Patent Publication Number: US-2022213195-A1

Title: BIFUNCTIONAL FUSION PROTEIN AGAINST PDL1 AND TGFß AND USE THEREOF

Description:
TECHNICAL FIELD 
     The invention relates to the technical field of biomedicine or biopharmaceuticals. Specifically, the present invention relates to anti-PDL1 antibodies, bifunctional fusion proteins against PDL1 and TGFβ, and variants and antigen-binding fragments thereof, as well as a preparation method thereof and methods and uses for treating cancer. 
     BACKGROUND 
     PD1(programmed cell death-1) and PDL1(programmed cell death-1 ligand 1) are an important kind of immune checkpoint proteins, wherein PD1 is expressed on the surface of activated T cells and NK cells, and tumor cell inhibits maturation and proliferation of T cell and NK cell after bonding to PD1 by expressing PDL1 in large quantities, and then escapes from immune surveillance (Iwai et al., PNAS 99:12293-7, 2002) (Ohigashi et al., Clin Cancer Res 11:2947-53, 2005). Therefore, by blocking the interaction between PD1 and PDL1, T cells can be reactivated, and the tumor cells can be identified and killed to benefit the patient. 
     TGFβ can inhibit the growth of early tumors, but as the tumor continues to progress, the tumor cells become no longer sensitive to TGFβ, and at this time, TGFβ in turn promotes tumor growth by promoting epithelial-stromal transfer and suppressing the immune system (Bierie et al., Nat Rev Cancer. 2006; 6:506-20). 
     The extracellular domain of TGFβRII is only 136 amino acid residues in length and the TGFβRII can bind TGFβ1 (Lin et al., J Biol Chem. 1995; 270:2747-54). Soluble TGFβRII-Fc has been tested as an anticancer agent and has been shown that it can inhibit the growth of malignant mesothelioma in a murine model (Suzuki et al., Clin Cancer Res. 2004; 10:5907-18) (Suzuki et al., Clin Cancer Res. 2004; 10:5907-18). 
     M7824 bifunctional fusion protein from the company Merck (Chinese application number CN201580007865.3) is anti-PDL1 antibody Avelumab linked to TGFβRII by (G 4 S) 4 G, some phase I and phase II clinical trials have been completed, and it has performed well in a variety of solid tumors (MSB0011359C). 
     However, in the face of the patient&#39;s medicinal demands for disease treatment, especially demands for antibody drugs, there is still an urgent clinical need to provide an anti-PDL1 antibody or bifunctional fusion protein against PDL1 and TGFβ with higher binding activity and better efficacy. 
     SUMMARY 
     In the full text of the present invention, various implementations regarding VL (light chain variable region), VH (heavy chain variable region), LCDR (light chain complementarity determining region), HCDR (heavy chain complementarity determining region), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 can be implemented individually or in any combination. 
     The present invention relates to an antibody or antigen-binding fragment thereof, or bifunctional fusion protein and the preparation method and use thereof. 
     In an aspect of the present invention, the present invention relates to an antibody or antigen-binding fragment thereof including three heavy chain complementarity determining regions, wherein HCDR1 amino acid sequence is represented by SEQ ID NO: 8, HCDR2 amino acid sequence is represented by SEQ ID NO: 9, and the HCDR3 amino acid sequence is represented by SEQ ID NO: 10. Further, the antibody or antigen-binding fragment thereof also includes three light chain complementarity determining regions, wherein the LCDR1 amino acid sequence is represented by SEQ ID NO: 5, the LCDR2 amino acid sequence is represented by SEQ ID NO: 6, and the LCDR3 amino acid sequence is represented by SEQ ID NO: 7. 
     In an aspect of the present invention, the antibody or antigen-binding fragment thereof provided in the present invention includes a heavy chain variable region represented by SEQ ID NO: 2; preferably, further includes a light chain variable region represented by SEQ ID NO: 1. 
     In an aspect of the present invention, the present invention relates to an antibody or antigen-binding fragment thereof including three light chain complementarity determining regions, wherein the LCDR1 amino acid sequence is represented by SEQ ID NO: 11, the LCDR2 amino acid sequence is represented by SEQ ID NO: 6, and the LCDR3 amino acid sequence is represented by SEQ ID NO: 12; and/or three heavy chain complementarity determining regions, wherein the HCDR1 amino acid sequence is represented by SEQ ID NO: 13, the HCDR2 amino acid sequence is represented by SEQ ID NO: 14, and the HCDR3 amino acid sequence is represented by SEQ ID NO: 15. 
     In another aspect, the present invention relates to an antibody or antigen-binding fragment thereof including the light chain variable region of the amino acid sequence represented by SEQ ID NO: 3, and/or the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: 4. 
     In another aspect, the sequence of the light chain constant region of the antibody or antigen-binding fragment thereof of any one of the preceding aspects is SEQ ID NO: 16. 
     In another aspect, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof of any one of the preceding aspects is SEQ ID NO: 17. 
     Specifically, the antibody or antigen-binding fragment thereof provided in the present invention preferably includes the light chain variable region of the amino acid sequence represented by SEQ ID NO: 1, the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: 2, the light chain constant region of the amino acid sequence represented by SEQ ID NO: 16 and the heavy chain constant region of the amino acid sequence represented by SEQ ID NO: 17. Alternatively, the antibody or antigen-binding fragment thereof provided in the present invention preferably includes the light chain variable region of the amino acid sequence represented by SEQ ID NO: 3, the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: 4, the light chain constant region of the amino acid sequence represented by SEQ ID NO: 16 and the heavy chain constant region of the amino acid sequence represented by SEQ ID NO: 17. 
     In another aspect, the present invention relates to the antibody or antigen-binding fragment thereof of any one of the preceding aspects including a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, a scFv fragment, a dsFv fragment or the like. 
     In another aspect, any one of the above antibodies or antigen-binding fragments thereof binds PDL1. 
     In another aspect, the present invention relates to a bifunctional fusion protein including the antibody or antigen-binding fragment thereof of any one of the preceding aspects and a TGFβRII fragment, and preferably the TGFβRII fragment includes a TGFβRII extracellular domain. 
     In another aspect, in the bifunctional fusion protein, the antibody or antigen-binding fragment thereof of any one of the preceding aspects is linked to the TGFβRII fragment by a linker, preferably, the TGFβRII fragment is linked to a C-terminus of the heavy chain constant region of the antibody or antigen-binding fragment thereof, preferably by a (G 4 S) 4 G (i.e., GGGGSGGGGSGGGGSGGGGSG) linker, preferably, the sequence of the heavy chain constant region of the bifunctional fusion protein is SEQ ID NO: 22, and preferably the sequence of the TGFβRII fragment of the bifunctional fusion protein is SEQ ID NO: 23; and specifically, the structure of the bifunctional fusion protein is: an antibody or antigen-binding fragment thereof-(G 4 S) 4 G-TGFβRII fragment. 
     In another aspect, in the bifunctional fusion protein, the sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 1, the sequence of the heavy chain variable region is SEQ ID NO: 2, preferably, in the bifunctional fusion protein, the sequence of the light chain constant region is SEQ ID NO: 16, the sequence of the heavy chain constant region is SEQ ID NO: 22, and the sequence of the TGFβRII fragment is SEQ ID NO: 23; and specifically, the sequences of the heavy chain constant region and the TGFβRII fragment which are linked by (G 4 S) 4 G are SEQ ID NO: 18. 
     In another aspect, in the bifunctional fusion protein, the sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 3, the sequence of the heavy chain variable region is SEQ ID NO: 4, preferably, in the bifunctional fusion protein, the sequence of the light chain constant region is SEQ ID NO: 16, the sequence of the heavy chain constant region is SEQ ID NO: 22, and the sequence of the TGFβRII fragment is SEQ ID NO: 23; and specifically, the sequences of the heavy chain constant region and the TGFβRII fragment which are linked by (G 4 S) 4 G are SEQ ID NO: 18. 
     In another aspect, any one of the above bifunctional fusion proteins binds PDL1 and TGFβ. 
     In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof or the bifunctional fusion protein of any one of the preceding aspects. 
     In another aspect, the present invention relates to a vector including the nucleic acid of the preceding aspect, or the vector can express the antibody or antigen-binding fragment thereof or the bifunctional fusion protein of any one of the preceding aspects. Preferably, the vector may be a viral vector; preferably the viral vector includes but is not limited to a lentiviral vector, an adenovirus vector, an adeno-associated viral vector, a retroviral vector, or the like; preferably the vector may be a non-viral vector; preferably the vector may be a mammalian cell expression vector; preferably the expression vector may be a bacterial expression vector; and preferably the expression vector may be a fungal expression vector. 
     In another aspect, the present invention relates to a cell that can express a cell of the antibody or antigen-binding fragment thereof or the bifunctional fusion protein of any one of the preceding aspects. Preferably, the cell is a bacterial cell; preferably the bacterial cell is an  E. coli  cell and the like; preferably the cell is a fungal cell; preferably the fungal cell is a yeast cell; preferably the yeast cell is a Pichia pastoris and the like; preferably the cell is a mammalian cell; preferably the mammalian cell is a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, a NK cell, or the like. 
     In another aspect, the present invention relates to a pharmaceutical composition including the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid, the vector or the cell of any one of the preceding aspects, preferably, the pharmaceutical composition further includes a pharmaceutically acceptable excipient, and preferably, the pharmaceutically acceptable excipient includes one or more of the following: a solvent, a dispersant, an additive, a plasticizer and the like which are pharmaceutically acceptable. 
     In another aspect, the pharmaceutical composition may further include other therapeutic agents. In some implementations, the other therapeutic agents include chemotherapeutic agents, immunotherapeutic agents, or hormonal therapeutic agents. The combined administration of the antibody or antigen-binding fragment and the other therapeutic agents can enhance the therapeutic effect. 
     In another aspect, the “enhance the therapeutic effect” refers to enhancing the therapeutic effect of other therapeutic agents or therapies. The antibody or antigen-binding fragment provided in the present invention can be administered individually or in combination with other therapeutic agents or therapies. In some implementations, the other therapeutic agents or therapies include chemotherapeutic agents, immunotherapeutic agents, hormonal therapeutic agents, radiation therapy and surgery. 
     In another aspect, there is provided a kit including the antibody or antigen-binding fragment thereof of the present invention, or including the bifunctional fusion protein, or including the nucleic acid encoding the antibody or antigen-binding fragment thereof or the bifunctional fusion protein. 
     In another aspect, the present invention relates to an application of the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid, the vector or the cell of any one of the preceding aspects in preparation of medicaments for treatment or prophylaxis of diseases. 
     In another aspect, the present invention relates to an application of the antibody or antigen-binding fragment thereof, the bifunctional fusion protein or the nucleic acid of any one of the preceding aspects in preparation of a diagnostic or detection kit. 
     In another aspect, there is provided a method of treating or preventing a disease including administering the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid, the vector, the cell or the pharmaceutical composition of any one of the preceding aspects to a subject in need. 
     In another aspect, there is provided a method of diagnosis or detection including administering the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid or the kit of any one of the preceding aspects to a subject in need or sample. Preferably, the method is a method of diagnosing or detecting diseases. 
     In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid, the vector, the cell or the pharmaceutical composition of any one of the preceding aspects for the treatment or prophylaxis of diseases. 
     In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof, the bifunctional fusion protein, the nucleic acid, or the kit of any one of the preceding aspects for detection or diagnosis. Preferably, the use is to diagnose or detect diseases. 
     In another aspect, the disease is cancer. 
     In another aspect, the cancer includes gastric cancer, esophageal cancer, head-and-neck cancer, bladder cancer, cervical cancer, sarcoma, cytoma, lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia, lymphoma and the like. 
     In another aspect, the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof, or the bifunctional fusion protein of any one of the preceding aspects, which includes transfecting cells with the above vectors, and expressing the antibody or antigen-binding fragment thereof, or the bifunctional fusion protein by the transfected cells; or includes expressing the antibody or antigen-binding fragment thereof, or the bifunctional fusion protein with the above cells. 
     The antibody or antigen-binding fragment thereof or the bifunctional fusion protein provided in the present invention has one or more of the following advantages: enhanced TGFβ1 binding activity, enhanced PDL1 affinity, enhanced ability of blocking the binding of PDL1 to PD1, enhanced activity of blocking TGFβ1 function, enhanced ability of promoting T cells to secrete IFN-γ, better immunoregulation effect, and better tumor suppression effect. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the serum titers of BoAn-hMab1 mice after five immunizations (2500-fold dilution). 
         FIG. 2  shows the binding of the bifunctional fusion protein to the TGFβ1 protein. 
         FIG. 3  shows that the bifunctional fusion protein promotes secretion of IFN-γ by CD4+T. 
         FIG. 4  shows the blocking effect of the bifunctional fusion protein on the TGFβ1 function. 
         FIG. 5A  shows body weights of the MC38-hPD-L1 tumor model mice; 
         FIG. 5B  shows tumor volumes of the MC38-hPD-L1 tumor model mice; and  FIG. 5C  shows tumor weights of the MC38-hPD-L1 tumor model mice 
     
    
    
     DESCRIPTION 
     The present invention will be further described below in connection with specific embodiments. The described embodiments are a part of the embodiments of the present invention, but not all the embodiments. It should be understood that the following examples are given in order to provide a general disclosure and description of how to use the methods and compositions of the present invention to general professionals in the technical field to which the invention belongs, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those having ordinary skill in the art without the exercise of inventive faculty are within the scope of the present invention. 
     Materials and Reagents: PDL1 protein (Sinobiological, catalog number: 10084-H08H); PD1 protein (Sinobiological, catalog number: 10377-H08H); TGFβ1 (Sinobiological, catalog number: 10804-HNAC); NaHCO 3  (Sinopharm, 10018960); TMB (Beijing Makewonder, catalog number: 1001); STREP/HRP (R&amp;D, catalog number: 890803); HBS-EP +1×buffer (GE, catalog number: BR-1008-26); enzyme label plate (Suzhou Beaver, catalog number: 40301); biosensor chip (GE, catalog number: BR100530); 96-well round bottom plate (Corning, catalog number: 3799); anti Human IgG Fc amino coupling kit (GE, cat # BR-1008-39); 
     Example 1 Preparation of Bifunctional Fusion Proteins Against PDL1 and TGFβ 
     1.1 Immunization of PDL1 Protein 
     The PDL1 protein is emulsified with Freund&#39;s complete adjuvant (Sigma, catalog number: F5881-10ML), Freund&#39;s incomplete adjuvant (Sigma, catalog number: F5506-10ML) or gold adjuvant (Sigma, catalog number: T2684-1mL) to immunize fully human antibody transgenic mouse BoAn-hMab1 of Boan bio (prepared according to the method described in Chinese Patent CN103571872B). A total of 11 mice including 6 males and 5 females were immunized this time, the age of mice was 6-12 months, a total of 5 immunizations were performed, and 6 mice with higher serum titers were selected for booster immunization with the above PDL1 protein. The serum titers detected by ELISA (2500-fold dilution) are shown in  FIG. 1 . Herein, the PL1Q15, PL1Q16, PL1Q18, PL1Q19 and PL1Q21 mice were mice immunized with Freund&#39;s adjuvant, and the PL1Q24 mouse was a mouse immunized with gold adjuvant. 
     1.2 Establishment of Phage Library 
     Six mice with higher serum titers in 1.1 were sacrificed and the spleens were dissected out, the spleens were ground and broken with a syringe rubber stopper and filtered with a filter (FALCON, catalog number: 352350), and the filtered spleen cells were frozen for future use. After RNA extraction, reverse transcription was performed to obtain cDNA, the steps for establishing the phage library refer to the method described in Carlos F. Barbas III, Phage display: A laboratory manual, the variable regions of the heavy and light chains are obtained from the cDNA by PCR, and then single chain Fv (scFv) was obtained by overlapping extension PCR of the variable regions of the heavy chain and the light chain, scFv was digested with SfiI enzyme (NEB, catalog number: # R0123L) and then ligated with plasmid pCOMB3× (BIOVECTOR, Chinese Center, Plasmid Vector Strain Cell Line Gene Collection., 510837) by T4 DNA ligase (Sino Biological), the ligation product was electrotransfected into  E. coli  TG1 competent cells (Lucigen, catalog number: A96595-2), and after incubating the transfected TG1 in 37° C. 220 rpm shaker, the phage was added to infect, the supernatant of the culture was recovered, concentrated and purified to obtain a phage library. 
     1.3 Screening of Phage Library 
     Plate screening: preparation of CBS buffer: 1.59 g of Na 2 CO 3  (Sinopharm, 10019260) and 2.93 g of NaHCO 3  were weighed, and the distilled water was added to 1 L to prepare CBS buffer. The PDL1 protein was diluted to 10 μg/mL with CBS buffer, and then added to the screening plate (Costar, 42592) at 100 μg/well, 8 wells were used for each library and the screening plate was left overnight at 4° C.; the protein solution in the well plate was discard the next day, the plate was sealed with 2% BSA (Solarbio, A8010) for 1 h, and phage library (2×10 12 /well) samples was added to incubate at 37° C. for 2 h, PDL1 specific binding phage was eluted with elution buffer (add 4.2 ml of concentrated hydrochloric acid (Comeo) to 500 ml of ultrapure water and adjust the pH to 2.2 with glycine powder (Biotopped, BG0617-500)) after being washed 4-10 times with PBST (PBS+0.05% Tween20). 
     Magnetic bead screening: biotinylation of PDL1-mFc protein (Acrobiosystems, catalog number: PD1-H52A3) (molar ratio of protein to biotin is 1:2): the PDL1-mFc protein was changed into 0.1 M pH 8.0 NaHCO 3 , an appropriate amount of biotin (Thermo, 21335) was weighed by precision balance and then dissolved with the ultrapure water, the appropriate amount of dissolved biotin was immediately added to the PDL1-mFc protein solution, and incubated on a rotary mixer in the dark for 40 min, and then the PDL1-mFc protein solution was changed into PBS after labeling. The biotinylated PDL1-mFc protein (3 μg/library) was bound to magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, catalog number: 00355871) (10 μL/library) for 1 h at room temperature, and then incubated with the phage library at room temperature for 2h after sealing with 2% BSA, and PDL1 specific binding phage was eluted with elution buffer (pH 2.2) after being washed 4-10 times. 
     Phage clones express scFv through  E. coli , and then detect the blocking of PDL1/PD1 binding by scFv through ELISA, and clones blocking the activity are retained for constructing subsequent molecules. 
     ELISA detection of blocking of PDL1/PD1 binding by scFv: the PDL1 protein was diluted to 0.5 μg/mL with pH 9.6 CBS, coated with enzyme-labeled plate, 100 μL/well, and incubated overnight at 4° C.; and 3% defatted milk powder was used for sealing at 37° C. for 1 h after washing the plate. 50 μL of scFv periplasm was added to each well after washing the plate. Then, biotin-labeled PDL1-Fc protein (final concentration was 0.2 μg/mL) was added, 50 μL/well, and incubated at 37° C. for 1 h; STREP/HRP diluted with PBST was added after washing the plate, 100 μL/well, and incubated at 37° C. for 1 h. After washing the plate, 100 μL of TMB was added to each well for color development, after 10 min, 50 μL of 2M H 2 SO 4  was added to each well to stop the color development, and OD450 was read with a microplate reader. 
     1.4 Construction and Production of Bifunctional Fusion Protein 
     Magnetic bead screened clones BA533, BA603, BA613, BA623, BA649, BA669, BA446 and plate screened clone BA705 were sent to Invitrogen Biotechnology Ltd for sequencing. The amino acid sequences of the light chain variable region and the heavy chain variable region of each clone are presented in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 amino acid sequences of light chain variable region and heavy chain 
               
               
                 variable region of candidate clones (CDR underlined) 
               
            
           
           
               
               
               
            
               
                 Clone ID 
                 Light chain variable region sequence 
                 Heavy chain variable region sequence 
               
               
                   
               
               
                 BA533 
                 DIQMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSNYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS TREAGVPDRFSGSG 
                 VRQAPGKGLEWVAI ITYAGSNE YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSTPLT FGGGT 
                 DNSKNTLYLQMNSLRPEDTAVYYC ARDRIWVDY WGQ 
               
               
                   
                 KVEIK (SEQ ID NO: 1) 
                 GTLVTVSS (SEQ ID NO: 2) 
               
               
                   
                 CDR region: 
                 CDR region: 
               
               
                   
                 LCDR1: QSVLYSSNNKNY (SEQ ID NO: 5) 
                 HCDR1: GFTFSNYA (SEQ ID NO: 8) 
               
               
                   
                 LCDR2: WAS (SEQ ID NO: 6) 
                 HCDR2: ITYAGSNE (SEQ ID NO: 9) 
               
               
                   
                 LCDR3: QQYYSTPLT (SEQ ID NO: 7) 
                 HCDR3: ARDRIWVDY (SEQ ID NO: 10) 
               
               
                   
               
               
                 BA669 
                 DIVMTQSPDSLAMSLGERATINCKSS QSVLYNSNNK   
                 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLTY WAS TRESGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISYDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSLPLT FGGGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIYFDY WGQ 
               
               
                   
                 KVEIK (SEQ ID NO: 3) 
                 GTLVTVSS (SEQ ID NO: 4) 
               
               
                   
                 CDR region: 
                 CDR region: 
               
               
                   
                 LCDR1: QSVLYNSNNKNY (SEQ ID NO: 11) 
                 HCDR1: GFTFSSYA (SEQ ID NO: 13) 
               
               
                   
                 LCDR2: WAS (SEQ ID NO: 6) 
                 HCDR2: ISYDGSNK (SEQ ID NO: 14) 
               
               
                   
                 LCDR3: QQYYSLPLT (SEQ ID NO: 12) 
                 HCDR3: ARDRIYFDY (SEQ ID NO: 15) 
               
               
                   
               
               
                 BA603 
                 DIVMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS TRESGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISYDGSNK YYADSVKGRFITSR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSIPIT FGQGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIYFDY WGQ 
               
               
                   
                 KLEIK 
                 GTLVTVSS 
               
               
                   
               
               
                 BT613 
                 ELVLTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGKSLRLSCAAS GFTFSSYS MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS TRESGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISFDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSTPLT FGGGT 
                 DNSKNTLYLQMTSLRTEDTAVYYC ARDRIYLDY WGQ 
               
               
                   
                 KVDIK 
                 GTLVTVSS 
               
               
                   
               
               
                 BA623 
                 DIQMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSNYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS IRDSGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISYDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDAAVYYC QQFYSIPLT FGGGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIYFDY WGQ 
               
               
                   
                 KVEIK 
                 GTLVTVSS 
               
               
                   
               
               
                 BA649 
                 DIVMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS TRDSGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISYDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSIPLT FGGGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIYFDY WGQ 
               
               
                   
                 KVEIK 
                 GTLVTVSS 
               
               
                   
               
               
                 BA705 
                 DIVMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GITFSNYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLIY WAS TRESGVPDRFSGSG 
                 VRQAPGKGLEWVAL ISYDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSIPLT FGGGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIYFDY WGQ 
               
               
                   
                 KVEIK 
                 GTLVTVSS 
               
               
                   
               
               
                 BA466 
                 EIVMTQSPDSLAVSLGERATINCKSS QSVLYSSNNK   
                 EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYA MHW 
               
               
                   
                   NY LAWYQQKPGQPPKLLID WAS TRESGVPDRFSGSG 
                 VRQAPGKGLEWVAA ISYDGSNK YYADSVKGRFTISR 
               
               
                   
                 SGTDFTLTISSLQAEDVAVYYC QQYYSFPLT FGGGT 
                 DNSKNTLYLQMNSLRAEDTAVYYC ARDRIFFDY WGQ 
               
               
                   
                 KVEIK 
                 GTLVTVSS 
               
               
                   
               
            
           
         
       
     
     Through variable region gene amplification (2*Phanta Max Master Mix, manufacturer: Vazyme, catalog number: P515-AA), signal peptide and variable region overlap extension, homologous recombination (ClonExpress II One Step Cloning Kit, manufacturer: Vazyme, catalog number: C112-01) and other methods, the nucleotide sequence fragment encoding VH was finally inserted into the vector pCDNA3.4 (Life Technology) with the nucleotide sequence encoding SEQ ID NO: 18 wherein SEQ ID NO: 18 contains the sequences of the heavy chain constant region of the IgG1 antibody and the TGFβRII sequence which are linked by (G 4 S) 4 G, the nucleotide sequence fragment encoding VL was inserted into the vector pCDNA3.4 (Life Technology) with the nucleotide sequence encoding the light chain constant region amino acid sequence (SEQ ID NO: 16) of the antibody, then HEK293 cells were transfected, and incubated in 37° C.\8% CO 2\125  rpm shaker for 6 to 7 days, and finally the culture supernatant was purified by Protein A filler to obtain the clone of bifunctional fusion protein in Table 2 for subsequent detection. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Clones of bifunctional fusion protein against PDL1-TGFβ 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 PLTGBQ19-BA533-IgG1 
                 PLTGBQ24-BA603-IgG1 
                 PLTGBQ16-BA466-IgG1 
               
               
                 PLTGBQ21-BA649-IgG1 
                 PLTGBH03-BT613-IgG1 
                 PLTGBQ24-BA705-IgG1 
               
               
                 PLTGBQ21-BA669-IgG1 
                 PLTGBQ21-BA623-IgG1 
                 / 
               
               
                   
               
            
           
         
       
     
     Production of control bifunctional fusion protein: M7824 bifunctional fusion protein of the company Merck is anti-PDL1 antibody Avelumab linked to TGFβRII by (G 4 S) 4 G, some phase I and phase II clinical trials have been completed, and it has performed well in a variety of solid tumors. The amino acid sequence of the variable region of Avelumab of the company Merck (the sequence of the heavy chain variable region is SEQ ID NO: 19, the sequence of the light chain variable region is SEQ ID NO: 20) is determined by IMGT database, the complete gene is synthesized and then inserted into the vector pCDNA3.4 for expression in HEK293 cells, and the purified bifunctional fusion protein is named PLTGB-M7824-IgG1 (the sequence of the heavy chain variable region is SEQ ID NO: 19, the sequence of the light chain variable region is SEQ ID NO: 20, the sequence of the light chain constant region is SEQ ID NO: 21, and the sequence of the heavy chain constant region and TGFβRII is SEQ ID NO: 18). 
     1.5 ELISA Detection of Blocking of PDL1-PD1 Binding by Bifunctional Fusion Protein 
     The PD1 protein was diluted to 0.5 μg/mL with CBS at pH 9.6, and then was coated with enzyme-labeled plate, 100 μL/well, and incubated overnight at 4° C.; 3% defatted milk powder was used for sealing at 37 ° C. for 1 h after washing the plate; PBST (PBS+0.05% Tween20) was added to dilute the bifunctional fusion protein to different concentrations (0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.0625 μg/mL, 0.03125 μg/mL, 0.015625 μg/mL) after washing the plate, 50 μL/well, biotin-labeled PDL1-Fc protein (final concentration is 0.2 μg/mL) was then added, 50 μL/well, and incubated at 37° C. for 1 h; STREP/HRP diluted with PBST was added, 100 μL/well, and incubated at 37° C. for 1 h. TMB was added for color development after washing the plate, and 50 μL of 2M H 2 SO 4  was added to each well to stop the color development after 10 min, OD450 was read on a microplate reader. The results show that the candidate bifunctional fusion proteins of the present invention (PLTGBQ16-BA466-IgG1, PLTGBQ19-BA533-IgG1, PLTGBQ24-BA603-IgG1, PLTGBQ21-BA623-IgG1, PLTGBQ21-BA649-IgG1, PLTGBQ21-BA669-IgG1, PLTGBQ24- BA705-IgG1, PLTGBH03-BT613-IgG1) can effectively block the binding of PD1 to PDL1. 
     1.6 ELISA Detection of Binding of Bifunctional Fusion Protein to TGFβ1 Protein 
     The TGFβ1 was diluted to different concentrations (0.2 μg/mL, 0.05 μg/mL, 0.0125 μg/mL) with pH 9.6 CBS, and then was coated with enzyme-labeled plate respectively, 100 μL/well, and incubated overnight at 4° C.; 3% defatted milk powder was used for sealing at 37° C. for 1 h after washing the plate; 100 μL of PBST (PBS+0.05% Tween20) diluted candidate bifunctional fusion protein (1 μg/mL) was added to each well after washing the plate, and incubated at 37° C. for 1 h; then goat anti-human IgG/HRP was added and incubated at 37° C. for 1 h; TMB is added for color development after washing the plate, and 50 μL of 2M H2504 was added to each well to stop the color development after 10 min, OD450 was read on the microplate reader. The results are shown in Table 3 and  FIG. 2 . 
     The candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 have higher OD values at various concentrations than the control group PLTGB-M7824-IgG1, indicating that the candidate bifunctional fusion proteins have better binding ability for TGFβ1. 
     Furthermore, it is predicted that the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 can block the TGF pathway better than the control group PLTGB-M7824-IgG1, and have better immune regulation and anti-tumor properties in clinical. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Data of ELISA detection of binding of bifunctional fusion 
               
               
                 protein to TGFβ1 protein (corresponding to FIG. 2) 
               
            
           
           
               
               
            
               
                   
                 OD450 at different TGFβ1 concentrations 
               
            
           
           
               
               
               
               
            
               
                   
                 TGFβ1 
                 TGFβ1 
                 TGFβ1 
               
               
                   
                 concen- 
                 concen- 
                 concen- 
               
               
                   
                 tration 
                 tration 
                 tration 
               
               
                 Name 
                 0.2 μg/mL 
                 0.05 μg/mL 
                 0.0125 μg/mL 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 PLTGBQ16-BA466-IgG1 
                 3.829 
                 2.009 
                 0.88 
               
               
                 PLTGBQ19-BA533-IgG1 
                 3.699 
                 2.113 
                 0.758 
               
               
                 PLTGBQ24-BA603-IgG1 
                 3.921 
                 2.367 
                 0.861 
               
               
                 PLTGBQ21-BA623-IgG1 
                 3.854 
                 2.302 
                 0.813 
               
               
                 PLTGBQ21-BA649-IgG1 
                 4.122 
                 2.136 
                 0.636 
               
               
                 PLTGBQ21-BA669-IgG1 
                 3.93 
                 2.351 
                 0.728 
               
               
                 PLTGBQ24-BA705-IgG1 
                 4.038 
                 2.184 
                 0.836 
               
               
                 PLTGBH03-BT613-IgG1 
                 3.403 
                 1.46 
                 0.388 
               
               
                 PLTGB-M7824-IgG1 
                 3.572 
                 1.807 
                 0.537 
               
               
                   
               
            
           
         
       
     
     Example 2 Characterization of Bifunctional Fusion Protein 
     2.1 Production of Bifunctional Fusion Protein 
     ExpiCHO (Thermo, catalog number: A29129) express system is used to transfect and express the bifunctional fusion protein, and protein A (GE, Mabselect SuRe) column is used to purify the supernatant: the cell culture fluid was centrifuged at 4500 g and filtered with a 0.45 βm filter, the target bifunctional fusion protein was eluted with 0.1 M pH3.2 glycine buffer after loading and equilibrating, and neutralized with pH8.0 1 M Tris. Size exclusion chromatography (Shanghai Bestchrom, catalog number: AG319109) is used to purify: equilibrate the column with 1.5 CV of buffer, load the sample, the sample volume is not more than 3% CV, continue to rinse with buffer and collect the target bifunctional fusion protein after peak appears. 
     2.2 Mixed Lymphocyte Reaction/MLR detection of bifunctional fusion protein activity 
     RPMI 1640 (Gibco, catalog number: 11875-093), FBS (Gibco, catalog number: 10091-148), HEPES (Solarbio, catalog number: H1090) were mixed according to 90:10:1 to prepare a complete medium, DC cells (ALLCELLS, catalog number: PB-DC001F-0.3M) and CD4+T cells (ALLCELLS, catalog number: PB009-2F-C-3M) were resuspended with the complete medium respectively, and then added to 96-well round bottom plate according to a cell ratio of 1:10, and the final volume is 150 μL/well. The bifunctional fusion protein was diluted, with the complete medium, two concentrations: 4μg/mL and 0.4 μg/mL, and added into the cell wells respectively, 50 μL/well, and the final volume is 200 μL. After being cultured for 5 days, the culture supernatant was detected with IFN-γ kit (cisbio, catalog number: 62HIFNGPEG). The detection results are shown in Table 4 and  FIG. 3 . 
     The candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 have higher IFN-γ secretion values at the same concentration than the control group PLTGB-M7824-IgG1. 
     Furthermore, it is predicted that the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 may have better immune regulation and anti-tumor properties than the control group PLTGB-M7824-IgG1. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Data of Mixed Lymphocyte Reaction/MLR detection of bifunctional 
               
               
                 fusion protein activity (corresponding to FIG. 3) 
               
            
           
           
               
               
            
               
                   
                 IFN-γ secretion value at different 
               
               
                   
                 bifunctional fusion protein concentrations 
               
            
           
           
               
               
               
            
               
                   
                 bifunctional 
                 bifunctional 
               
               
                   
                 fusion protein 
                 fusion protein 
               
               
                   
                 concentration 
                 concentration 
               
               
                 Name 
                 (4 μg/mL) 
                 (0.4 μg/mL) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 PLTGBQ16-BA466-IgG1 
                 3029.907 
                 2262.317 
               
               
                 PLTGBQ19-BA533-IgG1 
                 3594.345 
                 2336.045 
               
               
                 PLTGBQ24-BA603-IgG1 
                 2983.494 
                 2355.715 
               
               
                 PLTGBQ21-BA623-IgG1 
                 3147.502 
                 2572.435 
               
               
                 PLTGBQ21-BA649-IgG1 
                 3589.663 
                 2269.363 
               
               
                 PLTGBQ21-BA669-IgG1 
                 5117.964 
                 2781.657 
               
               
                 PLTGBQ24-BA705-IgG1 
                 2801.897 
                 2550.59 
               
               
                 PLTGBH03-BT613-IgG1 
                 4328.078 
                 2365.794 
               
               
                 PLTGB-M7824-IgG1 
                 3423.783 
                 1996.965 
               
               
                   
               
            
           
         
       
     
     2.3 SPR Detection of Affinity of Bifunctional Fusion Protein and PDL1 Protein 
     Antibody binding kinetics adopts BIAcore8K instrument to detect. Anti-human IgG antibody was coupled to a CMS biosensor chip by the anti-human IgG Fc amino coupling kit to obtain approximately 1000 RU (response units). The PDL1 was diluted to 50 nM with HBS-EP +1×buffer, and then diluted 2-fold to a total of 5 concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM), and a blank control was set. The measurement steps and conditions were as follows: bifunctional fusion protein sample injection 2 μg/mL, sample injection time 70 s, flow rate 5 μl/min, stability time 5 s; PDL1 protein binding and dissociation: binding 60 s, flow rate 30 μl/min, dissociation 450 s; regeneration: regeneration was performed for 30 s with 3M MgCl 2  buffer, starting for 3 times. The association constant (ka) and dissociation constant (kd) were calculated using a one-to-one Languir model (BIAcore Evaluation Software version 3.2), and the equilibrium dissociation constant KD is calculated from kd/ka. The affinity data of each bifunctional fusion protein is shown in Table 5. 
     The results show that the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 of the present invention have a PDL1 affinity that is substantially equivalent to that of the control group PLTGB-M7824-IgG1. Wherein, PLTGBQ19-BA533-IgG1 has better PDL1 affinity than the control group. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Affinity of bifunctional fusion protein and PDL1 protein 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 ka (1/Ms) 
                 kd(1/s) 
                 KD(M) 
               
               
                   
               
               
                 PLTGBQ16-BA466-IgG1 
                 1.14E+06 
                 2.32E−03 
                 2.03E−09 
               
               
                 PLTGBQ19-BA533-IgG1 
                 1.50E+06 
                 7.95E−04 
                 5.29E−10 
               
               
                 PLTGBQ24-BA603-IgG1 
                 1.70E+06 
                 1.64E−03 
                 9.63E−10 
               
               
                 PLTGBQ21-BA623-IgG1 
                 1.34E+06 
                 1.79E−03 
                 1.34E−09 
               
               
                 PLTGBQ21-BA649-IgG1 
                 1.85E+06 
                 1.67E−03 
                 9.04E−10 
               
               
                 PLTGBQ21-BA669-IgG1 
                 1.78E+06 
                 1.36E−03 
                 7.64E−10 
               
               
                 PLTGBQ24-BA705-IgG1 
                 1.45E+06 
                 8.37E−04 
                 5.77E−10 
               
               
                 PLTGBH03-BT613-IgG1 
                 1.39E+06 
                 1.01E−03 
                 7.30E−10 
               
               
                 PLTGB-M7824-IgG1 
                 4.98E+05 
                 3.00E−04 
                 6.03E−10 
               
               
                   
               
            
           
         
       
     
     2.4 Detection of Blocking Effect of Bifunctional Fusion Protein on TGFβ1 Function by MV-1-Lu cells 
     EMEM (ATCC, catalog number: 30-2003), FBS (Gibco, catalog number: 10099-141) and HEPES (Solarbio, catalog number: H1090) were mixed according to 90:10:1 to prepare a complete medium, MV-1-Lu cells were resuspended with the complete medium, and added to white 96-well plate (Corning, catalog number: 3917), 50 μL/well, 2000 cells/well. The bifunctional fusion protein was diluted to 333.3 ng/mL with the complete medium, and then diluted three times in sequence, a total of 6 concentrations, added into the cell wells, 25 μL/well. TGF β 1 was diluted to 2 ng/ml with complete medium and added into the cell wells, 25 μL/well. The final concentrations of the bifunctional fusion protein: 83.3 ng/mL, 27.8 ng/mL, 9.26 ng/mL, 3.09 ng/mL, 1.03 ng/mL and 0.34 ng/mL. After being cultured for 4 days, the fluorescence value (the fluorescence value can represent the number of cells) of the 96-well plate was detected with CellTiter-Glo kit (Promega, catalog number: G7571), and the detection results are shown in  FIG. 4   
     It can be seen that the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 have better activity in blocking TGF131 function than the control group PLTGB-M7824-IgG1. 
     Furthermore, it is predicted that the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 can block the TGF pathway better than the control group PLTGB-M7824-IgG1, and have better immune regulation and anti-tumor properties. 
     Example 3 Efficacy test of Anti-PD-L1 Monoclonal Antibody in Mouse MC38-hPDL1 Colon Cancer Model 
     The IgG1 monoclonal antibody corresponding to the bifunctional fusion protein was constructed, the monoclonal antibody corresponding to PLTGBQ19-BA533-IgG1 is BA533-IgG1 (the sequence of the heavy chain variable region is SEQ ID NO: 2, the sequence of the light chain variable region is SEQ ID NO: 1, the sequence of the light chain constant region is SEQ ID NO: 16, and the sequence of the heavy chain constant region is SEQ ID NO: 17); the monoclonal antibody corresponding to PLTGBQ21-BA669-IgG1 is BA669-IgG1 (the sequence of the heavy chain variable region is SEQ ID NO: 4, the sequence of the light chain variable region is SEQ ID NO: 3, the sequence of the light chain constant region is SEQ ID NO: 16, and the sequence of the heavy chain constant region is SEQ ID NO: 17); and the monoclonal antibody corresponding to PLTGB-M7824-IgG is Avelumab (the sequence of the heavy chain variable region is SEQ ID NO: 19, the sequence of the light chain variable region is SEQ ID NO: 20, the sequence of the light chain constant region is SEQ ID NO: 21, and the sequence of the heavy chain constant region is SEQ ID NO: 17). 
     Mouse colon cancer MC38-hPD-L1 cells was purchased from Beijing Biocytogen Co., Ltd. The culture conditions are that 10% FBS (Gibco, catalog number: 10099-141C) and 1% P/S (Gibco, catalog number: 15070-063) are added in DMEM (Gibco, catalog number: 11965-092) medium, and cultured in an incubator with 5% CO 2  at 37° C. The subculture is performed 2-3 times per week. B-hPD-L1 humanized mouse, female, 6-8 weeks old, purchased from Jiangsu Biocytogen Co., Ltd. 
     The MC38-hPD-L1 cells in good condition were collected, resuspended and mixed in PBS, the cell concentration was adjusted to 5×10 6 /ml, and 0.1 ml of cell suspension was inoculated subcutaneously on the dorsal limb of each mouse. When the average tumor volume reached 87 mm3, randomization was started. There were 7 groups of 6 mice each, administration was started on the day of grouping, and anti-PD-L1 monoclonal antibody was diluted with PBS, each antibody had two doses: 3 mg/kg and 10 mg/kg, administered by intraperitoneal injection once every 2 days, the same volume of PBS was given in solvent control group as a control. The mice were weighted and the tumor volume was measured 2-3 times per week, tumor volume (mm 3 ) =0.5 ×long diameter×short diameter 2 . When the tumor volume of the control group reached 2000 mm 3 , the mice were euthanized, and the tumors were stripped and weighed. The results are shown in  FIG. 5A ,  FIG. 5B ,  FIG. 5C  and Tables 6 and 7. 
     As shown in  FIG. 5A , the body weight of the mice increases steadily without any adverse reactions. 
     As shown in  FIG. 5B  and  FIG. 7C , when BA533-IgG1, BA669-IgG1, and Avelumab were administered at a dose of 10 mg/kg, they could significantly inhibit the growth of the tumors of the mice MC38-hPD-L1, and the relative tumor suppression rate TGI (%)≥80%, there is no statistical difference between the three. When BA533-IgG1, BA669-IgG1, and Avelumab were administered at a dose of 3 mg/kg, the tumor suppression rates of BA533-IgG1 and BA669-IgG1 were superior to Avelumab. 
     The above results indicate that, at low doses, the candidate antibodies have better tumor suppression effect than the control antibody, and at high doses, they also have comparable tumor suppression effects. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Tumor volume data of MC38-hPD-L1 tumor model mice 
               
               
                 (average value ± SEM) (corresponding to FIG. 5B) 
               
            
           
           
               
               
            
               
                 Group 
                 Tumor volume at endpoint (mm 3 ) 
               
               
                   
               
               
                 Vehicle(Q2D, i.p.) 
                 1972 ± 224  
               
               
                 BA533-IgG1(3 mg/kg, Q2D, i.p.) 
                 780 ± 185 
               
               
                 BA669-IgG1(3 mg/kg, Q2D, i.p.) 
                 679 ± 113 
               
               
                 Avelumab(3 mg/kg, Q2D, i.p.) 
                 969 ± 208 
               
               
                 BA533-IgG1(10 mg/kg, Q2D, i.p.) 
                 465 ± 131 
               
               
                 BA669-IgG1(10 mg/kg, Q2D, i.p.) 
                 392 ± 83  
               
               
                 Avelumab(10 mg/kg, Q2D, i.p.) 
                 408 ± 90  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Tumor weight data of MC38-hPD-L1 tumor model (average 
               
               
                 value ± SEM) (corresponding to FIG. 5C) 
               
            
           
           
               
               
            
               
                 Group 
                 Tumor weight at endpoint (g) 
               
               
                   
               
               
                 Vehicle (Q2D, i.p.) 
                 2.12 ± 0.24 
               
               
                 BA533-IgG1 (3 mg/kg, Q2D, i.p.) 
                 0.92 ± 0.26 
               
               
                 BA669-IgG1 (3 mg/kg, Q2D, i.p.) 
                 0.77 ± 0.13 
               
               
                 Avelumab (3 mg/kg, Q2D, i.p.) 
                 1.22 ± 0.33 
               
               
                 BA533-IgG1 (10 mg/kg, Q2D, i.p.) 
                 0.46 ± 0.12 
               
               
                 BA669-IgG1 (10 mg/kg, Q2D, i.p.) 
                 0.39 ± 0.08 
               
               
                 Avelumab (10 mg/kg, Q2D, i.p.) 
                 0.42 ± 0.09