WO2022105751A1

WO2022105751A1 - TGFβRII FUSION PROTEIN AND USE THEREOF

Info

Publication number: WO2022105751A1
Application number: PCT/CN2021/130930
Authority: WO
Inventors: 张少飞; 付凤根; 唐蓉
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2020-11-19
Filing date: 2021-11-16
Publication date: 2022-05-27

Abstract

A new fusion protein including a PD-L1 antibody and a TGFβ receptor, a nucleic acid encoding the fusion protein or a fragment thereof, a host cell comprising same, and the relevant use. In addition, the present invention also relates to the therapeutic use of said fusion proteins or a fragment thereof.

Description

TGFβRII fusion protein and use thereof

The present invention relates to novel fusion proteins comprising PD-L1 antibody and TGFβ receptor. Furthermore, the present invention relates to nucleic acids encoding the fusion proteins or fragments thereof and host cells comprising the same, and related uses. Furthermore, the present invention relates to the therapeutic use of these fusion proteins or fragments thereof.

Background of the Invention

Programmed death ligand 1 (PD-L1) is a protein involved in suppressing immune system responses during chronic infection, pregnancy, tissue allotransplantation, autoimmune disease and cancer. PD-L1 regulates immune responses by binding to an inhibitory receptor called programmed death 1 (PD-1) expressed on the surface of T cells, B cells and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of PD-L1/PD-1 and PD-L1/B7.1 complexes negatively regulates T-cell receptor signaling, leading to subsequent downregulation of T-cell activation and suppression of antitumor immune activity. PD-L1 is overexpressed in many cancers. PD-L1 overexpression in tumor cells promotes tumor invasion and is often associated with poor prognosis.

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that regulates cell growth and differentiation, motility, extracellular matrix production and immune function. TGF[beta] has three mammalian isoforms, TGF[beta]-1, TGF[beta]-2 and TGF[beta]-3, each with distinct functions in vivo. All 3 TGFβ use the same receptor signaling system. Binding of TGFβ to TGFβRII is a critical step in the initiation of activation of the TGFβ signaling pathway, which leads to phosphorylation of Smad2 and translocation of the activated Smad2/Smad4 complex to the nucleus to regulate gene expression. Therefore, blocking the binding of human TGFβ1, TGFβ2 and TGFβ3 to human TGFβRII can inhibit angiogenesis and inhibit tumor cell growth, which is one of the research directions of cancer treatment.

It is known that inhibiting the PD-1/PD-L1 pathway on the basis of targeting TGF-β that neutralizes the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting tumor occurrence and development. Although there are a variety of anti-PD-L1 and TGF-βRII fusion proteins, such as Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β (Yan Lan et al., Science Translational Medicine 17 Jan 2018 ), those disclosed in WO 2015/118175 A2 or WO 2018/205985 A1, but these existing fusion proteins all have certain problems. For example, the molecule in the WO 2015/118175 A2 patent has a relatively low affinity for the anti-PD-L1 end, and there is a problem of fragmentation in the protein expression and purification; while the WO 2018/205985 A1 patent solves the problem of molecular fragmentation, it is carried out at the N-terminus of TGFβRII. truncation, but this truncation affects the affinity and blocking activity of the molecule for TGFβ.

Therefore, there is still a need in the art for new fusion protein molecules comprising PD-L1 and TGFβ receptors, which have a high affinity for PD-L1, ensuring a high occupancy ratio for PD-L1 and for PD-1/PD- Efficient blocking of L1 interaction; and still maintains TGFβ binding activity similar to TGFβ receptor, and has high stability.

SUMMARY OF THE INVENTION

In some aspects, the present invention relates to a novel fusion protein molecule comprising PD-L1 and TGFβRII, which has high affinity for PD-L1 to ensure a high occupancy ratio for PD-L1 and for PD-1/PD-L1 interaction Efficient blocking of the action, high binding activity to TGFβ, and high stability at the same time.

In some aspects, the present invention relates to the following specific embodiments:

1. A fusion protein molecule comprising

(i) anti-PD-L1 antibody or antigen-binding fragment thereof;

(ii) the ECD or C-terminal truncated ECD of the TGFβRII molecule;

Wherein, (i) and (ii) are not directly connected by a joint;

Wherein, the anti-PD-L1 antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3 respectively shown in the following amino acid sequences: SEQ ID NOs: 1, 2 and 3, and respectively as shown in the following amino acid sequences LCDR1, LCDR2 and LCDR3: SEQ ID NOs: 8, 9 and 10;

2. The fusion protein molecule of embodiment 1, wherein the anti-PD-L1 antibody is directly linked to the ECD of the TGFβRII molecule at the C-terminus of the heavy chain.

3. The fusion protein molecule of

embodiment

1 or 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises:

comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or A VH consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 11 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith or an amino acid sequence of 99% identity or a VL consisting of said amino acid sequence.

4. The fusion protein molecule of

embodiment

heavy chain constant region, which

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5 a specific amino acid sequence or consist of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 5; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 5;

and / or

light chain constant region, which

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 a specific amino acid sequence or consist of said amino acid sequence;

(ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 12.

5. The fusion protein molecule of

embodiment

1 or 2, wherein the anti-PD-L1 antibody is a monoclonal antibody.

6. A fusion protein molecule comprising

(i) chain A: anti-PD-L1 antibody light chain;

(ii) Chain B: an anti-PD-L1 antibody heavy chain directly linked at the C-terminus to the ECD of TGFβRII or to a C-terminally truncated ECD;

Wherein, the anti-PD-L1 antibody light chain comprises LCDR1, LCDR2 and LCDR3 as respectively shown in the following amino acid sequences: SEQ ID NO: 8, 9 and 10, and the anti-PD-L1 antibody heavy chain comprises respectively as shown in the following amino acid sequences HCDR1, HCDR2, HCDR3: SEQ ID NOs: 1, 2 and 3.

7. The fusion protein of embodiment 6, which consists of 2 chains A and 2 chains B.

8. The fusion protein of embodiment 6, wherein the anti-PD-L1 antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence of 96%, 97%, 98% or 99% identity or a VH consisting of said amino acid sequence, and the anti-PD-L1 antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 11 and has at least 90% therewith , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or VL consisting of said amino acid sequences. 9. The fusion protein of embodiment 6, wherein

Chain A comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14 a specific amino acid sequence or consist of said amino acid sequence;

and / or

Chain B comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7 a specific amino acid sequence or consist of said amino acid sequence.

10. The fusion protein molecule of

embodiment

1 or 2 or 6, wherein the ECD

(i) comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 41;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 41; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 5, 4, 3, 2, 1 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 41.

11. The fusion protein molecule of

embodiment

1 or 2 or 6, wherein the C-terminally truncated ECD is a C-terminal truncated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14 or 15 amino acid ECD.

12. The fusion protein molecule of

embodiment

1 or 2 or 6, wherein the C-terminally truncated ECD

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence selected from SEQ ID NO: 42 or 43 % identical amino acid sequences or consist of said amino acid sequences;

(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 42 or 43; or

(iii) comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) compared to the amino acid sequence selected from SEQ ID NO: 42 or 43 The amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) of or consisting of said amino acid sequences.

13. The fusion protein molecule of any one of embodiments 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv), (Fab') ₂ , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.

14. An isolated nucleic acid encoding the amino acid sequence shown in SEQ ID NO:7.

15. A vector comprising the nucleic acid of embodiment 14, preferably the vector is an expression vector.

16. A host cell comprising nucleic acids encoding chain A and chain B of the fusion protein molecule of any one of embodiments 6-12, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from CHO -S cells or HEK293 cells or other cells suitable for the production of antibodies or antigen-binding fragments thereof.

17. A method of making a molecule of a fusion protein comprising culturing the host cell of embodiment 16 under conditions suitable for expressing nucleic acids encoding strand A and strand B of the fusion protein molecule of any one of embodiments 6-12 , optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the fusion protein molecule from the host cell.

18. A pharmaceutical composition comprising the fusion protein molecule of any one of embodiments 1 to 13, and optionally one or more other therapeutic agents such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small Molecular drugs or immunomodulators, and optionally pharmaceutical excipients.

19. A pharmaceutical combination comprising the fusion protein molecule of any one of embodiments 1 to 13, and one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immune regulator.

20. A method of preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the fusion protein molecule of any one of embodiments 1 to 13, or the pharmaceutical composition of embodiment 18, or the pharmaceutical combination of embodiment 19.

21. The method of embodiment 20, wherein the tumor is a cancer, preferably the cancer has elevated levels (eg nucleic acid or protein levels) of TGFβ and/or PD-L1 (or PD-1 or PD-L2) ), for example the cancer is colon cancer.

22. The method of

embodiment

20 or 21, wherein the method further comprises administering to the patient one or more therapies, such as a therapeutic modality and/or other therapeutic agents, preferably the therapeutic modality includes surgery and/or radiation therapy, The other therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents.

Description of drawings:

FIG. 1A shows the NMR structure of TGFβRII, and FIG. 1B shows a schematic diagram of the fusion protein of the present invention.

Figure 2 shows that the fusion protein molecule PTR2, together with two control molecules, M7824 and SHR1701, blocks the signal generated by SMAD2 reporter cells upon stimulation of TGFβ.

Figure 3 shows the fusion protein molecule PTR2, with two control molecules M7824 and SHR1701, to block PD-1 binding to PD-L1 to restore the signal intensity of PD-1 overexpressing reporter cells.

Figure 4 shows the reversal of TGFβ1 inhibition of T cell proliferation by the fusion protein molecule PTR2, with two control molecules, M7824 and SHR1701.

Figure 5 shows the activation of the T-cell factor IFN-γ by the fusion protein molecule PTR2, with two control molecules, M7824 and SHR1701, and by anti-PD-L1 mAb.

Figure 6 shows the anti-tumor activity of the fusion protein molecule PTR2, with two control molecules M7824 and SHR1701, and anti-PD-L1 mAb.

Figure 7 shows the restoration of the signal intensity of PD-1 overexpressing reporter cells by blocking the binding of PD-1 to PD-L1 by the fusion protein molecule PTR2.

Figure 8 shows the effect of the fusion protein molecule PTR2 on blocking the signal generated by SMAD2 reporter cells stimulated by TGF[beta].

Detailed description of the invention

I. Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which shall be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For the purpose of interpreting this specification, the following definitions will be used and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps. Herein, when the terms "comprising" or "comprising" are used, unless otherwise indicated, combinations of the stated elements, integers or steps are also encompassed. For example, reference to an antibody variable region that "comprises" a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.

The terms "programmed cell death 1 ligand 1", "PD-L1", "programmed death ligand 1", "cluster of differentiation 274", "CD274" or "B7 homolog 1" as used herein refer to those derived from Any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). The term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 produced by processing in a cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information on the human PD-L1 gene, including genomic DNA sequence, can be found under NCBI Gene ID No. 29126. Additional information on the mouse PD-L1 gene, including genomic DNA sequence, can be found under NCBI Gene ID No. 60533. Amino acid sequences of exemplary full-length human PD-L1 proteins can be found, for example, under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7, while exemplary full-length mouse PD-L1 proteins can be found, for example, under NCBI Accession No. NP_068693 or Uniprot Accession No. Q9EP73. L1 protein sequence.

The terms "anti-PD-L1 antibody", "anti-PD-L1", "PD-L1 antibody" or "PD-L1 binding antibody" as used herein refer to an antibody that is capable of binding PD with sufficient affinity -L1 protein or a fragment thereof. In one embodiment, the anti-PD-L1 antibody binds to a non-PD-L1 protein to a degree that is less than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1 , about 60%, about 70%, about 80%, or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or bioluminescence interferometry or MSD assay. In some embodiments, the binding is performed, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), biolight interferometry, MSD assay or surface plasmon resonance (SPR) or flow cytometry technically measured.

The terms "full-length antibody", "complete antibody" and "intact antibody" are used interchangeably herein to refer to an antibody that is substantially structurally similar in structure to a native antibody or has a heavy chain comprising an Fc region as defined herein.

The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

An "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; dAb (domain antibody); linear antibody; single chain antibody (eg, scFv); single domain antibody such as VHH ; a dAb; or a camelid antibody.

"Complementarity determining regions" or "CDR regions" or "CDRs" are loops in the variable domains of antibodies that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ( "antigen contact point"). The CDRs are mainly responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and based on affinity propagation clustering using a large number of crystal structures North CDR Definition.

For example, according to different CDR determination schemes, the residues of each CDR are as follows.

A CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

Unless otherwise stated, in the present invention, when referring to a residue position in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering system according to the Kabat ( Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In one embodiment, the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:

HCDR1 was determined according to the AbM rule, and HCDR2 and HCDR3 were determined according to the Kabat rule.

In one embodiment, the light chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:

LCDR1, LCDR2 and LCDR3 are determined according to Kabat's rule.

It should be noted that the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Thus, when referring to antibodies defined by specific CDR sequences as defined in the present invention, the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the region of minimum overlap can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. The residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.

"An antibody in the IgG format" refers to the IgG format to which the heavy chain constant region of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in the IgG4 format means that its heavy chain constant region is derived from IgG4, or an antibody in the IgG1 format means that its heavy chain constant region is derived from IgG1.

A "humanized" antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of the non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

"Human antibody" or "fully human antibody" or "fully human antibody" are used interchangeably and refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a human Either human cells are generated or derived from non-human sources using human antibody repertoires or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

As used herein, the term "binding" or "specific binding" means that binding is selective for an antigen or target and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or by bioluminescence interferometry or by thin biofilms Interferometry or MSD assay or Surface Plasmon Resonance (SPR) assay.

"TGFβRII" or "TGFβ receptor II" means having the wild-type human TGFβ receptor type 2 isoform A sequence (eg, the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 or the amino acid sequence of Uniprot ID: P37173) , or a polypeptide having a wild-type human TGFβ receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233), or a polypeptide having substantially the same sequence as said wild-type amino acid sequence. TGF[beta]RII can retain at least 75%, 80%, 85%, 90%, 95%, or 99% of the TGF[beta]-binding activity of the wild-type sequence, or equal to or higher than the binding activity of the wild-type sequence. In one embodiment, the TGFβRII of the present invention comprises the amino acid sequence shown in SEQ ID NO:45, or has at least 85%, 86%, 87%, 88%, 89% with the amino acid sequence shown in SEQ ID NO:45 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

"Extracellular domain of TGFβRII" or "ECD of TGFβRII" or "ECD" refers to the extracellular domain of TGFβRII that can bind TGF-β and block its activity. In some embodiments, the ECD is determined according to the Topological domain of Uniprot. The ECD of TGFβRII is generally set forth in SEQ ID NO: 41 and has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. Typically, ECDs can be truncated at the C-terminus to obtain "C-terminally truncated ECDs" without affecting their ability to bind TGF-beta. For example, 1-30 amino acids can be terminally truncated without significantly affecting its activity. Preferably, the C-terminal truncated ECD is an ECD truncated at the terminal by 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5 amino acids, eg at the C-terminal Truncating the ECD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid (including any range thereof as an endpoint). In some embodiments, the C-terminal truncated ECD of the invention comprises the amino acid sequence set forth in SEQ ID NO:42, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In some embodiments, when ECD is used to construct a fusion protein, typically 1 or 2 amino acids at the N-terminus can be mutated to a G to increase its flexibility for attachment to other molecules (eg, antibodies, eg, anti-PD-L1 antibodies). Thus, references herein to "ECD" or "C-terminally truncated ECD" generally also encompass ECDs or C-terminally truncated ECDs that have 1-2 amino acid mutations at the N-terminus for the purpose of constituting a fusion protein.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (eg, immunosuppressive agents) ).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.

"Chemotherapeutic agents" include chemical compounds useful in the treatment of diseases of the immune system.

The term "small molecule drug" refers to low molecular weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less susceptible to eliciting an immune response than macromolecules.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants.

As used herein, an "immunosuppressant," "immunosuppressive drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition or combination of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention .

A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the required dose and for the required period of time. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition or combination thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject .

A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because a prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.

"Isolated nucleic acid encoding a fusion protein molecule or fragment thereof" refers to one or more nucleic acid molecules that encode the heavy or light chain (or fragment thereof, eg, heavy or light variable region) of a fusion protein molecule ), including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

Calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and

gap weights

16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and

gap weights

40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty of 12, gap penalty of 4), using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determining the percent identity between two amino acid sequences or nucleotide sequences. Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.

The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.

The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is usually characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include colon cancer.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

The term "pharmaceutical adjuvant" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.

The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients.

The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to kits, pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (eg, (i) a fusion protein of the invention, and (ii) other therapeutic agents) are combined as separate entities simultaneously, without specific time constraints or at the same or different time intervals, Administration to the patient is performed sequentially, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient. In some embodiments, the fusion proteins of the invention and other therapeutic agents used in pharmaceutical combinations are administered at levels no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The doses and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts produces a greater effect in the treatment of a disease or condition than either component alone can achieve. The ingredients may each be in separate formulations, which may be the same or different.

The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat the diseases described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the individual active ingredients in multiple or separate containers such as tablets, capsules, powders and liquids. Powders and/or liquids can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also includes the sequential use of each type of therapeutic agent at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.

As used herein, "treating" refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

A "subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue like from a fresh, frozen and/or preserved organ or tissue sample or biopsy or biopsy; blood or any blood component; bodily fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid); ), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

II. Antibody fusion proteins

In some embodiments, fusion protein molecules of the invention or fragments thereof bind PD-L1 (eg, human PD-L1 ) with high affinity. In some embodiments, the fusion protein molecules or fragments thereof of the invention bind TGF[beta] (eg, human TGF[beta]) with high affinity.

In some embodiments, the fusion protein molecules or fragments thereof of the invention bind human PD-L1 with higher affinity than known anti-PD-L1 antibodies. In some embodiments, the fusion protein molecules or fragments thereof of the invention bind human TGF[beta] with higher affinity than known fusion protein molecules. In some embodiments, the affinity of the fusion protein molecule or fragment thereof is determined by biolight interferometry.

In some embodiments, the fusion protein molecules of the invention, or fragments thereof, bind human TGFβ1 with an equilibrium dissociation constant (K _D ) that is less than or equal to about 10 nM, ₉ nM, 8 nM, 7 nM, 6 nM, 5 nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM or 0.5nM.

In some embodiments, the fusion protein molecules of the invention, or fragments thereof, bind to human PD-L1 with an equilibrium dissociation constant (K _D ) that is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, ₆ nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM.

In some embodiments, the molecules of the invention or fragments thereof are capable of blocking TGFβ1 signaling while blocking the binding of PD-1 to PD-L1.

In some embodiments, the molecules of the invention or fragments thereof are capable of reversing the inhibition of T cells by TGF[beta]1, and/or activating T cells (eg, increasing the secretion of cytokines such as IFN-[gamma]).

In some embodiments, the molecules of the invention or fragments thereof are more stable than known fusion protein molecules.

In some embodiments, the molecules or fragments thereof of the invention are capable of inhibiting tumor cells, eg, tumor cells expressing PD-1 or PD-L1 or PD-L2, and/or TGFβ.

In some embodiments, the molecules of the invention or fragments thereof can be used to treat cancer. In some embodiments, the molecules of the present invention or fragments thereof can effectively inhibit tumor growth, and the tumor inhibition rate is greater than or equal to about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.

In some embodiments, fusion protein molecules of the invention comprise

(i) anti-PD-L1 antibodies or fragments thereof;

(ii) the ECD or C-terminal truncated ECD of the TGFβRII molecule;

However, (i) and (ii) are directly connected without a linker.

In some embodiments, the anti-PD-L1 antibody is directly linked to the TGFβRII molecule at the C-terminus of the heavy chain.

In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is a complete antibody.

In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.

In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region ).

In some aspects, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL). In some aspects, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region HC and a light chain constant region LC.

In some embodiments, the heavy chain variable regions of the invention

(i) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:4 sequence or consists of said amino acid sequence; or

(ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4; or

(iii) comprising 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably The amino acid sequence of amino acid substitutions, more preferably conservative substitutions of amino acids, consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the light chain variable regions of the invention

(i) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 11 sequence or consists of said amino acid sequence; or

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 11; or

(iii) comprising 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably) compared to the amino acid sequence selected from SEQ ID NO: 11 The amino acid sequence of amino acid substitutions, more preferably conservative substitutions of amino acids, consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the invention, HCDR1, HCDR2 and HCDR3, are selected from the three complementarity determining regions contained in the VH as set forth in SEQ ID NO:4 HCDR1, HCDR2 and HCDR3

In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region of the invention, LCDR1, LCDR2 and LCDR3, are selected from the three complementarity determining regions contained in the VL as set forth in SEQ ID NO: 11 LCDR1, LCDR2, and LCDR3.

In some embodiments, HCDRl comprises or consists of the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:3.

In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8.

In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9.

In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody heavy chain constant region CH of the invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some embodiments, the antibody light chain constant region CL of the invention is a lambda or Kappa light chain constant region.

In some preferred embodiments, the antibody heavy chain constant region CH of the present invention

(iii) comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from SEQ ID NO: 5 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.

In some embodiments, the amino acid change occurs in the Fc region. In one embodiment, the amino acid change is N297A, L234A or L235A. In one embodiment, the amino acid changes are L234A and L235A. In one embodiment, the amino acid change in the Fc region attenuates the CDC or ADCC activity of the antibody.

In some embodiments, the antibody light chain constant region CL of the invention

(iii) comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 12 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.

In some specific embodiments of the present invention, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises: the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 4, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 11.

In some specific embodiments of the invention, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises:

HCDR1, HCDR2, HCDR3, respectively, as shown in the following amino acid sequences: SEQ ID NOs: 1, 2, and 3, and LCDR1, LCDR2, and LCDR3, respectively, as shown in the following amino acid sequences: SEQ ID NOs: 8, 9, and 10.

In some embodiments, the anti-PD-L1 antibody of the present invention is an antibody in the form of IgGl or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4, preferably an antibody in the form of IgGl.

In some embodiments, the anti-PD-L1 antibody is humanized.

In some embodiments, the anti-PD-L1 antibody is a human antibody.

In some embodiments, the anti-PD-L1 antibody is a chimeric antibody.

In one embodiment, the anti-PD-L1 antibodies of the present invention also encompass antibody fragments thereof (eg, antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') ₂ , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.

In some embodiments, the ECD or C-terminally truncated ECD of the TGFβRII molecules of the invention is mutated to a G at 1 or 2 amino acids at the N-terminus. In a preferred embodiment, the ECD or C-terminally truncated ECD of the TGFβRII molecule of the invention is mutated to a G at 1 amino acid at the N-terminus.

In some embodiments, the ECD of the TGFβRII molecules of the invention

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO:41 a specific amino acid sequence or consist of said amino acid sequence;

(iii) comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 41 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.

In some embodiments, the C-terminally truncated ECD is one that is truncated at the C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids ECD.

In some embodiments, the C-terminally truncated ECD of the TGFβRII molecules of the invention

In a preferred embodiment, the fusion protein molecule of the invention comprises

(i) chain A: anti-PD-L1 antibody light chain;

(ii) Chain B: an anti-PD-L1 antibody heavy chain with the ECD of TGFβRII or a C-terminally truncated ECD linked at the C-terminus.

In one embodiment, the fusion protein molecule of the invention consists of 2 chains A and 2 chains B.

In an embodiment of the invention, the anti-PD-L1 antibody light chain in chain A is as defined above. In some embodiments of the invention, the anti-PD-L1 antibody heavy chain in chain B is as defined above. In some embodiments of the invention, the ECD or C-terminally truncated ECD of TGFβRII is as defined above.

In some specific embodiments of the invention, chain A of the fusion protein molecule of the invention:

comprising or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14 amino acid sequence or consist of said amino acid sequence.

In some specific embodiments of the invention, chain B of the fusion protein molecule of the invention:

comprising or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7 amino acid sequence or consist of said amino acid sequence.

In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain. In some embodiments, the amino acid changes described herein occur in the Fc region of an antibody heavy chain constant region, and in one embodiment, the amino acid changes are N297A, L234A, L235A, or L234A and L235A. . In preferred embodiments, the amino acid changes in the Fc region attenuate the ADCC and/or CDC effects of the antibody.

In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in the following table:

原始残基original residue	示例性置换Exemplary permutation	优选的保守氨基酸置换Preferred conservative amino acid substitutions
Ala(A)Ala(A)	Val、Leu、IleVal, Leu, Ile	ValVal
Arg(R)Arg(R)	Lys、Gln、AsnLys, Gln, Asn	LysLys
Asn(N)Asn(N)	Gln、His、Asp、Lys、ArgGln, His, Asp, Lys, Arg	GlnGln
Asp(D)Asp(D)	Glu、AsnGlu, Asn	GluGlu
Cys(C)Cys(C)	Ser、AlaSer, Ala	SerSer
Gln(Q)Gln(Q)	Asn、GluAsn, Glu	AsnAsn
Glu(E)Glu(E)	Asp、GlnAsp, Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn、Gln、Lys、ArgAsn, Gln, Lys, Arg	ArgArg
Ile(I)Ile(I)	Leu、Val、Met、Ala、Phe、正亮氨酸Leu, Val, Met, Ala, Phe, Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸、Ile、Val、Met、Ala、PheNorleucine, Ile, Val, Met, Ala, Phe	IleIle
Lys(K)Lys(K)	Arg、Gln、AsnArg, Gln, Asn	ArgArg
Met(M)Met(M)	Leu、Phe、IleLeu, Phe, Ile	LeuLeu
Phe(F)Phe(F)	Trp、Leu、Val、Ile、Ala、TyrTrp, Leu, Val, Ile, Ala, Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val、SerVal, Ser	SerSer
Trp(W)Trp(W)	Tyr、PheTyr, Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp、Phe、Thr、SerTrp, Phe, Thr, Ser	PhePhe
Val(V)Val(V)	Ile、Leu、Met、Phe、Ala、正亮氨酸Ile, Leu, Met, Phe, Ala, Norleucine	LeuLeu

In certain embodiments, the substitutions occur in the CDR regions of the antibody. Typically, the variant obtained has a modification (eg, improvement) in certain biological properties (eg, increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody. Exemplary substitutional variants are affinity matured antibodies.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the carbohydrate attached to it can be varied. In some embodiments, the Fc region effector function is eliminated by mutating the glycosylation site, eg, N297A, or L234A and L235A mutations, thereby reducing ADCC activity or CDC activity.

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants, to alter one or more functional properties of the antibody, such as serum half-life, serum half-life, Complement fixation, complement dependent cytotoxicity, Fc receptor binding and/or antibody dependent cytotoxicity. Fc region variants can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid changes (eg, substitutions) at one or more amino acid positions.

In one embodiment of the invention, the antibodies described herein introduce changes in the Fc region to attenuate the ADCC activity or CDC activity of the antibody.

III. Nucleic Acids of the Invention and Host Cells Containing the Same

In one aspect, the present invention provides nucleic acids encoding any of the above fusion protein molecules. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector such as pcDNA3.1. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells (eg, CHO-S) or 293 cells (eg, 293F or HEK293 cells)), or other cells suitable for the production of antibodies or fragments thereof. In another embodiment, the host cell is prokaryotic.

For example, the nucleic acid of the present invention comprises a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 7, or encoding the amino acid sequence shown in SEQ ID NO: 7 with at least 85%, 90%, 91%, 92%, 93%, A nucleic acid having an amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity.

In one embodiment, one or more vectors are provided comprising the nucleic acid. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In one embodiment, the vector is pV120 or pCHO1.0.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the vector encoding the fusion protein include the prokaryotic or eukaryotic cells described herein.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of fusion proteins or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the encoded vector. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in fusion proteins that produce antibodies with partially or fully human glycosylation patterns. Host cells suitable for expression of fusion proteins of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered for growth in suspension can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (HEK293, 293F or 293T cells) and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2/0. Suitable mammalian host cell lines for the production of fusion proteins are known in the art.

IV. Production and Purification of Fusion Protein Molecules of the Invention

In one embodiment, the present invention provides a method of making a fusion protein molecule of the invention or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises under conditions suitable for expression of a nucleic acid encoding a fusion protein molecule of the invention or a fragment thereof The host cell is cultured and optionally the fusion protein or fragment thereof is isolated. In a certain embodiment, the method further comprises recovering the fusion protein molecule or fragment thereof of the invention from the host cell.

In one embodiment, there is provided a method of making a fusion protein molecule of the present invention, wherein the method comprises, under conditions suitable for expression of the fusion protein, culturing a fusion protein containing chain A and chain B (optionally in one) encoding the fusion protein. A nucleic acid in a vector) or a host cell comprising an expression vector of the nucleic acid, as provided above, and optionally the fusion protein is recovered from the host cell (or host cell culture medium).

For recombinant production of the fusion protein molecules of the invention, the nucleic acid encoding the strand of the fusion protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using routine procedures.

Fusion protein molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

V. Assay

The fusion proteins provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the fusion proteins of the invention are tested for their PD-L1 and/or TGF[beta] binding activity, eg, by known methods such as bio-optical interference and the like.

The invention also provides assays for identifying biologically active fusion proteins. Biological activities can include, for example, binding PD-L1 or TGFβ (eg, binding human PD-L1 or TGFβ), blocking TGFβ1 signaling, blocking PD-1 binding to PD-L1, reversing TGFβ1 inhibition of T cells, and /or activation of T cells (eg, increased secretion of cytokines such as IFN-γ), inhibition of tumor cells, etc.

In certain embodiments, antibodies of the invention are tested for such biological activity.

Cells for use in any of the above in vitro assays include cell lines that either naturally express PD-L1 or TGFβ or are engineered to express or overexpress PD-L1 or TGFβ. Such cells also include cell lines transfected with DNA encoding PD-L1 or TGFβ that express PD-L1 or TGFβ and do not normally express PD-L1 or TGFβ.

VI. PHARMACEUTICAL COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS

In some embodiments, the present invention provides compositions comprising any of the fusion protein molecules described herein or fragments thereof, preferably the compositions are pharmaceutical compositions. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, a composition, eg, a pharmaceutical composition, comprises a fusion protein molecule of the invention, or a fragment thereof, in combination with one or more other therapeutic agents.

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising the fusion protein molecules described herein or fragments thereof, or compositions comprising polynucleotides encoding the fusion protein molecules or fragments thereof described herein (including pharmaceutical composition or pharmaceutical preparation). These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C.Rowe, P.J.Seskey and S.C.Owen, Pharmaceutical Press, London, Chicago.

The compositions of the present invention may be in a variety of forms. Such forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

Pharmaceutical formulations comprising the antibodies described herein can be prepared by admixing an antibody of the invention of the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it would be desirable to also provide other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs or immunomodulatory agents, and the like. The active ingredients are suitably combined in amounts effective for the intended use.

VII. Drug Combinations and Kits

In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a fusion protein molecule or fragment thereof of the present invention, and one or more other therapeutic agents (eg, chemotherapeutic agents, cytokines, cytotoxic agents) , other antibodies, small molecule drugs or immunomodulators, etc.).

Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit. Dosage units can thus be provided according to the dosing regimen or the interval between drug administrations.

In one embodiment, the kit of parts of the present invention contains in the same package:

- a first container containing a pharmaceutical composition comprising a fusion protein molecule described herein or a fragment thereof;

- a second container containing a pharmaceutical composition comprising other therapeutic agents.

VIII. Uses and methods

One aspect of the present invention provides a method of preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of a fusion protein molecule or fragment thereof, pharmaceutical composition, pharmaceutical combination or kit of the present invention .

In some embodiments, the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) PD-L1 or PD-L2 or PD-1.

In some embodiments, the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) TGF[beta].

In some embodiments, the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) TGFβ and PD-L1 (or PD-1 or PD-L2).

In some embodiments, the tumor, eg, cancer, includes solid and hematological tumors and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in early, intermediate or advanced stages or metastatic.

In some embodiments, the tumor therapy will benefit from inhibition of PD-L1 or PD-L2 or PD-1, and/or TGFβ at the nucleic acid or protein level.

In a specific embodiment, the fusion protein molecules or fragments thereof of the present invention are capable of inhibiting tumor cell proliferation, such as tumor cells expressing TGFβ and/or PD-L1 (or PD-1 or PD-L2).

In some embodiments, the tumor is tumor immune escape.

In some embodiments, the tumor is cancer, such as colon cancer.

The subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual having or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (eg, cancer). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

In other aspects, the present invention provides the use of a fusion protein molecule or fragment thereof or a pharmaceutical composition or pharmaceutical combination or kit in the manufacture or manufacture of a medicament for the use described herein, eg for prophylaxis or treatment herein The related disease or condition mentioned.

In some embodiments, a fusion protein molecule or fragment thereof or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of the disorder and/or symptoms associated with the disorder.

In some embodiments, the fusion protein molecules or fragments thereof or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, eg, therapeutic modalities and/or other therapeutic agents, for the uses described herein, eg For the prevention and/or treatment of the related diseases or conditions mentioned herein.

In some embodiments, the treatment modality includes surgery; radiation therapy, localized or focused radiation, and the like.

In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulatory agents.

Exemplary immunomodulatory agents include immunosuppressive or anti-inflammatory agents.

Such combination therapy encompasses combined administration (eg, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the additional therapeutic agents and/or agents may be administered Administration of the fusion protein molecules or fragments thereof of the invention occurs prior to, concurrently with, and/or subsequent to.

The route of administration of the pharmaceutical composition is according to known methods, eg, orally, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Routes; via sustained release systems or via implanted devices. In certain embodiments, the compositions can be administered by bolus injection or by continuous infusion or by implanted devices.

These and other aspects and embodiments of the invention are described in the accompanying drawings (the Brief Description of the Drawings immediately follows) and the following Detailed Description and are exemplified in the following Examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Example

Example 1. Preparation of fusion protein molecules

(1) Molecular sequence design

The bifunctional antibody fusion protein of the present invention is composed of 1) anti-PD-L1 antibody and 2) ECD of TGFβ receptor 2 (TGFβRII). The anti-PD-L1 antibody part adopts the published antibody sequence (WO2019129136A1, HZ3266-IgG1N297A molecule). The N-terminus of TGFβRII (Uniprot ID: P37173) is prone to breakage (WO 2018/205985 A1). In order to analyze the possible reasons for the breakage, we fitted the conformations of 10 models in the NMR structure of TGFβRII.

As shown in Figure 1A, the N-terminus of TGFβRII is structurally different in 10 conformations, indicating that its conformation is very active. Therefore, we speculate that if the flexible GGGGS linking peptide is used to connect it to the antibody, the activity will be further increased, which may be the reason for the breakage.

The inventors of the present application directly linked the PD-L1 antibody domain TGFβRII ECD (TGFβRII extracellular domain) for the first time without using an additional linker. The schematic diagram of the obtained fusion protein is shown in FIG. 1B .

(2) Expression and purification of the molecule of the present invention

The CDR regions, light chain variable regions and heavy chain variable regions, light chain and heavy chain amino acid sequences, and corresponding nucleotide sequences of the three fusion proteins (PTR2, M7824, SHR1701) exemplified in the present application are described in this application. listed in the Sequence Listing section. in

The PTR2 fusion protein is constructed from the HZ3266-IgG1 N297A molecule disclosed in WO2019129136A1, which is directly linked to the ECD region of TGFβRII at the C-terminus of the heavy chain (shown in SEQ ID NO: 43), wherein the N297A amino acid mutation is replaced by L234A and L235A mutations.

The M7824 fusion protein is disclosed by WO2015118175A2 and is named Anti-PDL1/TGFβTrap in this patent application, wherein between Anti-PDL1 and TGFβRII ECD is linked with GGGGSGGGGSGGGGGSGGGGSG;

The SHR1701 fusion protein is disclosed by WO2018205985A1 and named fusion protein 9 in this patent application, wherein the anti-PD-L1 and TGFβRII ECD are linked by GGGGSGGGGSGGGGSGGGGSG.

The three exemplary fusion proteins described above were expressed and purified in Expi293 cells. For transient expression of antibodies in Expi293 (Gibco) cells, the vector pcDNA3.1 (Invitrogen) was used. First, the complete heavy chain and complete light chain of the fusion protein were cloned into separate pcDNA3.1 vectors to obtain plasmid DNA. The pcDNA3.1 vector with the complete heavy chain of the fusion protein molecule and the pcDNA3.1 vector with the complete light chain were co-transfected into Expi293 cells by chemical transfection. The chemical transfection reagent used was PEI (purchased from Polysciences), and the cultured Expi293 was transiently transfected according to the protocol provided by the manufacturer. The specific steps are as follows:

First, prepare plasmid DNA and transfection reagents in an ultra-clean workbench, add Opti-MEM medium (Gibco) (1/10 of the transfection volume) into a 50ml centrifuge tube, add plasmid DNA (100μg/100ml), The Opti-MEM medium containing the plasmid was filtered into a new 50ml centrifuge tube, and filtered PEI (1 g/L, Polysciences) was added to the centrifuge tube (mass ratio (plasmid:PEI)=1:3), Mix well and let stand for 20min. Gently pour the DNA/PEI mixture into Expi293 cells and mix well, incubate at 37°C under 8% CO2 conditions, 14h after transfection, add VPA (2.2M, Sigma) according to 0.1% of the transfected cell volume, respectively. 2.5% glucose (200g/L, Sigma) and 2.5% Feed were added, and the cells were cultured again under the conditions of 37°C and 8% CO2.

After 6 days or when the cells were continuously cultured until the cell viability was less than or equal to 60%, centrifuge at 13000 rpm for 20 min. Take the supernatant and purify the supernatant with HiTrap MabSelect SuRe (GE) packing column to make the purity of the fusion protein molecule > 95%.

Example 2. Fusion protein affinity determination experiment

Biofilm thin-layer interferometry (BLI) was used to determine the equilibrium dissociation constant (KD) of the antibody of the present invention binding to human PD-L1 and TGFβ. The BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AHC (18-5060, Fortebio) sensors and soak them in SD buffer (1x PBS, BSA 0.1%, Tween-20 0.05%).

Take 100 μl of SD buffer, 100 nM of the fusion protein molecule prepared in Example 1, 100 nM of human PD-L1 (PD1-H5229, Acro biosystem), and 100 nM of human TGFb (TG1-H4214, Acro biosystem) were added to 96 wells of black polystyrene. Styrene half volume microplates (Greiner, 675076). Detection was performed using Fortebio Octet Red96e, and the sensor position was selected according to the sample position. After immobilizing the antibody by the AHC sensor, it binds to PD-L1 and TGFb. The instrument setting parameters are as follows: running steps: Baseline, Antibody Loading～1nm, Baseline, Antigen Association and Dissociation; the running time of each step depends on the speed of sample binding and dissociation, the rotation speed is 1000rpm, and the temperature is 30℃. KD values were analyzed using ForteBio Octet analysis software.

Table 1. Summary of the affinity of fusion protein molecules to recombinant human TGFβ1

样本sample	KD(M)KD(M)	Kon(1/ms)Kon(1/ms)	Kdis(1/s)Kdis(1/s)
PTR2PTR2	4.73E-104.73E-10	1.02E+061.02E+06	4.82E-044.82E-04
M7824M7824	8.82E-108.82E-10	7.30E+057.30E+05	6.44E-046.44E-04
SHR1701SHR1701	1.71E-091.71E-09	8.26E+058.26E+05	1.41E-031.41E-03

It can be seen from the above table that the PTR2 molecule of the present invention and the M7824 control molecule show extremely high affinity for recombinant human TGFβ1. However, in the control molecule of SHR1701, the binding efficiency of TGFβ1 decreased due to the truncation of the extracellular segment of TGFβRII.

The ForteBio affinity assay was performed again as described above, except that the target recombinant human TGFβ1 protein was replaced with recombinant human PD-L1.

Table 2. Summary of the affinity of fusion protein molecules to recombinant human PD-L1

样本sample	KD(M)KD(M)	Kon(1/ms)Kon(1/ms)	Kdis(1/s)Kdis(1/s)
PTR2PTR2	8.35E-118.35E-11	4.86E+054.86E+05	4.05E-054.05E-05
M7824M7824	2.66E-102.66E-10	2.84E+052.84E+05	7.57E-057.57E-05
SHR1701SHR1701	6.71E-116.71E-11	1.08E+061.08E+06	7.28E-057.28E-05

It can be seen from the above table that the PTR2 molecule of the present invention and the SHR1701 control molecule show extremely high affinity for recombinant human PD-L1. While the M7824 control molecule had a slightly lower binding affinity for PD-L1 than the other two molecules.

Example 3. The fusion protein molecule of the present invention blocks the experiment of SMAD2 reporter cell signal

In order to verify the blocking effect of the fusion protein of the present invention on TGFβ, the molecular PTR2 of the present invention, together with two control molecules M7824 and SHR1701, were detected to block the effect of the SMAD2 reporter cell on the signal generated under the stimulation of TGFβ. The detailed experimental process is as follows:

The 4T1 cell line (ATCC: CRL-2539) was infected with the SMAD2 luciferase reporter system (Qiagen) in the form of lentivirus, and the 4T1-SMAD2 reporter cell line was obtained by screening. In the experiment, 50,000 cells were plated in each well, and then recombinant human TGFβ1 (R&D Systems) was added to each well to a final concentration of 2ng/ml. Next, the fusion protein molecules to be tested prepared in Example 1 were added to the cells at a final concentration of up to 80 nM, diluted 4 times, with a total of 9 gradient points, and incubated overnight in a cell incubator.

Thaw the configured Bio-Glo luciferase reporter system substrate (Promega, G7940), take out the cell plate and equilibrate at room temperature for 30 minutes, add 100ul Bio-Glo substrate to each well, and stand still for 10 minutes in the dark. Using a microplate reader (Molecular Devices, Spectra MAXi3), according to the manufacturer's instructions, the full wavelength scan was used to read the value. The results are shown in Figure 2.

The following conclusions can be drawn from Fig. 2: (1) the molecule PTR2 of the present invention and the control molecule M7824 can effectively block the signal of TGFβ1; (2) the signal inhibition efficiency of SHR1701 to TGFβ1 is low. This experiment reproduced the difference in the affinity of the fusion protein molecule of the present invention and the control molecule for TGFβ1 at the protein level, again indicating that the truncation of the N-terminus of the extracellular segment of TGFβRII affects the binding to TGFβ.

Example 4. The fusion protein of the present invention blocks the experiment that Jurkat PD-1 reporter cells are inhibited by PD-L1

To verify the ability of the molecules of the present invention to block the binding of PD-1 to PD-L1 and restore the activity of T cells, we established an in vitro PD-1 reporter cell experimental system. The specific experimental steps are as follows:

(1) One day in advance, plate PD-L1 aAPC/CHO-K1 cells (PROMEGA, J1252) in the middle 60 wells of a white flat-bottomed 96-well plate, 40,000/well, 100ul system, and the culture condition is 10% FBS (HyClone, SH-30406.05)+Ham's F12 (GIBCO, 11765-054); 100ul PBS (GIBCO, 10010-049) was added to the outer circle to prevent edge effects; cultured overnight.

(2). Dilute the fusion protein molecule prepared in Example 1 with Assay buffer (H1640 medium+1% FBS), to configure a series of fusion protein molecule solutions, the highest concentration is 400nM, four times serial dilution, 9 concentrations, The last concentration is 0.

(3). Aspirate the cell supernatant in the white 96-well plate and wash the cells once with Assay buffer.

(4). Add the fusion protein molecule solution prepared in (2) into the cells, 40ul per well; add 40ul Assay buffer to the outer circle.

(5). Take an appropriate amount of PD-1 Effector cells (Promega) for counting, resuspend in Assay buffer, 1.25 million/ml; add 40ul of cell suspension to the well plate in the previous step, so that each well contains 50,000 PD-1 cells. 1 Effector cells.

(6). After mixing, put it back into the incubator and continue to incubate at 37°C, 5% CO ₂ for 6 hours.

(7) Return the configured Bio-Glo Reagent (PROMEGA, G7940) to room temperature, add 80ul to the well plate in step (6), and incubate at room temperature for about 5 minutes in the dark, then the microplate reader (Molecular Devices, Spectra MAXi3) detects chemiluminescence. Set up a control well (40ul Assay buffer+40ul Bio-Glo Reagent) for background subtraction. See Figure 3 for the results.

It can be seen that the molecule PTR2 of the present invention can efficiently block the combination of PD-L1 and PD-1, and restore the signal intensity of reporter cells overexpressing PD-1. In addition, the efficiency of PTR2 and SHR1701 in blocking the binding of PD-L1 to PD-1 was slightly higher than that of M7824, which is consistent with the difference in the affinity of PD-L1 at the protein level.

Example 5. The fusion protein molecule of the present invention reverses the proliferation of CD4+ T cells

In order to verify the ability of the fusion protein molecule of the present invention to reverse the ability of TGFβ to inhibit the proliferation of T cells, the experimental steps are as follows:

(1) 100ul/well Anti-CD3 antibody (Biolegend, 317325) 0.5ug/ml was coated in a 96-well plate, overnight at 4°C.

(2). The next day, human CD4+ T cells were isolated and purified from the recovered PBMCs (ALLCELLS, PB005F-C) using the CD4+ T cell purification kit (Stem cell, 19052).

(3). Use Cell Trace CFSE Cell Proliferation kit (Invitrogen, C34554), label the isolated and purified T cells in (2) according to the instructions, and count them; use AIM-V medium (GIBCO, A3021002) to dilute the cells to 1.2*10 ^6/ml, cells were plated on 96-well plates, 100ul/well, and incubated at 37°C for 1 hour.

(4) Use AIM-V medium to dilute the fusion protein molecule PTR2 and control molecules M7824 and SHR1701 prepared in Example 1 (starting at 100nM, 3-fold dilution, 50ul/well), and then add TGF at a final concentration of 20ng/ml -β protein (R&D, 7754-BH), 50ul/well, incubate at 37°C for 30 minutes after mixing.

(5). Transfer the fusion protein molecule and protein mixed solution obtained in (4) to the cell culture plate in step (3), 100ul/well, and put it into an incubator for 4 days after mixing.

(6). After 4 days, the proportion of proliferating cells was detected by flow cytometry.

See Figure 4 for the results. It can be seen that adding TGFβ1 after activation of CD4+ T cells can inhibit cell activation, that is, reduce cell proliferation rate. However, adding the molecule PTR2 of the present invention and the control molecule M7824 or SHR1701 can reverse the inhibition of TGFβ1 on the proliferation of CD4+ T cells to different degrees. Among them, PTR2 and M7824 could reverse T cell proliferation to the control level, but SHR1701 only weakly reversed the proliferation inhibition, indicating that the truncated TGFβRII extracellular segment decreased the binding activity of TGFβ.

Example 6. Activation experiment of human T cells by the molecules of the present invention

In order to verify the in vitro activation effect of the molecules of the present invention on human T cells, we established an MLR (mixed lymphocyte reaction) experiment. The detailed experimental process is as follows:

(1). Recover 1 branch of mature dendritic cells (DC, ALLCELLS, PB-DC002F-C) and 1 branch of PBMC (ALLCELLS, PB005F-C) according to the instructions;

(2). Extract CD4+T cells from PBMC according to CD4+T cell purification kit (Stem cell, 19052);

(3). Dilute the cells with AIM-V (Gibco) medium so that the density of DC cells is 400,000/ml and the density of CD4+ T cells is 2,000,000/ml;

(4) The fusion protein molecule PTR2 prepared in Example 1 was diluted with AIM-V medium, the control molecule M7824 or SHR1701 and the anti-PD-L1 antibody (WO2019129136A1, HZ3266-IgG1N297A molecule, prepared according to the method in this document), the highest The concentration is 400nM, 4-fold serial dilution;

(5). In the first 96-well plate, mix 50ul DC+50ul CD4+T cells+50ul fusion protein molecule+50ul AIM-V;

(6). In the second 96-well plate, mix 50ul DC+50ul CD4+ T cells+50ul fusion protein molecule+50ul (20ng/ml TGF-β in SFM).

(7). Put the first 96-well plate in (5) and the second 96-well plate in (6) back into the incubator at 37°C and continue to culture for 6 days in 5% CO ₂ .

(8). Centrifuge the two cell culture plates in (7) at 400g for 5min, and draw 50ul of supernatant into two new 96-well plates respectively;

(9). Use Human Th1/Th2/Th17 Kit (BD, 560484), configure the detection solution according to the instructions, mix with the supernatant obtained in (8), incubate at room temperature for 3h, and detect by flow cytometry.

The results are shown in Figure 5. The experimental results show that after adding the molecule PTR2 of the present invention and the control molecules M7824 and SHR1701 to the mixed lymphocyte reaction experimental system, the activation of T cells is improved compared with adding anti-PD-L1 monoclonal antibody alone. Among them, SHR1701 has a weaker effect on T cell activation, while PTR2 and M7824 can significantly increase the degree of T cell activation.

Example 7. The efficacy of the fusion molecule of the present invention in the MC38 tumor-bearing human PD-L1 knock-in mouse model

Human PD-L1 knock-in C57 mice (female mice, 4-6 weeks old, Shanghai Southern Model Center) were divided into 6 groups of 9 mice, and each mouse was subcutaneously injected with 10 ⁶ MC38-hPDL1 mice. Murine colon cancer cells (Nanjing Galaxy). On the 7th day after cell injection (day7), 5 mg/kg hIgG (Equitech-Bio, Cat. No.: 161206-0656), PTR2, M7824 and SHR1701 prepared in Example 1, or 2 mg/kg were injected intraperitoneally, respectively. PTR2 prepared in Example 1. Anti-PD-L1 antibody (WO2019129136A1, HZ3266-IgG1N297A molecule, prepared according to the method in the literature) was intraperitoneally administered in equimolar number at 4.11 mg/kg.

Dosing twice a week for a total of five doses. The tumor size was measured twice a week, and the largest long axis (L) and the largest wide axis (W) of the tumor were measured with vernier calipers, and the tumor volume was calculated according to the following formula: V=L×W ² /2.

The results are shown in Figure 6. Compared with mice injected with hIgG, the anti-PD-L1 antibody at 4.11 mg/kg had only a weak tumor-suppressive effect (TGI was 19.3%), and the PTR2 at 5 mg/kg (high dose of PTR2) or the control molecules M7824 and SHR1701 had no effect. It showed a more significant tumor inhibition efficiency (TGI was 76.1%, 52.3% and 40.8%, respectively), indicating that these molecules have the effect of blocking TGFβ, but at the same dose, the efficacy of PTR2 is significantly better than the two controls molecular. In addition, the efficacy of PTR2 at 2 mg/kg (low dose of PTR2) is similar to that of the control molecules M7824 and SHR1701 at a high dose (5 mg/kg), indicating that it can achieve better tumor-inhibiting efficacy at low doses. Known reference molecules have certain advantages.

Example 8. In vitro activity detection of the fusion protein molecule of the present invention before and after being placed at 40°C for 14 days

The ExpiCHO-S ^™ cell line expressing the fusion protein molecule was generated using the ExpiFectamine ^™ CHO Transfection Kit (Gibco) according to the manufacturer's instructions.

The complete heavy and light chains of PTR2 were first cloned into separate pcDNA3.1 (Invitrogen) vectors to obtain plasmid DNA. Afterwards, the respectively constructed pcDNA3.1 plasmids were co-transfected into ExpiCHO-S ^TM cell line by chemical transfection method. Specific steps are as follows:

First, prepare plasmid DNA and transfection reagents in an ultra-clean workbench, add OptiPRO ^™ -SFM medium (Gibco) (volume is 8% of the transfection volume) into a 50ml centrifuge tube, add plasmid DNA (80μg/100ml), The OptiPRO ^™ -SFM medium containing the plasmid was filtered into a new 50ml centrifuge tube, and the ExpiFectamine ^™ CHO Reagent in the kit was added to the centrifuge tube (mass ratio (plasmid: Reagent)=1:4), and mixed well Pour the DNA/Reagent mixture into ExpiCHO-S ^TM cells as soon as possible (no more than 5 minutes) and mix well. Incubate at 37°C under 8% CO2 conditions. After 18-22h after transfection, the volume of transfected cells is 0.6% respectively. The ExpiFectamine ^TM CHO Enhancer in the kit was supplemented, and 30% ExpiCHO ^TM Feed was added, and the cells were again cultured under the conditions of 37°C and 8% CO2.

After 6 days or when the cells were continuously cultured until the cell viability was less than or equal to 60%, centrifuge at 13000 rpm for 20 min. Take the supernatant and purify the supernatant with HiTrap MabSelect SuRe (GE) packing column, so that the purity of the fusion protein molecule PTR2 is more than 95%.

As described in Example 1, the molecule PTR2 of the present invention does not have an additional linker peptide added between anti-PD-L1 and TGFβRII to increase the stability of the molecule. To verify the stability, we concentrated the PTR2 prepared above to 10 mg/ml, placed it in a 40°C incubator for 14 days (T14d), and then compared it with the sample placed at -80°C (T0d).

Experiments to block the inhibition of Jurkat PD-1 reporter cells by PD-L1 were first performed as described in Example 4. The experimental results are shown in Figure 7.

Next, experiments were performed to molecularly block SMAD2 reporter cell signaling. The experimental method is as described in Example 3. The experimental results are shown in Figure 8.

As shown in the above two experimental results, the PTR2 samples placed at 40°C for 14 days completely maintained the activities of blocking PD-1/PD-L1 and blocking TGFβ, showing good molecular stability.

Serial information:

Claims

A fusion protein molecule comprising

(i) anti-PD-L1 antibody or antigen-binding fragment thereof;

(ii) the ECD or C-terminal truncated ECD of the TGFβRII molecule;

Wherein, (i) and (ii) are not directly connected by a joint;

Wherein, the anti-PD-L1 antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3 respectively shown in the following amino acid sequences: SEQ ID NOs: 1, 2 and 3, and respectively as shown in the following amino acid sequences LCDR1, LCDR2 and LCDR3: SEQ ID NOs: 8, 9 and 10;
The fusion protein molecule of claim 1, wherein the anti-PD-L1 antibody is directly linked to the ECD of the TGFβRII molecule at the C-terminus of the heavy chain.
The fusion protein molecule of claim 1 or 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises:

comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or A VH consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 11 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith or an amino acid sequence of 99% identity or a VL consisting of said amino acid sequence.
The fusion protein molecule of claim 1 or 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises:

heavy chain constant region, which

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5 a specific amino acid sequence or consist of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 5; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 5;

and / or

light chain constant region, which

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 a specific amino acid sequence or consist of said amino acid sequence;

(ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 12.
2. The fusion protein molecule of claim 1 or 2, wherein the anti-PD-L1 antibody is a monoclonal antibody.
A fusion protein molecule comprising

(i) chain A: anti-PD-L1 antibody light chain;

(ii) Chain B: an anti-PD-L1 antibody heavy chain directly linked at the C-terminus to the ECD of TGFβRII or to a C-terminally truncated ECD;

Wherein, the anti-PD-L1 antibody light chain comprises LCDR1, LCDR2 and LCDR3 as respectively shown in the following amino acid sequences: SEQ ID NO: 8, 9 and 10, and the anti-PD-L1 antibody heavy chain comprises respectively as shown in the following amino acid sequences HCDR1, HCDR2, HCDR3: SEQ ID NOs: 1, 2 and 3.
The fusion protein of claim 6, which consists of 2 chains A and 2 chains B.
The fusion protein of claim 6, wherein the anti-PD-L1 antibody heavy chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence shown in SEQ ID NO: 4 , 97%, 98% or 99% identical amino acid sequence or VH consisting of said amino acid sequence, and the anti-PD-L1 antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 11 and has at least 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or VL consisting of said amino acid sequences.
The fusion protein of claim 6, wherein

Chain A comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14 a specific amino acid sequence or consist of said amino acid sequence;

and / or

Chain B comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7 a specific amino acid sequence or consist of said amino acid sequence.
The fusion protein molecule of claim 1 or 2 or 6, wherein ECD

(i) comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 41;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 41; or

(iii) an amino acid sequence comprising or consisting of an amino acid sequence having no more than 5, 4, 3, 2, 1 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 41.
The fusion protein molecule of claim 1 or 2 or 6, wherein the C-terminally truncated ECD is a C-terminal truncated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14 or 15 amino acid ECD.
The fusion protein molecule of claim 1 or 2 or 6, wherein the C-terminally truncated ECD

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence selected from SEQ ID NO: 42 or 43 % identical amino acid sequences or consist of said amino acid sequences;

(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 42 or 43; or

(iii) comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) compared to the amino acid sequence selected from SEQ ID NO: 42 or 43 The amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) of or consisting of said amino acid sequences.
The fusion protein molecule of any one of claims 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab ') 2. Single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
An isolated nucleic acid encoding the amino acid sequence shown in SEQ ID NO:7.
A vector comprising the nucleic acid of claim 14, preferably the vector is an expression vector.
A host cell comprising nucleic acids encoding chain A and chain B of the fusion protein molecule of any one of claims 6-12, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from CHO-S cells or HEK293 cells or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
A method of making a molecule of a fusion protein comprising culturing the host cell of claim 16 under conditions suitable for expressing nucleic acids encoding strand A and strand B of the fusion protein molecule of any one of claims 6-12, any Optionally the antibody or antigen-binding fragment thereof is isolated, optionally the method further comprises recovering the fusion protein molecule from the host cell.
A pharmaceutical composition comprising the fusion protein molecule of any one of claims 1 to 13, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
A pharmaceutical combination comprising the fusion protein molecule of any one of claims 1 to 13, and one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents .
A method of preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the fusion protein molecule of any one of claims 1 to 13, or the pharmaceutical composition of claim 18, or the right Requires 19 drug combinations.
The method of claim 20, wherein the tumor is a cancer, preferably, the cancer has elevated levels (eg nucleic acid or protein levels) of TGFβ and/or PD-L1 (or PD-1 or PD-L2), For example the cancer is colon cancer.
The method of claim 20 or 21, wherein the method further comprises administering to the patient one or more therapies, such as a treatment modality and/or other therapeutic agents, preferably, the treatment modality includes surgery and/or radiation therapy, other treatments The agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents.