Human TIGIT-specific single domain antibody and its application

Abstract

The present invention provides anti-TIGIT (T cell immunoglobulin and ITIM domain) single domain antibodies and variants thereof. The present invention also includes a method for administering immunotherapy to a subject suffering from a disease such as cancer or an infectious disease. The method comprises administering to the subject a drug containing an effective therapeutic dose of an anti-TIGIT single domain antibody or a fusion molecule thereof, to enhance its endogenous immune response by promoting the activation of an endogenous response or the inhibition of an endogenous response.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 012,221, filed on April 19, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to therapeutic antibodies, and in particular to an anti-TIGIT single domain antibody and applications thereof.

[0004] Sequence Listing

[0005] This application includes a sequence listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy created on April 18, 2021 is named AG3-021US_ST25.txt and is 135KB in size. Background Art

[0006] Cancer is generally defined as a group of diseases involving abnormal cell growth that may invade or spread to other parts of the body. In 2019, approximately 1.8 million people were diagnosed with cancer in the United States. Approximately 606,880 people die from cancer each year in the United States, with lung and bronchial cancers being the leading causes of death. Colorectal cancer and pancreatic cancer are the second and third leading causes of cancer death, respectively.

[0007] Cancer is associated with several factors, including smoking, obesity, poor diet, lack of physical activity, and excessive alcohol consumption. Other factors include certain infections, exposure to ionizing radiation, and environmental pollution. Some cancers are associated with infections, such as Helicobacter pylori, hepatitis B, hepatitis C, human papillomavirus infection, Epstein–Barr virus infection, and human immunodeficiency virus infection (HIV).

[0008] Traditional cancer treatments directly remove tumor tissue and inhibit its spread. Such treatments include surgery, chemotherapy, radiotherapy, hormone therapy, targeted therapy, and palliative care. Treatments are usually selected based on the type, location, grade of cancer, and the patient's health status and preferences. All of the above methods have their limitations, especially when the cancer has metastasized, these therapies may be ineffective. In addition, chemotherapy and radiotherapy have a series of side effects related to cytotoxicity.

[0009] Because cancer cells divide faster than most normal cells, they are more sensitive to chemotherapy drugs. However, chemotherapy drugs also attack other cells in the body, especially rapidly dividing cells, such as blood cells and cells lining the mouth, stomach and intestines. Therefore, there is a need for more targeted drugs and cancer treatments with fewer toxic side effects. Developing a treatment that harnesses the immune system to attack and kill tumor cells is a promising direction.

[0010] Human cancers will have a large number of genetic and epigenetic changes, and the resulting new antigens may be recognized by the immune system (Sjoblom et al., Science 314: 268-74 (2006)). Although endogenous immune responses to tumors can be observed in preclinical models and patients, this immune response is ineffective, and the tumors formed will be recognized as "self" and tolerated by the immune system. Tumor cells may suppress the host's immune response through a variety of mechanisms, ultimately leading to immune tolerance (Topalian et al., J Clin Oncol 29: 4828-36 (2011); Mellman et al., Nature 480: 480-489 (2011)). Among these mechanisms, endogenous "immune checkpoints" usually terminate the immune response to reduce damage to adjacent tissues, and cancer can use this checkpoint to escape the killing of the immune system. Considerable effort has been invested in developing immune checkpoint specific inhibitors, which have shown initial success and can provide new immunotherapies for cancer treatment, such as anti-CTLA-4 antibodies and ipilimumab for the treatment of advanced melanoma (Hodi et al., New Engl J Med 363:711-23 (2010)).

[0011] T cell immunoglobulin and ITIM domain (TIGIT) is a new target for immune checkpoint therapy (Dougall et al., Immunol Rev. 2017; 276: 112–20. doi: 10.1111 / imr.12518. PMID: 28258695). It is an immunosuppressive receptor expressed by lymphocytes, and its role in autoimmunity, viral immunity and tumors has been studied. TIGIT is associated with human cancer and T cell exhaustion. TIGIT acts as a ligand, receptor or competitor of the co-stimulatory receptor CD226, and inhibition of its activity can enhance T cell anti-tumor responses.

[0012] Many important immune cells can express TIGIT, and TIGIT expressed in different cell types has different functions. In the analysis of whole transcriptome sequencing (RNA-Seq; ~200x106reads / tumor) of 1467 unscreened clinical cases, high expression of PD-L1 was closely associated with high expression of TIGIT, FOXP3 and LAG3 (each P < 0.001) (Pal et al., Annals of Oncology (2018) 29 (suppl_8): viii400-viii441.10.1093 / annonc / mdy288).

[0013] Single checkpoint blockade is associated with upregulation of other inhibitory checkpoints and progression of resistance to therapy (Koyama et al., Nat Commun. 2016; 7:10501. doi:10.1038 / ncomms10501. PMID:26883990). Dual checkpoint blockade of TIGIT and PD-1 enhances antitumor immunity and survival in GBM mouse models (Hung et al., Oncoimmunology. 2018; 7(8):e1466769).

[0014] Therefore, there is a need for single-domain antibodies against TIGIT, which can effectively treat cancer and other immune-related diseases by themselves or as part of a bispecific molecule. The present application can meet various needs related to this and also provides methods of using antibodies and antibody conjugates, pharmaceutical compositions and products to treat diseases such as cancer. SUMMARY OF THE INVENTION

[0016] The invention described and claimed herein has numerous features and embodiments, including but not limited to those illustrated or described or mentioned in the Summary of the Invention. The invention described and claimed herein is not limited to the features or embodiments identified in the Summary of the Invention, which is intended to be illustrative and not limiting.

[0017] Specific embodiments include single domain antibodies that bind to human TIGIT. In one aspect, the single domain antibody comprises any one amino acid sequence selected from SEQ ID NO: 1-5 and 6.

[0018] In some embodiments, the CDR domain of the single domain antibody comprises one, two, three, four or five amino acid mutations, deletions or additions.

[0019] In some embodiments, the single domain antibody comprises CDR1, CDR2, and CDR3 identical to any one of the sequences derived from SEQ ID NOs: 1-5 and 6.

[0020] In some embodiments, the single domain antibody is humanized, in some embodiments, the single domain antibody is in a mature state, and has an enhanced affinity for human PD-1 relative to the parent antibody. In some embodiments, the single domain antibody is humanized and mature.

[0021] In some specific embodiments, the single domain antibody is human and comprises an amino acid sequence selected from SEQ ID NO:34, 35, 36, 37 and 38.

[0022] In some specific embodiments, the single domain antibody is human and comprises an amino acid sequence that is at least 90%, at least 95% or at least 98% homologous to any sequence selected from SEQ ID NO:34, 35, 36, 37 and 38.

[0023] In some embodiments, the single domain antibody is fused to the N-terminus or C-terminus of the antibody light chain or heavy chain.

[0024] In some embodiments, the single domain antibody is optionally fused to the N-terminus or C-terminus of the antibody light chain or heavy chain via a peptide connector, wherein the antibody binds to an antigen specifically expressed by a tumor cell or immune cell. In some embodiments, the single domain antibody is fused to another single domain antibody.

[0025] In some specific embodiments, the single domain antibody and the second antibody are fused to form a single domain antibody-antibody fusion molecule, wherein the second antibody binds to an antigen selected from: human PD-1, human PD-L1, CTLA4, HER2, EGFR, VEGFR2, VEGF, Claudin18.2, FAP, CD20, mesothelin, CMET, and 5T4.

[0026] In some specific embodiments, the antibody binds to PD-L1, wherein the anti-PD-L1 antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 19 or 22, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 20, 21 or 23.

[0027] In some specific embodiments, the antibody binds to PD-1, wherein the anti-PD-1 antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 15 or 17, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 16 or 18.

[0028] In some specific embodiments, the antibody binds to CTLA4, wherein the anti-CTLA-4 antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO:32, and a heavy chain with an amino acid sequence as shown in SEQ ID NO:33.

[0029] In some specific embodiments, the single-domain antibody-antibody fusion molecule further comprises a TGFβ receptor II extracellular domain or a functional analog thereof.

[0030] In some specific embodiments, the TGFβ receptor II extracellular domain comprises the amino acid sequence shown in SEQ ID NO:11, 12, 13 or 14.

[0031] In some embodiments, the single domain antibody can be selectively fused to the N-terminus or C-terminus of an Fc polypeptide or an albumin molecule or a fragment thereof via a peptide connector. In some embodiments, the fusion molecule further comprises a TGFβ receptor II extracellular domain or a functional analog thereof. In some embodiments, the TGFβ receptor II extracellular domain comprises an amino acid sequence as shown in SEQ ID NO: 11, 12, 13, or 14.

[0032] In some specific embodiments, the present application provides a completely new fusion molecule, which includes the above-mentioned single-domain antibody, optionally including an antigen binding portion that binds to human PD-1 or PD-L1, and also includes a TGFβ receptor II extracellular domain or a functional analog thereof. In some specific embodiments, the antigen binding portion binds to human PD-1 or PD-L1, and a cytokine, the cytokine is selected from: IL-2, IL-2 functional analogs, IL-15, IL-15 functional analogs, IL-12, IL-12 functional analogs, IL-21, IL-21 functional analogs, interferon α, interferon α functional analogs, interferon γ, interferon γ functional analogs, TNFα and TNFα functional analogs. In some specific embodiments, the cytokine portion is masked, wherein the masking portion is fused to the other parts of the fusion molecule through a cleavable peptide connector.

[0033] The present application also provides a fusion molecule comprising the above-mentioned single-domain antibody and a bifunctional molecule, wherein the bifunctional molecule simultaneously binds to PD-L1 and TGF-β. The bifunctional molecule comprises two identical antibody light chains and two identical heavy chain polypeptide chains. The light chain comprises the amino acid sequence shown in SEQ ID NO: 19; the heavy chain polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 24.

[0034] The present application also provides a fusion molecule comprising the above-mentioned single-domain antibody and a bifunctional molecule, wherein the bifunctional molecule simultaneously binds to PD-1 and TGFβ. The bifunctional molecule comprises two identical antibody light chains and two identical heavy chain polypeptide chains. The light chain comprises the amino acid sequence as described in SEQ ID NO: 15; the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 25, 26, 27, 28, 29, 30 or 31.

[0035] In some specific embodiments, the fusion molecule capable of binding to PD-L1 and TIGIT includes two identical light chains, whose amino acid sequence is SEQ ID NO: 19, and two identical heavy chain polypeptide chains, whose amino acid sequence has at least 95%, at least 99% or 100% homology with SEQID NO: 39.

[0036] In some specific embodiments, the fusion molecule comprises an amino acid sequence selected from SEQ ID NO:40, 41, 42 and 43.

[0037] In some specific embodiments, the fusion molecule comprises an amino acid sequence selected from SEQ ID NO: 42 and 43. The fusion molecule further comprises a heavy chain having an amino acid sequence of SEQ ID NO: 44 and a light chain having an amino acid sequence of SEQ ID NO: 32.

[0038] In some specific embodiments, the fusion molecule comprises an amino acid sequence having at least 95% or at least 99% homology to an amino acid sequence selected from SEQ ID NO: 42 and 43. The fusion molecule further comprises a heavy chain having an amino acid sequence having at least 99% homology to SEQ ID NO: 44 and a light chain having an amino acid sequence of SEQ ID NO: 32.

[0039] In some specific embodiments, the fusion molecule comprises an amino acid sequence selected from SEQ ID NO:46, wherein the fusion molecule further comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:45 and a light chain with an amino acid sequence as shown in SEQ ID NO:32.

[0040] In some specific embodiments, the fusion molecule includes an amino acid sequence that is at least 95% or at least 99% homologous to SEQ ID NO:46, wherein the fusion molecule further includes a heavy chain that has an amino acid sequence that is at least 99% homologous to SEQ ID NO:45 and a light chain that has an amino acid sequence as shown in SEQ ID NO:32.

[0041] Likewise, the present application provides a polynucleotide encoding any of the above single domain antibodies or any of the above fusion molecules. The present invention also discloses an expression vector comprising the above polynucleotide, and a host cell comprising the above expression vector.

[0042] Furthermore, the present application provides a method for preparing the single-domain antibody or the fusion molecule. The method comprises the following steps: 1) culturing host cells under conditions that allow the expression of the single-domain antibody or the fusion molecule; 2) isolating the single-domain antibody or the fusion molecule.

[0043] Furthermore, the present application provides a pharmaceutical composition, which includes the single domain antibody or the fusion molecule and a pharmaceutically acceptable excipient.

[0044] Furthermore, the present application provides a method for treating cancer or infectious diseases or activating the immune system of a patient in need thereof, comprising administering the above-mentioned pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings that accompany this application illustrate various aspects of the invention, in which:

[0046] Figure 1 Blocking report analysis results for selected single domain antibodies, wherein 41.1D11, 41.2D1, 41.3B4, 42.2D6, 41.1B4 mutant and 42.3D6 mutant are fusion proteins of D11, D1, B4, D6, B4 mutant and D6 mutant clones fused to the C-terminus of Fc (SEQ ID NO:7) via a linker as shown in SEQ ID NO:8.

[0047] Figure 2 Kinetic binding data of fusion molecules fused to the N-terminus and C-terminus of the Fc domain of human D1 molecules. The results showed that all human D1 molecules retained strong affinity for human TIGIT, with KD values ​​below 3 nM.

[0048] Figure 3 The structure of a bispecific molecule that can bind to human TIGIT and CTLA-4 is listed. The molecule includes two human D1s and a Fab of an anti-CTLA4 antibody, both of which are fused to the N-terminus of the Fc domain. DETAILED DESCRIPTION OF THE INVENTION

[0050] definition

[0051] "One specific embodiment / aspect" mentioned in the specification means that at least one specific embodiment / aspect of the present invention includes the specific features, structures or characteristics described in the specific embodiment / aspect. The use of the phrase "in one specific embodiment / aspect" or "in another specific embodiment / aspect" in multiple places in the specification does not necessarily refer to the same specific embodiment / aspect, nor is it necessarily independent or mutually exclusive of other specific embodiments / aspects. In addition, various features are only included in some specific embodiments / aspects and are not applicable to other specific embodiments / aspects. Similarly, various requirements are limited to certain specific embodiments / aspects for which they are required and are not applicable to other specific embodiments / aspects. In some specific embodiments, specific embodiments and aspects can be used interchangeably.

[0052] In general, the terms used in the present specification can be interpreted as the meaning commonly understood in the art in the text disclosed in this application and in the specific text in which each term is used. The specific terms used in the present invention are discussed below or elsewhere in the specification to provide further guidance to the relevant personnel on the description of the present invention. It should be understood that the same reference can be expressed in more than one way.

[0053] Therefore, alternatives and synonyms can be used in the present invention to any one or more terms. Whether a term is elaborated or discussed in detail here does not have any special meaning. The present invention provides synonyms for certain terms. One or more synonyms do not exclude replacement with other synonyms. The embodiments involved anywhere in this specification, including examples of any term discussed herein, are merely illustrative and are not intended to limit the scope and meaning of the present disclosure or any examples. Similarly, the present disclosure is not limited to the various specific embodiments given in this specification.

[0054] The following embodiments of the equipment, devices, methods and related results disclosed in the present invention are not intended to further limit the scope of the present application. It should be noted that the titles or subtitles used in the examples for the convenience of the reader should not be construed as limiting the scope of the present invention. Unless otherwise specified, the meanings of all technical terms and scientific terms involved in this article should be consistent with the understanding of those skilled in the art. If there is a conflict of meaning, the application document including the definition in the text shall prevail.

[0055] As the case may be, the term "about" or "generally" used in the present specification and the appended claims refers to a range of + / -20%, unless otherwise specified. Similarly, as the case may be, the term "substantially" used in the present specification and the appended claims refers to a range of + / -10%, unless otherwise specified. It should be understood that not all of the above terms are quantitative expressions applicable to range descriptions.

[0056] The term "active agent" or "active ingredient" refers to a substance, compound or molecule that has biological or other properties, including substances, compounds or molecules that have biological or physiological functions on the subject to which the active agent or active ingredient is administered. In other words, "active agent" or "active ingredient" refers to a component or ingredients in a composition that contributes to all or part of the function of the composition. The active agent can be a primary active agent, or in other words, a component of the composition that contributes to all or part of the function of the composition. The active agent can be a secondary active substance, or in other words, a component of the composition that contributes to the additional function or other functions of the composition.

[0057] The term "subject" or "patient" refers to any animal that can be treated, particularly mammals (including non-human animals, such as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep and non-human primates). Most preferably, the patient referred to herein is a human.

[0058] The term "pharmaceutically acceptable carrier" used in the present invention refers to any and all solvents, dispersion media, coatings, isotonic agents and sustained-release agents that are consistent with drug administration methods. The use of media and agents in the above-mentioned pharmaceutically active substances is well known to those skilled in the art. The above-mentioned composition may also include other active ingredients to supplement, increase or enhance its therapeutic effect.

[0059] The term "pharmaceutically acceptable composition" used in the present invention refers to a composition comprising at least one compound disclosed in the present invention and one or more pharmaceutically acceptable carriers.

[0060] The term "programmed death-1 (PD-1)" is an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on the surface of activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" used in the present invention includes human PD-1 (hPD-1), hPD-1 variants, subtypes, homologs, and analogs that have at least one identical epitope to hPD-1. The complete hPD-1 sequence is available in Genebank Accession No. U64863.

[0061] The term "programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which can reduce T cell activity and downregulate cytokine secretion after binding to PD-1. The term "PD-L1" used in the present invention includes human PD-L1 (hPD-L1), hPD-L1 variants, subtypes, homologs, and analogs with at least one identical epitope to hPD-L1. The complete hPD-L1 sequence is shown in Genebank Accession No. Q9NZQ7.0020.

[0062] The "T cell immunoglobulin and ITIM domain" or "TIGIT" binds with high affinity to the poliovirus receptor (PVR), and this binding increases IL10 secretion, decreases IL12B secretion and inhibits T cell activation by promoting the generation of mature immunoregulatory dendritic cells.

[0063] The present invention uses the terms "polypeptide", "peptide" and "protein" interchangeably to refer to polymers of amino acids. These terms apply to amino acid polymers, which may be artificial chemical mimetics in which one or more amino acids correspond to natural amino acids, natural amino acid polymers, and non-natural amino acid polymers. Methods for obtaining polypeptides (e.g., production, separation, purification, synthesis, and recombinant production) are well known to those of ordinary skill in the art.

[0064] Amino acids refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid actives that have the same functions as naturally occurring amino acids to some extent. Naturally occurring amino acids can be encoded by the genetic code or modified amino acids, such as hydroxyproline, γ-hydroxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds with the same basic chemical structure as naturally occurring amino acids, with carbon bound to hydrogen, carboxyl, amino and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. These analogs have modified R groups such as norleucine or modified peptide polymer backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid actives refer to compounds that have a different general chemical structure from amino acids, but have the same functions as naturally occurring amino acids to some extent.

[0065] Amino acids are referred to herein by either their commonly used three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides are referred to by their commonly recognized single-letter codes.

[0066] The compositions of the present invention include amino acid substitutions of proteins and peptides, which generally do not change the activity of the proteins or peptides (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). In one embodiment, these substitutions are substitutions of conservative amino acids. Generally, most substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly, and the above substitutions are bidirectional.

[0067] For "conservatively modified variants" of amino acid sequences, those skilled in the art should understand that individual substitutions, deletions or insertions of nucleotides, peptides, polypeptides or protein sequences can change, add or delete a single amino acid or a small number of amino acids in the coding sequence, and these changes result in amino acids being replaced by amino acids with similar chemical properties, which are "conservatively modified variants". Conservative substitution tables for functionally similar amino acids are well known to those skilled in the art. In addition, these conservatively modified variants do not exclude polymorphic variants, interspecies homologs and alleles involved in this application.

[0068] Each of the following eight groups contains amino acids that can be conservatively substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamate (E); 3) asparagine (N) glutamate (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Crichton, Proteins (1984)).

[0069] The term "analog" refers to a peptide, polypeptide or protein sequence that is different from the related peptide, polypeptide or protein sequence. These differences can be insertion, deletion or substitution of amino acids, phosphorylation, sulfation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., the use of non-natural amino acid structures or other changes well known to those skilled in the art.

[0070] The term "antibody" refers to a polypeptide comprising an immunoglobulin gene or a fragment framework region thereof, which can specifically bind to or recognize an antigen. Immunoglobulin genes known in the art include κ, λ, α, γ, δ, ε and μ constant region genes, and numerous immunoglobulin variable region genes. Light chains are divided into κ or λ. Heavy chains are divided into γ, μ, α, δ or ε, which are named immunoglobulins IgG, IgM, IgA, IgD and IgE, respectively. Generally, the antigen binding domain of an antibody is the most decisive in terms of binding specificity and affinity.

[0071] A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair comprising a light chain (about 25 kD) and a heavy chain (50-70 kD). At the N-terminus of each chain is a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. Variable light chains (VL) and variable heavy chains (VH) correspond to these light and heavy chains, respectively.

[0072] Antibodies exist, for example, as intact immunoglobulins or as fragments of some characterized fragments produced by the degradation of various proteolytic enzymes. Thus, for example, pepsin degrades the disulfide bonds of antibodies in the hinge region to produce F(ab)'2, i.e., a dimer of Fab, where Fab is a light chain bound to VH--CH1 via a disulfide bond. Cutting the disulfide bonds in the hinge region under mild conditions may reduce F(ab)'2, and the F(ab)'2 dimer is converted into a Fab' monomer. The Fab' monomer is essentially a portion of the Fab and hinge region (see Fundamental Immunology, Paul ed., 3d ed. 1993). Various antibody fragments are defined based on the hydrolysis of intact antibodies, and those skilled in the art will appreciate that the fragments are resynthesized using chemical or recombinant DNA methods. Therefore, the term "antibody" as used in the present invention also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods (e.g., single domain Fv) or confirmed by phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0073] Affinity maturation is T FH The process by which antibodies produced by activated B cells increase their affinity for antigens during the immune response. Under repeated exposure to the same antigen, host cells continuously produce antibodies with higher affinity. The antibodies obtained by the second immune response have an affinity many times stronger than that of the initial response antibodies. The affinity maturation process mainly occurs on the surface of immunoglobulins in the germinal center of B cells and is the result of high-frequency mutations in somatic cells and T cells. FH A direct result of cell selection.

[0074] Diabodies can be prepared by chemical cross-linking or by hybridomas. Alternatively, the diclonal antibody molecules can be prepared by recombinant technology. Dimerization can be improved by shortening the length of the connector connecting the VH and VL regions, which is usually used to prepare scFv fragments, from about 15 amino acids to about 5 amino acids. These connectors help splicing within the VH and VL domain chains. Any suitable short connector can be used. Therefore, the two fragments are spliced ​​into a dimeric molecule. Further shortening the length of the connector to 0-2 amino acids can produce trimeric (tri-antibody) or tetrameric (tetra-antibody) molecules.

[0075] Methods for humanizing antibodies are known in the prior art. In general, a humanized antibody has one or more amino acid residues introduced from its non-human source. These non-human amino acid residues are usually considered to be import residues, which are usually derived from the import variable region. Humanization can be performed essentially according to the method of Winter and colleagues (see, for example, Jones et al., Natural Science, 321: 522-525 (1986); Riechmann et al., Natural Science, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988) and Presta, Curr. Op. Structural Biology, 2: 593-596 (1992)), by replacing rodent CDRs or CDR sequences with corresponding sequences of human antibodies. Therefore, this type of humanized antibody is a chimeric antibody (U.S. Patent No. 4,816,567), and a portion of a substantially complete human variable region will be replaced by a corresponding non-human corresponding sequence. In fact, humanized antibodies are typical human antibodies in which some CDR amino acid residues and possibly some FR amino acid residues are substituted by amino acid residues at analogous sites in rodent antibodies.

[0076] The term "antigen-binding fragment" or "Fab" refers to the region of an antibody that binds to an antigen, which includes a constant region and a variable region (e.g., four domains: VH, CH1, VL, and CL1) of the heavy and light chains. The variable region includes an antigenic determinant (antigen-binding site as described above), which includes a set of complementarity determining regions at the amino terminus of the monomer. Each arm of the Y structure binds to an antigenic epitope.

[0077] The term "Fc region" or "crystallizable fragment" refers to the tail region CH2-CH3 of an antibody, which interacts with cell surface receptors, the so-called Fc receptors and the complement system. Its "effector function" allows the antibody to activate the immune system, thereby causing cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP play a regulatory role through the binding of Fc to Fc receptors on the surface of immune cells. CDC plays a regulatory role through the binding of Fc to proteins of the complement system (such as C1q).

[0078] The phrase "specifically (or selectively) binds" refers to an antibody or "specifically (or selectively) immunoreactive," when referring to a protein or polypeptide, refers to the binding reaction of the specified protein, usually in heterologous copies of the protein and other biological agents. Thus, under specific immunoassay conditions, a specified antibody binds to a specific protein with at least two times the ability to bind to the background (background), and usually with an ability of 10 to 100 times. Specific binding of an antibody under such conditions requires screening for antibodies that are specific for a particular protein. For example, polyclonal antibodies can be screened to obtain those that are specifically immunoreactive to a selected antigen but not to other proteins. Such screening can be accomplished by removing antibodies that cross-react with other molecules. Various immunoassay methods can be used to screen for antibodies that are specifically immunoreactive to a particular protein. For example, solid phase ELISA immunoassays are commonly used to screen for antibodies that are specifically immunoreactive with a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0079] The term "immunotherapy" refers to the treatment of a patient who has, is at risk of developing, or is at risk of developing, developing, or recurring a disease by a method that includes enhancing, suppressing, or otherwise altering the immune response. "Treatment" or "therapy" refers to any type or course of action, or administration of an active agent, of the invention, that aims to alter, alleviate, ameliorate, inhibit, slow down, or prevent, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease.

[0080] The construction of appropriate vectors containing the target sequence and regulatory sequences employs standard restriction enzyme digestion and ligation techniques, which are well known in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cleaved, spliced, and religated in the desired format.

[0081] A nucleic acid is "linkable" when it is functionally associated with another nucleic acid sequence. For example, DNA for a propeptide sequence or secretory peptide can be linked to DNA for a polypeptide if it is expressed as a propeptide that participates in the secretion of the polypeptide; a promoter or enhancer can be linked to a coding sequence if the coding sequence affects the transcription of the sequence; or a ribose binding site can be linked to a coding sequence if the coding sequence promotes translation. In general, "linkable" means that the DNA sequences are adjacent and, in the presence of the secretory peptide, continuous in the reading frame. However, the enhancer needs to be continuous. Ligation is accomplished at appropriate restriction sites. If the above sites are not present, the linkers or connectors of the synthetic oligonucleotides are the same as those of conventional plasmids.

[0082] "Conservatively modified variants" apply to amino acid and nucleotide sequences. For a particular nucleotide sequence, conservatively modified variants refer to nucleotides that encode the same or essentially the same amino acid sequence, or that do not encode an amino acid sequence to achieve essentially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleotides encode a given protein. For example, the codons GCA, GCC, GCG, and GCU all encode alanine. Therefore, at each position where a codon specifies arginine, the codon can be changed to a matching codon without changing the encoded polypeptide. Nucleic acid variants are "silent variants," which are conservatively modified variants of the same species. Each nucleotide sequence encoding a polypeptide herein also refers to each possible silent variant of the nucleotide. Those skilled in the art can recognize that each codon in the nucleotide (except AUG and TGG, AUG is usually only a codon for methionine, and TGG is usually only a codon for tryptophan) can be modified to a functionally identical molecule. Therefore, each silent variant of the nucleotide encoding a polypeptide is implicitly recorded in the sequence of each described expression product, but is not implicit for the actual probe sequence.

[0083] The term "identical" or "percentage" "identity" in the context of describing two or more nucleotide or polypeptide sequences means that the two or more corresponding sequences are identical or have a certain percentage of identity in terms of amino acid residues or nucleotides (e.g., about 60% identity, more preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when the corresponding comparison window or specified region is compared and collated for maximum identity) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters as follows, or by manual collation and eyeballing. These sequences are said to be "substantially identical sequences". This definition also refers to, or applies to, a test sequence. The definition also includes sequences in which deletions and / or insertions occur, as well as sequences in which substitutions occur. As described below, the preferred algorithm can account for proteins, etc. Preferably, the region of identity is at least 25 amino acids or nucleotides in length, or more preferably 50-100 amino acids or nucleotides in length.

[0084] In sequence alignment, one sequence is usually used as a reference sequence to compare the test sequence. When using sequence alignment software, the test and reference sequences are input into a computer, the sequence coordinates are specified, and if necessary, the sequence algorithm program parameters are set. Preferably, the default program parameters can be used, or selected parameters can be specified. The sequence alignment algorithm calculates the percentage of identity between the test sequence and the reference sequence based on the program parameters.

[0085] The "comparison window" used in the present invention includes comparing any portion of the number of adjacent sites in a group selected from 20 to the full length of the reference sequence, usually about 25 to 100, or 50 to about 150, wherein the sequence may be compared with the reference sequence of the same number of adjacent sites after the two sequences are in the optimal alignment position. Methods for comparing sequence alignments are common knowledge in the art. Optimal sequence alignment can be determined by reference to, for example, the local homology algorithm, Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm, Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), similarity method studies, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of the algorithm (Wisconsin Genetics Package GAP, ESTFIT, FASTA, and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual and visual inspection (see, for example, Manual of Molecular Biology (Ausubel et al., eds. 1995 Supplement)).

[0086] Examples of software suitable for determining percent sequence homology and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 use the parameters described herein to determine the percent sequence identity of the nucleotide or protein of the present invention. Software for BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short letters W in the sequence to be tested, which match or meet a positive threshold T when aligned with letters of the same length in the database sequence. T refers to the score threshold as adjacent letters (Altschul et al., supra). These initial adjacent letters serve as seeds to initiate searches for longer HSPs containing them. These letter extensions are extended in both directions of each sequence so that the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; usually > 0) and N (penalty score for mismatching residues; usually < 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of letters in each direction ceases when the cumulative alignment score differs from the maximum achieved value X by an order of magnitude; when the cumulative score reaches zero or below due to the accumulation of one or more negative scoring residue sequences; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the speed and sensitivity of the sequence alignment. The BLASTN program (for nucleotide sequences) uses the default word length (W) of 11, expectation (E) of 10, M=5, N=-4, and compares both chains. For amino acid sequences, the BLASTP program uses the default word length of 3, expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) with sequence (B) of 50, expectation value (E) of 10, M=5, N=-4, and compares both chains.

[0087] "Nucleic acid" refers to a polymer of deoxynucleotides or ribonucleotides, in single- or double-stranded form, and their complements. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkers, which are synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties as a control nucleic acid and are metabolized in a manner similar to the control nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl nucleotides, and peptide-nucleic acids (PNAs).

[0088] Unless otherwise specified, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., substitutions of degenerate codons) and complementary sequences, as well as the sequence explicitly indicated. Specifically, substitutions of degenerate codons can be obtained by synthesizing sequences in which the third position of one or more (or all) codons is replaced with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleotide is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0089] A particular nucleic acid sequence also implies a "splice variant". Similarly, a particular protein encoded by a nucleic acid implies any protein encoded by a splice variant of the nucleic acid. A "splice variant," as the name implies, is a product of alternative splicing of a gene. After transcription, the original nucleic acid transcript may be spliced, with different (alternative) nucleic acid splicing products encoding different polypeptides. The mechanism of splice variant generation varies, including replacement of exon splicing. This definition also includes alternative polypeptides derived from the same nucleotide sequence by reading the transcript. This definition includes any splicing reaction product, including recombinant forms of the splicing product. For example, the discussion of splice variants of potassium ion channels is by Leicher et al., J. Biol. Chem. 273 (52): 35095-35101 (1998).

[0090] In other aspects, the present invention also provides the use of protein therapeutic agents or pharmaceutical compositions for preventing or treating cancer, neurodegenerative diseases or infectious diseases, including the same use in the preparation of drugs.

[0091] The terms "treating" or "treatment" refer to one or more of the following: (1) inhibiting a disease, condition or disorder in an individual who is suffering from or exhibiting the pathology or symptoms of a disease, condition or disorder (e.g., inhibiting further development of the relevant pathology and / or symptoms); and (2) alleviating symptoms; such as alleviating the relevant disease, condition or disorder state in a patient who is suffering from or exhibiting the pathology or symptoms of a disease, condition or disorder (e.g., eliminating the relevant pathology and / or symptoms, such as reducing the severity of the disease).

[0092] Other technical terms used in the present invention have the meanings commonly used in the technical field, such as the definitions in different technical dictionaries. The specific values ​​or configurations in the non-limiting examples of the present invention are variable and are only used to illustrate at least one embodiment and are not intended to limit the scope of protection. DETAILED DESCRIPTION

[0093] It should be understood that both the general description above and the detailed description below are exemplary and explanatory, and are used to further explain the subject-related technology of the claims. Other features and beneficial effects related to the subject technology are described in the following part of the specification. To some extent, these features or beneficial effects may be obvious from the specification, or may be obtained according to the customary understanding of the subject technology. The beneficial effects of the subject technology can be achieved or obtained by the structures clearly pointed out in the specification and claims of the present invention. The present invention also provides pharmaceutical compositions and methods for immunotherapy of patients suffering from some diseases (such as cancer or infectious diseases).

[0094] Single-domain antibodies are antibody fragments with a single variable antibody domain. Like whole antibodies, single-domain antibodies selectively bind to specific antigens. However, single-domain antibodies are smaller than ordinary antibodies and have a small molecular weight (e.g., 12-15 kDa). Ordinary antibodies include two heavy chains and two light chains. In contrast, single-domain antibodies are smaller than Fab fragments and single-chain variable region fragments.

[0095] The present invention provides anti-TIGIT (T cell immunoglobulin and ITIM domain) single domain antibodies and variants thereof. In particular, the anti-TIGIT antibodies and variants thereof provided have the following properties: for example, high affinity for human TIGIT; cross-reaction in human TIGIT; and biochemical and biophysical properties that make the antibodies and variants thereof highly stable. Specific embodiments include anti-TIGIT single domain antibodies as part of a bispecific molecule. The antibodies and bispecific molecules can be used to treat cancer and other immune-related diseases.

[0096] Embodiments include anti-TIGIT single domain antibodies having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100% homologous to any sequence selected from SEQ ID NOs: 1-5 and 6. The anti-TIGIT single domain antibody of the present invention includes a CDR domain derived from a single domain antibody selected from SEQ ID NOs: 1-5 and 6.

[0097] In one embodiment, the anti-TIGIT single domain antibody 379D1 is human, comprising an amino acid sequence selected from SEQ ID NO: 34, 35, 36, 37 and 38. In some embodiments, the human single domain antibody 379D1 comprises an amino acid sequence having at least 95% homology and at least 99% homology with any one selected from SEQ ID NO: 34, 35, 36 and 37.

[0098] The anti-TIGIT single domain antibody and the Fc fragment or albumin molecule form a fusion molecule. The anti-TIGIT single domain antibody can be fused to the N-terminus and / or C-terminus of the antibody heavy chain and / or light chain. In a specific embodiment, the fusion molecule includes an amino acid sequence selected from SEQ ID NO: 40, 41, 42 and 43.

[0099] In some embodiments, the anti-TIGIT single domain antibody is fused to the N-terminus of the antibody heavy chain and / or light chain, wherein the antibody binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, VEGF, VEGFR2, EGFR, HER2, CMET, 5T4, Trop-2 and Claudin 18.2.

[0100] In some embodiments, the anti-TIGIT single domain antibody is fused to the C-terminus of the antibody heavy chain and / or light chain, wherein the antibody binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, VEGF, VEGFR2, EGFR, HER2, CMET, 5T4, Trop-2 and Claudin 18.2. In a specific embodiment, the fusion molecule includes two identical light chains having an amino acid sequence as shown in SEQ ID NO: 19, and two identical heavy chain polypeptide chains having an amino acid sequence with at least 95% homology, at least 99% homology or 100% homology to SEQ ID NO: 39.

[0101] In some embodiments, the anti-TIGIT single domain antibody is fused to the N-terminus of the Fc fragment. The fusion molecule further includes a light chain and a heavy chain of an antibody, wherein the antibody binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, VEGF, VEGFR2, EGFR, HER2, CMET, 5T4, Trop-2, and Claudin 18.2. In some embodiments, the Fc fragment of the fusion polypeptide chain or the Fc domain of the heavy chain includes a "knobs-into-holes" mutation. In some specific embodiments, the fusion molecule includes an amino acid sequence selected from SEQ ID NO: 46. The fusion molecule further includes a heavy chain having an amino acid sequence as shown in SEQ ID NO: 45, and a light chain having an amino acid sequence as shown in SEQ ID NO: 32. In some embodiments, the fusion molecule includes an amino acid sequence having at least 90% homology, at least 95% homology, or at least 99% homology with SEQ ID NO: 46. The fusion molecule further comprises a light chain having an amino acid sequence as shown in SEQ ID NO:32, and a heavy chain having an amino acid sequence at least 90% homologous, at least 95% homologous or at least 99% homologous to SEQ ID NO:45.

[0102] Figure 3 According to one embodiment, a bispecific molecule is described. An anti-TIGIT single domain antibody and an N-terminal fusion of an Fc fragment. The Fc fragment of the fusion polypeptide chain and the Fc domain of the heavy chain include a spherical hole mutation. Wherein, the fusion molecule includes a light chain and a heavy chain of an antibody that can bind to CTLA-4.

[0103] Application and administration

[0104] In human and animal therapeutic applications, the compounds of the present invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration and the type of treatment (such as prevention, prevention or treatment), the compounds are used in accordance with the above parameters. A summary of the relevant technology can be found in Remington: The Science and Practice of Pharmacy, 21 "Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York. The above documents are all cited by reference.

[0105] This specification also provides a pharmaceutical composition for administration to a subject. The pharmaceutical composition disclosed herein may further include a pharmaceutically acceptable carrier, excipient or diluent. The term "pharmaceutically acceptable" as used herein means that the composition is sufficient to achieve a therapeutic effect without harmful side effects, and can be easily determined based on the type of disease, patient age, weight, health status, sex, and drug sensitivity, route of administration, mode of administration, frequency of administration, duration of treatment, drugs used in combination therewith or drugs consistent with the compounds disclosed in the present disclosure, and other factors known in medicine.

[0106] On the other hand, the examples of carriers, excipients and diluents for pharmaceutical preparations include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talcum powder, magnesium stearate and mineral oil. In addition, pharmaceutical preparations can further include fillers, anticoagulants, lubricants, wetting agents, spices and preservatives.

[0107] The pharmaceutical composition disclosed in the present invention may further be in any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations and suppositories.

[0108] The ingredients can be formulated into a single dosage form suitable for the patient's body, preferably, according to the classical methods of the pharmaceutical field, formulated into a preparation that can be used for peptide drugs, so as to be administered by oral or parenteral routes, such as through the skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intracerebroventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal or rectal administration, but not limited thereto.

[0109] The total effective dose of the drug disclosed in the present application can be administered to the patient in a single dose, or can be administered in multiple doses for a period of time according to a fractionated treatment regimen. In the drug disclosed in the present application, the content of the active ingredient can vary according to the severity of the disease. Preferably, the total daily dose of the peptide disclosed in the present application can be about 0.0001 μg to 500 mg per 1 kg of patient body weight. However, in addition to the route of administration and treatment frequency of the drug, the determination of the effective dose of the peptide needs to consider various factors including patient age, weight, health status, gender, severity of disease, diet and secretion rate. In view of this, a technician in the field can easily determine the effective dose suitable for the specific use of the drug disclosed herein. The drug disclosed herein does not particularly limit the preparation, route of administration and mode, as long as it shows a beneficial effect.

[0110] Furthermore, the drug may be administered alone, or in combination or simultaneously with other pharmaceutical agents having preventive or therapeutic effects.

[0111] In another aspect, the present specification provides a method for preventing or treating cancer, infectious diseases or neurodegenerative diseases, comprising the step of administering the chimeric protein or a pharmaceutical composition comprising the same to a subject.

[0112] The active ingredient may be included in a pharmaceutical composition for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to adjust the toxicity of a solution having a concentration of 0-300 mM (the optimal concentration for liquid medicaments is 150 mM). The freeze-dried preparation also includes a freeze-dried protective agent, the main component of which is 0-10% sucrose (optimum 0.5-1.0%), and other optional freeze-dried protective agents include trehalose and lactose. The freeze-dried preparation also includes a bulking agent, the main component of which is 1-10% mannitol (optimum 2-4%). ​​The liquid preparation and the freeze-dried preparation also include a stabilizer, the main component of which is 1-50 mM L-methionine (optimum concentration 5-10 mM). Other bulking agents include glycine, arginine, and may also include >0-0.05% polysorbate 80 (optimally 0.005-0.01%), and additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.

[0113] The solution containing the active ingredient can be administered to the patient in a single dose or in a course of treatment, and can be administered to the patient at any time after diagnosis. The solution can be administered alone or in combination with other drugs or treatment methods that have a therapeutic effect on the condition.

[0114] Certain aspects of the present specification disclose, to some extent, treating a human or non-human mammal individual suffering from a disease (e.g., cancer). As used herein, the term "treat" refers to reducing or eliminating clinical symptoms of cancer in a human or non-human mammal; or delaying or preventing clinical symptoms of cancer in a human or non-human mammal. For example, the term "treat" can refer to alleviating symptoms characteristic of cancer, including but not limited to reducing the severity of the disease, for example, reducing by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, or at least 100%. The actual symptoms associated with cancer are well known and can be determined by a person of ordinary skill in the art by considering factors including but not limited to the site of the disease, the cause of the disease, the severity of the disease, and / or the tissue or organ affected by the disease. Those skilled in the art understand the appropriate symptoms or indicators associated with a particular type of disease and are able to know how to determine whether an individual is suitable for the treatment disclosed in the present invention.

[0115] In other aspects of this embodiment, the effective therapeutic dose of the pharmaceutical composition disclosed herein generally ranges from about 0.001 mg / kg to about 100 mg / kg, and the frequency of administration can be, for example, every 3, 5, 7, 10 or 14 days. In one aspect of this embodiment, the application discloses an effective dose of a pharmaceutical composition, for example, can be at least 0.001 mg / kg, at least 0.01 mg / kg, at least 0.1 mg / kg, at least 1.0 mg / kg, at least 5.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, or at least 50 mg / kg, and the frequency of administration can be, for example, every 3, 5, 7, 10 or 14 days. In other aspects of this embodiment, the application discloses an effective dose of the pharmaceutical composition, which can be, for example, about 0.001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 15 mg / kg, about 0.001 mg / kg to about 20 mg / kg, about 0.001 mg / kg to about 25 mg / kg, about 0.001 mg / kg to about 30 mg / kg, about 0.001 mg / kg to about 35 mg / kg, about 0.001 mg / kg to about 40 mg / kg, about 0.001 mg / kg to about 45 mg / kg, about 0.001 mg / kg to about 50 mg / kg, about 0.001 mg / kg to about 75 mg / kg, or about 0.001 mg / kg to about 100 mg / kg, and the frequency of administration can be, for example, every 3, 5, 7, 10 or 14 days. In other aspects of this embodiment, the application discloses an effective dosage range of the pharmaceutical composition, which can be, for example, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 35 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 45 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.01 mg / kg to about 75 mg / kg, or about 0.01 mg / kg to about 100 mg / kg, and the frequency of administration can be, for example, every 3, 5, 7, 10 or 14 days.In other aspects of this embodiment, the application discloses an effective dosage range of the pharmaceutical composition, which can be, for example, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 15 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 35 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 45 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 75 mg / kg, or about 0.1 mg / kg to about 100 mg / kg, and the frequency of administration can be, for example, every 3, 5, 7, 10 or 14 days.

[0116] The dosage can be a single dose or a cumulative dose (continuous administration) and can be easily determined by those skilled in the art. For example, the treatment of cancer may include the administration of a single effective dose of the therapeutic compound or pharmaceutical composition disclosed in the present application. Alternatively, the treatment of cancer may include multiple administrations according to the effective dose of the pharmaceutical composition within a certain period of time, for example, once a day, twice a day, three times a day, once every few days, or once a week. The administration time varies from person to person, depending on factors such as the severity of individual symptoms. For example, the effective dose of the pharmaceutical composition disclosed in the present application can be given to an individual once a day at irregular intervals until the individual no longer needs treatment. Those of ordinary skill in the art can confirm that the individual's condition will be monitored throughout the treatment process, and the effective amount of the antibody administration disclosed in the present application will be adjusted accordingly.

[0117] Example

[0118] The non-limiting examples provided below are only for illustrative purposes, so that the representative embodiments completed are more fully understood. These embodiments are only used to illustrate the application and are not used to limit the scope of the application. Therefore, these embodiments should not be construed as limiting any embodiment described in the application, especially the type and quantity of formulation compounds and / or preparation methods and applications thereof.

[0119] Example 1

[0120] Immune response

[0121] TIGIT-specific single domain antibodies (MAbs) were prepared by immunizing alpacas. Two alpacas were immunized with human TIGIT-his (Acrobiosystems cat.#TIT-H52H3) antigens, and serum samples were collected after the immunization step. The isolated PBMCs can be used to construct a phage library. The phage library was panned and initially screened by ELISA, and the positive clones were sequenced. The protein sequences of the selected clones are shown in Table 1.

[0122] The corresponding genes of these proteins were fused to the C-terminus of the IgG1 Fc domain and expressed by HEK293 and further confirmed by TIGIT / CD155 blocking reporter assay.

[0123] Example 2

[0124] Transient transfection

[0125] HEK293 cells were plated at 1.5 × 10 5 Cells / well were cultured overnight in 24-well tissue culture dishes in 1 mL complete medium. Transfection was performed with 500 ng DNA using Lipofectamine 3000 (Life Technologies, cat. # L3000015) according to the manufacturer's instructions. Supernatants were collected 3-5 days later and their binding activity was tested by ELISA.

[0126] Large-scale transfections were performed to produce purified material using Lipofectamine 3000 with 5% ultra-low IgG fetal bovine serum (Life technologies, cat. #16250-078) according to the manufacturer's instructions.

[0127] Example 3

[0128] TIGIT / CD155 blocking reporter gene assay

[0129] The TIGIT / CD155 blocking bioassay (a–e) (Cat.# J2201, J2205) is a cell-based bioluminescent assay that overcomes the limitations of other assays and can be used to measure the ability and stability of antibodies or other biologics to target TIGIT (6,7). The assay involves two genetically engineered cell lines:

[0130] a. TIGIT effector cell line: Jurkat T cells are engineered to express human TIGIT together with a luciferase reporter gene, the expression of which is driven by its own promoter, which responds to TCR activation and co-stimulation of CD226; and

[0131] b. CD155aAPC / CHO-K1 cell line: CHO-K1 cells are engineered to express human CD155, and this genetically engineered cell surface protein is designed to activate the T cell receptor (TCR) complex in an antigen-independent manner.

[0132] The TIGIT effector cell line and CD155aAPC / CHO-K1 cell line are provided in a thaw-ready format, and the cryopreserved cells can be directly thawed, plated and used in the assay without the need for cell culture and expansion. When the two cells are co-cultured, TIGIT inhibits CD226 activity and promoter-mediated fluorescence. The added anti-TIGIT antibody blocks the interaction between TIGIT and CD155 or inhibits the effect of TIGIT on blocking CD266 homodimers, thereby generating promoter-mediated fluorescence. The TIGIT / CD155 blocking assay involves fusing, plating and assaying the cells using essential medium and serum. Using Bio-Glo TM Luciferase assay system and standardized luminometer such as Discover System quantitatively detects bioluminescent signals.

[0133] The TIGIT / CD155 blocking reporter gene assay was used as a cell-based functional assay to study the single domain antibody. The single domain antibody was cloned and fused to the C-terminus of IgG1 Fc. The fusion molecule was transiently expressed and purified. Figure 1 The results of TIGIT reporter gene assay. Different curves are used to compare the following single domain antibodies by reflecting the relationship between fluorescence value (relative light value) and concentration:

[0134] a)41.1 D11

[0135] b)41.2 D1

[0136] c)41.3 B4

[0137] d)42.2 D6

[0138] e) 41.1 B4 mutant

[0139] f) 42.3 D6 mutant

[0140] Each fusion molecule is D11, D1, B4, D6, B4 mutant and D6 mutant clones fused to the C-terminus of Fc (SEQ ID NO: 7) via a linker as shown in SEQ ID NO: 8. Anti-human TIGIT antibody purchased from R&D Systems (Catalog # MAB7898) was used as a positive control for cell-based functional assays.

[0141] Example 4

[0142] Humanization

[0143] The D1 VHH homology model was established using pdb 3H6 as a structural model, and its humanized design was reconfirmed using another structural model pdb 4p59. The humanized D1 sequence (SEQ ID NO: 34-47) was fused to the N-terminus and C-terminus of IgG1 Fc, and the molecule was cloned, expressed, and purified. The affinity of the humanized D1-Fc fusion molecule to human TIGIT was tested using the ForteBio instrument, and the results are as follows: Figure 2 As shown. The data show that human D1 molecules have a strong affinity for human TIGIT, with a KD value of less than 3nM. Specifically, all human D1 molecules have a strong affinity for human TIGIT, with a KD value of less than 3nM.

[0144] Finally, it should be understood that although certain aspects of this specification are highlighted by reference to specific embodiments, it will be readily understood by those skilled in the art that these disclosed embodiments are only used to illustrate the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methods, procedures and / or reagents described herein, unless otherwise expressly stated. In addition, it should be recognized that certain modifications, changes or substitutions of the common techniques in the art can be obtained according to the teachings herein without departing from the subject matter of this specification. The terms used in the present invention are only for the purpose of describing specific embodiments and are not used to limit the scope of protection of the present invention, which is only defined by the claims, and therefore, the present invention is not limited to the precise display or description above.

[0145] Table 1: Protein sequences of single domain antibodies

[0146]

[0147] Table 2: Additional sequences

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Sequence Listing <110> Ogilvy Pharmaceuticals Ltd. <120> Human TIGIT-specific single domain antibody and its application <130> AG3-021WO <140> 63012221 <141> 2020-04-19 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Clone 379-D1; Single domain antibody protein sequence <400> 1 Gln Val Gln Val Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Thr Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Pro Ile 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Clone 379-D11; Single domain antibody protein sequence <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Thr Glu Arg Thr Phe Asn Thr Tyr 20 25 30 Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Pro Ile 35 40 45 Ala Ala Ile Thr Arg Gly Ser Arg Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Tyr Arg Asp Asp Ala Lys Ser Ala Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr His Pro Val Arg Pro Ile Val Asp Ser 100 105 110 Trp Gly Gln Gly Thr His Val Thr Val Thr Ser 115 120 <210> 3 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Clone 379 - B4; Single - domain antibody protein sequence <400> 3 Gln Val Gln Val Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Gly Arg Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Arg Ala Thr Asn Thr Gly Arg Thr Thr Thr Thr Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Ser Thr 65 70 75 80 Val Asp Leu Gln Met Asn Ser Leu Ile Leu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Ala Asp Ile Ile Ser Asp Gly Lys Thr Val Asn Arg Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Clone 379 - D6; Single - domain Antibody Protein Sequence <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Val Met 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Val Ser Arg Gly Gly Gly Gly Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Arg Cys 85 90 95 Ala Ala Asp Ser Lys Arg Gly Ala His Met Met Ser Ala His Ala Ser 100 105 110 Glu Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 123 <212> PRT <213> Artificial sequence <220> <223> 379-B4 mutant clone; single domain antibody protein sequence <400> 5 Glu Val Gln Val Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Gly Arg Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Arg Ala Thr Asn Thr Gly Arg Thr Thr Thr Thr Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Ser Thr 65 70 75 80 Val Asp Leu Gln Met Asn Ser Leu Ile Leu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Ala Asp Ile Ile Ser Asp Gly Lys Thr Val Asn Arg Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 125 <212> PRT <213> Artificial sequence <220> <223> 379-D6 mutant clone; single domain antibody protein sequence <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Val Met 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Val Ser Arg Gly Gly Gly Gly Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Arg Cys 85 90 95 Ala Ala Asp Ser Lys Arg Gly Ala His Met Met Ser Ala His Ala Ser 100 105 110 Glu Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG1 Fc - Single Domain Antibody Protein Sequence <400> 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 8 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 9 <211> 209 <212> PRT <213> Artificial sequence <220> <223> VEGFR2 extracellular domain fragment containing the second and third IgC2-like (immunoglobulin-like) domains of VEGF receptor II <400> 9 Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly 1 5 10 15 Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys 20 25 30 Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro 35 40 45 Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys 50 55 60 Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val 65 70 75 80 Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr 85 90 95 Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro 100 105 110 Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys 115 120 125 Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr 130 135 140 Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys 145 150 155 160 Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile 165 170 175 Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser 180 185 190 Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu 195 200 205 Lys <210> 10 <211> 205 <212> PRT <213> Artificial sequence <220> <223> It includes the second IgC2-like (immunoglobulin-like) domain of VEGF receptor I and the second IgC2-like (immunoglobulin-like) domain of VEGF receptor II. VEGF-binding portion of the third IgC2-like (immunoglobulin-like) domain <400> 10 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys 195 200 205 <210> 11 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Extracellular domain of TGF-β receptor II (TGFβRII) <400> 11 Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 12 <211> 117 <212> PRT <213> Artificial sequence <220> <223> TGF-β1 binding portion comprising a fragment of the extracellular domain of TGFβ receptor II <400> 12 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 1 5 10 15 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 20 25 30 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 35 40 45 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 50 55 60 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 65 70 75 80 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 85 90 95 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 100 105 110 Thr Ser Asn Pro Asp 115 <210> 13 <211> 112 <212> PRT <213> Artificial sequence <220> <223> TGF-β1 binding portion comprising a fragment of the extracellular domain of TGFβ receptor II <400> 13 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 1 5 10 15 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 20 25 30 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 35 40 45 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 50 55 60 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 65 70 75 80 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 85 90 95 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 100 105 110 <210> 14 <211> 279 <212> PRT <213> Artificial sequence <220> <223> TGF-β1 binding portion comprising two TGFβ receptor II extracellular domain fragments <220> <221> Repeating Unit <222> (138)..(142) <223> n = 1, 2, 3, 4 or 5 <400> 14 Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Ile 130 135 140 Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp 145 150 155 160 Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val 165 170 175 Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser 180 185 190 Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 195 200 205 Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 210 215 220 Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys 225 230 235 240 Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys 245 250 255 Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu 260 265 270 Tyr Asn Thr Ser Asn Pro Asp 275 <210> 15 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Light chain of the PD-1 antibody Nivolumab <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 440 <212> PRT <213> Artificial sequence <220> <223> PD-1 antibody Nivolumab heavy chain amino acid sequence <400> 16 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 17 <211> 218 <212> PRT <213> artificial sequence <220> <223> PD-1 antibody Pembrolizumab <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 18 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Sequence of Antibody Pembrolizumab <400> 18 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 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 Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe 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 Gly Leu Pro Ser Ser 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 Gln 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 Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu 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 Leu Gly Lys 435 440 445 <210> 19 <211> 214 <212> PRT <213> artificial sequence <220> <223> PD-L1 antibody ASKB1296 <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 20 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> PD-L1 Antibody ASKB1296 Heavy Chain <400> 20 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Tyr Phe Tyr Gly Ser Ser Phe Tyr Ala Val Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Lys 450 <210> 21 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of PD-L1 antibody ASKB1296 <400> 21 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Glu Asp 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Tyr Phe Tyr Gly Ser Ser Phe Tyr Ala Val Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Lys 450 <210> 22 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Atezolizumab light chain sequence <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 23 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Atezolizumab Heavy Chain Sequence <400> 23 Glu Val Gln Leu Val 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 Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 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 Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> twenty four <211> 596 <212> PRT <213> Artificial sequence <220> <223> PD-L1 antibody heavy chain polypeptide chain <220> <221> MISC_FEATURE <222> (31)..(31) <223> X is E or D <220> <221> Repeating Unit <222> (455)..(459) <223> n is 1, 2, 3, 4 or 5 <400> twenty four Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Xaa Asp 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Tyr Phe Tyr Gly Ser Ser Phe Tyr Ala Val Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Ile Pro Pro His 450 455 460 Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly 465 470 475 480 Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser 485 490 495 Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser 500 505 510 Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn 515 520 525 Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro 530 535 540 Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met 545 550 555 560 Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser 565 570 575 Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr 580 585 590 Ser Asn Pro Asp 595 <210> 25 <211> 582 <212> PRT <213> Artificial sequence <220> <223> PD-1 antibody heavy chain polypeptide chain <220> <221> Repeating Unit <222> (441)..(445) <223> n is 1, 2, 3, 4 or 5 <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Ile Pro 435 440 445 Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn 450 455 460 Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 465 470 475 480 Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 485 490 495 Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 500 505 510 Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 515 520 525 Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys 530 535 540 Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser 545 550 555 560 Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 565 570 575 Asn Thr Ser Asn Pro Asp 580 <210> 26 <211> 719 <212> PRT <213> Artificial Sequence <220> <223> hPD1(IgG1)-(G4S)4G-2xshorterTGFbRIIECD <400> 26 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 465 470 475 480 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 485 490 495 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 500 505 510 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 515 520 525 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 530 535 540 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 545 550 555 560 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 565 570 575 Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Val Lys Phe Pro 595 600 605 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 610 615 620 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 625 630 635 640 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 645 650 655 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 660 665 670 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 675 680 685 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 690 695 700 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 705 710 715 <210> 27 <211> 600 <212> PRT <213> Artificial Sequence <220> <223> hPD1(IgG1)-(G4S)4G-1xTGFbRIIECD <400> 27 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 465 470 475 480 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 485 490 495 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 500 505 510 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 515 520 525 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 530 535 540 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 545 550 555 560 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 565 570 575 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 580 585 590 Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 <210> 28 <211> 581 <212> PRT <213> Artificial Sequence <220> <223> hPD1(IgG1)-(G4S)4G-1xshorterTGFbRIIECD <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 465 470 475 480 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 485 490 495 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 500 505 510 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 515 520 525 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 530 535 540 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 545 550 555 560 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 565 570 575 Thr Ser Asn Pro Asp 580 <210> 29 <211> 582 <212> PRT <213> Artificial Sequence <220> <223> PD-1 heavy chain polypeptide chain <220> <221> REPEAT <222> (445)..(449) <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Ile Pro 435 440 445 Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn 450 455 460 Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg 465 470 475 480 Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile 485 490 495 Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg 500 505 510 Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys 515 520 525 Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys 530 535 540 Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser 545 550 555 560 Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr 565 570 575 Asn Thr Ser Asn Pro Asp 580 <210> 30 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> PD-1 heavy chain polypeptide chain <400> 30 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 450 455 460 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 465 470 475 480 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 485 490 495 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 500 505 510 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 515 520 525 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 530 535 540 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 545 550 555 560 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 565 570 575 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 580 585 590 <210> 31 <211> 744 <212> PRT <213> Artificial sequence <220> <223> hPD1(IgG4)-2xTGFBRIIECD <400> 31 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser 450 455 460 Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe 465 470 475 480 Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn 485 490 495 Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys 500 505 510 Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile 515 520 525 Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe 530 535 540 Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys 545 550 555 560 Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys 565 570 575 Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 595 600 605 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 610 615 620 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 625 630 635 640 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 645 650 655 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 660 665 670 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 675 680 685 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 690 695 700 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 705 710 715 720 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 725 730 735 Glu Tyr Asn Thr Ser Asn Pro Asp 740 <210> 32 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the light chain of Ipilimumab <400> 32 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 33 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Sequence of the Heavy Chain of Ipilimumab <400> 33 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser 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 Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 34 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Human 379D1 amino acid sequence, version 1 <400> 34 Gln Val Leu Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Pro Val 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val 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 Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Human 379D1 amino acid sequence, version 3 <400> 35 Gln Val Leu Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Pro Ile 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val 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 Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Human 379D1 amino acid sequence, version 3 <400> 36 Gln Val Val Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Pro Ile 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val 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 Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Human 379D1 amino acid sequence, version 4 <400> 37 Gln Val Val Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Pro Ile 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Human 379D1 amino acid sequence, version 5 <220> <221> MISC_FEATURE <222> (3) <223> X is L or V <220> <221> MISC_FEATURE <222> (44)..(44) <223> X is G or D <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is V or I <220> <221> MISC_FEATURE <222> (88)..(88) <223> X is A or P <400> 38 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 584 <212> PRT <213> Artificial sequence <220> <223> ASKB1296 HV_379D1 fusion molecule amino acid sequence <220> <221> MISC_FEATURE <222> (31)..(31) <223> X is E or D <220> <221> Repeating Unit <222> (455)..(459) <223> n is 0, 1, 2, 3, 4 or 5 <220> <221> MISC_FEATURE <222> (462)..(462) <223> X is L or V <220> <221> MISC_FEATURE <222> (503)..(503) <223> X is G or D <220> <221> MISC_FEATURE <222> (509)..(509) <223> X is V or I <220> <221> MISC_FEATURE <222> (549)..(549) <223> X is A or P <400> 39 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Xaa Asp 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Tyr Phe Tyr Gly Ser Ser Phe Tyr Ala Val Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gln Val Xaa Val Val 450 455 460 Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 465 470 475 480 Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr Pro Val Gly Trp Phe 485 490 495 Arg Gln Ala Pro Gly Lys Xaa Ala Ala Arg Glu Pro Xaa Ala Ala Ile 500 505 510 Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 515 520 525 Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr Leu Gln Met 530 535 540 Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Thr 545 550 555 560 Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe Trp Asn Gln 565 570 575 Gly Thr Leu Val Thr Val Ser Ser 580 <210> 40 <211> 355 <212> PRT <213> Artificial sequence <220> <223> 379D1- IgG1 Fc fusion molecule amino acid sequence, version 1 <220> <221> MISC_FEATURE <222> (3) <223> X is L or V <220> <221> MISC_FEATURE <222> (44)..(44) <223> X is G or D <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is V or I <220> <221> MISC_FEATURE <222> (88)..(88) <223> X is A or P <220> <221> Repeating Unit <222> (124)..(128) <223> n is 0, 1, 2, 3, 4 or 5 <400> 40 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 130 135 140 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 145 150 155 160 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 165 170 175 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 180 185 190 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 195 200 205 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 210 215 220 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 225 230 235 240 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 245 250 255 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 260 265 270 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 275 280 285 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 290 295 300 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 305 310 315 320 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 325 330 335 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 340 345 350 Pro Gly Lys 355 <210> 41 <211> 483 <212> PRT <213> Artificial sequence <220> <223> 379D1- IgG1 Fc fusion molecule amino acid sequence, version 2 <220> <221> MISC_FEATURE <222> (3) <223> X is L or V <220> <221> MISC_FEATURE <222> (44)..(44) <223> X is G or D <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is G or D <220> <221> MISC_FEATURE <222> (88)..(88) <223> X is A or P <220> <221> Repeating Unit <222> (124)..(128) <223> n = 0, 1, 2, 3, 4 or 5 <220> <221> MISC_FEATURE <222> (131)..(131) <223> X is L or V <220> <221> MISC_FEATURE <222> (172)..(172) <223> X is G or D <220> <221> MISC_FEATURE <222> (176)..(176) <223> X is V or I <220> <221> MISC_FEATURE <222> (216)..(216) <223> X is A or P <220> <221> Repeating Unit <222> (254)..(258) <223> n = 0, 1, 2, 3, 4 or 5 <400> 41 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 130 135 140 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 145 150 155 160 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 165 170 175 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 195 200 205 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 225 230 235 240 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 245 250 255 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 260 265 270 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 290 295 300 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 Pro Gly Lys <210> 42 <211> 355 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of 379D1- IgG1 Fc fusion molecule with knob mutation, version 3 <220> <221> MISC_FEATURE <222> (3) <223> X is L or V <220> <221> MISC_FEATURE <222> (44)..(44) <223> X is G or D <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is V or I <220> <221> MISC_FEATURE <222> (88)..(88) <223> X is A or P <220> <221> repeating unit <222> (124)..(128) <223> n is 0, 1, 2, 3, 4 or 5 <400> 42 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 130 135 140 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 145 150 155 160 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 165 170 175 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 180 185 190 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 195 200 205 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 210 215 220 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 225 230 235 240 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 245 250 255 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 260 265 270 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 275 280 285 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 290 295 300 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 305 310 315 320 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 325 330 335 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 340 345 350 Pro Gly Lys 355 <210> 43 <211> 483 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of 379D1- IgG1 Fc fusion molecule with knob mutation, version 4 <220> <221> MISC_FEATURE <222> (3) <223> X is L or V <220> <221> MISC_FEATURE <222> (44)..(44) <223> X is G or D <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is V or I <220> <221> MISC_FEATURE <222> (88)..(88) <223> X is A or P <220> <221> Repeating Unit <222> (124)..(128) <223> n is 0, 1, 2, 3, 4 or 5 <220> <221> MISC_FEATURE <222> (131)..(131) <223> X is L or V <220> <221> MISC_FEATURE <222> (172)..(172) <223> X is G or D <220> <221> MISC_FEATURE <222> (176)..(176) <223> X is V or I <220> <221> MISC_FEATURE <222> (216)..(216) <223> X is A or P <220> <221> Repeating Unit <222> (252)..(256) <223> n is 0, 1, 2, 3, 4 or 5 <400> 43 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gln Val Xaa Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 130 135 140 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 145 150 155 160 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Pro Xaa 165 170 175 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 195 200 205 Leu Gln Met Asn Ser Leu Arg Xaa Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 225 230 235 240 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 245 250 255 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 260 265 270 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 290 295 300 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 Pro Gly Lys <210> 44 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Ipilimumab heavy chain amino acid sequence with hole mutation <400> 44 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser 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 Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 355 360 365 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 45 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Ipilimumab heavy chain amino acid sequence with knos mutation <400> 45 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser 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 Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 46 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> (D1-V1)2-Fc, Hole <400> 46 Gln Val Leu Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 20 25 30 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Pro Val 35 40 45 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val 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 Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 100 105 110 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gln Val Leu Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 130 135 140 Ser Leu Arg Leu Ser Cys Ala Gly Ser Glu Arg Thr Phe Ser Ser Tyr 145 150 155 160 Pro Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Pro Val 165 170 175 Ala Ala Ile Thr Arg Gly Ala Arg Ser Thr Tyr Tyr Ala Asp Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Thr Val Tyr 195 200 205 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Ala Thr Trp Gly Thr Thr Pro Pro Val Gln Pro Ile Ala Asp Phe 225 230 235 240 Trp Asn Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Glu Pro Lys 245 250 255 Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 260 265 270 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 275 280 285 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 290 295 300 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 305 310 315 320 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 325 330 335 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 340 345 350 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 355 360 365 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 370 375 380 Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 385 390 395 400 Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 405 410 415 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 420 425 430 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 435 440 445 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 450 455 460 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 465 470 475 480 Leu Ser Pro Gly Lys 485

Claims

1. A single domain antibody that binds to human TIGIT protein, characterized in that: The single domain antibody comprises the amino acid sequence of SEQ ID NO:

1.

2. A humanized single domain antibody that binds to human TIGIT protein, characterized in that: The humanized single domain antibody comprises an amino acid sequence selected from SEQ ID NO: 34, 35, 36, 37.

3. A single domain antibody-antibody fusion molecule, characterized in that: The single-domain antibody-antibody fusion molecule comprises the single-domain antibody of any one of claims 1 to 2, wherein the single-domain antibody is fused to the N-terminus or C-terminus of the light chain or heavy chain of a second antibody via a peptide connector.

4. A fusion molecule, characterized in that The fusion molecule comprises the single domain antibody according to any one of claims 1 to 2, wherein the single domain antibody is fused to the N-terminus or C-terminus of an Fc polypeptide or an albumin molecule via a peptide connector.

5. The single domain antibody-antibody fusion molecule according to claim 3, characterized in that: The second antibody binds to an antigen selected from human PD-1, human PD-L1, CTLA4, HER2, EGFR, VEGFR2, VEGF, Claudin18.2, FAP, CD20, mesothelin, CMET, and 5T4.

6. The single domain antibody-antibody fusion molecule according to claim 3, characterized in that: The second antibody binds to PD-L1, and the second antibody comprises a light chain having an amino acid sequence of SEQ ID NO: 19 and a heavy chain having an amino acid sequence of SEQ ID NO: 20 or 21; Or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 22 and a heavy chain having the amino acid sequence of SEQ ID NO:

23.

7. The single domain antibody-antibody fusion molecule according to claim 3, characterized in that: The second antibody binds to PD-1, and the second antibody comprises a light chain having an amino acid sequence of SEQ ID NO: 15 and a heavy chain having an amino acid sequence of SEQ ID NO: 16; Or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 17 and a heavy chain having the amino acid sequence of SEQ ID NO:

18.

8. The single domain antibody-antibody fusion molecule according to claim 3, characterized in that: The second antibody binds to CTLA4, and the second antibody includes a light chain having an amino acid sequence of SEQ ID NO: 32 and a heavy chain having an amino acid sequence of SEQ ID NO:

33.

9. The single domain antibody-antibody fusion molecule according to any one of claims 5 to 8, characterized in that The fusion molecule further comprises a TGFβ receptor II extracellular domain.

10. The single domain antibody-antibody fusion molecule according to claim 9, characterized in that: The TGFβ receptor II extracellular domain comprises the amino acid sequence of SEQ ID NO: 11, 12, 13 or 14.

11. The fusion molecule according to claim 4, characterized in that The fusion molecule further comprises a TGFβ receptor II extracellular domain.

12. The fusion molecule according to claim 11, characterized in that The TGFβ receptor II extracellular domain includes the amino acid sequence shown in SEQ ID NO:11, 12, 13 or 14.

13. A fusion molecule, characterized in that The fusion molecule comprises the single domain antibody according to any one of claims 1 to 2 and a bifunctional molecule capable of binding to PD-L1 and TGF-β, The bifunctional molecule comprises two identical light chains and two identical heavy chain polypeptide chains, The light chain comprises the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

24.

14. A fusion molecule, characterized in that The fusion molecule comprises the single domain antibody according to any one of claims 1 to 2 and a bifunctional molecule capable of binding to PD-L1 and TGF-β, The bifunctional molecule comprises two identical light chains and two identical heavy chain polypeptide chains, The light chain comprises the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30 or 31.

15. The fusion molecule according to claim 4, characterized in that The fusion molecule binds to PD-L1 and TIGIT, wherein the fusion molecule comprises two identical light chains having an amino acid sequence of SEQ ID NO: 19, and two identical heavy chain polypeptide chains having an amino acid sequence of SEQ ID NO:

39.

16. The fusion molecule according to claim 4, characterized in that The fusion molecule comprises an amino acid sequence selected from SEQ ID NO: 40, 41, 42 and 43.

17. The fusion molecule according to claim 4, characterized in that The fusion molecule comprises the amino acid sequence of SEQ ID NO: 42 and 43, The fusion molecule further comprises a heavy chain having an amino acid sequence of SEQ ID NO: 44 and a light chain having an amino acid sequence of SEQ ID NO:

32.

18. A polynucleotide encoding the single domain antibody according to any one of claims 1-2.

19. A polynucleotide encoding the single domain antibody-antibody fusion molecule according to claim 3.

20. An expression vector comprising the polynucleotide of any one of claims 18 or 19.

21. A host cell comprising the expression vector according to claim 20.

22. A method for preparing a single domain antibody according to any one of claims 1 to 2 or a single domain antibody-antibody fusion molecule according to claim 3, characterized in that: The method comprises culturing the host cell of claim 21 under conditions allowing expression of the single domain antibody or the single domain antibody-antibody fusion molecule, and isolating the single domain antibody or the single domain antibody-antibody fusion molecule.

23. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the single domain antibody according to any one of claims 1 to 2 or the single domain antibody-antibody fusion molecule according to claim 3, and a pharmaceutically acceptable excipient.

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