regulatory proteins including PD-L1 and MHC. Both preclinical and early clinical studies suggest that ADCs and immunotherapy are promising combination options for tumor treatment. IBI3014 is an innovative TROP2 and PD-L1 dual-targeting ADC, comprising a humanized bispecific antibody linked to a novel DNA topoisomerase I inhibitor, NT1, via a cleavable linker. In vitro studies demonstrated the unconjugated bispecific antibody of IBI3014 has potent internalization activity potent immune checkpoint blocking activity. IBI3014 demonstrated superior cytotoxicity to benchmark and parental ADCs across several cell line-derived xenograft (CDX) models with different TROP2 and PD-L1 expression pattern, offering broader tumor type coverage. IBI3014 showed acceptable stability profile in mice and monkeys and can be well tolerated in monkeys with a highest non-severely toxic dose (HNSTD) of 50 mg/kg. Conclusion: IBI3014 represents a first-in-class bispecific ADC that integrates targeted cytotoxicity with checkpoint blockade within a single molecule. This dual mechanism of action not only enhances therapeutic efficacy but also maintains a favorable safety profile, which provides a promising approach for cancer therapy. Xiguang Zhang, Yanping Jin, Jinchang Lu, Ninghuan Li, Zhihai Wu, Min Wu, Yan Bian, Fenggen Fu, Weiwei Wu, Shuaixiang Zhou, Huizhong Xiong. IBI3014, a TROP2xPD-L1 bi-specific ADC integrating ADC killing with checkpoint blockade within one molecule, exhibits promising efficacy and safety in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 344.

Immune checkpoint blockade (ICB) therapies blocking PD1/PDL1 signaling have shown unprecedented clinical efficacy in multiple indications. However, the overall response rate for ICB-sensitive tumors are only 10-50%. Moreover, many types of cancer, e.g. pancreatic, ovarian, and MSS colorectal cancer, remain highly refractory to ICB therapies. Thus, it remains a significant unmet medical need to improve the responsiveness and durability of current ICB therapies. Based on emerging clinical evidence, it is possible to address this challenge by combining PD1 or PDL1 mAbs with drugs targeting complementary mechanisms. Indeed, next-generation ICB therapies, e.g. PD1 x IL2 fusion protein, PD1 x CTLA4 BsAb, and PD1 x VEGF BsAb, have shown promising clinical signals and improved efficacy compared to PD1 mAb. This underlines the potential of incorporating two clinically validated anti-cancer mechanism-of-action into one drug. IFNα has shown clinical efficacy and potential synergistic anti-cancer effect with PD1 mAb. Indeed, soluble IFNα has been approved to treat cancer. Similar to the development of other cytokine-based therapies, it is necessary and challenging to overcome off-tumor activity of IFNα to achieve efficacy-safety balance, limit systemic toxicity, and improve pharmacokinetics profile and drug exposure. Here, we engineered a PD1-IFNα fusion protein composed of a functionally attenuated IFNα and a clinically validated PD1 mAb, Sintilimab, aiming to specifically stimulate PD1-positive immune cells. This PD1-IFNα fusion protein utilized bivalent PD1 binders and induced potent PD1/PDL1 blockade. To minimize systemic toxicity induced by IFNα and improve developability, we engineered human IFNα-2b to decrease the affinity between IFNα and IFNα receptors. The PD1-IFNα fusion protein elicited highly PD1-dependent IFNα signaling, selectively activated in PD1-high cells compared to PD1-low cells. Systemic administration of a mouse surrogate molecule showed superior anti-tumor efficacy compared to PD1 mAb, with undetectable toxicity, in several mouse syngeneic tumor models. This PD1-IFNα fusion protein showed favorable pharmacokinetics profile in mice and drug developability. Together, our pre-clinical data demonstrated that a bifunctional PD1-IFNα fusion protein elicits robust PD1/PDL1 signaling blockade and PD1-dependent IFNα signaling activation to achieve anti-tumor efficacy, with potential to expand the benefits of ICB therapies to more cancer patients. Jinyang Li, Yi Wang, Mengqiang Di, Min Wu, Xueying Feng, Yiyi Li, Weiwei Wu, Xiguang Zhang, Shuaixiang Zhou, Huizhong Xiong. A PD1-IFNα fusion protein, with an attenuated IFNα fused to a clinically validated PD1 mAb, elicited PD1-dependent IFNα signaling and superior anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4881.

A leading HIV vaccine strategy requires a priming immunogen to induce broadly neutralizing antibody (bnAb) precursors, followed by a series of heterologous boosters to elicit somatic hypermutation (SHM) and produce bnAbs. In two randomized, open-label phase 1 human clinical trials, IAVI G002 in the United States and IAVI G003 in Rwanda and South Africa (IAVI, International Aids Vaccine Initiative), we evaluated the safety and immunogenicity of mRNA-encoded nanoparticles as priming immunogens (both trials) and first-boosting immunogens (IAVI G002). The vaccines were generally safe and well tolerated, except that 18% of IAVI G002 participants experienced skin reactions. Priming induced bnAb precursors with substantial frequencies and SHM; heterologous boosting elicited increased SHM, affinity, and neutralization activity toward bnAb development; and elicited antibodies exhibited precise bnAb structural mimicry. The results establish clinical proof of concept that heterologous boosting can advance bnAb precursor maturation and demonstrate bnAb priming in Africa, where the HIV burden is highest.

T cell engager (TCE) therapies targeting specific tumor associated antigens (TAAs) have shown clinical efficacy in multiple types of solid tumors. Indeed, TCE has been approved to treat melanoma and small cell lung cancer. Nevertheless, it remains necessary and challenging to improve the efficacy and safety profile of TCE to deliver benefits to more cancer patients. Recurrent and chemotherapy resistant ovarian cancer remains an urgent global unmet medical need. These is limited therapeutic options for this highly lethal and refractory disease. Recently, a MUC16 TCE demonstrated promising and durable clinical efficacy against heavily pretreated MUC16-high ovarian cancer, with acceptable safety profile. This underlines the potential to develop a MUC16 TCE with favorable safety profile and enhanced anti-tumor efficacy to possibly expand the benefits to MUC16-medium and MUC16-low ovarian cancer patients. MUC16 is a glycoprotein overexpressed in several types of solid tumors, especially in the majority of ovarian cancer. Here, we developed a 2+1 format MUC16 TCE, with improved tumor cell binding affinity, enhanced tumor cell killing potency, and MUC16-dependent T cell binding and activation. This MUC16 TCE targets membrane-proximal domains of MUC16 and achieves avidity-driven tumor cell binding in the presence of soluble CA125. Moreover, this MUC16 TCE elicited limited T cell binding and activation in the absence of MUC16, compared to benchmark molecule, to minimize off-target activity. This 2+1 format MUC16 TCE also showed favorable pharmacokinetics profile in mice. Functionally, systemic administration of this MUC16 TCE showed superior anti-tumor efficacy, with undetectable toxicity, in xenograft tumor models. Importantly, this MUC16 TCE demonstrated potent and synergistic anti-tumor efficacy in combination with clinically validated therapeutic strategies for ovarian cancer in pre-clinical tumor models. This provides rationale to develop combination therapies in future clinical trials. Indeed, combination of TCE with other therapies, e.g. ADC, chemotherapy, PD1/PDL1 mAb, are being tested clinically. Together, our pre-clinical data demonstrated that a 2+1 MUC16 TCE elicits potent anti-tumor efficacy and minimized off-target T cell activation, with potential to deliver benefits to ovarian cancer patients and patient with other MUC16-positive cancer. Jinyang Li, Yang Li, Nan Gao, Yi Wang, Mengqiang Di, Min Wu, Yiyi Li, Ran Xue, Shiyu Hao, Weiwei Wu, Xiguang Zhang, Li Li, Shuaixiang Zhou, Huizhong Xiong. A 2+1 format MUC16 targeting T cell engager induces MUC16-dependent T cell activity and superior anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3510.

Folate Receptor alpha (FRα) is a validated target that is overexpressed in multiple cancers with unmet medical need, including ovarian, lung and breast cancers, while restricted expression in normal tissues. Although mirvetuximab soravtansine (IMGN853), an antibody-drug conjugate (ADC) with a microtubule inhibitor, has been approved from the FDA for the treatment of FRα-expressing platinum-resistant ovarian cancer. There is still a large proportion of patients who do not respond to treatment, especially those with low FRα expression. We have developed a next-generation anti-FRα ADC, IBI3010, which consists of an FRα-targeting biparatopic antibody that is conjugated to a novel topoisomerase I inhibitor, NT1, with a homogeneous drug-to-antibody ratio of 8 (DAR8). Here, we describe the key pharmacological experiments, in vivo efficacy and results from the good laboratory practice (GLP) toxicology studies. In vitro studies have shown that the biparatopic antibody exhibits higher binding and internalization in tumor cells. IBI3010 shows comparable or better cytotoxicity to IMGN853 in various FRα expressing cancer cell lines. In addition, IBI3010 has superior in vitro bystander effect compared to IMGN853. In vivo efficacy was evaluated in various cell line-derived xenograft models with different FRα expression levels. IBI3010 exhibits superior antitumor activity to IMGN853, especially in the FRα low-expression models. In the GLP toxicology study, IBI3010 was tolerated in cynomolgus monkeys at all dose levels, with the highest dose of 60 mg/kg, defined as the highest non-severely toxic dose (HNSTD). Our findings support the clinical development of IBI3010 to evaluate its potential as an ADC therapeutic for FRα-expressing solid tumors. Bo Wang, Jiakai Xing, Shuaixiang Zhou, Weiwei Wu, Min Wu, Rongmei Zuo, Chenchen Zhu, Menghui Zhao, Li Li, Chengjie Cai, Nan Gao, Zhihai Wu, Ninghuan Li, Zeyu Liu, Jinling Xu, Bingliang Chen, Kaijie He. Preclinical characterization of IBI3010, a FRα targeting biparatopic antibody-drug conjugate (ADC), for the treatment of FRα expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB222.

Inhibition of EGFR signaling is clinically effective in colorectal cancer (CRC) as evidenced by the approval for Cetuximab in combination with chemotherapy or Encorafenib, a BRAF inhibitor, and the promising clinical results when combined with KRAS inhibitors, such as Adagrasib or Sotorasib. This underscores the clinical versatility of EGFR antibodies. However, these strategies are inevitably associated with high target-related toxicities, which limit the maximal potential of the drugs. Here, we designed IBI3019, a next-generation EGFR blocker that targets EGFR, CDH17 and CD16A. By targeting CDH17, an adhesion molecule overexpressed in CRC, we enhanced EGFR inhibition capabilities of the tri-specific antibody on the tumor while sparing normal tissues, particularly skin tissues, which are commonly associated with severe toxicities from EGFR blockers. Additionally, IBI3019 incorporates a high affinity CD16A single domain antibody that mediates stronger antibody-dependent cell cytotoxicity (ADCC) than IgG1 used in Cetuximab or the low fucosylated Fc of Amivantamab, a bispecific antibody targeting c-MET and EGFR, both in vitro and in vivo. IBI3019 demonstrated more potent in vitro efficacy than Cetuximab and Amivantamab in CRC cell lines, regardless of KRAS mutation status or the expression of CDH17, c-MET and EGFR. It exhibited strong synergy with Fulzerasib, a KRAS G12C inhibitor and MRTX1133, a KRAS-G12D inhibitor. In NCI-H508 and CCK81 CRC cell line-derived xenograft (CDX) models, IBI3019 suppressed tumor growth more effectively than Cetuximab and Amivantamab. Importantly, in a CRC patient-derived xenograft (PDX) model that is insensitive to Cetuximab, IBI3019 achieved near complete eradication of tumors in all mice. In pre-toxicity studies, cynomolgus monkeys were administrated 4 weekly doses of 50 mg/kg or 150 mg/kg of IBI3019 intravenously, and no mortality or serious toxicity was observed in either group. The highest non-severely toxic dose (HNSTD) of IBI3019 is 150 mg/kg and the calculated therapeutic index is 66. In contrast, previous reports indicated that 5 out of 10 monkeys treated with 75 mg/kg/week of Cetuximab exhibited early mortalities associated with excessive dermatologic toxicities. These results underscore the high safety profile of IBI3019. IBI3019 is a highly optimized FIC molecule with enhanced EGFR blocking capability and ADCC, maximizing tumor inhibition while minimizing toxicity. Its superior efficacy and safety profile support further clinical evaluation. Tiongsun Chia, Weiwei Wu, Li Li, Shuaixiang Zhou, Jia Lu, Shuming Lin, Ming Wu, Jia Liu, Feifei Wang, Ya Liu, Chenjuan Zhu, Ying Zhang, Yayao Yan, Huisi Liu, Jian Guan, Junjie Deng, Ninghuan Li, Zhihai Wu, Bin Li, Enhong Zhong, Bingliang Chen, Kaijie He. IBI3019, a first-in-class EGFR/CDH17/CD16A tri-specific antibody, demonstrated potent efficacy against CRC and an excellent safety profile in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4249.

A protective HIV vaccine will need to induce broadly neutralizing antibodies (bnAbs) in humans, but priming rare bnAb precursor B cells has been challenging. In a double-blinded, placebo-controlled phase 1 human clinical trial, the recombinant, germline-targeting envelope glycoprotein (Env) trimer BG505 SOSIP.v4.1-GT1.1, adjuvanted with AS01B, induced bnAb precursors of the VRC01-class at a high frequency in the majority of vaccine recipients. These bnAb precursors, which target the CD4 receptor binding site, had undergone somatic hypermutation characteristic of the VRC01-class. A subset of isolated VRC01-class monoclonal antibodies neutralized wild-type pseudoviruses and was structurally extremely similar to bnAb VRC01. These results further support germline-targeting approaches for human HIV vaccine design and demonstrate atomic-level manipulation of B cell responses with rational vaccine design.