A bispecific antibody and preparation and use thereof

Abstract

The application provides a kind of bispecific antibody and its preparation and application, the bispecific antibody includes two chains: from N end to C end in order: VH1 (or VL1), L1, VH2, CH1, the first polypeptide chain of Fc, and from N end to C end in order: VL1 (or VH1), L2, VL2, CL, second polypeptide chain.It is realized tumor selective accumulation by being located in the N end of double antibody structure of the binding domain targeted to tumor associated antigen (TAA), and its shield is located in the inside immune checkpoint antibody fragment to the binding activity of immune checkpoint to reduce systemic activity.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a bispecific antibody and its preparation and application. Background Technology

[0002] Cancer immunotherapy has revolutionized clinical practice in oncology, with immune checkpoint blockade therapy becoming a cornerstone treatment for advanced malignancies. Programmed death-ligand 1 (PD-L1) is an inhibitory receptor expressed on the surface of tumor cells that binds to the programmed death 1 (PD-1) receptor on T lymphocytes. The PD-L1 / PD-1 binding axis constitutes a key immunosuppressive mechanism, whereby PD-L1 on the surface of tumor cells binds to the PD-1 receptor on T cells, thereby effectively suppressing the anti-tumor immune response. PD-L1-targeting antibodies, such as atezolizumab (ATE), as representative drugs of immune checkpoint blockade therapy, reverse this immunosuppression by blocking the interaction between PD-L1 and PD-1, enabling cytotoxic T cells to regain their ability to recognize and eliminate tumor cells [Trends Mol Med. 2015;21:24–33;Nat Rev Immunol. 2018;18:153–67;International Journal of Molecular Sciences, 2020. 21(15)].

[0003] However, despite the significant success of PD-1 / PD-L1 inhibitors in the treatment of various cancers, their clinical application is still limited by immune-related adverse events (irAEs). After PD-1 / PD-L1 blockade, overactivation of the immune system may trigger multi-organ toxicity reactions such as gastrointestinal, liver, or skin [Ann Oncol, 2015. 26(12): p. 2375-91; SciRep, 2022. 12(1): p. 20038; Front Immunol, 2022. 13: p. 1070961; Cutan OculToxicol, 2022. 41(1): p. 73-90]. Importantly, a meta-analysis of clinical trials further showed that the incidence of grade 3-4 adverse events was higher (55%) with combination therapy, significantly higher than with ipilimumab (27%) or nivolumab (15%) monotherapy [J Emerg Med, 2018. 55(4): p. 489-502; JAMA Oncol, 2016. 2(10): p. 1346-1353].

[0004] Bispecific antibodies have become a revolutionary treatment approach in precision immunotherapy, enhancing efficacy and safety by simultaneously targeting two different targets. Among them, tumor-associated antigen (TAA) × PD-(L)1 bispecific antibodies have attracted much attention due to their ability to simultaneously target TAA and PD-(L)1 and exhibit stronger anti-tumor activity [Transl Oncol, 2021. 14(1): p. 100916; Acta Pharmacol Sin, 2022. 43(3): p. 672-680; Oncoimmunology, 2018. 7(8): p. e1466016; Clin Cancer Res, 2020. 26(15): p.4154-4167]. However, whether TAA × PD-L1 bispecific antibodies can accurately distinguish between target tumor tissue and non-target normal tissue in vivo (which is crucial for improving the safety of PD-(L)1 inhibitors) remains a major challenge. Brief Description of the Invention

[0005] To enable the drug to accumulate in specific tumor sites enriched with tumor-associated antigens (TAAs) and reduce immune-related adverse events caused by immune checkpoint inhibitors, this invention provides a bispecific antibody that places the binding domain targeting the TAA at the N-terminus of the bispecific antibody structure to achieve selective accumulation at tumor sites (significantly improving the accuracy of distinguishing between tumor tissue and non-target normal tissue), and uses it to mask the binding activity of the immune checkpoint antibody fragment located on the inner side to immune checkpoints to reduce systemic activity.

[0006] A first aspect of the present invention provides a bispecific antibody comprising two chains:

[0007] From N-terminus to C-terminus, the sequence is: VH1 (or VL1), L1, VH2, CH1, Fc, the first polypeptide chain, and...

[0008] From the N-terminus to the C-terminus, the second polypeptide chain consists of VL1 (or VH1), L2, VL2, and CL.

[0009] The first binding domain, composed of VH1 and VL1, binds to tumor cell surface antigens, and the second binding domain, composed of VH2 and VL2, binds to immune checkpoint antigens. The EC50 of the second binding domain binding to immune checkpoint antigens is 2 to 50 times that of the first binding domain binding to tumor cell surface antigens. Preferably, the EC50 of the second binding domain binding to immune checkpoint antigens is 40 to 50 times, 30 to 50 times, 30 to 40 times, 20 to 30 times, 10 to 20 times, 2 to 10 times, 5 to 10 times, or 2 to 5 times that of the first binding domain binding to tumor cell surface antigens.

[0010] The bispecific antibody of the present invention contains an immune checkpoint antigen selected from, but not limited to, PD-L1, PD-1, Tim3, LAG-3, 41BB, etc.; preferably, the immune checkpoint antigen is selected from PD-L1.

[0011] In a specific implementation, the second binding domain of the immune checkpoint protein PD-L1 has VH2 as shown in SEQ ID NO.30 and VL2 as shown in SEQ ID NO.31, or has VH2 as shown in SEQ ID NO.32 and VL2 as shown in SEQ ID NO.33.

[0012] In specific embodiments, the tumor-associated antigen is selected from, but not limited to, GPC3, EGFR, HER2, Nectin4, Trop2, PSMA, MUC14, etc. In one specific embodiment, the tumor-associated antigen is EGFR; preferably, the first binding domain of the tumor-associated antigen EGFR has VH1 as shown in SEQ ID NO.34 and VL1 as shown in SEQ ID NO.35. In one specific embodiment, the tumor-associated antigen is GPC3; preferably, the first binding domain of the tumor-associated antigen GPC3 has VH1 as shown in SEQ ID NO.36 and VL1 as shown in SEQ ID NO.37.

[0013] In specific embodiments, L1 and L2 are optionally selected from the linker peptides shown in SEQ ID NO.38-SEQ ID NO.41. Preferably, L1 linking VH1 and VH2 has the amino acid sequence shown in SEQ ID NO.38, and L2 linking VL1 and VL2 has the amino acid sequence shown in SEQ ID NO.39; preferably, L1 linking VH1 and VH2 has the amino acid sequence shown in SEQ ID NO.38, and L2 linking VL1 and VL2 has the amino acid sequence shown in SEQ ID NO.41; preferably, preferably, L1 linking VH1 and VH2 has the amino acid sequence shown in SEQ ID NO.40, and L2 linking VL1 and VL2 has the amino acid sequence shown in SEQ ID NO.39; preferably, L1 linking VH1 and VH2 has the amino acid sequence shown in SEQ ID NO.40, and L2 linking VL1 and VL2 has the amino acid sequence shown in SEQ ID NO.41.

[0014] In a specific embodiment, the Fc of the first polypeptide chain and the second polypeptide chain is selected from IgG1, IgG2, IgG3, or IgG4. The Fc can be derived from human, mouse, or monkey. In one specific embodiment, the Fc is a wild-type Fc or an Fc variant; preferably, the Fc has an amino acid sequence as shown in any of SEQ ID NO. 42-44.

[0015] In specific embodiments, the bispecific antibody of the present invention comprises a heavy chain and a light chain having the following amino acid sequences respectively: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:4 and SEQ ID NO:2; SEQ ID NO:4 and SEQ ID NO:3; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:6; SEQ ID NO:10 and SEQ ID NO:11; SEQ ID NO:10 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:14; SEQ ID NO:20 and SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23; SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:24 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:26; SEQ ID NO:28 and SEQ ID NO:29 ... NO:45; SEQ ID NO:46 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:41.

[0016] In a second aspect, the present invention provides a polynucleotide encoding a first polypeptide chain and / or a second polypeptide chain of a bispecific antibody as described above.

[0017] A third aspect of the invention provides a carrier comprising the polynucleotides described above.

[0018] In a fourth aspect, the present invention provides a host cell comprising the bispecific antibody as described above.

[0019] A fifth aspect of the invention provides a pharmaceutical composition comprising the bispecific antibody as described above.

[0020] A sixth aspect of the invention provides the use of the bispecific antibody as described above in the preparation of a medicament for tumor treatment. Preferably, the tumor is a solid tumor or a hematologic malignancy; further, the solid tumor is selected from non-small cell lung cancer, colorectal cancer, or liver cancer.

[0021] The TAA-targeting bispecific antibody of this invention can achieve tumor-site-specific immune activation through antigen-driven cell membrane enrichment, dependent on TAA expression levels. The enhanced affinity resulting from dual targeting compensates for the fine-tuning of PD-L1 affinity, thereby achieving localized immune checkpoint blockade. Since TAA expression levels are low in normal tissues, its immune checkpoint blockade effect is negligible, thus minimizing the off-target toxicity of PD-L1 antagonists. Attached Figure Description

[0022] Figure 1 Schematic diagram of bispecific antibody architecture

[0023] Figure 2 This is an SDS-PAGE electrophoresis image of different bispecific antibodies under reducing and non-reducing conditions. Lane "+" indicates reducing conditions, lane "-" indicates non-reducing conditions, and lane "M" is a molecular weight marker for the pre-stained protein.

[0024] Figure 3 Size exclusion chromatography results of different double-antibody gels

[0025] Figure 4 The solutions were represented by different dual-antibody solutions (Tm). The Cross configuration showed a higher Tm value, indicating stronger conformational stability under thermal stress.

[0026] Figure 5 Binding of the double antibody for FACS detection to MC38 cells (5A), CHO-K-hPDL1 (5B), MC38-hEGFR (5C), and MC38-hGPC3 cells (5D).

[0027] Figure 6 To assess the sequential binding capacity of Cross-LS / HS-GPC3 to antigens (PDL1 and GPC3) by bridging ELISA.

[0028] Figure 7 The blocking ability of different antibodies against the PD1 / PDL1 pathway, as detected by ELISA.

[0029] Figure 8 To determine the titer of anti-drug antibodies (ADA) by ELISA after four consecutive injections of cross-LS / HS-GPC3 DVD-Ig bispecific antibody at 2 mg / kg or 1 mg / kg every three days in mice, the antibody was administered at this dose.

[0030] Figure 9 To investigate the inhibitory effects of different doses of bispecific antibodies on tumors in MC38-hGPC3 tumor-bearing mice.

[0031] Figure 10The inhibitory effect of cross-LS / HS-GPC3 on tumors in MC38-hEGFR (10A) or MC38-hGPC3 (10B) tumor-bearing mice.

[0032] Figure 11 To investigate the effect of reducing the frequency of Cross-LS / HS-GPC3 administration on tumor suppression in different tumor-bearing mice.

[0033] Figure 12 Tumor volume data for tumor-infiltrating lymphocyte (TIL) analysis in MC38-hGPC3 models treated with cross-LS / HS-GPC3 (2 mg / kg) or control.

[0034] Figure 13 In MC38-hGPC3 models treated with cross-LS / HS-GPC3 (2 mg / kg) or control, the percentages of mCD3+ T cells, mCD8+ T cells, and mCD4+ T cells in the tumor microenvironment (TME), as well as the CD8+ / CD4+ ratio, were measured.

[0035] Figure 14 To demonstrate that cross-LS / HS-GPC3 loses its inhibitory effect on tumor growth in the absence of mCD8+ T cells, mice carrying MC38-hGPC3 tumors were treated with either DPBS or Cross-LS / HS-GPC3 on days 6, 8, 10, and 12, respectively. To eliminate CD8+ T cells, mice were injected with 200 µg of aCD8 antibody on days 4, 8, and 12. Detailed Implementation

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0037] Example

[0038] The embodiments are for illustrative purposes only and are not intended to limit the invention in any way.

[0039] Example 1: Construction, expression, and purification of bispecific antibodies

[0040] Genes encoding the VH and VL domains of panitumumab (aEGFR mAb), atezolizumab (ATE) (aPDL1), MDX1105 (aPDL1 mAb), and GC33 (aGPC mAb) were synthesized by Genwiz and amplified by PCR using 2×Phanta Flash Master Mix (Vazyme). The amplified PCR products were cloned into pFuse-hIgG1-Fc (InvivoGen). The constructed antibody heavy and light chain genes were co-transfected into FreeStyle 293-F cells according to the manufacturer's protocol. Five days after transfection, the culture supernatant containing the secreted proteins was collected and purified using Protein A (GenScript). The purified proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions. Figure 2 The analysis was performed using size exclusion chromatography (SEC)-HPLC (GE Healthcare) to analyze protein aggregates. Figure 3 The dissolution temperature (Tm value) of the bispecific antibody was calculated using the Applied Biosystems™ Protein Thermal Shift Assay (Thermo Fisher). Figure 4 The structure of the bispecific antibody is shown in [reference needed]. Figure 1 The sequence is shown in Table 1.

[0041]

[0042] Example 2: ELISA Combination Experiment

[0043] The binding affinity of antibodies (including bispecific antibodies and control antibodies) to PD-L1, EGFR, and GPC3 antigens was assessed using ELISA. Antigen proteins (1 µg / mL) were diluted in DPBS (pH 7.4) and coated into 96-well plates, incubated overnight at 4°C. After blocking with 3% skim milk (dissolved in DPBS, pH 7.4) at 37°C for 1 hour, the bispecific antibodies and control antibodies (panitumumab, atezolizumab, and GC33) were serially diluted 5-fold and added to the plates, incubated at 37°C for 1 hour. After washing three times with DPBS containing 0.05% Tween-20, a 1:500 dilution of HRP-labeled goat anti-human IgG Fc secondary antibody (Southern Biotech) was added, and the plates were incubated at room temperature for 1 hour. Finally, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added, and the plates were incubated at room temperature in the dark for 10 minutes. The absorbance was measured at 650 nm using a CLARIOstar Plus microplate reader, and the curves were plotted and the data analyzed using GraphPad Prism software. The EC50 of the bispecific antibody binding to the antigen is shown in Table 2-5.

[0044] The results are shown in Tables 2-5. All bispecific antibody constructs bound tumor surface antigens with high affinity, and their EC50 binding values ​​were similar to those of the parent monoclonal antibodies (aEGFR or aGPC3 mAb). The affinity for PD-L1 was closely related to the parent monoclonal antibody (ATE or MDX1105) used and the linker. The EC50 of ATE-based bispecific antibodies binding immune checkpoint inhibitors was 2–47.61 times higher than that binding tumor surface antigens, while the EC50 of MDX-based bispecific antibodies binding immune checkpoint inhibitors was 2.33–2.79 times higher than that binding tumor surface antigens.

[0045]

[0046]

[0047]

[0048]

[0049] Example 3: Flow cytometry combined with affinity analysis

[0050] The binding ability of bispecific antibodies was assessed by flow cytometry. Experimental cells were incubated with serially diluted bispecific antibodies (panitumumab, atezolizumab, and GC33) at 4°C for 1 hour. Cells were washed three times with DPBS containing 2% fetal bovine serum (FBS), and then incubated with mouse anti-human IgG Fc APC-labeled secondary antibody at 4°C for 40 minutes. After washing three times with DPBS containing 2% FBS, cell binding was detected by flow cytometry. GraphPad Prism software was used to plot curves and analyze the data.

[0051] The results are as follows Figure 5As shown, the binding activity of the four bispecific antibodies on MC38 (endogenously expressing mPD-L1) and CHO-K-hPDL1 (exogenously expressing hPD-L1) cells was significantly reduced (EC50 values ​​were all greater than 25 nM), representing a reduction of more than 185-fold compared to ATE monoclonal antibodies (EC50 = 0.047 nM for MC38; EC50 = 0.149 nM for CHO-K-hPDL1). Subsequently, the binding of these antibodies to tumor antigens was studied using double-positive cells co-expressing mPD-L1 with human EGFR (MC38-hEGFR) or co-expressing mPD-L1 and human GPC3 (MC38-hGPC3). Compared to binding on MC38 cells, the four bispecific antibodies showed significantly enhanced binding to human EGFR or human GPC3 antigens on double-positive cells: EC50 values ​​showed a >625-fold increase in binding activity, and the median fluorescence intensity (MFI) increased by 14-fold. The antigen affinity exhibited by all four bispecific antibodies remained strongly correlated with their parent monoclonal antibodies. These functional studies suggest that bispecific antibodies targeting tumor-associated antigens can achieve efficient aggregation, potentially contributing to the blocking of PD1 / PD-L1 interactions on the surface of specific tumor cells.

[0052] Example 4: Dual-target bridging ELISA experiment

[0053] To verify that the aggregation of bispecific antibodies (Cross-LS / HS-GPC3) on the surface of specific tumor cells can promote PD-1 / PD-L1 blockade, we first used a bridging ELISA method to assess the dual-target binding ability of the bispecific antibodies. hPD-L1-his or hGPC3-his protein (2 µg / ml) was coated onto 96-well immunoassay plates and incubated overnight at 4°C. After washing with PBS, the plates were blocked with PBS solution containing 1% BSA for 1 hour, and then incubated at 37°C with 5-fold serially diluted bispecific antibodies for 1 hour. After washing three times with PBS, 2 µg / ml of biotinylated GPC3 or biotinylated PD-L1 protein was added, and the plates were incubated at 37°C for 1 hour. After washing, streptavidin-HRP was added, and the plates were incubated at room temperature for 1 hour. After washing again, the plates were developed with TMB substrate. The reaction was terminated by adding 1 M H2SO4, and the absorbance was measured at 450 nm using a CLARIOstar Plus reader.

[0054] After the bispecific antibody binds to the coated hPD-L1-his, the co-binding is then detected using the hGPC3-his antigen. Figure 6The binding activity shown is comparable to that detected based on the anti-Fc fragment, indicating that after the bispecific antibody binds to PDL1, the GPC3 binding domain remains accessible, forming a PDL1-bispecific antibody-GPC3 trimer complex, and this binding conforms to the classic antibody-antigen interaction mechanism. Similarly, when GPC3 is captured first, the PD-L1 detection results show that while the binding ability of ATE is retained, it is weakened, consistent with the conclusion of weaker PD-L1 binding observed in previous ELISA studies (Tables 2-5). Regardless of the antigen capture order, the PDL1-bispecific antibody-GPC3 crosslinking can be achieved, demonstrating that the GPC3xPDL1 construct Cross-LS / HS-GPC3 possesses an intrinsic dual-target co-binding capability.

[0055] Example 5: Detection of PD1 / PD-L1 blocking bioactivity

[0056] Blockage of PD1 / PD-L1 interaction was assessed using an alternative method. This assay used Jurkat-NFAT-Luc-hPD1 cells (expressing PD1 and transfected with GPC3 CAR) as effector cells and HepG2-hPD-L1 cells (highly expressing GPC3, low EGFR, and high PD-L1) as target cells. Effector and target cells were seeded at a density of 10,000 cells per well and co-cultured with diluted antibody. After 24 hours of incubation, the supernatant was collected, and IL-2 levels were analyzed using a commercial ELISA kit according to the manufacturer's instructions. Increased IL-2 production following NFAT activation indicated that PD-1-mediated inhibition was reversed.

[0057] The results are as follows Figure 7As shown, HS / LS-GPC3 (EC50 = 0.59 nM) and cross-LS / HS-GPC3 (EC50 = 0.43 nM) exhibited significant PD-1 / PD-L1 inhibitory activity, comparable to the control Atezolizumab (EC50 = 0.17 nM). In contrast, HS / LS-EGFR (EC50 = 23.69 nM) showed a significantly reduced potency (a 139-fold decrease), consistent with the reduced cell membrane accumulation capacity observed in the flow cytometry analysis. The high consistency between the EC50 values ​​in the blocking assay and the antigen-binding affinity analysis establishes a direct structure-function relationship, confirming that the bispecific antibody targeting GPC3 achieves effective immune checkpoint inhibition through tumor-associated antigen (TAA)-driven localization. This restricted activity characteristic reflects the spatial selectivity advantage of the bispecific antibody architecture: precisely guiding immune checkpoint regulation to the tumor microenvironment by recognizing TAAs. The core of this TAA-dependent effect lies in the fact that high TAA binding affinity and cell surface density can compensate for the weakened PD-L1 binding affinity of bispecific antibodies, thereby enhancing their potential to selectively exert IC blocking function at TAA-positive tumor sites, while minimizing immune-related adverse reactions (irAEs) in other PD-L1-positive normal tissues.

[0058] Example 6 Pharmacokinetic Study

[0059] Pharmacokinetic studies were conducted in female Sprague-Dawley (CD-SD) rats. Following a single subcutaneous injection of Cross-LS / HS-GPC3 and control samples at a dose of 10 mg / kg into CD-SD rats, blood samples were collected at serial time points, and serum concentrations of the intact bispecific antibodies and their structural backbone were quantified by ELISA. GPC3 targeting capture assays and PD-L1 orientation assays were used, both quantified using anti-human Fc assays.

[0060] The results are shown in Table 6. The intact bispecific antibody exhibited rapid absorption kinetics, reaching its maximum serum concentration (Cmax: 31.32 μg / mL) 72 hours after administration. However, rapid elimination began on day 5, characterized by accelerated systemic elimination (serum concentration decreased by >64% compared to Cmax), ultimately resulting in a shorter terminal half-life compared to conventional monoclonal antibodies (T1 / 2: 48.9 h). Parallel PK curves of the intact bispecific antibody and its backbone antibody confirmed structural integrity, with no breakage or detectable dissociation, validating the stability of the engineered complementary site pairing.

[0061]

[0062] Example 7: In vivo immunogenicity assay

[0063] Given previous reports (Sci Rep 11, 5774 (2021)) of a high incidence of antidrug antibodies (ADA) in atezolizumab-treated mouse models, this study used C57BL / 6 mice to assess the ADA titer of bispecific antibodies. Four mice per group were administered the cross-LS / HS-GPC3 bispecific antibody every three days for a total of four doses. ADA titers in mouse blood were detected by ELISA. The procedure was briefly as follows: each well of a 96-well plate was coated overnight at 4°C with 1 µg / mL of cross-LS / HS-GPC3, HS / LS-EGFR, or atezolizumab in PBS. After washing three times with PBS, the plate was blocked for 1 hour at room temperature with 1% BSA in PBS. Serum samples were incubated with the pretreated serum in 1% BSA in PBS at room temperature for 1 hour. After washing three times with PBS, HRP-labeled goat anti-mouse IgG Fc specific secondary antibody (Invitrogen) was added, and the mixture was incubated in 1% BSA in PBS solution at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween 20 (PBST), HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) solution was added. After 30 minutes, the reaction was terminated with 1M H2SO4, and the absorbance was measured at 450 nm using a CLARIOstar Plus microplate reader.

[0064] The results are as follows Figure 8 As shown, all bispecific antibody constructs elicited significant ADA titers (OD > 0.4, compared to OD < 0.2 in the DPBS group) over four dosing cycles. The observed immunogenicity can likely be attributed to the shared humanized framework sequence between the GPC3 / EGFR targeting domain and the ATE-derived PD-L1 complementation site, sequences that are heterologous in mice. The observed ADA response was directly correlated with accelerated clearance kinetics, suggesting that immune-mediated clearance is a key factor contributing to the shortened half-life in pharmacokinetics.

[0065] Example 8: Therapeutic effect study in a tumor model

[0066] The therapeutic potential of the bispecific antibody was evaluated in immunocompetent C57BL / 6 mice carrying MC38-hGPC3 tumors (GPC3 overexpression in this model). Five × 10^6 MC38-hGPC3 cells were subcutaneously injected into the right abdomen of each mouse. When the tumor volume reached approximately 50–100 mm³, the mice were grouped and treated with either intraperitoneal (ip) bispecific antibody or DPBS. Mouse weight and tumor size were measured and recorded every two days using calipers. Tumor volume was calculated using the formula: length × 1 / 2 (width)².

[0067] Tumor tissue was collected and dissociated into single-cell suspensions using standard mechanical and enzymatic methods. Cells were blocked using mouse TruStain FcX™ following the manufacturer's instructions. Cells were stained with an antibody staining combination. Cells were analyzed using a BECKMAN flow cytometer. Data were analyzed using CytExpert and GraphPad Prism software.

[0068] The results are as follows Figure 9 As shown, when the tumor volume reached 50–100 mm³, intraperitoneal injection of bispecific antibodies (6, 3, and 1 mg / kg) was administered every other day, and no treatment-related adverse events were observed during the monitoring period. Compared with the solvent-controlled DPBS group on day 12, both bispecific antibodies potently inhibited tumor growth at all tested doses (1–6 mg / kg) (TGI 68.29%–75.84%). Comparable efficacy was observed at 1 mg / kg and 6 mg / kg doses, indicating that target saturation was reached at the lowest tested dose (1 mg / kg), which may be attributed to the high aggregation resulting from GPC3-guided tumor localization. The robust efficacy shown in in vivo was contrasted by the decreased PDL1 binding affinity observed in vitro. Figure 5 or Figure 7 In contrast, this means that TAA-mediated spatial enrichment compensates for moderate immune checkpoint targeting potency.

[0069] To elucidate the GPC3-dependent antitumor mechanism, a comparative efficacy study was conducted in MC38-hEGFR and MC38-hGPC3 homology models. In mice with established tumors, low-dose (1 mg / kg) bispecific antibodies against HS / LS-GPC3 or cross-LS / HS-GPC3 were administered, with HS / LS-EGFR serving as a non-GPC3 but EGFR-specific control. Results showed that both GPC3 bispecific antibodies exhibited tumor growth inhibition (TGI) in the MC38-hGPC3 model. Figure 10 B). By day 8 post-treatment, both HS / LS-GPC3 and cross-LS / HS-GPC3 showed moderate nonspecific tumor suppression in the EGFR-overexpression model (mean TGI: 34%). Figure 10 A). However, with systemic accumulation of the drug, by day 10, HS / LS-GPC3 showed tumor suppressor activity comparable to the control HS / LS-EGFR (TGI: 55%). Notably, cross-LS / HS-GPC3 exhibited strict antigen selectivity, showing potent efficacy in GPC3-overexpressing tumors (TGI: 73%), but weaker activity in EGFR-overexpressing models (TGI: 32%), highlighting its structural precision.

[0070] To investigate whether in vitro GPC3-targeted immune checkpoint blockade can translate into in vivo antitumor selectivity, we introduced a model system to test the ability of GPC3×PD-L1 to selectively target and eliminate double-positive tumors (rather than single-positive untargeted tumors). In this model system, we established the MC38-hEGFR model, characterized by high PD-L1 expression, low GPC3 expression, and high EGFR expression, to simulate untargeted “normal tissue,” while MC38-hGPC3 represented the “targeted tumor.” Based on previous evidence regarding GPC3-driven membrane enrichment, we hypothesized that spatially constrained PD-L1 blockade would produce GPC3-dependent therapeutic selectivity: a potent TGI in the MC38-hGPC3 “targeted tumor” model, while a very low TGI was observed in the MC38-hEGFR “normal tissue” control. We adjusted the onset time and frequency of administration, starting when the tumor volume reached 500 mm³, and extended the dosing interval from every other day to every 4 days. Starting from day 8, GPC3×PD-L1 (1-2 mg / kg) or a solvent was administered intraperitoneally every 4 days for a total of three administrations. In the MC38-hGPC3 model, the cross-LS / HS-GPC3 bispecific antibody showed dose-dependent selectivity (…). Figure 11 A): A dose of 2 mg / kg induced significant tumor suppression in MC38-hGPC3 “targeted tumor” (TGI: 80%; **p < 0.01 compared to DPBS), while a dose of 1 mg / kg also showed efficacy on day 16 (TGI: 65.30%, *p < 0.05), but tumor recurrence occurred after the last treatment. Figure 11 A). In the MC38-hEGFR model, neither dosage group altered tumor progression, and its growth kinetics were consistent with the DPBS control group. Figure 11 B). This significant difference in response between “targeted tumors” and “normal tissues” demonstrates the in vivo selectivity of GPC3×PD-L1 and confirms its GPC3-dependent therapeutic efficacy.

[0071] To directly monitor tumor-targeting distribution, cross-LS / HS-GPC3 labeled with Cy5.5 was injected into MC38-hGPC3 or MC38-hEGFR tumor-bearing mice, respectively. Quantitative analysis using regions of interest (ROIs) delineated in the tumor regions showed that Cy5.5-cross-LS / HS-GPC3 uptake in MC38-hGPC3 and MC38-hEGFR tumor tissues at 8 and 12 hours was significantly different (*P < 0.05). Figure 11C). The mean radiation efficiency of the MC38-hGPC3 tumor at 12 hours was (4.51 ± 0.25) × 10⁻⁶. 8 The value of [p / s / cm² / sr] / [μW / cm²] was lower in the control group, while the value of MC38-hEGFR tumors was (2.34 ± 0.48) × 10⁻⁶. 8 This indicates that it has selective accumulation properties in GPC3-positive tumors.

[0072] Semi-quantitative analysis of the MC38-hGPC3 and MC38-hEGFR groups also showed differences in mean radiation efficiency between tumors and organs. Figure 11 D). In the MC38-hEGFR group, hepatic Cy5.5 signaling uptake was higher (tumor: 4.42 ± 0.30 × 10⁻⁶). 8 vs. liver: 5.67 ± 0.08 ×10 8 *P < 0.05), while in the MC38-hGPC3 group, tumor and hepatic uptake levels were comparable (tumor: 4.78 ± 0.87 × 10⁻⁶). 8 vs. Liver: 5.02 ± 0.16 × 10 8 (No statistical difference, P = 0.80) further indicates that Cy5.5 accumulates very little in EGFR-positive tumors, providing evidence for the low accumulation of cross-LS / HS-GPC3 in "normal tissue".

[0073] Example 9 Analysis of tumor-infiltrating T lymphocytes

[0074] Based on the established mechanism of atezolizumab—namely, restoring CD8+ T cell-mediated anti-tumor immunity by blocking the PD-1 / PD-L1 signaling pathway and promoting T cell infiltration—we aimed to investigate whether a bispecific GPC3×PD-L1 antibody could induce a similar atezolizumab-derived immune response in MC38-hGPC3 tumors. Mice carrying MC38-hGPC3 tumors were treated with GPC3×PD-L1 (2 mg / kg, every other day from day 6 to day 12) or with a solvent. Tumor tissue was collected on day 14 post-implantation (tumor growth inhibition rate TGI: 75%, ****P < 0.0001). Figure 12 Immunophenotypic analysis of tumor-infiltrating lymphocytes (TILs) was performed using flow cytometry. Subsequently, the animals were euthanized and tumor tissue was collected for flow cytometry analysis of bispecific antibody-driven lymphoid compartment remodeling.

[0075] like Figure 13As shown in Figure A, cross-LS / HS-GPC3 treatment significantly expanded CD3+ T cells in the CD45+ lymphocyte population (34.73% vs. 22.35% in the control group; **p < 0.01), consistent with potent antitumor activity. In particular, the CD8+ / CD4+ T cell ratio increased 3.1-fold from 1.78 in the control group to 5.31 in the cross-LS / HS-GPC3 group (P < 0.01). Figure 13 (D) This change was driven by a 2.3-fold increase in tumor-infiltrating CD8+ cytotoxic T lymphocytes (CTLs) (41.9% in the cross-LS / HS-GPC3 group and 15.2% in the control group; ***p < 0.001) Figure 13 B), while the proportion of CD4+ T lymphocytes did not change significantly (8.87% in the cross-LS / HS-GPC3 group and 8.44% in the control group; p=0.77). Figure 13 C). These results indicate that cross-LS / HS-GPC3 therapy achieves GPC3-guided PD-L1 blockade and reprograms the tumor microenvironment (TME) into a pro-inflammatory state dominated by effector CTLs, thereby effectively promoting tumor regression in vivo.

[0076] To verify the functional necessity of CTLs, antibody-mediated CD8+ T cell exhaustion experiments were performed in mice carrying MC38-hGPC3 tumors. Figure 14 As shown, in the absence of CD8+ T cells, cross-LS / HS-GPC3 induced transient tumor suppression during the initial dosing period (days 6–8), but the therapeutic effect completely disappeared by day 10. The loss of tumor growth inhibition (TGI: <10% vs. 55% in the aforementioned immunocompetent mice) directly validates that CTLs are the main mediators of bsAb-driven antitumor activity, consistent with the TME remodeling results observed in flow cytometry.

Claims

1. A bispecific antibody comprising two chains: From the N-terminus to the C-terminus, the sequence is: VH1 (or VL1), L1, VH2, CH1, and the first polypeptide chain of Fc. From the N-terminus to the C-terminus, the second polypeptide chain consists of VL1 (or VH1), L2, VL2, and CL. in, The first binding domain, composed of VH1 and VL1, binds to tumor cell surface antigens, while the second binding domain, composed of VH2 and VL2, binds to immune checkpoint antigens. The EC50 of the second binding domain binding to immune checkpoint antigens is 2 to 50 times that of the first binding domain binding to tumor cell surface antigens.

2. The bispecific antibody according to claim 1, wherein the immune checkpoint antigen is PD-L1; preferably, the second binding domain of PD-L1 has VH2 as shown in SEQ ID NO.30 and VL2 as shown in SEQ ID NO.31, or has VH2 as shown in SEQ ID NO.32 and VL2 as shown in SEQ ID NO.

33.

3. The bispecific antibody according to claim 1 or 2, wherein the tumor cell surface antigen is selected from GPC3 or EGFR.

4. The bispecific antibody according to claim 3, wherein the first binding domain for EGFR has VH1 as shown in SEQ ID NO. 34 and VL1 as shown in SEQ ID NO. 35; and the first binding domain for GPC3 has VH1 as shown in SEQ ID NO. 36 and VL1 as shown in SEQ ID NO.

37.

5. The bispecific antibody according to any one of claims 1-4, wherein the L1 and L2 are optionally selected from the linker peptides shown in SEQ ID NO. 38-41.

6. The bispecific antibody according to any one of claims 1-5, wherein the Fc of the first polypeptide chain and the second polypeptide chain is selected from IgG1, IgG2, IgG3 or IgG4; preferably, the Fc has an amino acid sequence as shown in SEQ ID NO.42-44.

7. The bispecific antibody according to any one of claims 1-6, wherein the bispecific antibody comprises a heavy chain and a light chain having the following amino acid sequences respectively: SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:20 and SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23; SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:4 and SEQ ID NO:2; SEQ ID NO:4 and SEQ ID NO:3; SEQ ID NO:7 and SEQ ID NO:6; SEQ ID NO:10 and SEQ ID NO:11; SEQ ID NO:10 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:14; SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:24 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:26; SEQ ID NO:28 and SEQ ID NO:29; SEQ ID NO:1 SEQ ID NO:20 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:

41.

8. A polynucleotide encoding a first polypeptide chain and / or a second polypeptide chain of a bispecific antibody according to any one of claims 1-8.

9. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-8 and a pharmaceutically usable carrier.

10. Use of the antibody according to any one of claims 1-9 in the preparation of a medicament for treating solid tumors; preferably, the solid tumor is selected from non-small cell lung cancer, colorectal cancer, or liver cancer.

Images