In the past two decades, advances in biotechnology have brought improvements in protein engineering and manufacturing technology, and promoted the development of increasingly complex bispecific antibodies (BsAbs). At present, more than 100 kinds of BsAbs are undergoing clinical evaluation for cancer patients (including the marketed Blinatumomab and Catumaxomab). Among these BsAbs, bispecific immune cell binding proteins account for the majority. They have one arm targeting immune cell surface receptors and the other arm targeting tumor cell surface receptors. They can redirect specific immune effector cells to tumor cells. Although there are not many ongoing clinical trials of dual BsAb for tumor-associated antigen (TAA), the dual TAA strategy of BsAb still has many advantages, such as improving tumor selectivity, while regulating two functional pathways in tumor cells, and improving delivery and selectivity of payloads for ADC.

Fig.1 The mechanism of BsAb targeting dual TAAs

  • Improve Tumor Selectivity

Many tumor-targeted mAbs can eliminate tumor cells, but sometimes can also cause severe toxicity to healthy tissues. For example, anti-CD47 monoclonal antibody can block the “don’t eat me” signal expressed on tumor cells to avoid macrophage-mediated phagocytosis, but it also exists on red blood cells, platelets and other healthy cells. Anti-CD47 mAb causes severe anemia and thrombocytopenia, which may be the main reason for Celgene’s decision to terminate the phase I clinical study of CC-90002 (NCT02641002). In order to avoid this, BsAb is designed as a tumor-specific targeting arm to drive the selective binding of the second arm to the tumor after the affinity for CD47 is optimized. For example, TG-1801 (NI-1701) developed by TG Therapeutics is a BsAb (Fab×Fab-Fc, IgG1, 1+1) against CD19 and CD47. CD19 is a biomarker expressed only on normal B cell and B cell line malignant tumors. TG-1801 combines the low-affinity CD47 arm with the high-affinity CD19 arm, which can specifically block the “don’t eat me” signal on B cells, and potentially overcome the limitations of CD47 monospecific targeted therapy, as well as reduce the risk of unnecessary CD47 blockade in healthy cells. Similarly, the CD20 x CD47 BsAb IMM0306 (Fab×Ligand-Fc, IgG1, 2+2) developed by ImmuneOnco has achieved significant therapeutic effects in various tumor models, and it has not been shown to be combined with human red blood cells in preclinical studies.

In addition to hematological malignancies, there are also some BsAbs that have a similar mode of action in solid tumors to improve the blocking/activation specificity, such as IBI322 and RO6874813 (RO7386). IBI322 is a CD47 × PDL-1 BsAb developed by Innovent Biologics. It preferentially accumulates in PD-L1 positive solid tumors, thereby reducing the potential side effects of CD47 pathway blocking healthy cells.

  • Block Two Functional Pathways in Tumor Cells

Cancer is a highly complex and multifactorial disease involving multiple pathogenic proteins and crosstalk pathways. The interaction between different pathways supports a complex molecular network that may mediate tumor escape. Due to the inherent tumor heterogeneity, drug resistance is often observed in patients who relapse after single-molecule targeted therapy.

Epidermal Growth Factor Receptor (EGFR) is the first receptor tyrosine kinase discovered, which plays an important role in regulating cell proliferation, survival and differentiation. EGFR overexpression is related to the development of epithelial malignancies, such as non-small cell lung cancer, ovarian cancer, colorectal cancer and prostate cancer. Tyrosine kinase inhibitors (TKI) targeting the EGFR signaling cascade, such as Gefitinib and Erlotinib, have achieved clinical success in the past two decades, but they are also facing the challenge of drug resistance. For example, in patients with non-small cell lung cancer (NSCLC), the first-generation EGFR TKI showed a clinically meaningful response, but drug resistance was found to occur within a year or less. Although the second/third-generation TKI showed activity in drug-resistant patients, they will eventually develop acquired resistance due to new EGFR mutations. Another important reason for TKIs resistance is the activation of the RTK (receptor tyrosine kinase) pathway. For example, activation of the hepatocyte growth factor/mesenchymal-epithelial transformation factor (HGF/MET) pathway has been shown to often bypass EGFR TKI inhibitors. Considering this, two BsAbs targeting EGFR and c-met (JNJ61186372, Janssen; EMB01, EpimAb) have been under clinical studies.

JNJ-61186372, a humanized EGFR×c-met BsAb (Fab×Fab-Fc, IgG1, 1+1) was designed by Genmab and Janssen Biotech using DuoBody technology. JNJ-61186372 blocks the ligand-induced phosphorylation of epidermal growth factor receptor and c-met, and enhances antibody-dependent cellular cytotoxicity (ADCC) activity through FC engineering with low fucose content. In addition, JNJ-61186372 down-regulates the expression of receptors on tumor cells, thereby preventing drug resistance mediated by emerging EGFR or c-met mutations. In a phase I study (NCT02609776) involving 108 patients with NSCLC, JNJ-61186372 showed controllable safety and broad-spectrum anti-tumor effects in patients with drug resistance in EGFR exon 20 insertion, EGFR C797S mutation, MET amplification or third-generation EGFR TKI Osimertinib. Based on these data, on March 10, 2020, the FDA granted the new lung cancer drug JNJ-61186372 (JNJ-6372) Breakthrough Therapy Designation. Once approved, this will be the first targeted therapy for lung cancer patients with EGFR exon 20 insertion mutations.

In another example, a HER2 × HER3 BsAb (Zenocutuzumab, Fab × Fab-Fc, IgG1, 1+1) is undergoing clinical evaluation for the treatment of neuromodulin 1 (NRG1) fusion solid tumor patients. The NRG1 gene encodes neuregulin, the ligand of HER3, which binds to HER3, resulting in the formation of a heterodimeric complex of HER2 and HER3. It’s found in patients with HER2-driven cancers that it escapes HER2-targeted drugs through NRG1 activation of the HER3 pathway. NRG1 and partner gene fusion is a rare oncogene event that occurs in ~3% of NSCLC, ~1.5% of pancreatic cancer and less than 1% of other cancers, and in KRAS-wild-type pancreatic ductal adenocarcinoma, it appears frequently and provides a potential drug target for patients who do not benefit from KRAS inhibitors. Due to the high affinity of MCLA-128 to HER2, MCLA-128 binds to HER2 that is expressed on tumor cells and prevents the formation of HER2/3 heterodimers and the binding of neuregulin to HER3, thereby inhibiting tumors proliferation of cells.

  • Increase the Selectivity of ADC Payload Delivery

Antibody-drug conjugate (ADC) therapy combines the targeting accuracy of antibodies with the cytotoxic activity of highly effective cytotoxic payloads by combining with monoclonal antibodies. Once the drug-bound antibody binds to the antigen on the tumor cell surface, the ADC is internalized by receptor-mediated endocytosis, and the toxic payload is released. In the case that tumor-specific mAb targets are significantly absent or tumor-selective targets are not always well internalized, compared to ADCs based on monospecific antibodies selectivity, ADC-BsAb can be designed to increase payload delivery thus to enhance its internalization or overcome the escape mechanism of tumor cells.

The approval of Enmetrastuzumab for the treatment of metastatic breast cancer confirms that HER2 can be an effective ADC target. However, the internalization of the ADC for HER2 usually relies on the cross-linking of HER2 molecules, and the monomeric HER2 cannot be internalized well. In order to improve the internalization ability of HER2 targeting ADC, a BsAb-based CD63 and HER2 targeting ADC was designed. CD63, also known as lysosomal-associated membrane protein 3 (LAMP3), is a member of the Tetraspanin superfamily. It shuttles between cell membranes and cells, and is highly expressed in pancreatic cancer, gastric cancer and melanoma. HER2×CD63 BsAb showed strong internalization, lysosomal accumulation and cytotoxicity in HER2-positive tumor cells, while its internalization effect on HER2-negative cells was weak.

CD19 and CD22 targeted therapies have successfully treated B-cell lymphoma and rare hairy cell leukemia (HCL), respectively. However, for CD19 targeted therapy, a group of cancer cells in B-lineage leukemia patients turned to express CD22, thus escaping the killing mediated by CD19 targeted therapy. For CD22 targeted therapy, HCL accounts for only a small part of leukemia patients, and it is crucial to expand the use of the drug to a wider range of patients. In order to overcome these resistance mechanisms, GT Biopharma has developed OXS-1550 (scFv×scFv, 1+1), which is a CD19 × CD22 BsAb linked to a modified form of diphtheria toxin and is currently under phase I studies of treating patients suffering from relapsed or refractory B-cell lymphoma or leukemia.

Although there are not many clinical trials targeting BsAbs targeting dual TAA, early clinical studies have proven to have good anti-tumor effects in hematological malignancies and solid tumors. It’s believed that based on the unique advantages, this method can unveil great potential in the future.