HK1252955B - Single domain antibody for programmed death-ligand (pd-l1) and derived protein thereof - Google Patents

Description

Single domain antibodies and their derivative proteins targeting programmed death ligand (PD-L1)

Technical Field

The present invention relates to the field of medicine and biology, and discloses single-domain antibodies and derivative proteins thereof against programmed death ligand (PD-L1). Specifically, the present invention discloses a programmed death ligand 1 (PD-L1) binding molecule and its use, particularly in the treatment and/or prevention, or diagnosis of PD-L1-related diseases such as tumors.

Background Art

Programmed death-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The original members of this family, CD28 and ICOS, were discovered by their ability to enhance T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999), Nature 397:263-266; Hansen et al. (1980), Immunogenics 10:247-260). Two cell surface glycoprotein ligands of PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation and cytokine secretion after binding to PD-1 (Freeman et al. (2000), J Exp Med 192:1027-34; Latchman et al. (2001), Nat Immunol 2:261-8; Cater et al. (2002), Eur J Immunol 32:634-43; Ohigashi et al. (2005), Clin Cancer Res 11:2947-53). Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1 but not to other CD28 family members (Blank et al. 2004). PD-L1 expression on the cell surface has also been shown to be upregulated by IFN-γ stimulation.

PD-L1 expression has been found in several murine and human cancers, including lung, ovarian, colon, melanoma, and various myeloma tumors (Iwai et al. (2002), PNAS 99:12293-7; Ohigashi et al. (2005), Clin Cancer Res 11:2947-53). Existing results indicate that high expression of PD-L1 by tumor cells plays a key role in tumor immune evasion by increasing T cell apoptosis. Researchers have found that the P815 tumor cell line transfected with PD-L1 resists lysis by specific CTLs in vitro and exhibits enhanced tumorigenicity and invasiveness when inoculated into mice. These biological properties can be reversed by blocking PD-L1. In mice with PD-1 knockout, which blocks the PD-L1/PD-1 pathway, inoculation with tumor cells prevents tumor formation (Dong et al. (2002), Nat Med 8:793-800). It has also been suggested that PD-L1 may be involved in intestinal mucosal inflammation, and inhibition of PD-L1 prevents the wasting disease associated with colitis (Kanai et al. (2003), J Immunol 171:4156-63)

There is still a need in the art for anti-PD-L1 antibodies that can bind to PD-L1 with high affinity and block the binding of PD-1 to PD-L1, particularly anti-PD-L1 heavy chain single domain antibodies.

SUMMARY OF THE INVENTION

The inventors of the present invention used phage display technology to screen and obtain an anti-PD-L1 heavy chain single domain antibody (VHH) with high specificity, high affinity and high stability.

In a first aspect, the present invention provides a PD-L1 binding molecule comprising an immunoglobulin single variable domain consisting of an amino acid sequence selected from SEQ ID NOs: 1-6 and a human immunoglobulin Fc region.

In another aspect, the present invention relates to nucleic acid molecules encoding PD-L1 binding molecules, and expression vectors and host cells containing the nucleic acid molecules.

The present invention also relates to pharmaceutical compositions comprising the PD-L1 binding molecules of the present invention.

The present invention also relates to methods of making the PD-L1 binding molecules described herein.

The present invention also relates to uses of the PD-L1 binding molecules and pharmaceutical compositions of the present invention, in particular uses and methods for preventing and/or treating diseases associated with PD-L1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the blocking effect of PD-L1 heavy chain single domain antibody on PD-1/PD-L1 interaction.

FIG2 shows the binding curve of the PD-L1 heavy chain single domain antibody to the PD-L1 antigen protein.

FIG3 shows the blocking curve of the PD-L1 heavy chain single domain antibody on the interaction between PD-1 and PD-L1.

FIG4 shows the sequence alignment of five humanized variants of antibody strain 56.

FIG5 shows the binding curve of PD-L1 single-domain antibody Fc fusion protein to PD-L1 (ELISA method).

FIG6 shows the blocking curve of PD-L1 single domain antibody Fc fusion protein on PD-L1/PD-1 interaction (competitive ELISA method).

FIG7 shows the blocking curve of PD-L1 single domain antibody Fc fusion protein on PD-L1/CD80 interaction (competitive ELISA method).

Figure 8 shows the blocking curve of PD-L1 single domain antibody Fc fusion protein on 293-PDL1/PD1 interaction (FACS method)

Figure 9 shows the blocking curve of PD-L1 single domain antibody Fc fusion protein on Jurket-PD1/PDL1 interaction (FACS method)

Figure 10. Flow cytometry was used to detect the specificity of PD-L1 single domain antibody Fc fusion protein binding to PD-L1 protein.

Figure 11. Flow cytometry detection of the binding of PD-L1 single domain antibody Fc fusion protein to monkey PD-L1 protein.

FIG12 shows that PD-L1 single domain antibody Fc fusion protein recognizes PD-L1 positive cell populations on patient tissue sections.

FIG13 shows the activation effect of PD-L1 single-domain antibody Fc fusion protein on PBMC.

FIG14 shows the activation effect of PD-L1 single domain antibody Fc fusion protein on CD4+ T cells.

Figure 15 shows that PD-L1 single domain antibody Fc fusion protein stimulates IL-2 secretion.

FIG16 shows the CDC and ADCC activities of PD-L1 single domain antibody Fc fusion proteins with mutant Fc.

Figure 17 shows the tumor growth curve after treatment with PD-L1 single domain antibody Fc fusion protein.

FIG18 shows the inhibitory activity of PD-L1 single domain antibody Fc fusion protein on tumor growth at different administration times.

Figure 19 shows a comparison of the tumor growth inhibitory activities of hu56V2-Fc and 2.41. A: A375:PBMC = 5:1; B: A375:PBMC = 1:1.

FIG20 shows the effects of alkaline and oxidative treatments on the activity of PD-L1 single domain antibody Fc fusion protein.

Detailed Description of the Invention

definition

Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to heavy chain antibodies or conventional four-chain antibodies, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and/or the domain.

As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule, consisting of a two-layer sandwich of about seven antiparallel beta-sheet strands arranged in two beta sheets, optionally stabilized by conserved disulfide bonds.

As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site.

As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. One example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody," such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae, as defined below.

"VHH domain", also known as heavy chain single domain antibody, VHH, _VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional four-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional four-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain.

In the context of the present invention, the terms "heavy chain single domain antibody", "VHH domain", "VHH", " _VHH domain", "VHH antibody fragment", "VHH antibody" and "domain"("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.

For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domains of Camelidae are numbered according to the general numbering scheme for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). According to this numbering scheme,

- FR1 comprises the amino acid residues at positions 1-30,

- CDR1 comprises the amino acid residues at positions 31-35,

- FR2 comprises the amino acids at positions 36-49,

- CDR2 comprises the amino acid residues at positions 50-65,

- FR3 comprises the amino acid residues at positions 66-94,

- CDR3 comprises the amino acid residues at positions 95-102, and

- FR4 comprises the amino acid residues at positions 103-113.

It should be noted, however, that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

Alternative methods for numbering the amino acid residues of VH domains are known in the art and can also be applied analogously to VHH domains. However, unless otherwise indicated, in the present specification, claims and drawings, the numbering according to Kabat as described above and applicable to VHH domains will be followed.

The total number of amino acid residues in a VHH domain will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

Other structural characteristics and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

VHH domains (which are naturally "designed" to functionally bind to antigens in the absence of, and without interacting with, light chain variable domains) can be used as single, relatively small, functional antigen-binding structural units, domains, or polypeptides. This distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies, which are generally unsuitable for practical application as single antigen-binding proteins or immunoglobulin single variable domains by themselves, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFvs consisting of a VH domain covalently linked to a VL domain).

Due to these unique properties, the use of VHH domains—alone or as part of a larger polypeptide—offers a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):

Only a single domain is required to bind the antigen with high affinity and selectivity, thus eliminating the need for two separate domains and ensuring that the two domains are in the proper spatial conformation and configuration (e.g., scFv generally requires the use of a specially designed linker);

- VHH domains can be expressed from a single gene and do not require post-translational folding or modification;

- VHH domains can be easily engineered into multivalent and multispecific formats (formatting);

- VHH domains are highly soluble and have no tendency to aggregate;

- VHH domains are highly stable to heat, pH, proteases, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thereby achieving cost, time, and environmental savings;

- VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

- The VHH domain is relatively small compared to conventional four-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa or 1/10 the size of conventional IgG), and therefore exhibits higher tissue penetration and can be administered at higher doses than conventional four-chain antibodies and antigen-binding fragments thereof;

- VHH domains may display so-called cavity-binding properties (especially due to their extended CDR3 loop compared to conventional VH domains), thereby being able to access targets and epitopes that are inaccessible to conventional 4-chain antibodies and antigen-binding fragments thereof.

Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185–195; Li et al., J Biol Chem., 287 (2012) 13713–13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94/04678.

A VHH domain derived from Camelidae can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a conventional human four-chain antibody VH domain. The humanized VHH domain may contain one or more fully human framework region sequences, and in a specific embodiment, may contain human framework region sequences from IGHV3.

In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., immunoglobulin single variable domain of the present invention) molecule can bind to. Specificity can be determined based on the affinity and/or avidity of the antigen binding molecule. Avidity, represented by the dissociation equilibrium constant (KD) of the antigen and the antigen binding protein, is a measure of the binding strength between the epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding molecule (or, affinity can also be expressed as an association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding molecule (e.g., immunoglobulin, antibody, immunoglobulin single variable domain, or a polypeptide containing it) and a related antigen. Avidity is related to both: the affinity between the antigen binding site on its antigen binding molecule, and the number of related binding sites present on the antigen binding molecule.

Typically, a specific binding molecule will bind to the desired antigen with a dissociation constant (KD) of preferably ^10-7 to ^10-11 moles/liter (M), more preferably ^10-8 to ^10-11 moles/liter, even more preferably ^10-9 to ^10-11 , even more preferably ^10-10 to ^10-11 or lower, and/or with an association constant (KA) of at least ¹⁰⁷ M ^-1 , preferably at least ¹⁰⁸ M ^-1 , more preferably at least ¹⁰⁹ M ^-1 , more preferably at least ¹⁰¹⁰ M ^-1 , for example at least ¹⁰¹¹ M ^-1 , as measured in a Biacore or KinExA assay. Any KD value greater than ^10-4 M is generally considered to indicate nonspecific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays as described herein.

Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substitutions of an amino acid within the following groups (i) to (v) by another amino acid residue within the same group: (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component with which it is normally associated in that source or medium (e.g., another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) compared to its natural biological source and/or the reaction medium or culture medium from which it was obtained. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. A "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).

As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

PD-L1 binding molecules of the present invention

In a first aspect, the present invention provides a PD-L1 binding molecule that can specifically bind to PD-L1 and consists of an amino acid sequence of the formula A-L-B, wherein A represents an immunoglobulin single variable domain, L represents an amino acid linker or is absent, and B represents a human immunoglobulin Fc region,

wherein the immunoglobulin single variable domain consists of an amino acid sequence selected from SEQ ID NO: 1-6,

The PD-L1 binding molecule can form a homodimer in the form of (ALB) ₂ through the human immunoglobulin Fc region.

As used herein, the term "human immunoglobulin Fc region" refers to the Fc region of the constant region of human IgG1, IgG2, IgG3, or IgG4 (the amino acid sequences of which are referenced in the protein database www.uniprot.org, entries P01857, P01859, P01860, and P01861, respectively), comprising the hinge region (or a portion thereof), the CH2 region, and the CH3 region of the immunoglobulin constant region. In the present invention, the amino acid sequence of the "human immunoglobulin Fc region" may be modified by mutating 1-5 amino acids in the CH2 region to increase or eliminate Fc-mediated ADCC or CDC activity or enhance or decrease FcRn affinity; or by mutating 1-4 amino acids in the hinge region to increase protein stability. However, in the present invention, the "human immunoglobulin Fc region" does not include mutations that promote or prevent the formation of Fc heterodimers, nor does it include modifications that add other functional protein sequences to the nitrogen-terminus or carbon-terminus of the Fc fragment.

In a preferred embodiment, the human immunoglobulin Fc region is mutated to remove ADCC and CDC activities. In a specific embodiment, the amino acid sequence of the immunoglobulin Fc region is selected from SEQ ID NOs: 7-9.

Those skilled in the art will understand that, under the action of the human immunoglobulin Fc region, the PD-L1 binding molecules of the present invention will generally exist in the form of homodimers.

As used in the present invention, the term "amino acid linker" refers to a non-functional amino acid sequence of 1-20 amino acid residues in length and without secondary or higher structure. The amino acid linker is, for example, a flexible linker, such as GGGGS, GS, GAP, etc.

In some embodiments, the PD-L1 binding molecule of the invention consists of an amino acid sequence selected from SEQ ID NOs: 10-27.

The PD-L1 binding molecules of the present invention have at least one of the following characteristics:

(a) KD value for binding to human PD-L1 is less than 1×10 ^-7 M;

(b) blocking the interaction between PD-L1 and PD-1;

(c) enhance the activation of PBMCs and T cells;

(d) Inhibit tumor growth.

The PD-L1 binding molecule of the present invention may bind to PD-L1 with a KD value of less than 1× ^10-7 M, preferably less than 1× ^10-8 M, more preferably less than 1× ^10-9 M, even more preferably less than 1× ^10-10 M, and even more preferably less than 1× ^10-11 M.

In some embodiments, the PD-L1 binding molecules of the present invention can specifically bind to human PD-L1 and block the interaction between PD-L1 and PD-1. In some embodiments, the PD-L1 binding molecules of the present invention can specifically bind to human PD-L1 and block the interaction between PD-L1 and CD80.

The PD-L1 binding molecules of the present invention are capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%.

Furthermore, the PD-L1 binding molecules of the present invention are resistant to alkaline and oxidative treatments. For example, after treatment with a strong base (e.g., 500 mM ammonium bicarbonate) for about 8 hours, preferably about 16 hours, more preferably about 24 hours, or more preferably about 32 hours, the activity of the PD-L1 binding molecules of the present invention remains unchanged. Alternatively, after treatment with an oxidant (1% hydrogen peroxide) for about 2 hours, preferably about 4 hours, or more preferably about 8 hours, the activity of the PD-L1 binding molecules of the present invention remains unchanged.

In addition, the PD-L1 binding molecules of the present invention are stable at high concentrations. For example, at a concentration of about 100 mg/ml, more preferably about 150 mg/ml, more preferably about 200 mg/ml, or more preferably about 250 mg/ml, the PD-L1 binding molecules of the present invention remain stable and do not aggregate.

Nucleic acids, vectors, host cells

In another aspect, the present invention relates to a nucleic acid molecule encoding a PD-L1 binding molecule of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

The nucleic acids of the present invention may also be in the form of, present in, and/or part of a vector, such as a plasmid, cosmid, or YAC. The vector may particularly be an expression vector, i.e., a vector that provides for expression of the PD-L1 binding molecule in vitro and/or in vivo (i.e., in a suitable host cell, host organism, and/or expression system). The expression vector typically comprises at least one nucleic acid of the present invention, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Specific examples of regulatory elements and other elements that are useful or necessary for expression of the PD-L1 binding molecules of the present invention include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

The nucleic acids of the invention can be prepared or obtained by known means (e.g. by automated DNA synthesis and/or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the invention given herein, and/or can be isolated from suitable natural sources.

In another aspect, the present invention relates to a host cell that expresses or is capable of expressing one or more PD-L1 binding molecules of the invention and/or contains a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

However, the present invention can also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

The present invention also provides a method for producing the PD-L1 binding molecules of the present invention, the method generally comprising the following steps:

- culturing the host cells of the invention under conditions that allow expression of the PD-L1 binding molecules of the invention; and

- recovering the PD-L1 binding molecule expressed by the host cells from the culture; and

- Optionally further purifying and/or modifying the PD-L1 binding molecules of the invention.

In a preferred embodiment, the PD-L1 binding molecules of the present invention are produced using mammalian cells. The PD-L1 binding molecules of the present invention can be highly expressed in mammalian cells. For example, the expression level can reach about 5 g/L, preferably about 6 g/L, preferably about 7 g/L, preferably about 8 g/L, more preferably about 9 g/L, or more preferably about 10 g/L or higher.

The PD-L1-binding molecules of the present invention can be produced intracellularly in the cells described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods for producing the PD-L1-binding molecules of the present invention are well known to those skilled in the art.

However, the PD-L1 binding molecules of the present invention can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

Pharmaceutical composition

In another aspect, the present invention provides a composition, such as a pharmaceutical composition, comprising one or a combination of PD-L1 binding molecules of the present invention formulated together with a pharmaceutically acceptable carrier. Such a composition may comprise one or a combination (e.g., two or more different) of PD-L1 binding molecules of the present invention. Those skilled in the art will appreciate that, under the action of the human immunoglobulin Fc region, the PD-L1 binding molecules of the present invention will generally exist as homodimers.

The pharmaceutical compositions of the present invention can also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy can include a PD-L1 binding molecule of the present invention in combination with at least one other anti-tumor agent. For example, a PD-L1 binding molecule of the present invention can be used in combination with an antibody that targets other tumor-specific antigens. Such antibodies targeting other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, antibodies against EGFR variants, anti-VEGFa antibodies, anti-HER2 antibodies, or anti-CMET antibodies. Preferably, the antibody is a monoclonal antibody.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, e.g., Berge, S.M. et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and salts derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. In many cases, it is preferred to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium hydroxide in the composition. Prolonged absorption of injectable drugs can be achieved by including agents that delay absorption, such as monostearate and gelatin in the composition.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except to the extent that they are incompatible with the active compound, may be included in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained.

Sterile injections can be prepared by mixing the active compound in a suitable solvent in the required amount and adding one or a combination of the ingredients listed above as needed, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injections, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), from which a powder of the active ingredient plus any additional required ingredients is obtained from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specific specification of the dosage unit form of the present invention is limited by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art for formulating such active compound for treating individual sensitivity.

For the administration of antibody molecules, dosage range is about 0.0001 to 100 mg/kg, more generally 0.01 to 20 mg/kg recipient body weight. For example, dosage can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or within the range of 1-20 mg/kg. Exemplary treatment regimens require weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or initial dosing interval slightly shorter (such as once a week to once every three weeks) later dosing interval lengthening (such as once a month to once every 3-6 months).

Alternatively, the antibody molecule can also be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. Generally, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are given at less frequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, higher dosages need to be given at shorter intervals sometimes until the progression of the disease is alleviated or stopped, preferably until the patient shows partial or complete improvement in symptoms of the disease. Afterwards, the patient can be administered with a preventive regimen.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention being employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds, and/or materials being used in combination with the particular composition being employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

A "therapeutically effective amount" of a PD-L1 binding molecule of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the suffering of the disease. For example, for the treatment of PD-L1-associated tumors, a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. One skilled in the art can determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

The compositions of the present invention can be administered by one or more routes of administration using one or more methods known in the art. It will be understood by those skilled in the art that the route and/or mode of administration will vary depending on the desired outcome. Preferred routes of administration for the PD-L1 binding molecules of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the PD-L1 binding molecules of the invention can also be administered via a non-parenteral route, such as a topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The therapeutic composition can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention can be administered using a needle-free subcutaneous injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of known implants and modules that can be used in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a medical infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the PD-L1 binding molecules of the present invention can be formulated to ensure proper distribution in the body. For example, the blood-brain barrier (BBB) blocks many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention can cross the BBB (if necessary), they can be formulated in, for example, liposomes. For methods of preparing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548 and 5,399,331. Liposomes contain one or more targeting moieties that can be selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Disease prevention and treatment

In another aspect, the present invention provides uses and methods of the PD-L1-binding molecules, nucleic acid molecules, host cells, and pharmaceutical compositions described herein for preventing and/or treating diseases associated with PD-L1. PD-L1-related diseases that can be prevented and/or treated using the PD-L1-binding molecules of the present invention are described in detail below.

cancer

The blocking of PD-L1 by the PD-L1 binding molecules of the present invention can enhance the immune response to cancer cells in patients. PD-L1 is enriched in a variety of human cancers (Dong et al. (2002) Nat Med. 8: 787-9). The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune escape of cancer cells (Dong et al. (2003) J Mol Med 81: 281-7; Blank et al. (2004) Cancer Immunol Immunother [epub]; Konishi et al. (2004) Clin Cancer Res 10: 5094-5100). Inhibiting the local interaction of PD-L1 with PD-1 can reverse immunosuppression, and when the interaction of PD-L2 with PD-1 is also blocked, the effect is synergistic (Iwai et al. (2002) PNAS 99: 12293-7; Brown et al. (2003) J Immunol 170: 1257-66). The PD-L1 binding molecules of the present invention can be used alone to inhibit the growth of cancerous tumors. Alternatively, as described below, the PD-L1 binding molecules of the present invention can be used in combination with other anti-tumor treatments, such as other immunogenic agents, standard cancer therapies, or other antibody molecules.

Therefore, in one embodiment, the present invention provides a method for preventing and/or treating cancer, comprising administering to the subject a therapeutically effective amount of a PD-L1 binding molecule of the present invention to inhibit the growth of tumor cells in the subject.

Preferred cancers that can be prevented and/or treated using the PD-L1 binding molecules of the present invention include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred treatable cancers include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumors, and osteosarcoma. Examples of other cancers that can be treated with the methods of the present invention include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic ... Myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of said cancers. The present invention can also be used to treat metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al. (2005) Int Immunol 17: 133-144).

Optionally, the PD-L1 binding molecules of the present invention can be combined with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol 173: 4919-28). Non-limiting examples of immunogenic agents that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

In humans, some tumors, such as melanoma, have been shown to be immunogenic. It is expected that by blocking PD-L1 using the PD-L1 binding molecules of the present invention to promote T cell activation, tumor responses in the host can be activated. PD-L1 blockers (such as anti-PD-L1 antibodies, such as the PD-L1 binding molecules of the present invention) may be most effective when combined with tumor vaccination regimens. Many experimental strategies for tumor vaccination have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043, DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. 5th ed.). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proa Natl. Acad. Sci U.S.A. 90:3539-43).

Studies of gene expression and large-scale gene expression patterns in various tumors have identified many so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens have been shown to be targets of tumor-specific T cells found in the host. The PD-L1 binding molecules of the present invention can be used in combination with recombinantly produced tumor-specific proteins and/or peptides to generate an immune response against these proteins. These proteins are normally seen by the immune system as self-antigens and are therefore tolerated. Tumor antigens can also include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and is expressed in more than 85% of human cancers, but only in a limited number of self-tissues (Kim, N et al. (1994) Science 266: 2011-2013). Tumor antigens can also be “neoantigens” expressed by cancer cells, for example due to somatic mutations that alter the protein sequence or produce a fusion protein of two unrelated sequences (e.g., bcr-abl in the Philadelphia chromosome).

Other tumor vaccines can include proteins from viruses associated with human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in combination with PD-L1 blockers (such as anti-PD-L1 antibodies, such as the PD-L1 binding molecules of the present invention) is purified heat shock proteins (HSPs) isolated from tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells, and these HSPs are very effective in delivering them to antigen-presenting cells to elicit tumor immunity (Suot, R and Srivastava, P (1995) Science 269: 1585-1588; Tamura, Y. et al. (1997) Science 278: 117-120).

Dendritic cells (DCs) are strong antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be generated in vitro and loaded with various protein and peptide antigens and tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have been directly fused to tumor cells for immunization (Kugler, A. et al. (2000) Nature Medicine 6: 332-336). As a vaccination method, DC immunization can be effectively combined with PD-L1 blockers (such as anti-PD-L1 antibodies, such as the PD-L1 binding molecules of the present invention) to activate a stronger anti-tumor response.

CAR-T, whose full name is Chimeric Antigen Receptor T-Cell Immunotherapy, is another effective cell therapy for malignant tumors. Chimeric antigen receptor T cells (CAR-T cells) are a chimeric protein that couples the antigen binding portion of an antibody that can recognize a certain tumor antigen with the intracellular portion of the CD3-ζ chain or FcεRIγ in vitro. The patient's T cells are transfected by gene transduction to express the chimeric antigen receptor (CAR). At the same time, co-stimulatory molecule signal sequences can also be introduced to increase the cytotoxic activity, proliferation and survival time of T cells and promote the release of cytokines. After the patient's T cells are "recoded", they can be expanded in vitro to generate a large number of tumor-specific CAR-T cells and then infused back into the patient's body to achieve the purpose of tumor treatment. PD-L1 blockers (such as anti-PD-L1 antibodies, such as the PD-L1 binding molecules of the present invention) can be combined with CAR-T cell therapy to activate a stronger anti-tumor response.

The PD-L1 binding molecules of the present invention can also be combined with standard cancer treatments. The PD-L1 binding molecules of the present invention can be effectively combined with chemotherapy regimens. In these cases, it can reduce the dose of the chemotherapeutic agent administered (Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of an anti-PD-L1 antibody and decarbazine for the treatment of melanoma. Another example of such a combination is the combination of an anti-PD-L1 antibody and interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for combining the PD-L1 binding molecules of the present invention with chemotherapy is that cell death, which is a consequence of the cytotoxic effects of most chemotherapeutic compounds, should result in elevated levels of tumor antigens in the antigen presentation pathway. Other combined therapies that can synergize with PD-L1 blockade through cell death include radiotherapy, surgery, and hormone deprivation. These regimens all generate sources of tumor antigens in the host. Angiogenesis inhibitors can also be combined with the PD-L1 binding molecules of the present invention. Inhibition of angiogenesis leads to tumor cell death, which can provide tumor antigens to the host's antigen presentation pathway.

The PD-L1 binding molecules of the present invention can also be used in combination with antibodies targeting other tumor-specific antigens. Such antibodies targeting other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, anti-EGFR variant antibodies, anti-VEGFα antibodies, anti-HER2 antibodies, or anti-CMET antibodies. Preferably, the antibody is a monoclonal antibody.

The PD-L1 binding molecules of the present invention can also be used in combination with bispecific antigens that target Fcα or Fcγ receptor-expressing effector cells to tumor cells (see, for example, US Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can also be used to target two different antigens. For example, anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The use of PD-L1 blockers can enhance the T cell aspects of these responses. Alternatively, bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers can be used to deliver antigens directly to DCs.

Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by tumors. These include, in particular, TGF-β (KehrL J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274: 1363-1365). Antibodies to each of these can be used in combination with the PD-L1 binding molecules of the present invention to counteract the effects of immunosuppressive agents and facilitate the host's tumor immune response.

Other antibodies that can be used to activate a host immune response can be used in combination with the PD-L1 binding molecules of the present invention. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in combination with the PD-L1 binding molecules of the present invention (Ito, N. et al. (2000) Immunobiology 201(5): 527-40). In order to increase the level of T cell activation, it is also possible to combine activating antibodies against T cell co-stimulatory molecules such as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) and antibodies that block the activity of negative co-stimulatory molecules such as CTLA-4 (e.g., U.S. Patent No. 5,811,097) or BTLA (Watanabe, N. et al. (2003) Nat Immunol 4: 670-9), B7-H4 (Sica, GL et al. (2003) Immunity 18: 849-61).

Bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. Graft-versus-host disease is a consequence of this treatment, and graft-versus-tumor responses can provide therapeutic benefits. PD-L1 blockers can be used to increase the effectiveness of tumor-specific T cells. There are also several experimental treatment options involving ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to fight tumors with antigen-specific T cells (Greenberg, R. and Riddell, S. (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. It is expected that ex vivo activation in the presence of the PD-L1 binding molecule of the present invention can increase the frequency and activity of adoptively transferred T cells. Therefore, the present invention also provides a method for activating immune cells (such as PBMCs or T cells) in vitro, comprising contacting the immune cells with the PD-L1 binding molecule of the present invention.

Infectious diseases

Other methods of the present invention are used to treat patients exposed to specific toxins or pathogens. Therefore, another aspect of the present invention provides a method for preventing and/or treating an infectious disease in a subject, comprising administering a PD-L1 binding molecule of the present invention to the subject, such that the infectious disease in the subject is prevented and/or treated.

Similar to the application for tumors as described above, PD-L1 blockers can be used alone or as an adjuvant in combination with vaccines to stimulate immune responses to pathogens, toxins and self-antigens. Examples of pathogens for which this treatment method is particularly applicable include pathogens for which there are currently no effective vaccines, or pathogens for which conventional vaccines are not completely effective. These include but are not limited to HIV, hepatitis viruses (A, B, C), influenza viruses, herpes viruses, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. PD-L1 blockers are particularly useful against established infections with pathogens such as HIV, which present altered antigens during the infection process. When anti-human PD-L1 antibodies are administered, these new epitopes are recognized as foreign substances, thereby causing a strong T cell response that is not affected by the negative signal of PD-L1.

Some examples of pathogenic viruses that cause infectious diseases that can be treated using the methods of the present invention include HIV, hepatitis (A, B, C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

Some examples of pathogenic bacteria that cause infectious diseases that can be treated using the methods of the present invention include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Ralstonia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Anthrax, Yersinia pestis, Leptospira, and Lyme disease bacteria.

Some examples of pathogenic fungi that cause infectious diseases that can be treated with the methods of the present invention include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

Some examples of pathogenic parasites that cause infectious diseases that can be treated using the methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba species, Giardia lamblia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia gonorrhoeae, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

In all of the above approaches, PD-L1 blockade can be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

Autoimmune reaction

Anti-PD-L1 antibodies can stimulate and amplify autoimmune responses. Therefore, it is possible to consider designing vaccination regimens using anti-PD-L1 antibodies in combination with multiple self-proteins to effectively generate immune responses against these self-proteins for disease treatment.

For example, Alzheimer's disease involves the inappropriate accumulation of Aβ peptides in amyloid deposits in the brain; antibody responses to amyloid can clear these amyloid deposits (Schenk et al. (1999) Nature 400: 173-177). Other self-proteins can also be used as targets, such as IgE, which is involved in the treatment of allergies and asthma, and TNFα, which is involved in rheumatoid arthritis. Finally, anti-PD-L1 antibodies can be used to induce antibody responses to various hormones. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors required for the growth of specific tumors can also be considered as possible vaccination targets.

As described above, a similar approach using anti-PD-L1 antibodies could be used to induce a therapeutic autoimmune response to treat patients with inappropriate accumulation of self-antigens, such as amyloid deposits including Aβ in Alzheimer's disease, cytokines such as TNFα, and IgE.

Chronic inflammatory diseases

Anti-PD-L1 antibodies can also be used to treat the following diseases, such as chronic inflammatory diseases, such as lichen planus and T cell-mediated chronic inflammatory skin and mucosal diseases (Youngnak-Piboonratanakit et al. (2004) Immunol Letters 94; 215-22). Therefore, in one aspect, the present invention provides a method for eliminating chronic inflammatory diseases using T cells, comprising administering a PD-L1 binding molecule of the present invention to a subject.

vaccine adjuvants

One aspect of the present invention provides the use of the PD-L1 binding molecules of the present invention as vaccine adjuvants. By co-administering an anti-PD-L1 antibody and a target antigen (e.g., a vaccine), the anti-PD-L1 antibody can be used to enhance the specific immune response against the antigen.

Thus, one aspect of the present invention provides a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) a PD-L1 binding molecule of the present invention, such that the immune response to the antigen in the subject is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of these antigens include those described in the above sections, such as the tumor antigens (or tumor vaccines) described above, or antigens from the above-mentioned viruses, bacteria, or other pathogens.

Reagent test kit

Also within the scope of the present invention are kits comprising a PD-L1 binding molecule, immunoconjugate, or pharmaceutical composition of the invention, and instructions for use. The kit may further comprise at least one additional reagent or one or more additional PD-L1 binding molecules of the invention (e.g., binding molecules that bind to different epitopes of PD-L1). Kits generally include a label indicating the intended use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit.

Example

The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples.

Example 1: Screening of heavy chain single domain antibodies against PD-L1

1.1 Library construction:

The PDL1-Fc fusion protein (SEQ ID NO: 28) used for immunization was expressed in CHO cells (pCDNA4, Invitrogen, Cat V86220) and purified by Protein A affinity chromatography. A Xinjiang Bactrian camel (Camelus bactrianus) was selected for immunization. After four immunizations, 100 ml of peripheral blood lymphocytes were extracted from the camel and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit according to the manufacturer's instructions. Nested PCR was used to amplify the nucleic acid fragment encoding the variable region of the heavy chain antibody:

First round of PCR:

Upstream primer: GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 29);

Downstream primer: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 30).

Second round of PCR:

The first-round PCR product was used as a template.

Upstream primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 31);

Downstream primer: GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 32).

The target heavy chain single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pCDisplay-3 (Creative Biolabs, Cat: VPT4023) using the restriction endonucleases PstI and NotI (purchased from NEB). The product was then electroporated into E. coli electrocompetent cells TG1 to construct a heavy chain single-domain antibody phage display library targeting PD-L1 and assay the library. By serial dilution plating, the library capacity was calculated to be 1.33×10 ⁸ . To test the insertion rate of the library, 24 clones were randomly selected for colony PCR. The results showed that the insertion rate had reached 100%.

1.2 Selection of heavy chain single domain antibodies targeting PD-L1:

The plates were coated with 10 μg/well of PDL1-Fc fusion protein and incubated at 4°C overnight. The next day, after blocking with 1% skim milk at room temperature for 2 hours, 100 μl of phage (8×10 ¹¹ tfu, from the camel heavy chain single-domain antibody phage display library constructed in 1.1) was added and allowed to react at room temperature for 1 hour. The plates were then washed five times with PBST (PBS containing 0.05% Tween 20) to remove unbound phage. Finally, phage that specifically bound to PD-L1 were dissociated with triethylamine (100 mM) and infected with Escherichia coli TG1 in logarithmic phase growth to produce and purify phage for the next round of screening. The same screening process was repeated 3-4 times. Thus, positive clones were enriched, achieving the goal of using phage display technology to screen PD-L1-specific antibodies from the antibody library.

1.3 Screening of specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA)

After 3-4 rounds of panning, the PD-L1-binding-positive phage obtained were infected with blank E. coli and plated. Subsequently, 96 single colonies were selected and cultured separately to produce and purify phage. Plates were coated with PDL1-Fc fusion protein overnight at 4°C, and the obtained sample phage (blank phage control group) was added and allowed to react at room temperature for 1 hour. After washing, a primary mouse anti-HA tag antibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added and allowed to react at room temperature for 1 hour. After washing, a secondary goat anti-mouse alkaline phosphatase-labeled antibody (purchased from Aimejie Technology Co., Ltd.) was added and allowed to react at room temperature for 1 hour. After washing, an alkaline phosphatase colorimetric solution was added, and the absorbance was read at 405 nm. A positive clone well was identified when the OD value of the sample well was more than 3 times that of the control well. The bacteria from the positive clone well were transferred to LB liquid containing 100 μg/mL ampicillin for culture to extract the plasmid and perform sequencing.

The protein sequences of each clone were analyzed using the sequence alignment software Vector NTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same antibody strain, while clones with different CDR sequences were considered different antibody strains.

Example 2: Preliminary evaluation and identification of heavy chain single domain antibodies targeting PD-L1

2.1 Expression and purification of heavy chain single domain antibodies in host bacteria Escherichia coli:

The coding sequence of the heavy chain single-domain antibody obtained by sequencing analysis was subcloned into the expression vector pET32b (Novagen, product number: 69016-3), and the recombinant plasmid identified as correct by sequencing was transformed into the expression host bacteria BL1 (DE3) (Tiangen Biochemical Technology, product number: CB105-02), which was spread on a plate of LB solid medium containing 100 micrograms per milliliter of ampicillin and cultured at 37°C overnight. A single colony was selected for inoculation and cultured overnight. The next day, the overnight bacteria were transferred and amplified, cultured in a 37°C shaker until the OD value reached 0.6-1, induced with 0.5mM IPTG, and cultured in a 28°C shaker overnight. The next day, the bacteria were harvested by centrifugation and the cells were disrupted to obtain a crude antibody extract. The antibody protein was then purified using a nickel ion affinity chromatography column. Ultimately, an antibody protein with a purity of over 90% was obtained.

2.2 Detection of specific binding of candidate PD-L1 heavy chain single domain antibodies to human PD-L1 protein

Coat the plate with PDL1-Fc fusion protein overnight at 4°C. Add 100 ng of the heavy chain single-domain antibody obtained in Example 2.1 to each well (the control group is a single-domain antibody that does not bind to the PDL1-Fc protein) and react at room temperature for 1 hour. After washing, add the primary mouse anti-His tag antibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) and react at room temperature for 1 hour. After washing, add the secondary goat anti-mouse horseradish peroxidase-conjugated antibody (Sino Biological, Cat: SSA007200) and react at room temperature for 1 hour. After washing, add the colorimetric solution and read the absorbance at 405 nm.

Coat the plate with Fc protein overnight at 4°C. Add 100 ng of the heavy chain single-domain antibody obtained in Example 2.1 to each well (the control group is a single-domain antibody against an unrelated target) and react at room temperature for 1 hour. After washing, add the primary rabbit anti-human Fc antibody (purchased from Shanghai Puxin Biotechnology Co., Ltd.) and react at room temperature for 1 hour. After washing, add the secondary goat anti-rabbit horseradish peroxidase-labeled antibody (purchased from Shanghai Puxin Biotechnology Co., Ltd.) and react at room temperature for 1 hour. After washing, add the colorimetric solution and read the absorbance at 405 nm.

When the ratio of the OD value of the PDL1-Fc protein divided by the OD value of the blank control is greater than or equal to 4, the candidate antibody is judged to be able to bind to the PDL1-Fc protein; at the same time, for the above-mentioned antibodies that can bind to the PDL1-Fc antigen protein, if the ratio of the OD value of the PDL1-Fc binding to the OD value of the Fc protein binding is greater than or equal to 5, the candidate antibody is considered to be able to specifically bind to the PD-L1 part but not the Fc part.

The results showed that one of the screened antibody strains, antibody strain 56, could specifically bind to PD-L1 but not to Fc. The specific results are shown in Table 1 below:

Table 1

Antibody strain OD (for PD-L1) OD (to Fc) OD(PD-L1/Fc) OD (PD-L1/blank) SEQ ID NO PDL1-56dAb 2.931 0.068 43.10294118 54.27777778 1 blank 0.054 0.072 0.75 1

2.3 Binding of PD-L1 heavy chain single domain antibody to mouse PD-L1 protein

Mouse PDL1-Fc protein (SEQ ID NO: 33) was expressed in HEK293 cells (pCDNA4, Invitrogen, Cat V86220).

The plate was coated with 0.5 μg/well of mouse PDL1-Fc fusion protein at 4°C overnight. 100 ng of the heavy chain single-domain antibody obtained in Example 2.1 was added to each well (the control group was a single-domain antibody against other unrelated targets) and reacted at room temperature for 1 hour. After washing, the primary mouse anti-His tag antibody was added and reacted at room temperature for 1 hour. After washing, the secondary goat anti-mouse horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 405 nm. The results are shown in Table 2.

Table 2

Antibody strain OD (for mouse PD-L1) PDL1-56dAb 0.075 blank 0.096

It can be seen that the human PD-L1 heavy chain single domain antibody of the present invention does not bind to the mouse PDL1-Fc protein.

2.4 Competitive ELISA to investigate the blocking effect of PD-L1 heavy chain single domain antibody on the interaction between PD-1 and PD-L1

PDL1-Fc protein and PD1-Fc protein (SEQ ID NO: 34) were expressed in HEK293 cells (pCDNA4, Invitrogen, Cat V86220). Biotinylated protein PD1-Fc-Biotin was obtained using the Biotinlylation Kit from Thermo Fisher Scientific.

The plate was coated with 0.5 μg/well of PDL1-Fc fusion protein at 4°C overnight, followed by addition of 100 ng of the heavy chain single-domain antibody obtained in Example 2.1 (the control group was a single-domain antibody against other unrelated targets, or just buffer) and 10 μg of PD1-Fc-Biotin to each well (the blank group did not add any antibody or protein, only an equal volume of buffer was added), and the reaction was carried out at room temperature for 1 hour. SA-HRP (purchased from Sigma) was then added and the reaction was carried out at room temperature for 1 hour. The colorimetric solution was then added, and the absorbance was read at a wavelength of 405 nm. When the sample OD value was <0.8 compared to the control OD value, the antibody was considered to have a blocking effect.

The results are shown in Table 3 , and antibody strain 56 exhibited a blocking effect on the PD-1/PD-L1 interaction.

Table 3

sample OD Control 1 2.335 Control 2 2.413 blank 0.079 PDL1-56 0.129

2.5 Investigating the blocking effect of PD-L1 heavy chain single domain antibody on the interaction between cell surface PD-L1 and PD-1 by FACS

HEK293 cells transiently expressing human PD-L1 protein on the membrane (293-PDL1 cells) were obtained by transiently transfecting human HEK293 cells with a plasmid carrying the full-length human PD-L1 protein gene (pCDNA4, Invitrogen, Cat V86220).

293-PDL1 cells were taken and resuspended in 0.5% PBS-BSA buffer in a 96-well plate. The above-mentioned antibodies to be tested were added. A negative control was also set up, which consisted of 2μg of single-domain antibodies targeting other targets. 0.3μg of hPD-1-Fc-biotin was added to all samples, and the secondary antibody was SA-PE from eBioscience. After staining, the samples were detected by flow cytometry. If the fluorescence value after adding the antibody shifted towards the blank direction compared to the group without antibody, the antibody was considered to be able to block the interaction between cell surface PD-L1 and PD-1. This method was used to identify antibodies that can block the binding between cell surface PD-L1 antigen and PD-1.

The results are shown in Figure 1 , and antibody strain 56 exhibits a blocking effect on the PD-1/PD-L1 interaction.

2.6 Binding curve of PD-L1 heavy chain single domain antibody to PD-L1 antigen protein

Plates were coated with 0.5 μg/well of the resulting PD-L1 heavy chain single-domain antibody at 4°C overnight. A serial dilution series of PDL1-Fc fusion protein was then added and allowed to react at room temperature for 1 hour. After washing, goat anti-human IgG-Fc horseradish peroxidase-labeled antibody was added and allowed to react at room temperature for 1 hour. After washing, horseradish peroxidase colorimetric solution was added, and absorbance was read at 405 nm. Softmax Pro v5.4 software was used for data processing and graphical analysis. Four-parameter fitting was used to obtain the antibody binding curve for PD-L1 and the EC50 value (approximately 5 ng/ml). The results are shown in Figure 2.

2.7 Blockade Curve of PD-1 Heavy Chain Single Domain Antibody on the Interaction between PD-1 and PD-L1

Plates were coated with 0.5 μg/well of PDL1-Fc fusion protein overnight at 4°C. Then, 100 μL of a serial dilution series of PD-L1 blocking single-domain antibody Fc fusion protein (containing 100 μg/mL PD1-Fc-Biotin) was added to each well and allowed to react at room temperature for 1 hour. SA-HRP (Sigma) was then added and allowed to react at room temperature for 1 hour. The colorimetric solution was then added, and the absorbance was read at 405 nm.

Data processing and graphical analysis were performed using Softmax Pro v5.4. Four-parameter fitting was used to obtain the PD-L1/PD-1 blocking curves and IC50 values (143 ng/mL) for 56 antibody pairs. The results are shown in Figure 3.

Example 3: Humanization of PD-L1 single domain antibody

The humanization method uses the protein surface amino acid humanization (resurfacing) method and VHH humanization universal framework transplantation method (CDR grafting to a universal framework) to complete.

The humanization steps were as follows: Homology modeling was performed on antibody strain 56 using Modeller 9. The reference homology sequence was the NbBcII10 antibody (PDB ID: 3DWT). The relative solvent accessibility of the amino acids was calculated based on the protein's three-dimensional structure. If an amino acid in antibody strain 56 was solvent-exposed, it was replaced with the amino acid at the same position in the reference human antibody DP-47 sequence, ultimately completing the replacement.

The specific steps for the universal framework transplantation method for VHH humanization are as follows: First, obtain the universal humanized VHH framework h-NbBcII10FGLA (PDB ID: 3EAK) designed by Cécile Vincke et al. based on sequence homology. This framework design is based on the nanobody NbBcII10 antibody (PDB ID: 3DWT). The protein surface amino acid humanization was performed with reference to the human antibody DP-47, and the amino acids FGLA of the VHH sequence framework 2 (framework-2) were modified to complete the humanization. We directly used h-NbBcII10FGLA as the framework and replaced the CDRs with the CDR regions of antibody strain 56 to complete the antibody humanization.

Humanization of antibody strain 56 was performed to obtain five humanized variants of antibody strain 56. Table 4 lists the sequence numbers of these humanized variants and the amino acid changes therein, where the amino acid residue numbering is based on Kabat numbering. Figure 4 shows the alignment of the humanized sequences.

Table 4

Example 4: Preparation of PD-L1 blocking antibody protein using mammalian cells

4.1 Preparation of Fc Fusion Protein of PD-L1 Single Domain Antibody

Based on the amino acid sequence of the constant region of human immunoglobulin gamma 1 (IgG1) (P01857) on the protein database Uniprot, the amino acid sequence of the human IgG1-Fc region was obtained (SEQ ID NO: 7). A nucleic acid fragment encoding human IgG1-Fc was obtained from total RNA of human PBMCs by reverse transcription PCR. Overlapping PCR was then used to obtain a nucleic acid fragment encoding the fusion protein of the PD-L1 single-domain antibody and Fc obtained in the above example. This fragment was then subcloned into the vector pCDNA4 (Invitrogen, CatV86220). Alternatively, Fc region sequences that have been subjected to site-directed mutagenesis to remove ADCC activity or CDC activity, such as SEQ ID NO: 8 or 9, can be used.

The recombinantly constructed single-domain antibody-Fc fusion protein plasmid was transfected into HEK293 cells for antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid/PEI mixture was added to the HEK293 cell suspension and cultured at 37°C, 10% CO2, and 90 rpm; 50 μg/L IGF-1 was also added. Four hours later, EX293 medium, 2 mM glutamine, and 50 μg/L IGF-1 were added, and cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target PD-L1 single-domain antibody-Fc fusion protein.

The sequences of the obtained PD-L1 single domain antibody-Fc fusion proteins are shown in SEQ ID NOs: 10-27.

4.2 Preparation of PD-L1 Antibodies from MedImmune LLC and Roche

MedImmune LLC's anti-PD-L1 antibody was cloned using the method described in patent 2.14H9 in US20130034559 and then cloned into the pCDNA4 vector. TM004, Roche's anti-PD-L1 antibody, was cloned using the method described in patent YW243.55.S70.hIgG in US20130045201A1 and then cloned into the pCDNA4 vector.

The recombinant plasmid was transiently transfected and expressed in HEK293 cells using the same method as in 4.1. The anti-PD-L1 antibody obtained from MedImmune LLC was renamed 2.41H90P; the anti-PD-L1 antibody from Roche was renamed 243.55.

4.3 Comparison of expression results between PD-L1 single-domain antibody Fc fusion protein and two known PD-L1 antibodies

Using the same expression system and transient transfection conditions, the expression levels of the PD-L1 single-domain antibody Fc fusion protein of the present invention were above 200 mg/L, while the expression level of antibody 2.41H90P was approximately 80 mg/L, and the expression level of antibody 243.55 was approximately 40 mg/L. This result demonstrates that the PD-L1 single-domain antibody Fc fusion protein of the present invention is more structurally stable than the other two known PD-L1 antibodies and can achieve higher expression levels.

Example 5: Identification of the function of PD-L1 single domain antibody Fc fusion protein

5.1 ELISA method for the identification of PD-L1 binding ability of PD-L1 single-domain antibody Fc fusion protein

PDL1-Chis protein (SEQ ID NO: 35) was obtained by transient expression in HEK293 and purification by nickel column affinity chromatography. 0.5 μg/well of the obtained PDL1-Chis protein was coated on the plate at 4°C overnight, and then a gradient dilution series of the PD-L1 single-domain antibody Fc fusion protein obtained in the above example was added and reacted at room temperature for 1 hour. After washing, goat anti-human IgG-Fc horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, the color developing solution was added and the absorbance was read at a wavelength of 405 nm. The software SotfMax Pro v5.4 was used for data processing and graphical analysis. By four-parameter fitting, the antibody binding curve to PD-L1 and the EC50 value (the EC50 value of all test antibodies was approximately 150 ng/mL) were obtained to reflect the affinity of the antibody to PD-L1.

The results are shown in Figure 5, where the ordinate represents OD405 and the abscissa represents the concentration of the PD-L1 single-domain antibody Fc fusion protein (unit: ng/mL). The inverted triangle, right triangle, and square represent three different humanized Fc fusion proteins of antibody strain 56: hu56v1-Fc, hu56v2-Fc, and hu56v5-Fc. The three proteins have comparable affinity for PD-L1.

5.2 Identification of the PD-L1 Binding Ability of Single Domain Antibody Fc Fusion Protein to PD-L1 (SPR Method) and Comparison with Known Antibodies

The binding kinetics of the PD-L1 single-domain antibody-Fc fusion protein obtained in the above example against recombinant human PD-L1 were measured using the surface plasmon resonance (SRP) method using a BIAcore X100 instrument. Recombinant human PD-L1-Fc was directly coated onto a CM5 biosensor chip to obtain approximately 1000 response units (RU). For kinetic measurements, the antibody was serially diluted threefold (1.37 nm to 1000 nm) in HBS-EP + 1× buffer (GE, cat# BR-1006-69) and injected at 25°C for 120 s. The dissociation time was 30 min, followed by regeneration in 10 mM glycine-HCl (pH 2.0) for 120 s. The association rate (k on ) and dissociation rate (k off ) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (k D ) was calculated as the ratio k off / k on .

The measured binding affinities of the anti-PD-L1 antibodies are shown in Table 5. The results show that the affinity of the PDL1-56-Fc protein for the PD-L1 target protein is significantly higher than that of two known PD-L1 antibodies in the art. Its higher Ka and lower Kd values indicate that the antibody fusion protein can bind to the PD-L1 antigen more rapidly and is difficult to dissociate from it. This further demonstrates that PDL1-56-Fc, as a blocking antibody, has properties that are superior to the two known PD-L1 antibodies.

Table 5

Antibody Ka Kd KD PDL1-56-Fc 1.796E+6 1.432E-5 7.975E-12 PDL1-hu56V2-Fc 2.123E+6 1.820E-5 8.573E-12 PDL1-56 3.323E+6 8.213E-4 2.472E-10 2.41H90P 7.949E+5 6.160E-5 7.750E-11 243.55 4.481E+5 6.055E-5 1.351E-10

5.3 Identification of the Blocking Effect of PD-L1 Single Domain Antibody Fc Fusion Protein on PDL1-PD1 Interaction (Competitive ELISA)

Coat the plates with 0.5 μg/well of PDL1-Fc fusion protein overnight at 4°C. Then, add a serial dilution series of the PD-L1 single-domain antibody Fc fusion protein obtained in the above example, 100 μL per well (the dilution solution contains 100 μg/mL PD1-Fc-Biotin), and react at room temperature for 1 hour. After washing, add SA-HRP (purchased from Sigma) and react at room temperature for 1 hour. After washing, add the colorimetric solution and read the absorbance at 405 nm.

Data processing and graphical analysis were performed using Softmax Pro v5.4. Four-parameter fitting was used to generate the PDL1-PD1 blocking curves and IC50 values. The results are shown in Figure 6, where the ordinate represents OD405 and the abscissa represents the concentration of the PD-L1 single-domain antibody Fc fusion protein (in ng/mL). The inverted triangle, upright triangle, and square represent the three different humanized Fc fusion proteins of antibody strain 56: hu56v1-Fc, hu56v2-Fc, and hu56v5-Fc, respectively. The three proteins demonstrated comparable blocking abilities against the PDL1-PD1 interaction.

5.4 Identification of the Blocking Ability of PD-L1 Single Domain Antibody Fc Fusion Protein on PD-L1/CD80 Interaction (Competitive ELISA)

CD80-Fc protein (SEQ ID NO: 36) was expressed in HEK293 cells and biotinylated protein CD80-Fc-Biotin was obtained using the Biotinlylation kit from Thermo Fisher Scientific.

Coat the plates with 0.5 μg/well of PDL1-Fc fusion protein overnight at 4°C. Then, add a serial dilution series of the PD-L1 single-domain antibody Fc fusion protein obtained in the above example (100 μL per well) (the dilution solution contains 300 μg/mL CD80-Fc-Biotin) and react at room temperature for 1 hour. After washing, add SA-HRP (purchased from Sigma) and react at room temperature for 1 hour. After washing, add the colorimetric solution and read the absorbance at 405 nm.

Data processing and graphical analysis were performed using Softmax Pro v5.4 software. Four-parameter fitting was used to obtain the PDL1-CD80 blocking curve and IC50 value. The results are shown in Figure 7, where the ordinate represents OD405 and the abscissa represents the concentration of the PD-L1 single-domain antibody Fc fusion protein hu56V2-Fc (in ng/mL). The results showed that the PD-L1 blocking single-domain antibody Fc fusion protein hu56V2-Fc can effectively block the interaction between PD-L1 and CD80.

5.5 Identification of the Blocking Ability of PD-L1 Single Domain Antibody Fc Fusion Protein on PD-L1/PD1 Interaction (FACS Method)

Human HEK293 cells were transiently transfected with a plasmid containing the full-length human PD-L1 gene to transiently express monkey PD-L1 protein on the membrane.

Cells were grouped at 5×105 cells/tube. PD1-muFc (SEQ ID NO: 39) at a working concentration of 2 μg/ml was added to each group, followed by different concentrations of KN035. The cells were incubated on ice for 30 minutes. After washing three times, PE-labeled goat anti-mouse secondary antibody was added as the detection antibody. After incubation on ice for 30 minutes, the fluorescence intensity was measured by flow cytometry.

GraphPad Prism software was used for data processing and graphical analysis. Four-parameter fitting was used to obtain the blocking curve and IC50 value for the direct interaction between PDL1 expressed on the 293 cell membrane and soluble PD1. The results are shown in Figure 8, where the ordinate is the MFI and the abscissa is the concentration of the PD-L1 single-domain antibody Fc fusion protein hu56V1-Fcm1 (in μg/mL). The results showed that the PD-L1 blocking single-domain antibody Fc fusion protein can effectively block the interaction between PDL1 expressed on the 293 cell membrane and PD1.

The Jurket cell line was transformed and integrated with a plasmid containing the full-length human PD1 gene to obtain a Jurket cell line that stably expresses human PD1 protein and was named Jurket-PD1.

Jurkat-PD1 cells were incubated with biotin-labeled PDL1-muFc (SEQ ID NO: 38) protein (30 μg/ml) on ice for 30 min, and then serially diluted PD-L1 single-domain antibody Fc fusion protein hu56V1-Fcm1 was added and incubated on ice for 1 h. The cells were washed three times with PBS, and then Streptavidin PE was added at a dilution of 1:250. The cells were incubated on ice for 30 min, washed three times with PBS, and the fluorescence intensity was detected by flow cytometry.

GraphPad Prism software was used for data processing and graphical analysis. Four-parameter fitting was used to obtain the blocking curve and IC50 value of the antibody against the direct interaction between Jurkat-PD1 and soluble PDL1-muFc protein. The results are shown in Figure 9, where the ordinate is the MFI and the abscissa is the concentration of the PD-L1 single-domain antibody Fc fusion protein hu56V1-Fcm1 (in μg/mL). The results show that the PD-L1 blocking single-domain antibody Fc fusion protein can effectively block the interaction between Jurkat-PD1 and PD-L1.

5.6 Analysis of the Specificity of PD-L1 Single Domain Antibody Fc Fusion Protein Binding to PD-L1 Protein

Human HEK293 cells were transiently transfected with a plasmid (pCDNA4, Invitrogen, Cat V86220) carrying the full-length gene for human B7 family proteins, resulting in transient expression of human PD-L1 protein on membranes. This plasmid also fused EGFP to the C-terminus of the target protein, allowing for monitoring B7 family protein expression levels on membranes by green fluorescence intensity. Constructed transiently transfected cell lines include: 293-PDL1-EGFP, 293-PDL2-EGFP, 293-B7H3-EGFP, and 293-B7H3-EGFP.

Resuspend the constructed cells in 0.5% PBS-BSA buffer and add hu56V2-Fc antibody. Also, set up a negative control with 2 μg of a single-domain antibody targeting an unrelated target. Incubate on ice for 20 minutes. After washing, add eBioscience secondary antibody anti-hIg-PE and incubate on ice for 20 minutes. After washing, resuspend the cells in 500 μl of 0.5% PBS-BSA buffer and analyze by flow cytometry.

The results are shown in Figure 10. The upper row is the control group, and the lower row is the sample group. It can be clearly seen that the hu56V2-Fc antibody specifically binds only to the human PD-L1 protein and does not bind to other B7 family proteins.

5.7 Binding of PD-L1 Single Domain Antibody Fc Fusion Protein to Monkey PD-L1 Protein

Human HEK293 cells were transiently transfected with a plasmid containing the full-length monkey PD-L1 gene, transiently expressing the monkey PD-L1 protein (SEQ ID NO: 37) on the membrane. This plasmid also fused the target protein to the C-terminus of the EGFP protein, allowing the expression level of the monkey PD-L1 protein on the membrane to be monitored by green fluorescence intensity.

Resuspend the constructed cells in 0.5% PBS-BSA buffer, add hu56V2-Fc antibody, and incubate on ice for 20 minutes. After washing, add eBioscience secondary antibody anti-hIg-PE and incubate on ice for 20 minutes. After washing, resuspend the cells in 500 μl of 0.5% PBS-BSA buffer and analyze by flow cytometry.

The results are shown in Figure 11. It can be clearly seen that the hu56V2-Fc antibody can effectively bind to the monkey PD-L1 protein.

5.8 PD-L1 single-domain antibody Fc fusion protein can effectively identify PD-L1 positive cell populations in patient tissue sections

Tumor tissue sections from patients with PD-L1-positive lung cancer were stained overnight with 5 μg/mL hu56V2-Fc antibody as the primary antibody, and then stained with goat anti-human HRP-labeled antibody (Perkin-Elmer, Cat: NEF802001EA) as the secondary antibody at room temperature for 1.5 hours before color development.

The results are shown in Figure 12. The hu56V2-Fc antibody can effectively identify PD-L1-positive cell populations on tissue sections of lung cancer patients, and can simultaneously identify PD-L1-positive tumor cells and PD-L1-positive immune cells.

5.9 Activation of PBMCs by PD-L1 Single Domain Antibody Fc Fusion Protein

Peripheral blood mononuclear cells (PBMCs) were isolated from concentrated leukocytes in peripheral blood of healthy donors using density gradient centrifugation with human lymphocyte separation medium (Tianjin Haoyang).

A serial dilution series of 2.5 μg/mL anti-CD3 antibody and PD-L1 single-domain antibody Fc fusion protein hu56V2-Fc (also designated KN035 in this experiment) was coated onto cell culture plates overnight at 4°C. The next day, 1 x 10 ⁵ PBMCs were added to each well. After 5 days of culture, the supernatant was collected and assayed for IFN-γ levels using an IFN-γ ELISA kit (ebioscience).

The results are shown in Figure 13. It can be seen that the PD-L1 single-domain antibody Fc fusion protein combined with anti-CD3 antibody can enhance the secretion of interferon-γ by PBMC cells, indicating that the PD-L1 single-domain antibody Fc fusion protein enhances the activation of PBMC cells. This activation effect is also concentration-dependent.

5.10 The activation of CD4+ T cells by PD-L1 single domain antibody Fc fusion protein was measured in a dendritic cell-T cell mixed lymphocyte reaction and compared with the anti-PD-L1 antibody from MedImmune LLC

Peripheral blood mononuclear cells (PBMCs) were isolated from concentrated leukocytes from healthy donors using density gradient centrifugation using human lymphocyte separation medium (Tianjin Haoyang). These cells were then cultured in serum-free RPMI1640 medium for 1-2 hours. Non-adherent cells were removed and the cells were cultured in RPMI supplemented with 10% FBS, 10 ng/ml GM-CSF, and 20 ng/mL IL-4. After 5-6 days of culture, 10 ng/ml TNF-α was added and incubated for 24 hours to obtain mature dendritic cells (DCs).

The DC cells obtained by this method were resuspended in RPMI complete medium at 2×10 ⁵ /ml, and 50 μl was added to each well of a 96-well U-bottom plate (Costar: 3799) and cultured in an incubator.

CD4+ T cells were isolated from PBMC of another donor using a magnetic bead separation kit (Miltenyi Biotec: 130-096-533) according to the manufacturer's instructions.

^1x10⁴ dendritic cells and 1x10⁵ ^CD4 + T cells obtained using the above method were mixed, resuspended in RPMI complete medium, and plated into a 96-well culture plate. 50 μl of the cell mixture was added to each well. 100 μl of hu56V2-Fc diluted in RPMI complete medium was added to each well to a final concentration of 0.1 μg/ml or 0 μg/ml. After 5-7 days of culture, the supernatant was collected and assayed for IFN-γ levels using an IFN-γ ELISA kit (eBioscience).

The results are shown in Figure 14A. It can be seen that the PD-L1 single domain antibody Fc fusion protein can enhance the secretion of gamma interferon by CD4+ T cells in mixed lymphocyte reactions, that is, the PD-L1 blocking single domain antibody Fc fusion protein enhances T cell activation.

PBMCs from subject 1 were cultured with 50 ng/ml GM-CSF and 25 ng/ml IL-4 for 6 days. After maturation with TNF-α (50 ng/ml) for 24 h, DCs were collected. CD4+ T cells were then sorted from PBMCs from subject 2 using a magnetic bead separation kit (Miltenyi Biotec: 130-096-533). DCs were added to a 96-well U-bottom plate at a density of ¹⁰⁴ /well, and 2-4 h later, ¹⁰⁵ cells/well of CD4+ T cells were added. Different concentrations of the PD-L1 single-domain antibody Fc fusion protein hu56V1-Fcm1 or the anti-PD-L1 antibody 2.41H90P from MedImmune LLC were added to each well. After 5 days of culture, IFN-γ levels in the supernatant were detected using an IFN-γ ELISA kit (ebioscience).

The results are shown in Figure 14B, where the gray histogram indicates IFN-γ secretion stimulated by the PD-L1 single-domain antibody Fc fusion protein, and the black histogram indicates MedImmune LLC's anti-PD-L1 antibody 2.41H90P. The PD-L1 single-domain antibody Fc fusion protein enhanced the ability of CD4+ T cells to secrete IFN-γ in mixed lymphocyte reactions with increasing concentration. At the same concentration, the PD-L1 single-domain antibody Fc fusion protein was slightly more potent in activating T cells than MedImmune LLC's anti-PD-L1 antibody 2.41H90P.

5.11 PD-L1 Single Domain Antibody Fc Fusion Protein Stimulates T Cell IL-2 Secretion in a Jurkat/Raji-PDL1 Mixed Culture System and Comparison with MedImmune LLC's Anti-PD-L1 Antibody

We constructed a T cell activation system, namely the Jurkat/Raji-PDL1 co-culture system, to detect the effect of PD-L1 single-domain antibody Fc fusion protein on T cell activation and compared it with the anti-PD-L1 antibody from MedImmune LLC.

This system uses Jurkat cells (T cells) as effector cells and anti-human CD3 Antibody as the first signal for Jurkat cell activation; Raji cells Raji-PDL1 cells are genetically modified to stably express human PDL1, and CD80 in the B7 family molecules on their surface provides a second co-stimulatory signal to activate Jurkat cells. At the same time, the highly expressed PDL1 on their cell surface acts as a negative regulator to inhibit Jurkat cell activation by binding to PD1.

Use 10% FBS + 1640 + 150ng/ml anti-CD3 to prepare gradient dilutions of hu56V1-Fcm1 and 2.41H90P antibody proteins; Jurkat and Raji-PDL1 cells are adjusted to 3×10 ⁶ cells/ml and 1.5×10 ⁶ cells/ml with 10% FBS, respectively, add 50ul to each well, incubate at 37°C for 24 hours, remove 100ul culture supernatant, and use a kit to detect IL-2 expression.

Figure 15 shows the stimulation of IL-2 secretion by PD-L1 single-domain antibody Fc fusion protein, with the black bar graph indicating IL-2 secretion by MedImmune LLC's anti-PD-L1 antibody 2.41H90P. PD-L1 single-domain antibody Fc fusion protein enhanced IL-2 secretion by Jurkat cells in this mixed lymphocyte reaction with increasing concentration. At the same concentration, its ability to activate Jurkat cells was slightly stronger than that of MedImmune LLC's anti-PD-L1 antibody 2.41H90P.

5.12 Affinity of PD-L1 Single Domain Antibody Fc Fusion Protein for FcRn

Biotinylated PD-L1 single-domain antibody Fc fusion proteins hu56V1-Fc, hu56V2-Fc, hu56V1-Fcm1, and hu56V2-Fcm1 were diluted to 10 μg/mL and immobilized on an SA biosensor. Human FcRn protein (RnD Systems, Cat. No. 8639-FC-050) was diluted to 5 levels of 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM. The interaction was detected using the Fortebio Octet K2, with an immobilization time of 100 s, an association time of 60 s, and a dissociation time of 30 s.

Table 6 shows that the average KD of the PD-L1 single-domain antibody Fc fusion protein to FcRn is approximately 5.1E-07 M. There is no significant difference in affinity between the mutant Fc (Fcm1) and the wild-type Fc.

Table 6

Sample name KD(M) kon(1/Ms) kdis(1/s) hu56V1-Fc 5.13E-07 1.95E+05 1.00E-01 hu56V2-Fc 5.05E-07 2.10E+05 1.06E-01 hu56V1-Fcm1 5.10E-07 1.89E+05 9.63E-02 hu56V2-Fcm1 5.10E-07 2.34E+05 1.19E-01

5.13 Evaluation of CDC and ADCC Activities of PD-L1 Single Domain Antibody Fc Fusion Proteins with Mutated Fc

PBMCs activated with 300 IU/ml IL-2 for 24 hours were used as effector cells at a cell number of 8×10 ⁵ cells/well. Raji-PDL1 cells stably expressing human PDL1 protein were used as target cells at a cell number of 2×10 ⁵ cells/well. Different concentrations of hu56V1-Fcm1 or Rituxan protein as a positive control were added and cultured at 37°C for 6 hours. ADCC activity (%) was measured using the CytoTox Non-Radioactive Cytotoxicity Assay Kit.

FIG16A shows that hu56V1-Fcm1 has no significant ADCC activity compared to the positive control Rituxan.

Raji-PDL1 cells were used as target cells at a cell number of 2×10 ⁴ cells/well. 5% cynomolgus macaque serum provided complement. Different concentrations of hu56V1-Fcm1 and the positive control Rituxan were added. After incubation at 37°C for 2 hours, CCK-8 was used to detect CDC activity of the samples.

FIG16B shows that compared with the positive control, hu56V1-Fcm1 had no CDC activity in the concentration range of 0.02 ug/ml to 20 ug/ml.

5.14 Inhibitory Activity of PD-L1 Single Domain Antibody Fc Fusion Protein on Tumor Growth

The in vivo activity of hu56V2-Fc, a single-domain antibody Fc fusion protein that lacks mouse PD-L1 recognition, was investigated in immunodeficient NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice. This study was performed by subcutaneously implanting NOD/SCID mice with the human PD-L1-expressing melanoma cell line A375 (ATCC, CRL-1619 ^™ ) and human peripheral blood mononuclear cells (PBMCs). A375 and PBMCs were mixed at a 5:1 ratio prior to injection, and a total volume of 100 μl (5 million A375 and 1 million PBMCs) was injected subcutaneously. The antibody was first administered intraperitoneally 24 hours after tumor inoculation and then weekly thereafter at a dose of 0.3 mg/kg. PBS was used as a negative control. Four to six mice were included in each experimental group. Tumor formation was observed twice a week, and the long and short diameters of the tumor were measured with a vernier caliper. The tumor volume was calculated and a tumor growth curve was plotted (see Figure 17A). It can be seen that the antibody hu56V2-Fc can significantly inhibit tumor growth at a dose of 0.3 mg/kg.

The same in vivo model was used to investigate the Fc fusion protein, hu56V1-Fcm1, a single-domain antibody against PD-L1 that also does not recognize mouse PD-L1. A375 and human PBMCs were mixed at a ratio of 4:1 and inoculated subcutaneously into NOD-SCID mice. Four hours later, different doses of hu56V1-Fcm1 (0.1, 0.3, 1, 3, and 10 mg/kg) were administered intraperitoneally. The inhibitory effect of weekly dosing for four consecutive weeks on A375/human PBMC xenografts in NOD-SCID mice was investigated. PBS served as a negative control. Four to six mice were included in each experimental group. Tumor formation was observed twice weekly, and tumor major and minor diameters were measured with a vernier caliper. Tumor volume was calculated, and tumor growth curves were plotted (see Figure 17B). Results showed that all doses of hu56V1-Fcm1 (0.1 to 10 mg/kg) exhibited significant inhibitory effects on A375/human PBMC xenografts in NOD-SCID mice, with no apparent dose-dependent effect. The antibody hu56V1-Fcm1 can significantly inhibit tumor growth at a dose of 0.1 mg/kg.

5.15 Inhibitory Activity of PD-L1 Single Domain Antibody Fc Fusion Protein on Tumor Growth at Different Dosing Times

The above-mentioned NOD-SCID mouse A375/human PBMCs xenograft tumor model was continued for investigation. A375 and human PBMCs were mixed at a ratio of 4:1 and inoculated subcutaneously into NOD-SCID mice. Four hours later, hu56V1-Fcm1 (0.3 mg/kg) was intraperitoneally injected, and then intraperitoneally administered once every three days. The final number of administrations was 1, 2, 3, and 4 times, respectively. Tumor formation was observed every three days, and the long and short diameters of the tumors were measured with a vernier caliper to calculate the tumor volume until the 33rd day from the first administration. A tumor growth curve was drawn (Figure 18), and the results showed that each number of administrations within the investigation period had a significant tumor inhibitory effect on the NOD-SCID mouse A375/human PBMCs xenograft tumor.

5.16 Inhibitory Activity of PD-L1 Single Domain Antibody Fc Fusion Protein on Tumor Growth and Comparison with MedImmune LLC's Anti-PD-L1 Antibody

This study aimed to address this issue by subcutaneously transplanting NOD/SCID mice with the human PD-L1-expressing melanoma cell line A375 (ATCC, CRL-1619 ^™ ) and human peripheral blood mononuclear cells (PBMCs). A375 and PBMCs were mixed at a 5:1 ratio prior to injection, with a total subcutaneous injection volume of 100 μl (containing 5 million A375 and 1 million PBMCs). Antibody was administered intraperitoneally 24 hours after tumor inoculation and then weekly at a dose of 1 mg/kg. Treatment groups included hu56V2-Fc (denoted as hu56) and an anti-PD-L1 antibody from MedImmune LLC (denoted as 2.41). PBS served as a negative control. Each experimental group consisted of 4-6 mice. Tumor formation was observed twice weekly, and tumor major and minor diameters were measured with a vernier caliper. Tumor volume was calculated and tumor growth curves were plotted (see Figure 19A). MedImmune LLC's anti-PD-L1 antibody was largely ineffective in this model, and tumor volume exceeded that of the negative control group on day 35, so dosing and tumor volume measurement were discontinued. This indicates that in this model, hu56V2-Fc, at a dose of 1 mg/kg, significantly outperformed MedImmune LLC's anti-PD-L1 antibody 2.41H90P in inhibiting A375 tumor growth.

Since MedImmune LLC's anti-PD-L1 antibody did not show tumor suppression effects in the aforementioned system, this may be due to insufficient activation of PBMCs within the system to inhibit tumor cell growth. Therefore, the tumor suppression effect of MedImmune LLC's anti-PD-L1 antibody was re-evaluated by increasing the PBMC content in the mixed cell population.

A375 and PBMC were mixed at a 1:1 ratio before injection, and a total volume of 100 μl (containing 5 million A375 and 5 million PBMC) was subcutaneously injected. MedImmune LLC's anti-PD-L1 antibody (2.41H90P) was administered intraperitoneally for the first time 24 hours after tumor inoculation and then once a week at a dose of 1 mg/kg; PBS was used as a negative control. Each experimental group consisted of 4-6 mice. Tumor formation was observed twice a week, and the long and short diameters of the tumors were measured with a vernier caliper. The tumor volume was calculated and a tumor growth curve was plotted (see Figure 19B). It can be seen that after increasing the proportion of PBMCs, MedImmune LLC's anti-PD-L1 antibody showed an anti-tumor effect in the in vivo model.

The average tumor inhibition rate of the antibody on day 42 (TGI = (1-tumor volume of the administration group/tumor volume of the control group) x 100%) was calculated and listed in Table 7 below:

Table 7

The above-mentioned in vivo tumor inhibition experimental results show that the PD-L1 blocking single domain antibody Fc fusion protein of the present invention exhibits a significantly better tumor inhibition effect than the known PD-L1 blocking antibody (anti-PD-L1 antibody from MedImmune LLC) in the nude mouse model of melanoma A375.

Example 6: Stability Study of PD-L1 Single Domain Antibody Fc Fusion Protein

6.1 Study on the resistance of PD-L1 single-domain antibody Fc fusion protein to alkaline and oxidative damage

500 mM ammonium bicarbonate was used as the alkaline destroying agent and treated at 37°C for 38 hours. 1% hydrogen peroxide was selected as the oxidizing agent and treated at room temperature for 8 hours.

Competitive ELISA was used to assess the biological activity of the PD-L1 single-domain antibody Fc fusion proteins obtained in the above examples before and after treatment. As shown in Figure 20 , alkalinity and oxidative damage did not affect the activity of the candidate PD-L1 single-domain antibody Fc fusion proteins. The competitive ELISA activity after 38 hours of alkaline treatment was 103% of that at 0 hours. The competitive ELISA activity after 8 hours of oxidation treatment was 106% of that at 0 hours.

6.2 Stability of PD-L1 Single Domain Antibody Fc Fusion Protein at High Concentration

The PD-L1 single-domain antibody Fc fusion protein was concentrated by UF/DF and exchanged into PBS buffer. Its aggregate formation tendency was detected by SE-HPLC.

When concentrated to 200 mg/mL, the PD-L1 single-domain antibody Fc fusion protein showed a purity of 96.8% as determined by SE-HPLC. Compared to the lower concentration (~2 mg/mL), aggregates increased by approximately 2.4%. The protein solution showed no turbidity or aggregation during the entire concentration process.

Sequence Listing

>SEQ ID NO:1 Antibody strain 56

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:2 Hu56V1

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS

>SEQ ID NO:3 Hu56V2

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS

>SEQ ID NO:4 Hu56V3

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKGLERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS

>SEQ ID NO:5 Hu56V4

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKGLERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS

>SEQ ID NO:6 Hu56V5

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS

>SEQ ID NO:7 IgG1-Fc

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:8 IgG1-Fc-m1

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAG IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:9 IgG1-Fc-m2

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:10 56-Fc

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:11 Hu56V1-Fc

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:12 Hu56V2-Fc

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:13 Hu56V3-Fc

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKGLERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:14 Hu56V4-Fc

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKGLERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:15 Hu56V5-Fc

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:16 56-Fc-m1

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:17 Hu56V1-Fc-m1

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:18 Hu56V2-Fc-m1

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:19 Hu56V3-Fc-m1

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKGLERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:20 Hu56V4-Fc-m1

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKGLERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:21 Hu56V5-Fc-m1

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:22 56-Fc-m2

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:23 Hu56V1-Fc-m2

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:24 Hu56V2-Fc-m2

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:25 Hu56V3-Fc-m2

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKGLERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:26 Hu56V4-Fc-m2

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRRCMAWFRQAPGKGLERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:27 Hu56V5-Fc-m2

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:28 Human PDL1-Fc

TVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVN APYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREENLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTDDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:29

GTCCTGGCTGCTCTTCTACAAGGC

>SEQ ID NO:30

GGTACGTGCTGTTGAACTGTTCC

>SEQ ID NO:31

GATGTGCAGCTGCAGGAGTCTGGRGGAGG

>SEQ ID NO:32

GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

>SEQ ID NO:33 mice PDL1-Fc

FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKV NAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:34 Human PD1-Fc

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:35 PDL1-Chis

TVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREENLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTDGSHHHHHHHH

>SEQ ID NO:36 CD80-Fc

VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADF PTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>SEQ ID NO:37 monkey PDL1

FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKSGIRVTNSKKQRDTQLEET

>SEQ ID NO:38 human PDL1-muFc

FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA PYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTDIEGRMD PKSSDKTHTCPPCPAPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIE KTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

>SEQ ID NO:39 Human PD1-muFc

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQIEGRMDPKSSDKTHTCPPCPAPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

Claims (22)

1. A programmed death-ligand 1 (PD-L1) binding molecule that specifically binds to PD-L1 and consists of an amino acid sequence of formula A-L-B, where A represents a single variable domain of an immunoglobulin, L is absent or represents an amino acid linker, and B represents the Fc region of human immunoglobulin. The immunoglobulin single variable domain described therein is composed of an amino acid sequence selected from SEQ ID NO:1-6. The PD-L1 binding molecule can form a homodimer through the Fc region of the human immunoglobulin. 2. The PD-L1 binding molecule of claim 1, wherein the human immunoglobulin Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. 3. The PD-L1 binding molecule of claim 1, wherein the human immunoglobulin Fc region is mutated to remove ADCC and CDC activity. 4. The PD-L1 binding molecule of claim 1, wherein the amino acid sequence of the Fc region of the immunoglobulin is selected from SEQ ID NO: 7-9. 5. The PD-L1 binding molecule of any one of claims 1-4, wherein the amino acid linker is 1-20 amino acids long. 6. The PD-L1 binding molecule of claim 1, comprising an amino acid sequence selected from SEQ ID NO:10-27. 7. The PD-L1 binding molecule of any one of claims 1-4, having at least one of the following characteristics: (a) The KD value of binding to human PD-L1 is less than 1 × ^10⁻⁷ M; (b) Block the interaction between PD-L1 and PD-1; (c) Enhance the activation of PBMCs and/or T cells; (d) Inhibit tumor growth. 8. The PD-L1 binding molecule of claim 7, wherein the KD value of binding PD-L1 is less than 1× ^10⁻⁸ M. 9. The PD-L1 binding molecule of claim 7, wherein the KD value of binding PD-L1 is less than 1× ^10⁻⁹ M. 10. The PD-L1 binding molecule of claim 7, wherein the KD value of binding PD-L1 is less than 1 × ^10⁻¹⁰ M. 11. The PD-L1 binding molecule of claim 7, wherein the KD value of binding PD-L1 is less than 1 × ^10⁻¹¹ M. 12. A nucleic acid molecule encoding a PD-L1 binding molecule of any one of claims 1-11. 13. An expression vector comprising the nucleic acid molecule of claim 12 operatively linked to an expression regulatory element. 14. A host cell comprising the nucleic acid molecule of claim 12 or transformed with the expression vector of claim 13, and capable of expressing the PD-L1 binding molecule. 15. A method for producing a PD-L1 binding molecule of any one of claims 1-11, comprising: a) Culture the host cells of claim 14 under conditions that allow the expression of the PD-L1 binding molecule; b) Recover the PD-L1 binding molecule expressed by the host cells from the culture obtained from step a); and c) Optional further purification and/or modification of the PD-L1 binding molecule obtained from step b). 16. A pharmaceutical composition comprising the PD-L1 binding molecule of any one of claims 1-11 and a pharmaceutically acceptable carrier. 17. Use of the PD-L1 binding molecule of any one of claims 1-11 or the pharmaceutical composition of claim 16 in the preparation of a medicament for the prevention and/or treatment of cancer in a subject. 18. The use of claim 17, wherein the prevention and/or treatment of cancer further includes administering other antitumor treatments to the subject. 19. The use of claim 18, wherein the other antitumor treatment means includes chemotherapy, radiotherapy or antibodies targeting other tumor-specific antigens. 20. The use of claim 19, wherein the antibody targeting other tumor-specific antigens includes an anti-EGFR antibody, an anti-EGFR variant antibody, an anti-VEGFa antibody, an anti-HER2 antibody, or an anti-CMET antibody. 21. The use of claim 20, wherein the antibody targeting other tumor-specific antigens is a monoclonal antibody. 22. The use of any one of claims 17-21, wherein the cancer is selected from lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial cancer, and osteosarcoma.