CN113754777A - anti-PD-L1/TGF-beta fusion protein - Google Patents

anti-PD-L1/TGF-beta fusion protein Download PDF

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CN113754777A
CN113754777A CN202010488804.6A CN202010488804A CN113754777A CN 113754777 A CN113754777 A CN 113754777A CN 202010488804 A CN202010488804 A CN 202010488804A CN 113754777 A CN113754777 A CN 113754777A
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黄浩旻
邓岚
李理
朱祯平
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Zi Da Biological Medicine Co.,Ltd.
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Abstract

The invention relates to the technical field of fusion proteins, in particular to an anti-PD-L1/TGF-beta fusion protein. The fusion protein comprises an anti-PD-L1 antibody, a peptide linker, and a TGF β rii extracellular domain linked to the C-terminus of the anti-PD-L1 antibody heavy chain by the peptide linker. The fusion protein has the potential of treating diseases related to PD-L1 and TGF-beta activity.

Description

anti-PD-L1/TGF-beta fusion protein
Technical Field
The invention relates to the technical field of fusion proteins, and in particular relates to an anti-PD-L1/TGF-beta fusion protein.
Background
Human programmed cell death receptor-1 (PD-1) is a 288 amino acid type I membrane protein and is one of the known major Immune checkpoints (Immune Checkpoint) (Blank et al,2005, Cancer Immunotherapy,54: 307-) -314. PD-1 is expressed in activated T lymphocytes, and binding of the ligands PD-L1 (programmed cell death receptor-Ligand 1) and PD-L2 (programmed cell death receptor-Ligand 2) can inhibit the activity of T lymphocytes and the related in vivo cellular immune response. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. Numerous studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain immune system balance in vivo, but also a major mechanism and cause PD-L1 expressing positive tumor cells to circumvent immune surveillance. By blocking the negative regulation and control of cancer cells to PD-1/PD-L1 signal channels, the immune system is activated, and the tumor specific cellular immune response related to T cells can be promoted, thereby opening a new tumor treatment method, namely a tumor immunotherapy.
PD-1 (encoded by gene Pdcd 1) is an immunoglobulin superfamily member that is associated with CD28 and CTLA-4. The results of the study show that PD-1 negatively regulates antigen receptor signaling when bound to its ligand (PD-L1 and/or PD-L2). The murine PD-1 structure and the cocrystal structure of murine PD-1 and human PD-L1 have been clarified (Zhang, X. et al, Immunity 20: 337-347 (2004); Lin et al, Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins that contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding to a signaling molecule. The PD-1 cytoplasmic tail contains two tyrosine-based signaling motifs, the ITIM (immunoreceptor tyrosine inhibition motif) and the ITSM (immunoreceptor tyrosine transduction motif).
PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, namely, cancer resistance by using the immune system of the human body, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to break the tumor microenvironment becomes the key point of anti-tumor research. The role of PD-1 in the tumor microenvironment has been determined by prior work: PD-L1 is expressed in a number of mouse and human tumors (and can be induced by IFN-. gamma. in most PD-L1 negative tumor cell lines) and is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al, Proc. Natl. Acad. Sci. U.S.A.99: 12293-12297 (2002); Strome S.E. et al, Cancer Res., 63: 6501-6505 (2003)). Biopsy evaluation by immunohistochemistry has revealed expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer, as well as head and neck tumors, among others. Therefore, the blocking of the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor specific T cells and is beneficial to the immune system to eliminate tumor cells, so that PD-L1 becomes a hot target for developing tumor immunotherapy drugs.
Transforming growth factor-beta (TGF-beta) belongs to the TGF-beta superfamily that regulates cell growth and differentiation. TGF-. beta.signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors. TGF- β is a multifunctional cytokine that exerts tumor-inhibiting or tumor-promoting effects in a cell-or background-dependent manner. The tumor-inhibiting effect of TGF- β signaling results from its ability to induce the expression of multiple genes, and when mutations or epigenetic modifications are introduced during tumor development, cancer cells gradually tolerate the inhibitory effect of TGF- β signaling, eventually leading to tumor development. It has been found that blocking the TGF- β signalling pathway can reduce metastasis of the tumour.
Disclosure of Invention
The invention aims to provide a novel anti-PD-L1/TGF-beta fusion protein which can block PD-L1 and TGF-beta signal channels at the same time. It is also an object of the present invention to provide polynucleotide molecules encoding said fusion proteins; providing an expression vector comprising said polynucleotide molecule; providing a host cell comprising the expression vector; providing a method for preparing the fusion protein; providing a pharmaceutical composition comprising the fusion protein; provides the application of the fusion protein or the pharmaceutical composition in preparing a medicament for treating cancer; methods of providing the fusion protein or the pharmaceutical composition for treating cancer are provided.
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the invention provides an anti-PD-L1/TGF-beta fusion protein having the general formula:
Ab-L-TGFβRII ECD (I)
wherein Ab is an anti-PD-L1 antibody, L is a peptide linker, and TGF beta RII ECD is a TGF beta RIII extracellular domain; the N-terminus of the TGF- β RII extracellular domain is linked to the C-terminus of the anti-PD-L1 antibody heavy chain by a peptide linker; the heavy chain of the anti-PD-L1 antibody comprises the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 11, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12 is shown in the specification; the light chain of the anti-PD-L1 antibody comprises the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 13, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 15.
In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is as set forth in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: shown at 17.
In a preferred embodiment, the heavy chain amino acid sequence of the anti-PD-L1 antibody is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO: 3, the light chain amino acid sequence of the anti-PD-L1 antibody is set forth in SEQ ID NO: 4, respectively.
In a preferred embodiment, the anti-PD-L1 antibody is a monoclonal antibody.
In a preferred embodiment, the anti-PD-L1 antibody is a humanized antibody.
In a preferred embodiment, the anti-PD-L1 antibody is an IgG class antibody.
In a preferred embodiment, the TGF β rii extracellular domain is selected from one or a combination of the following groups:
1) a full-length TGF-beta RII extracellular domain;
2) a TGF beta RII extracellular domain truncated by 6-10 amino acids at the C-terminus, more preferably a TGF beta RII extracellular domain truncated by 8 amino acids at the C-terminus;
3) a TGF-beta RII extracellular domain truncated by 18-22 amino acids at the N-terminus, more preferably a TGF-beta RII extracellular domain truncated by 20 amino acids at the N-terminus;
4) a full-length or truncated TGF β rii extracellular domain comprising at least 1 glycosylation site mutation selected from S8P, T16P, T16V, N71Q.
In a preferred embodiment, the amino acid sequence of the extracellular domain of TGF β RII is selected from the group consisting of SEQ ID NO: 5-SEQ ID NO: 9.
in a preferred embodiment, the peptide linker is selected from (G)4S)3T or (G)4S)3XDYTHTP, wherein X is G or S and Y is K or A.
In a more preferred embodiment, the fusion protein is selected from the group consisting of: 869. 869F, 869J15, 869J16, 869J17, 869M1, 869M 3.
In a more preferred embodiment, the fusion protein is selected from the group consisting of:
1) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 18, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
2) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 19, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
3) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 20, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
4) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 21, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
5) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 22, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
6) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 23, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
7) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 24, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4, respectively.
In a second aspect of the invention there is provided a polynucleotide molecule encoding the fusion protein.
Those skilled in the art will appreciate that the polynucleotide molecules encoding the amino acid sequences of the above fusion proteins may be suitably modified by the introduction of substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog.
In a third aspect, the present invention provides an expression vector comprising the polynucleotide molecule described above.
In a fourth aspect, the present invention provides a host cell comprising the above-described expression vector.
The fifth aspect of the present invention provides a method for preparing a fusion protein, comprising the steps of:
a) culturing a host cell as described above under expression conditions such that an anti-PD-L1/TGF- β fusion protein is expressed;
b) isolating and purifying the fusion protein of step a).
In a sixth aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the fusion protein described above and one or more pharmaceutically acceptable carriers, diluents or excipients.
The seventh aspect of the invention provides the use of the fusion protein and the pharmaceutical composition in the preparation of a medicament for treating cancer.
According to the invention, the cancer is selected from: colorectal cancer, bile duct cancer, gallbladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma and the like.
An eighth aspect of the present invention provides a method of treating cancer comprising administering to a subject in need thereof the fusion protein or the pharmaceutical composition described above.
According to the invention, the cancer is selected from: colorectal cancer, bile duct cancer, gallbladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma and the like.
The invention has the positive effects that: the fusion protein has improved charge heterogeneity, can be combined with PD-L1 and TGF-beta with high affinity, has the affinity with PD-L1 equivalent to that of anti-PD-L1 monoclonal antibody positive control M8, and has the affinity with TGF-beta equivalent to that of fusion protein positive control M7824. The fusion protein can effectively block the combination of PD-1 and PD-L1, the blocking capability is equivalent to that of anti-PD-L1 monoclonal antibody positive control M8, and the activity of a pSMAD3 reporter induced by TGF-beta can be inhibited in a dose-dependent manner, and the inhibiting activity is equivalent to that of fusion protein positive control M7824. The fusion protein has the potential of treating diseases related to PD-L1 and TGF-beta activity.
Drawings
FIG. 1A: schematic structure of fusion protein
FIG. 1B: charge heterogeneity detection map of fusion protein 869
FIG. 1C: charge heterogeneity detection map of fusion protein 869F
FIG. 1D: charge heterogeneity detection map of fusion protein 869M1
FIG. 1E: charge heterogeneity detection map of fusion protein 869J15
FIG. 2A: fusion protein 869M1HPLC assay
FIG. 2B: fusion protein 869J15HPLC assay
FIG. 3A: ELISA detection of the affinity of the fusion proteins 869M1, 869M3, 869J15 for PD-L1
FIG. 3B: affinity ELISA assays for fusion proteins 869J15, 869J16, 869J17 and TGF-. beta.1
FIG. 3C: affinity ELISA assays for fusion proteins 869M1, 869M3 and TGF-. beta.1
FIG. 3D: ELISA assays for fusion proteins 869J15, 869J16, 869J17 simultaneously binding to PD-L1 and TGF-. beta.1
FIG. 4: FACS detection of the affinity of fusion proteins 869J15, 869J16 for the target cell surface antigen PD-L1
FIG. 5A: cell experiment detection chart of fusion proteins 869J15 and 869J16 for blocking binding of PD-1 and PD-L1
FIG. 5B: cell experiment detection chart of fusion proteins 869M1 and 869M3 for blocking binding of PD-1 and PD-L1
FIG. 6: map of fusion proteins 869F, 869M1 inhibiting SMAD3 reporter gene
Detailed Description
The following experimental examples are further illustrative of the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods or methods conventional in the art, such as methods of preparing polynucleotide molecules, methods for constructing vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids or methods of introducing plasmids into host cells, methods of culturing host cells, and the like, such methods being well known to those having ordinary skill in the art and described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.
In the present invention, the term "fusion protein" refers to a novel polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fusion" refers to a linkage by peptide bonds either directly or by means of one or more connecting peptides (peptide linkers). The term "linker peptide" refers to a short peptide, typically a peptide of 2-30 amino acids in length, that can link two polypeptide sequences.
In the present invention, the term "Antibody (Ab for short)" is an isotetraglycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and the like. Heavy chain constant regions include IgG1, IgG2, IgG3, IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by interpoly disulfide bonds formed between the hinge regions.
In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
In the present invention, the term "humanized" means that the CDRs are derived from an antibody of a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework and constant regions) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.
In the present invention, the terms "anti-and" binding "refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. Typically, the antibody is present in an amount less than about 10-7M, e.g. less than about 10-8M、10-9M、10-10M、10-11M or less binds the antigen with an equilibrium dissociation constant (KD). The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, using surface plasmon co-polymerizationVibration (Surface plasma Resonance, abbreviated SPR) measures the binding affinity of an antibody to an antigen in a BIACORE instrument or the relative affinity of the binding of an antibody to an antigen using ELISA.
In the present invention, the term "expression vector" refers to an expression vector conventional in the art, which may be a virus or a plasmid, comprising appropriate regulatory sequences, such as a promoter, a terminator, an enhancer, and the like. The expression vector preferably comprises pDR1, pcDNA3.4(+), pDFFR or pTT 5.
In the present invention, the term "host cell" is a variety of host cells that are conventional in the art, as long as the vector can stably replicate autonomously and the polynucleotide molecule carried by the vector can be efficiently expressed. Wherein the host cell comprises prokaryotic expression cells and eukaryotic expression cells, preferably comprising: COS, CHO, NS0, sf9, sf21, DH5 alpha, BL21(DE3), TG1, BL21(DE3) or 293E cells.
In the present invention, the term "effective amount" refers to an amount or dose that, upon administration of a pharmaceutical composition of the invention to a patient, produces a desired effect in the treated subject, including an improvement in the condition of the subject.
The sequence information referred to in the following examples is summarized in table 1 of the sequence listing.
TABLE 1 sequence listing
Figure BDA0002519780100000071
Figure BDA0002519780100000081
Figure BDA0002519780100000091
Figure BDA0002519780100000101
Figure BDA0002519780100000111
The anti-human PD-L1 antibody positive control M8 used in the following examples was derived from PCT/CN2020/090442, and its heavy and light chain amino acid sequences are SEQ ID NOs: 1 and SEQ ID NO: 4.
the anti-PD-L1/TGF- β fusion protein positive control M7824 used in the following examples was derived from US20150225483a1, and its heavy and light chain amino acid sequences are SEQ ID NOs: 25 and SEQ ID NO: 26.
the reagents and starting materials used in the following examples are commercially available unless otherwise specified.
Example 1 anti-PD-L1/TGF-beta bifunctional fusion protein construction
The TGF beta RII extracellular domain is used as an immune regulatory molecule part in the fusion protein, and the anti-PD-L1 antibody M8 is used as a targeting part of the fusion protein to form the anti-PD-L1/TGF beta dual-function fusion protein (the structural schematic diagram is shown in figure 1A), and the structural general formula is as follows:
Ab-L-TGFβRII ECD (I)
wherein the TGF-beta RII ECD is a TGF-beta RII extracellular domain, including full-length, truncated, or mutant forms of the TGF-beta RII extracellular domain. Wherein Ab is a M8 antibody or a mutant of M8 antibody. Wherein L is a peptide linker, comprising (G)4S)3T or (G)4S)3XDYTHTP, wherein X is G or S and Y is K or A.
The inventors continuously studied and improved the structure of the fusion protein by first passing the C-terminal amino acid of the heavy chain of M8 of the anti-PD-L1 antibody through a peptide linker (G) using homologous recombination4S)3T is linked to the N-terminus of the extracellular domain of TGF-beta RII and is expressed along with the light chain of M8, resulting in fusion protein 869. The 869 charge heterogeneity was severe and was optimized by the inventors, and it was found that the extracellular domain of TGF-. beta.RII has 3N-glycosylation sites, N47, N71, N131 (underlined), and N-terminal O-glycosylation sites S8 and T16 (underlined). Glycosylation results in anti-PDThe charge heterogeneity of the-L1/TGF-beta bifunctional fusion protein is very complex. In order to reduce the complexity of glycosylation modification, the glycosylation site is subjected to N71Q mutation, and the C end is truncated by 8 amino acids to obtain a sequence 869F; carrying out S8P, T16P and N71Q mutation on the glycosylation sites, and truncating 8 amino acids at the C end to obtain a sequence 869M 1; carrying out S8P, T16V and N71Q mutation on the glycosylation site at the N end, and truncating 8 amino acids at the C end to obtain a sequence 869M 3; in addition, the N end is truncated by 20 amino acids, the C end is truncated by 8 amino acids, and different peptide linkers are added to obtain 869J15-17 series proteins. Addition of peptide linker (G)4S)3GDATHTP was obtained 869J15, and a peptide linker (G) was added4S)3GDATHTP was obtained 869J16, and a peptide linker (G) was added4S)3SDKTHTP was obtained 869J17 (the sequence of the fusion protein constructed above is described in table 2). The charge heterogeneity detection results (fig. 1B-fig. 1E) show that the charge heterogeneity of the fusion protein 869F is significantly improved compared to the fusion protein 869, while the improvement of the charge heterogeneity of the 869M1, 869M3, 869J15-17 fusion proteins is more significant.
TABLE 2 fusion protein sequence description
Examples of fusion proteins Description of the fusion protein sequences
869 M8-LSPGK(G4S)3T-ECD(1-136)
869F M8-LSPGK(G4S)3T-ECD(1-128)/N71Q
869J15 M8-LSPGG(G4S)3GDATHTP-ECD(21-128)/N71Q
869J16 M8-LSPG(G4S)3GDATHTP-ECD(21-128)/N71Q
869J17 M8-LSPG(G4S)3SDKTHTP-ECD(21-128)/N71Q
869M1 M8-LSPGK(G4S)3T-ECD(1-128)/S8P,T16P,N71Q
869M3 M8-LSPGK(G4S)3T-ECD(1-128)/S8P,T16V,N71Q
Note: M8-LSPGK represents the heavy chain of M8, M8-LSPGG represents the mutation of amino acid residue K at position 447 of the heavy chain of M8 to G, and M8-LSPG represents the deletion of amino acid residue K at position 447 of the heavy chain of M8.
ECD (1-136) represents the full 136 amino acid TGF β RII extracellular domain.
ECD (21-128) represents the extracellular domain of TGF-beta RII truncated by 20 amino acids at the N-terminus and 8 amino acids at the C-terminus.
ECD (1-128) represents the extracellular domain of TGF-. beta.RII truncated by 8 amino acids at the C-terminus.
Example 2 anti-PD-L1/TGF-beta bifunctional fusion protein expression and purification
The DNA fragments of the heavy chain and the light chain of the anti-PD-L1/TGF-beta bifunctional fusion protein constructed in the above way are respectively subcloned into a pTT5 vector, and recombinant plasmids are extracted to co-transfect CHO cells and/or 293F cells. After 7 days of cell culture, the culture fluid is subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, then loaded on a HiTrap MabSelect SuRe column, protein is eluted by an eluent with 100mM citric acid and pH3.5 in one step, and a target sample is recovered and dialyzed to change the fluid to PBS. The purified protein is detected by HPLC, and the detection results of fig. 2A-2B show that the molecular state of the fusion protein is uniform, and the purity of the monomer reaches more than 97%.
Example 3 enzyme-linked immunosorbent assay (ELISA) for determining the affinity of the anti-PD-L1/TGF-beta bifunctional fusion protein for antigen
3.1 ELISA detection of affinity of anti-PD-L1/TGF-beta bifunctional fusion protein to PD-L1
Recombinant PD-L1-ECD-Fc protein (see WO2018/137576A1 for preparation) was prepared and plated at 100 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The antibody was diluted to 100. mu.g/ml with a diluent, and diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the resultant mixture was sequentially added to the microplate after blocking, 100. mu.l/well, and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat. # ab87422) was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC50。
The experimental results are shown in fig. 3A, the anti-PD-L1/TGF β bifunctional fusion proteins 869M1, 869M3, 869J15 and the positive control M8 monoclonal antibodies can effectively bind to PD-L1-ECD, the EC50(nM) values are 0.2374, 0.3278, 0.399 and 0.3335, respectively, and the affinities are equivalent.
3.2 ELISA detection of affinity of anti-PD-L1/TGF-beta bifunctional fusion protein to TGF-beta 1
To test the binding capacity of the anti-PD-L1/TGF-. beta.bifunctional fusion protein to TGF-. beta.1, TGF-. beta.1 protein (purchased from microbiosystems, Cat. # TG1-H4212) was plated at 20 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. Diluting the anti-PD-L1/TGF-beta bifunctional fusion protein to 100 mu g/ml by using a diluent, diluting by 4 times to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml), sequentially adding the blocked ELISA plates, placing at 100 mu L/hole, and standing at 37 ℃ for 1 hour. PBST wash plate 3 times, addHRP-labeled mouse anti-human Fab antibody was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well2SO4Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC50。
The experimental results are shown in fig. 3B and 3C, the anti-PD-L1/TGF- β bifunctional fusion protein can specifically bind TGF- β 1, and the EC50(nM) values of the anti-PD-L1/TGF- β bifunctional fusion proteins 869J15, 869J16, 869J17, 869M1, 869M3 and the positive control M7824 are 1.466, 1.508, 1.552, 0.8196, 0.8983 and 1.475 respectively, and the affinity is equivalent to that of the positive control M7824.
3.3 ELISA detection of the ability of the anti-PD-L1/TGF-. beta.bifunctional fusion protein to bind both PD-L1 and TGF-. beta.1
Steric hindrance may affect the ability of the anti-PD-L1/TGF- β bifunctional fusion protein to bind both antigens simultaneously. To test the ability of the anti-PD-L1/TGF-. beta.bifunctional fusion protein to bind both PD-L1 and TGF-. beta.1, TGF-. beta.1 was plated at 20 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. Diluting the anti-PD-L1/TGF-beta bifunctional fusion protein to 100 mu g/ml by using a diluent, diluting by 4 times to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml), sequentially adding the blocked ELISA plates, placing at 100 mu L/hole, and standing at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, PD-L1-ECD-Fc-biotin was added at 150 ng/well and left at 37 ℃ for 1 hour. After 3 PBST washes, HRP-labeled Streptavidin was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well2SO4Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC50。
The experimental results are shown in FIG. 3D, the anti-PD-L1/TGF-beta bifunctional fusion proteins 869J15, 869J16 and 869J17 can simultaneously bind to PD-L1 and TGF-beta 1, and the EC50(nM) values are 1.556, 1.303 and 0.9251 respectively.
Example 4 measurement of binding affinity of anti-PD-L1/TGF- β bifunctional fusion proteins to the target cell surface antigen PD-L1 by FACS method
In this experiment, PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat. # J1252) expressing PD-L1 on the cell surface were used as target cells, and the target cells were arranged in a 2X 10 order5Perwell was inoculated in a 96-well plate, washed three times with PBS containing 0.5% BSA, centrifuged at 300g each for 5 minutes, and the supernatant was discarded. Mu.l of 12-gradient antibody serially diluted from 3. mu.g/ml in a 3-fold gradient was added as a primary antibody to a 96-well plate, and the cells were suspended and incubated at 4 ℃ for 1 h. The cells were washed twice with PBS containing 0.5% BSA to remove unbound antibody and incubated with 100. mu.l of 1g/ml goat anti-human IgG-FITC (purchased from sigma, # Cat. # F9512) for 30 minutes at 4 ℃. The cells were centrifuged at 300g for 5min, washed twice with PBS containing 0.5% BSA to remove unbound secondary antibodies, and finally resuspended in 200 μ Ι PBS and the binding affinity of the antibody to CHO cell surface PD-L1 was determined by Beckman Co μ lter CytoFLEX flow cytometer. The data obtained were analyzed by GraphPad Prism6 software fitting.
The experimental results are shown in FIG. 4, the anti-PD-L1/TGF-beta bifunctional fusion proteins 869J15, 869J16 and the positive control M8 can both specifically bind to cell surface-expressed PD-L1, and EC50(nM) is 0.4600, 0.5763 and 0.7123 respectively, and the affinities are equivalent.
Example 5 cell experiment of anti-PD-L1/TGF-beta bifunctional fusion protein blocking the binding of PD-1 to PD-L1
Taking PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat. # J1252) growing in logarithmic phase, trypsinizing into single cells, transferring to a white bottom-transparent 96-well plate, 100. mu.l/well, 40000 cells/well, placing at 37 ℃ and 5% CO2And incubated overnight. The anti-PD-L1/TGF-beta bifunctional fusion protein, the positive control M8 and the negative control NC (isotype negative control antibody IgG1) were diluted in a triple gradient to 2 Xworking solution with a maximum concentration of 600nM and a minimum concentration of 0.09nM for a total of 9 concentration gradients. Simultaneously, the density is 1.4-2 multiplied by 106PD-1 effector cells (from promega, Cat. # J1252) with a cell viability of 95% or more were trypsinized to 1.25X 106Single cell suspension of cells/ml. PD-L1aAPC/CHO-K1 cells plated on the previous day were taken, the supernatant was discarded, 40. mu.l of antibody working solution diluted in a gradient was added, and an equal volume of PD-1 effector cells was added. Standing at 37 deg.C for 5% CO2And incubated for 6 hours. After incubation of the cells at 37 ℃ for 6 hours, 80. mu.l of detection reagent Bio-Glo (purchased from promega, Cat. # G7940) was added to each well. After incubation for 10 min at room temperature, luminescences were read with SpectraMaxi3 x. All data were in duplicate wells and the signal values averaged and fitted to the 4-parameter method and analyzed using GraphPad Prism 6.
The experimental results are shown in fig. 5, in fig. 5A, the anti-PD-L1/TGF- β bifunctional fusion proteins 869J15, 869J16 and the positive control M8 all effectively block the interaction between PD-1 and PD-L1, IC50(nM) is 0.1977, 0.1592 and 0.154, respectively, and the blocking abilities of the three are equivalent. In FIG. 5B, the anti-PD-L1/TGF- β bifunctional fusion proteins 869M1, 869M3 and the positive control M8 were all able to effectively block the interaction between PD-1 and PD-L1, and the IC50(nM) values were 1.559, 1.846 and 1.169, respectively, which were comparable in blocking ability.
Example 6 SMAD3 reporter Gene inhibition assay
SBE Reporter HEK293Cell (available from BPS bioscience, Cat. #60653) expresses Smad3 binding element (SEB) with a luciferase Reporter gene, and the in vitro activity of antibodies can be evaluated by studying the inhibitory effect of antibody proteins on TGF- β 1-induced activation of Smad3 in this Cell. The SBE293 cells grown in the logarithmic phase in adherent culture were washed once with DPBS, digested with pancreatin, and the pancreatin was neutralized. Trypan blue cells were counted and centrifuged at 300g for 5 min. Plates were counted after resuspension in MEM (10% FBS, 1% non-essential amino acids, 1mM sodium pyruvate) medium (all from Gibico, Cat. #10095-2Incubate overnight for about 24 h. TGF-. beta.1 (10ng/ml) was diluted in MEM (0.5% FBS, 1% nonessential amino acids, 1mM sodium pyruvate) medium, and the fusion protein was diluted to 100nM, 3-fold dilution, 10 gradients, and left at room temperature for 1h, after which the original medium of the cell plate was replaced, and incubated at 37 ℃ overnight. Adding 100 μ L/well detection reagent Bio-Glo (30 min in advance and 25 deg.C water bath thawing equilibrium temperature)Degree). After incubation for 10 min at room temperature, the luminescences were read with spectramaxi 3.
The results of the experiment are shown in fig. 6, the fusion protein inhibits the activity of the pSMAD3 reporter induced by TGF-beta in a dose-dependent manner, the IC50(nM) values of the fusion proteins 869F, 869M1 and the positive control M7824 are 0.236, 0.1324 and 0.1404 respectively, and the inhibition activity is equivalent.
Sequence listing
<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH
<120> an anti-PD-L1/TGF-beta fusion protein
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Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
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Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
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Ile Pro Pro His Val Gln Lys Pro Val Asn Asn Asp Met Ile Val Pro
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Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
<210> 9
<211> 128
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Ile Pro Pro His Val Gln Lys Pro Val Asn Asn Asp Met Ile Val Val
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Phe Ser Leu Thr Ser Tyr Gly Val His
1 5 10
<210> 11
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Leu Gly Leu Arg Ala Met Asp Tyr
1 5
<210> 13
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His
1 5 10
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Tyr Ala Ser Gln Ser Phe Ser
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gln Gln Ser Asn Ser Trp Pro Leu Thr
1 5
<210> 16
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 17
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr
20 25 30
Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 18
<211> 599
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile
450 455 460
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp
465 470 475 480
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
485 490 495
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
500 505 510
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp
515 520 525
Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro
530 535 540
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys
545 550 555 560
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
565 570 575
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu
580 585 590
Tyr Asn Thr Ser Asn Pro Asp
595
<210> 19
<211> 591
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile
450 455 460
Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp
465 470 475 480
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
485 490 495
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
500 505 510
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp
515 520 525
Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro
530 535 540
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys
545 550 555 560
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
565 570 575
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585 590
<210> 20
<211> 577
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp
450 455 460
Ala Thr His Thr Pro Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
465 470 475 480
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
485 490 495
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
500 505 510
Val Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His
515 520 525
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
530 535 540
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
545 550 555 560
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
565 570 575
Glu
<210> 21
<211> 576
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Ala
450 455 460
Thr His Thr Pro Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
465 470 475 480
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
485 490 495
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
500 505 510
Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp
515 520 525
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
530 535 540
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
545 550 555 560
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
565 570 575
<210> 22
<211> 576
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Lys
450 455 460
Thr His Thr Pro Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
465 470 475 480
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
485 490 495
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
500 505 510
Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp
515 520 525
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
530 535 540
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
545 550 555 560
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
565 570 575
<210> 23
<211> 591
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile
450 455 460
Pro Pro His Val Gln Lys Pro Val Asn Asn Asp Met Ile Val Pro Asp
465 470 475 480
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
485 490 495
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
500 505 510
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp
515 520 525
Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro
530 535 540
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys
545 550 555 560
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
565 570 575
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585 590
<210> 24
<211> 591
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile
450 455 460
Pro Pro His Val Gln Lys Pro Val Asn Asn Asp Met Ile Val Val Asp
465 470 475 480
Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val
485 490 495
Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser
500 505 510
Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp
515 520 525
Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro
530 535 540
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys
545 550 555 560
Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys
565 570 575
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585 590
<210> 25
<211> 607
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val
465 470 475 480
Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
485 490 495
Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln
500 505 510
Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
515 520 525
Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr
530 535 540
Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
545 550 555 560
Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
565 570 575
Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
580 585 590
Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
595 600 605
<210> 26
<211> 216
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215

Claims (14)

1. An anti-PD-L1/TGF-beta fusion protein having the general formula:
Ab-L-TGFβRII ECD(I)
wherein Ab is an anti-PD-L1 antibody, L is a peptide linker, and TGF beta RII ECD is a TGF beta RIII extracellular domain; the N-terminus of the TGF- β RII extracellular domain is linked to the C-terminus of the anti-PD-L1 antibody heavy chain by a peptide linker; the heavy chain of the anti-PD-L1 antibody comprises the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 11, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12 is shown in the specification; the light chain of the anti-PD-L1 antibody comprises the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 13, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 15.
2. The fusion protein of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is as set forth in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: shown at 17.
3. The fusion protein of claim 2, wherein the heavy chain amino acid sequence of the anti-PD-L1 antibody is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO: 3, the light chain amino acid sequence of the anti-PD-L1 antibody is set forth in SEQ ID NO: 4, respectively.
4. The fusion protein of claim 1, wherein the TGF β RII extracellular domain is selected from the group consisting of one or a combination of:
1) a full-length TGF-beta RII extracellular domain;
2) c-end truncates TGF beta RII extracellular domain of 6-10 amino acids;
3) the N-terminal truncates the extracellular domain of TGF beta RII with 18-22 amino acids;
4) a full-length or truncated TGF β rii extracellular domain comprising at least 1 glycosylation site mutation selected from S8P, T16P, T16V, N71Q.
5. The fusion protein of claim 4, wherein the amino acid sequence of the extracellular domain of TGF β RII is selected from the group consisting of SEQ ID NO: 5-SEQ ID NO: 9.
6. the fusion protein of claim 1, wherein the peptide linker is selected from the group consisting of (G)4S)3T or (G)4S)3XDYTHTP, wherein X is G or S and Y is K or A.
7. The fusion protein of any one of claims 1-6, wherein the fusion protein is selected from the group consisting of:
1) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 18, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
2) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 19, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
3) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 20, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
4) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 21, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
5) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 22, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
6) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 23, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4 is shown in the specification;
7) the heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 24, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 4, respectively.
8. A polynucleotide molecule encoding the fusion protein of any one of claims 1-7.
9. An expression vector comprising the polynucleotide molecule of claim 8.
10. A host cell comprising the expression vector of claim 9.
11. A method for preparing a fusion protein according to any one of claims 1 to 7, comprising the steps of:
a) culturing the host cell of claim 10 under expression conditions to express an anti-PD-L1/TGF- β fusion protein;
b) isolating and purifying the fusion protein of step a).
12. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-7 and one or more pharmaceutically acceptable carriers, diluents, or excipients.
13. Use of the fusion protein of any one of claims 1-7, or the pharmaceutical composition of claim 12, in the manufacture of a medicament for the treatment of cancer.
14. The use of claim 13, wherein the cancer is selected from the group consisting of: colorectal cancer, bile duct cancer, gallbladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
CN202010488804.6A 2020-06-02 2020-06-02 anti-PD-L1/TGF-beta fusion protein Pending CN113754777A (en)

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CN202180039999.9A CN115943166A (en) 2020-06-02 2021-06-02 anti-PD-L1/TGF-beta fusion protein
TW110120000A TW202146458A (en) 2020-06-02 2021-06-02 Anti-PD-L1/TGF-[beta] fusion protein containing an anti-PD-L1 antibody, a peptide linker, and a TGF[beta]RII extracellular structural domain
PCT/CN2021/098012 WO2021244587A1 (en) 2020-06-02 2021-06-02 ANTI-PD-L1/TGF-β FUSION PROTEIN

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