CN115916247A - Human TIGIT (tungsten inert gas) specific single-domain antibody and application thereof - Google Patents

Abstract

The invention provides anti-TIGIT (T cell immunoglobulin and ITIM domain) single domain antibodies and variants thereof. The invention also includes methods of administering immunotherapy to a subject afflicted with a disease, such as cancer or an infectious disease. The method comprises administering to the subject a medicament comprising a therapeutically effective amount of an anti-TIGIT single domain antibody or fusion molecule thereof to enhance an endogenous immune response by promoting activation of the endogenous response or inhibition of the endogenous response.

Description

Human TIGIT specific single domain antibody and application thereof

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 63/012,221, filed on 19/4/2020, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to a therapeutic antibody, in particular to an anti-TIGIT single domain antibody and application thereof.

Sequence listing

This application includes a listing of sequences that have been submitted electronically in ASCII format, and the sequence listing is incorporated by reference herein in its entirety. The ASCII copy created at 18.4.2021 was named AG3-021US_ST25. Txt, and was 135KB in size.

Background

Cancer is generally defined as a group of diseases associated with abnormal cell growth that may invade or spread to other parts of the body. Approximately 180 million people in the united states were diagnosed with cancer in 2019. Approximately 606,880 die of cancer each year in the united states, with lung and bronchial cancer being the leading cause of death. Rectal and pancreatic cancer are the second and third leading causes of cancer lethality, respectively.

Cancer is associated with several factors, including smoking, obesity, poor diet, lack of physical exercise, and excessive alcohol consumption. Other factors including certain infections, exposure to ionizing radiation, and environmental contamination are also included. Certain cancers are associated with infections such as helicobacter pylori, hepatitis b, hepatitis c, human papilloma virus infection, epstein-Barr virus infection, and Human Immunodeficiency Virus (HIV) infection.

Traditional cancer treatments directly resect tumor tissue and inhibit its spread. Such treatment methods include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and palliative treatment. The method of treatment is typically selected based on the type, location, grade of cancer and the health and preference of the patient himself. All of the above methods have their limitations, especially when the cancer has metastasized, and these therapies may be ineffective. In addition, chemotherapy and radiation therapy have a series of side effects associated with cytotoxicity.

Since cancer cells divide faster than most normal cells, they are more sensitive to chemotherapeutic drugs. However, chemotherapeutic drugs also attack other cells in the body, especially rapidly dividing cells such as blood cells and cells of the oral, gastric and intestinal walls. Therefore, a more targeted drug and a cancer treatment method with less toxic and side effects are needed. It is a promising direction to develop a therapeutic approach that uses the immune system to attack and kill tumor cells.

Human cancers undergo a number of genetic and epigenetic changes, and the resulting neoantigens may be recognized by the immune system (Sjoblom et al, science 314. Although endogenous immune responses to tumors are observed in preclinical models and patients, such immune responses are ineffective and the resulting tumors are recognized as "self and are 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 (2011); mellman et al, nature480:480-489 (2011)). Among these mechanisms, endogenous "immune checkpoints" typically terminate the immune response to mitigate tissue damage beside the cancer, and cancers can use such checkpoints to evade killing by the immune system. In developing immune checkpoint specific inhibitors, considerable effort has been devoted and primary efforts have been made to provide New immunotherapies for cancer treatment, such as anti-CTLA-4 antibodies and ipilimumab for advanced melanoma treatment (Hodi et al, new Engl J Med 363 711-23 (2010))

T-cell immunoglobulin and ITIM domains (TIGIT) are novel targets for immune checkpoint therapy (Dougall et al, immunol Rev.2017; 276-112.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 depletion. TIGIT acts as a ligand, receptor or competitor for the co-stimulatory receptor CD226, and inhibition of its activity potentiates the T cell anti-tumor response.

TIGIT is expressed by a variety of important immune cells, with different functions being expressed by different cell types. In the analysis of whole transcriptional genome sequencing (RNA-Seq; -200X 106 reads/tomor) of 1467 unscreened clinical cases, high expression of PD-L1 was closely related to 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/mdy 288).

Single checkpoint blockade is associated with the up-regulation of other inhibitory checkpoints and the progression of resistance to therapy (Koyama et al, nat commu.2016; 7 10501. Doi. TIGIT and PD-1 dual checkpoint blockade enhance the anti-tumor immunity and survival of GBM mouse models (Hung et al, oncoimmunology.2018;7 (8): e 1466769).

There is therefore a need for anti-TIGIT single domain antibodies that are effective by themselves or as part of bispecific molecules in the treatment of cancer and other immune related diseases. The present application can satisfy various needs associated therewith and also provides methods of treating diseases, such as cancer, using the antibodies and antibody conjugates, pharmaceutical compositions and articles of manufacture.

Summary of The Invention

The invention described and claimed herein has many characteristics and embodiments, including but not limited to those illustrated or described or referenced in the summary of the invention. The invention described and claimed herein is not limited to the features or specific embodiments identified in the summary of the invention, which is intended to be illustrative only and not limiting

Particular embodiments include single domain antibodies that bind to human TIGIT, in one aspect, the single domain antibody comprises any one of the amino acid sequences selected from SEQ ID NOs 1-5 and 6.

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

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

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

In some embodiments, the single domain antibody is of human origin and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34,35,36,37, and 38.

In some embodiments, the single domain antibody is of human origin and comprises an amino acid sequence having at least 90%, at least 95%, or at least 98% homology to any one of the sequences selected from SEQ ID NOs: 34,35,36,37, and 38.

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

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

In some 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 the group consisting of: human PD-1, human PD-L1, CTLA4, HER2, EGFR, VEGFR2, VEGF, claudin18.2, FAP, CD20, mesothelin, CMET, and 5T4.

In some embodiments, the antibody binds to PD-L1, wherein the anti-PD-L1 antibody includes a light chain having an amino acid sequence as set forth in SEQ ID NO 19 or 22 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO 20,21 or 23.

In some embodiments, the antibody binds to PD-1, wherein the anti-PD-1 antibody comprises a light chain having an amino acid sequence as set forth in SEQ ID NO. 15 or 17 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO. 16 or 18.

In some embodiments, the antibody binds to CTLA4, wherein the anti-CTLA-4 antibody comprises a light chain having an amino acid sequence as set forth in SEQ ID NO:32 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 33.

In some embodiments, the single domain antibody-antibody fusion molecule further comprises a TGF β receptor II extracellular domain or a functional analog thereof.

In some embodiments, the TGF β receptor II extracellular domain comprises an amino acid sequence set forth as SEQ ID NO 11,12,13 or 14.

In some embodiments, the single domain antibody is fused to the N-terminus or C-terminus of the Fc polypeptide or albumin molecule or fragment thereof, optionally via a peptide linker. In some embodiments, the fusion molecule further comprises a TGF β receptor II extracellular domain or a functional analog thereof. In some embodiments, the TGF β receptor II extracellular domain comprises an amino acid sequence as set forth in SEQ ID NO 11,12,13, or 14.

In some embodiments, the present application provides novel fusion molecules comprising the above-described single domain antibodies, optionally including an antigen binding portion that binds to human PD-1 or PD-L1, and further including a TGF-beta receptor II extracellular domain or a functional analog thereof. In some embodiments, the antigen binding portion binds to human PD-1 or PD-L1, and a cytokine selected from the group consisting of: IL-2, IL-2 functional analogs, IL-15 functional analogs, IL-12 functional analogs, IL-21 functional analogs, interferon alpha functional analogs, interferon gamma functional analogs, TNF alpha, and TNF alpha functional analogs. In some embodiments, the cytokine moiety is masked, wherein the masking moiety is fused to the other moiety of the fusion molecule via a cleavable peptide linker.

The present application also provides fusion molecules comprising the above-described single domain antibodies and a bifunctional molecule, wherein the bifunctional molecule binds both 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 an amino acid sequence shown as SEQ ID NO. 19; the heavy chain polypeptide chain includes an amino acid sequence as set forth in SEQ ID NO 24.

The present application also provides fusion molecules comprising the above-described single domain antibodies and a bifunctional molecule, wherein the bifunctional molecule binds both PD-1 and TGF-beta. The bifunctional molecule comprises two identical antibody light chains and two identical heavy chain polypeptide chains. The light chain comprises an amino acid sequence as set forth in SEQ ID NO. 15; the heavy chain comprises an amino acid sequence as shown in SEQ ID NO 25,26,27,28,29,30 or 31.

In some embodiments, the fusion molecule capable of binding to PD-L1 and TIGIT comprises two identical light chains having the amino acid sequence of SEQ ID NO. 19 and two identical heavy chain polypeptide chains having an amino acid sequence at least 95%, at least 99% or 100% homologous to SEQ ID NO. 39.

In some embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 40,41,42 and 43.

In some embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 42 and 43. The fusion molecule further comprises a heavy chain having the amino acid sequence of SEQ ID NO. 44 and a light chain having the amino acid sequence of SEQ ID NO. 32.

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

In some embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO 46, wherein the fusion molecule further comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO 45 and a light chain having an amino acid sequence as set forth in SEQ ID NO 32.

In some embodiments, the fusion molecule comprises an amino acid sequence having at least 95% or at least 99% homology with SEQ ID No. 46, wherein the fusion molecule further comprises a heavy chain having an amino acid sequence having at least 99% homology with SEQ ID No. 45 and a light chain having an amino acid sequence as set forth in SEQ ID No. 32.

Similarly, the present application provides polynucleotides encoding any of the single domain antibodies described above or any of the fusion molecules described above. Also disclosed are expression vectors comprising the above polynucleotides, and host cells comprising the above expression vectors.

Further, the present application provides a method of preparing said single domain antibody or said fusion molecule. The method comprises the following steps: 1) Culturing the host cell under conditions that allow expression of the single domain antibody or fusion molecule; 2) Isolating the single domain antibody or fusion molecule.

Further, the present application provides a pharmaceutical composition comprising the single domain antibody or the fusion molecule and a pharmaceutically acceptable excipient.

Further, the present application provides a method of treating cancer or an infectious disease or activating the immune system of a patient in need thereof comprising administering the above pharmaceutical composition.

Drawings

The accompanying drawings provided with this application illustrate various aspects of the invention, in which:

FIG. 1 is the results of blocking reporter assays for selected single domain antibodies, in which the 41.1D11,41.2D1,41.3B4,42.2D6,41.1B4 mutant and the 42.3D6 mutant were D11, D1, B4, D6, B4 mutant and D6 mutant cloned fusion proteins fused via the linker shown in SEQ ID NO:8 and the C-terminus of Fc (SEQ ID NO: 7).

Figure 2 is the kinetic binding data for fusion molecules in which the human D1 molecule and the N-and C-termini of the Fc domain are fused. The results show that all the human D1 molecules retain strong affinity for human TIGIT with KD values below 3nM.

Figure 3 lists the structure of bispecific molecules that can bind to human TIGIT and CTLA-4. The molecule comprises two human D1 s and one Fab of an anti-CTLA 4 antibody, both portions fused to the N-terminus of the Fc domain.

Detailed Description

Definition of

Reference in the specification to "one embodiment/aspect" means that at least one embodiment/aspect of the invention includes a particular feature, structure or characteristic described in connection with the embodiment/aspect. The use of the phrases "in one embodiment/aspect" or "in another embodiment/aspect" in various places in the specification are not necessarily all referring to the same embodiment/aspect, nor are they necessarily independent or mutually exclusive of other embodiments/aspects. In addition, various features are included in only some embodiments/aspects and not in others. Likewise, various requirements are limited to only certain specific embodiments/aspects that are required and do not apply to other specific embodiments/aspects. In certain embodiments, embodiments and aspects are used interchangeably.

In general, the terms used in the specification of the present invention are to be interpreted in the text disclosed in the present application and in the specific text where the terms are used, as the meanings commonly understood in the art. Certain terms employed by the present invention are discussed below or elsewhere in the specification to provide further guidance to the relevant person regarding the description of the invention. It is to be understood that the same is intended to be illustrative of but not limited to one expression.

Accordingly, alternatives and synonyms can be used with the present invention to any one or more of the terms. It has no special meaning to specify or discuss a term herein. The present invention provides synonyms for certain terms. One or more synonyms does not preclude substitution of other synonyms. Examples of embodiments referred to anywhere in this specification, including any terms discussed herein, are illustrative only and are not intended to limit the scope or meaning of the disclosure or any examples. As such, the present disclosure is not limited to the various specific embodiments set forth in this specification.

The following examples disclosing specific embodiments of the apparatus, devices, methods and their associated results are not intended to limit the scope of the present application further. It should be noted that the headings or sub-headings used in the examples for the convenience of the reader should not be construed as limiting the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

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

The term "active agent" or "active ingredient" refers to a substance, compound or molecule that has a biological activity or other property, including a substance, compound or molecule that has a biological or physiological function to the subject to which the active agent or active ingredient is administered. In other words, "active agent" or "active ingredient" refers to an ingredient (components) or ingredients (components) in a composition that contributes to all or part of the function of the composition. The active agent may be the primary active agent or, in other words, an ingredient in the composition that contributes to all or part of the function of the composition. The active agent may be a minor active or, in other words, an ingredient in the composition that contributes to additional or other functions of the composition.

The term "subject" or "patient" refers to any animal, particularly a mammal (including non-human animals such as dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep and non-human primates) for which treatment may be administered, most preferably, the patient referred to herein is a human.

The term "pharmaceutically acceptable carrier" as used herein refers to any and all solvents, dispersion media, coatings, isotonic and sustained release agents and the like, consistent with methods of drug administration. The use of the above-mentioned media and agents in pharmaceutically active substances is well known to the person skilled in the art. The compositions may also include other active ingredients to supplement, augment or potentiate their therapeutic effects.

The term "pharmaceutically acceptable composition" as used herein refers to a composition comprising at least one compound of the present disclosure and one or more pharmaceutically acceptable carriers.

The term "programmed death-1 (PD-1)" is an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed predominantly on the surface of activated T cells in vivo and binds to both ligands PD-L1 and PD-L2. The term "PD-1" as used in the present invention includes human PD-1 (hPD-1), hPD-1 variants, isoforms, homologs, and analogs having at least one epitope in common with hPD-1. The complete hPD-1 sequence is found in Genebank access No. U64863.

The term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which upon binding to PD-1 is capable of reducing T cell activity and down regulating cytokine secretion. The term "PD-L1" as used in the present invention includes human PD-L1 (hPD-L1), hPD-L1 variants, isoforms, homologs, and analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence is found in Genebank access No. Q9NZQ7.0020.

The "T cell immunoglobulin and ITIM domains" or "TIGIT" bind with high affinity to the poliovirus receptor (PVR), which increases IL10 secretion, decreases IL12B secretion and inhibits T cell activation by promoting the production of mature immunoregulatory dendritic cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer 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, as well as natural amino acid polymers and unnatural amino acid polymers. Methods for obtaining polypeptides (e.g., production, isolation, purification, synthesis, and recombinant production) are well known to those of ordinary skill in the art.

Amino acids refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid actives that are functionally equivalent to the naturally occurring amino acids to some extent. Naturally occurring amino acids may be encoded by the genetic code or may be later modified amino acids such as hydroxyproline, γ -hydroxyglutamic acid and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups such as norleucine or modified peptide polymeric molecular backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid actives are defined as having a chemical structure that is different from the general chemical structure of an amino acid, but that is somewhat functionally equivalent to a naturally occurring amino acid.

Amino acids are referred to herein by the commonly used three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, are represented by the accepted single-character code.

The compositions of The invention comprise amino acid substitutions of Proteins and peptides which do not normally alter The activity of The protein or peptide (h.neurath, r.l.hill, the Proteins, academic Press, new York, 1979). In one embodiment, these substitutions are conservative amino acid substitutions. Most substitutions are typically 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, which are bi-directional.

With respect to "conservatively modified variants" of an amino acid sequence, it will be understood by those skilled in the art that individual substitutions, deletions or insertions of nucleotides, peptide, polypeptide or protein sequences in the encoded sequence can alter, add or delete individual amino acids or small portions of amino acids, such alterations resulting in the substitution of an amino acid with a chemically similar amino acid, as such are "conservatively modified variants". Conservative substitution tables providing 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 contemplated herein.

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), glutamic acid (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., kristin, protein (1984)).

The term "analog" refers to a peptide, polypeptide or protein sequence that is different from the relevant peptide, polypeptide or protein sequence. These differences may be amino acid insertions, deletions or substitutions, phosphorylation, sulfation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., the use of unnatural amino acid structures or other modifications well known to those skilled in the art.

The term "antibody" refers to a polypeptide comprising the framework regions of immunoglobulin genes or fragments thereof, which can specifically bind to or recognize an antigen. Immunoglobulin genes known in the art include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, and a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The heavy chains are classified as gamma, mu, alpha, delta or epsilon, and are in turn designated as immunoglobulins IgG, igM, igA, igD and IgE, respectively. Generally, the antigen binding domain of an antibody is the most crucial in specificity and affinity of binding.

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

Antibodies exist, for example, as intact immunoglobulins or as some of the fragments that have been characterized as being produced by the breakdown of various proteolytic enzymes. Thus, for example, pepsin fractionationCleavage of disulfide bond of antibody at hinge region to produce F (ab)' ₂ I.e., a dimer of fabs, which are light chains disulfide-bonded to VH — CH1. Cleavage of the disulfide bond at the hinge region under mild conditions may result in F (ab)' ₂ Reduced, F (ab)' ₂ The dimer is converted to 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 in terms of hydrolysis of intact antibodies, which fragments are understood by those skilled in the art to be synthesized de novo using chemical or recombinant DNA methods. The term "antibody" as used herein therefore also encompasses antibody fragments produced by whole antibody modification or resynthesized using recombinant DNA methods (e.g., single domain Fv) or antibody fragments identified by phage display libraries (see, e.g., mcCafferty et al, nature 348.

Affinity maturation is T _FH The process by which antibodies produced by cell-activated B cells enhance their affinity for antigens during the course of an immune response. Under repeated exposure to the same antigen, the host cell continuously produces higher affinity antibodies. The secondary immune response results in antibodies with an affinity many times greater than that of the primary antibody. The affinity maturation process mainly occurs on the immunoglobulin surface of B cell germinal center, and is the high-frequency mutation and T of somatic cell _FH Direct results of cell selection.

Diabodies can be prepared by chemical cross-linking or by hybridoma. Alternatively, diabodies can be prepared by recombinant techniques. Dimerization can be enhanced by shortening the length of the linker connecting the VH and VL regions, typically from about 15 amino acids to about 5 amino acids used to make scFv fragments. These linkers facilitate splicing within the VH and VL domain chains. Any suitable short connector may be used. Thus, the two fragments are spliced into one dimeric molecule. Further shortening of the length of the linker to 0-2 amino acids can result in trimeric (triantion) or tetrameric (tetraantion) molecules.

Methods for humanizing antibodies are known in the prior art. Generally, a humanized antibody has one or more amino acid residues introduced from its non-human source. These non-human amino acid residues will generally be considered import residues, which are typically derived from import variable regions. Humanization can be essentially performed according to the methods of Winter and co-workers (see, e.g., jones et al, nature science, 321-525 (1986); riechmann et al, nature science, 332. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which a portion of the substantially intact human variable region is replaced by the corresponding non-human corresponding sequence. In fact, humanized antibodies are typically human antibodies in which certain CDR amino acid residues and possibly some FR amino acid residues are substituted by amino acid residues from analogous sites on rodent antibodies.

The term "antigen-binding fragment" or "Fab" refers to the region of an antibody that binds antigen, which includes one constant region and one variable region of the heavy and light chains (e.g., the four domains: VH, CH1, VL and CL 1). The variable region includes an antigenic determinant (an 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 epitope.

The term "Fc region" or "crystallizable fragment" refers to the antibody tail region CH2-CH3, which interacts with cell surface receptors, the so-called Fc receptor, and the complement system. Its "effector functions" cause the antibody to activate the immune system, which in turn causes cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ADCC and ADCP exert a regulatory effect through the binding of Fc to Fc receptors on the surface of immune cells. CDC exerts a regulatory effect through Fc binding to proteins of the complement system (e.g., C1 q).

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 defined protein, usually in a heterologous or other biological agent of the protein. Thus, under certain immunoassay conditions, a given antibody has at least a two-fold capacity to bind to a particular protein compared to background (background), and typically has a 10 to 100-fold capacity. Specific binding to antibodies under such conditions requires screening for antibodies specific for the particular protein. For example, polyclonal antibodies can be screened for specific immune responses to selected antigens rather than to other proteins. This screening can be accomplished by removing antibodies that cross-react with other molecules. Various immunoassay methods can be used to screen for antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to screen for Antibodies specifically immunoreactive with a protein (see, e.g., harlow & Lane, using Antibodies, A Laboratory Manual (1998) for a description of immunological formulations and conditions that can be used to determine specific immunological reactivity).

The term "immunotherapy" refers to the treatment of a disease that is suffering from or at risk of infection or that has relapsed by a method that includes enhancing, suppressing or otherwise modifying the immune response. "treatment" or "therapy" refers to any type or course of action, or administration of an active agent, alteration of a target, alleviation, amelioration, inhibition, deceleration, or prevention, progression, development, severity of symptoms or recurrence, complications, condition associated with a disease, or biochemical marker of the invention.

Construction of appropriate vectors containing the target sequence and the regulatory sequences is well known in the art using standard restriction and ligation techniques (see Maniatis et al, in Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, new York (1982)). The isolated plasmid, DNA sequence, or synthetic oligonucleotide is cleaved, spliced, and religated in the desired form.

A nucleic acid is "ligatable" when it is functionally linked to another nucleic acid sequence. For example, the DNA for the propeptide sequence or secretory peptide may be ligated to the DNA for the polypeptide if it is expressed as a propeptide that is involved in the secretion of the polypeptide; a promoter or enhancer may be linked to a coding sequence if the coding sequence affects the transcription of the sequence; alternatively, a ribose binding site can be linked to the coding sequence if the coding sequence can facilitate translation. Generally, "ligatable" means that the DNA sequences are linked in close proximity and, at the same time, are contiguous in reading frame in the presence of the secreted peptide. However, enhancers need to be contiguous. Ligation is accomplished at appropriate restriction sites. If the above sites are not present, the linker or linker of the synthesized oligonucleotide is the same as that of a conventional plasmid.

"conservatively modified variants" applies to amino acid and nucleotide sequences. For a particular nucleotide sequence, conservatively modified variants refers to nucleotides that encode identical or essentially identical amino acid sequences, or do not encode amino acid sequences such that essentially identical amino acid sequences are achieved. 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. Thus, at each position of the arginine designated by a codon, 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 is also referred to herein as each possible silent variant of a nucleotide. One skilled in the art will recognize that each codon in a nucleotide (except AUG, which is usually only a codon for methionine, and TGG, which is usually only a codon for tryptophan) can be modified to a functionally identical molecule. Thus, each silent variant of a nucleotide encoding a polypeptide is implicitly recited in the sequence of each described expression product, and is not implicit to the actual probe sequence.

The term "identity" or percent "homology" in the context of describing two or more nucleotide or polypeptide sequences refers to amino acid residues or nucleotides of which two or more corresponding sequences are identical or have a certain percentage of identity (e.g., about 60% identity, more preferably 65%,70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or designated regions are more identical when compared and aligned for maximum correspondence over a corresponding comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters or by manual alignment and targeting. These sequences are said to be "essentially identical sequences". This definition also refers to, or applies to, a sequence of tests. The definition also includes sequences in which deletions and/or insertions occur, as well as sequences in which substitutions occur. The preferred algorithm may account for proteins, etc., as described below. Preferably, the region where identity exists is over at least 25 amino acids or nucleotides in length, or more preferably over 50-100 amino acids or nucleotides in length.

Sequence alignments, typically one sequence acts as a control sequence to align the test sequences. When the sequence alignment software, test and control sequence input into the computer, assigned sequence coordinates, if necessary, and set sequence algorithm program parameters. Preferably, default program parameters may be used, or selected parameters may be specified. The sequence alignment algorithm calculates the percent identity of the test and control sequences based on the program parameters.

A "comparison window" as used herein includes comparing any portion of the number of adjacent sites in a population selected from 20 to the full length of the control sequence, typically from about 25 to 100, or from 50 to about 150, where the sequence may be compared to a control sequence of the same number of adjacent sites after both sequences are in the optimal alignment position. Methods for comparing sequence alignments are well known in the art. Optimal sequence alignment can be referenced, for example, by local homology algorithms, smith and Waterman, adv.appl.math.2:482 (1981), homology alignment algorithms, needleman and Wunsch, j.mol.biol.48:443 (1970), similar methods research, pearson and Lipman, proc.nat' l.acad.sci.usa 85.

Examples of suitable software for detecting 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 nucleotides or proteins of the invention. The software for BLAST analysis is publicly available through the national center for Biotechnology information. This algorithm involves first identifying high-ratio sequence pairs (HSPs) by identifying short letters W in the sequence to be tested, which match or satisfy some positive-valued threshold T when aligned with a letter of the same length in a database sequence. T refers to the score threshold as a neighbor (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 to enable the cumulative alignment score to 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 non-matching residues; usually < 0) of the nucleotide sequence. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. When the cumulative comparison score differs by an order of magnitude from the maximum realizations value X, the expansion of the letters will stop in each direction; accumulating scores to zero or below due to accumulation of one or more negative scoring residue sequences; or to the end of any one sequence. The BLAST algorithm parameters W, T, and X determine the speed and sensitivity of sequence alignment. The BLASTN program (for nucleotide sequences) used a default word length (W) of 11 and an expected value (E) of 10,m =5,n = 4 for two strand alignments. For amino acid sequences, the BLASTP program used a default word size of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, proc. Natl. Acad. Sci. Us 89 10915 (1989)) sequence (B) of 50 and an expected value (E) of 10, m =5, n = -4, for two strand alignments.

"nucleic acid" refers to deoxyribonucleotides or ribonucleotides, polymers in either single-or double-stranded form, and the complementary strand thereof. This term includes nucleic acids, including known nucleotide analogs or modified backbone residues or linkers, which are synthetic, naturally occurring, and non-naturally occurring, 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, methyl phosphates, chiral methyl phosphates, 2-O-methyl nucleotides, peptide-nucleic acids (PNAs).

Unless otherwise specified, a particular nucleic acid sequence likewise includes conservatively modified variants thereof (e.g., substitutions of degenerate codons) and complementary sequences, as well as the sequence explicitly indicated. Clearly, degenerate codon substitutions may be made by synthesizing sequences in which the third position of one or more (or all) of the selected codons is replaced with mixed base 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 (1994)). The term nucleotide is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

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. "splice variants," as the name implies, are the products of alternative splicing of genes. Following transcription, the initial nucleic acid transcript may be spliced, with different (alternative) nucleic acid splice products encoding different polypeptides. The mechanism by which splice variants arise is variable, including the replacement of exon splicing. This definition also encompasses alternative polypeptides derived from the same nucleotide by reading the transcript. Any splicing reaction product, including recombinant forms of the splicing product. For example, leicher et al, J.biol.chem.273 (52): 35095-35101 (1998) are discussed for splice variants of potassium ion channels.

In other aspects, the present specification also provides the use of a protein therapeutic or pharmaceutical composition for the prevention or treatment of cancer, neurodegenerative or infectious diseases, including the same use in the manufacture of a medicament.

The term "treating" or "treatment" refers to one or more of the following meanings: (1) Inhibiting a disease, condition, or disorder in an individual who is suffering from or presenting with a pathology or symptom of a disease, condition, or disorder, (e.g., inhibiting further development of the relevant pathology and/or symptom); and (2) relief of symptoms; such as relieving the relevant disease, condition or disorder state (e.g., eliminating the relevant pathology and/or symptom, such as reducing the extent of the condition) in a patient suffering from or presenting with the pathology or symptom of the disease, condition or disorder.

Other technical terms used in the present invention have meanings commonly used in the art, and may be, for example, paraphrases of different technical dictionaries. The particular values or configurations discussed in the present non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.

Detailed Description

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject matter related art of the claims. Additional features and advantages of the subject technology will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the subject technology. The advantages of the subject technology may be realized and attained by the structure particularly pointed out in the written description and claims hereof. The invention also provides pharmaceutical compositions and methods of immunotherapy for patients afflicted with certain diseases, such as cancer or infectious diseases.

Single domain antibodies are antibody fragments with a single variable antibody domain, which, like whole antibodies, selectively bind to a particular antigen. However, single domain antibodies are smaller than normal antibodies and have a small molecular weight (e.g., 12-15 kDa). Conventional antibodies comprise two heavy chains and two light chains, in contrast to single domain antibodies which are smaller than Fab fragments and single chain variable fragments.

The present invention provides anti-TIGIT (T cell immunoglobulin and ITIM domain) single domain antibodies and variants thereof. In particular, provided anti-TIGIT antibodies and variants thereof have the following properties: high affinity for human TIGIT, for example; cross-reaction occurs in human TIGIT; and biochemical and biophysical properties that confer high stability to the antibodies and variants thereof. Particular embodiments include anti-TIGIT single domain antibodies as part of a bispecific molecule. The antibodies and bispecific molecules are useful for treating cancer and other immune-related diseases.

Embodiments include anti-TIGIT single domain antibodies having an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100% homologous to any one of the sequences selected from SEQ ID NOs 1-5 and 6. The anti-TIGIT single domain antibodies of the invention comprise CDR domains derived from a single domain antibody selected from SEQ ID NOs 1-5 and 6.

In one embodiment, the anti-TIGIT single domain antibody 379D1 is human and comprises an amino acid sequence selected from SEQ ID NOs 34,35,36,37 and 38, and in some embodiments, the human single domain antibody 379D1 comprises an amino acid sequence at least 95% homologous, at least 99% homologous to any one selected from SEQ ID NOs 34,35,36 and 37.

The anti-TIGIT single domain antibody and the Fc fragment or the albumin molecule form a fusion molecule. The anti-TIGIT single domain antibody may be fused to the N-terminus and/or C-terminus of the antibody heavy and/or light chain. In particular embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 40,41,42 and 43.

In some embodiments, the anti-TIGIT single domain antibody is fused to the N-terminus of the heavy and/or light 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, herr 2, cmet,5t4, trop-2, and Claudin 18.2.

In some embodiments, the anti-TIGIT single domain antibody is fused to the C-terminus of an antibody heavy 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, herr 2, cmet,5t4, trop-2, and Claudin 18.2. In a particular embodiment, the fusion molecule comprises two light chains that are identical and have the amino acid sequence shown in SEQ ID No. 19, and two heavy chain polypeptide chains that are identical and have an amino acid sequence that is at least 95%, at least 99% or 100% homologous to SEQ ID No. 39.

In some embodiments, the anti-TIGIT single domain antibody is fused to the N-terminus of the Fc fragment. The fusion molecule further comprises a light chain and a heavy chain of an antibody, wherein the antibody binds to an antigen selected from the group consisting of PD-L1, PD-1, ctla-4, lag-3, tim-3, vegf, vegfr2, egfr, herr 2, cmet, 5tj4, 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 comprises a "knob-to-holes" mutation. In some particular embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO 46. The fusion molecule further comprises a heavy chain having the amino acid sequence shown as SEQ ID NO. 45 and a light chain having the amino acid sequence shown as SEQ ID NO. 32. In some embodiments, the fusion molecule comprises an amino acid sequence having at least 90% homology, at least 95% homology, or at least 99% homology to SEQ ID No. 46. The fusion molecule further includes a light chain having an amino acid sequence as set forth in SEQ ID NO. 32 and a heavy chain having an amino acid sequence at least 90%, at least 95% or at least 99% homologous to SEQ ID NO. 45.

Figure 3 depicts a bispecific molecule according to one embodiment. The N-terminus of the anti-TIGIT single domain antibody and Fc fragment were fused. The Fc fragment of the fusion polypeptide chain and the Fc domain of the heavy chain comprise sphere Kong Tubian. Wherein the fusion molecule comprises a light chain and a heavy chain of an antibody that binds to CTLA-4.

Application and administration mode

In human and animal therapeutic applications, the compounds of the invention may be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration and the type of treatment (e.g., prevention, prophylaxis or therapy), the compounds will remain consistent with the parameters set forth above throughout use. A summary of The related art can be found in Remington, the Science and Practice of Pharmacy,21 edition, lippincott Williams and Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds.J.Swarbrick and J.C.Boylan,1988-1999, marcel Dekker, new York. All of the above documents are incorporated by reference.

The present specification also provides a pharmaceutical composition for administration to a subject. The pharmaceutical compositions disclosed herein may further comprise a pharmaceutically acceptable carrier, excipient or diluent. The term "pharmaceutically acceptable" as used herein means a drug that is sufficient to allow the ingredients to achieve a therapeutic effect without adverse side effects, and can be readily determined according to the type of disease, the age, body weight, health condition, sex and drug sensitivity of the patient, the route of administration, the mode of administration, the frequency of administration, the duration of treatment, the drug used in combination therewith or the compound of the present disclosure, and other factors known in medicine.

On the other hand, examples of carriers, excipients and diluents for pharmaceutical preparations include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical preparation may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, and a preservative.

The pharmaceutical composition disclosed in the present invention may further be 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 formulations and suppositories.

The ingredients may be formulated into a single dosage form suitable for the patient's body, preferably, a formulation useful for peptide drugs according to the classical methods in the pharmaceutical field, so as to be administered by oral or parenteral routes, such as, but not limited to, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intracerebroventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal or rectal administration.

The total effective dose of the medicaments disclosed herein may be administered to a patient in a single dose, or may be administered in multiple doses over a period of time according to a fractionated treatment regimen. In the medicaments disclosed herein, the amount of active ingredient may vary depending on the severity of the disease. Preferably, the total daily dose of the peptides disclosed herein may be from about 0.0001. Mu.g to 500mg per 1kg of patient body weight. However, the effective dose of the peptide is determined in consideration of various factors including the age, body weight, health condition, sex, disease severity, diet and secretion rate of the patient, in addition to the administration route and treatment frequency of the drug. In view of this, one of ordinary skill in the art can readily determine an effective dose suitable for a particular use of the medicaments disclosed herein. The drug disclosed herein is not particularly limited in formulation, administration route and manner as long as it shows a beneficial effect.

In addition, the drugs may be administered alone, or in combination or simultaneously with other pharmaceutical preparations having prophylactic or therapeutic effects.

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

The active ingredient may be included in a pharmaceutical composition for parenteral administration. Suitable buffering agents 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 solutions having concentrations of 0-300mM (150 mM being the optimum concentration for liquid agents). The lyophilized preparation also comprises lyoprotectant with sucrose (0-10% (preferably 0.5-1.0%) as main ingredient, and other optional lyoprotectant including trehalose and lactose. The lyophilized preparation also comprises bulking agent, whose main ingredient is 1-10% mannitol (optimally 2-4%). The liquid preparation and lyophilized preparation also comprise stabilizer, whose main ingredient is L-methionine 1-50mM (optimally 5-10 mM). Other leavening 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.

The solution containing the active ingredient may be administered to the patient in a single administration or as a course of treatment, and may be administered to the patient at any time after the diagnosis is confirmed. The solutions can be administered alone or in combination with other drugs or treatments that are therapeutic to the condition.

Certain aspects of the specification disclose, to a certain extent, the treatment of a human or non-human mammalian subject suffering from a disease (e.g., cancer). The term "treating" as used herein refers to reducing or eliminating the clinical symptoms of cancer in a human or non-human mammal; or delaying or preventing the clinical symptoms of cancer in a human or non-human mammal. For example, the term "treating" can refer to alleviating a symptom characteristic of a cancer, including but not limited to alleviating the severity of a disease, e.g., 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 one of ordinary skill in the art by consideration of factors including, but not limited to, the site of the disease, the cause of the disease, the severity of the disease, and/or the effect of the tissue or organ being affected by the disease. Those skilled in the art are aware of the appropriate symptoms or indicators associated with a particular type of disease and are able to know how to determine whether an individual is eligible for the treatment disclosed herein.

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

The dosage may be a single dose or a cumulative dose (continuous administration) and can be readily determined by one skilled in the art. For example, treatment of cancer may comprise administration of one effective dose of a therapeutic compound or pharmaceutical composition disclosed herein. Alternatively, treatment of cancer may comprise multiple administrations of an effective dose of the pharmaceutical composition over a period of time, e.g., once a day, twice a day, three times a day, once every few days, or once a week. The time of administration varies from person to person and depends on factors such as the severity of the individual symptoms. For example, an effective dose of a pharmaceutical composition disclosed herein can be administered to an individual once a day at irregular intervals until the individual no longer requires treatment. One of ordinary skill in the art will recognize that the condition of an individual will be monitored throughout the course of treatment, and that the effective amount of antibody administered will be adjusted accordingly as disclosed herein.

Examples

The following non-limiting examples are provided for illustrative purposes only to provide a more complete understanding of the representative embodiments that have been completed. These examples are provided only for the purpose of illustration and are not intended to limit the scope of the present application. These examples should therefore not be construed as limiting any of the embodiments described herein, especially with respect to the type and number of formulation compounds and/or methods of preparation and use thereof.

Example 1

Immune response

TIGIT-specific single domain antibodies (MAbs) were prepared by a method of immunizing alpaca. Two alpacas were separately immunized with human TIGIT-his (antibodies cat. # TIT-H52H 3) antigen, serum samples were taken after the immunization step was completed, and the isolated PBMCs were used to construct phage libraries. Panning was performed on the phage library and primary screening was performed using ELISA in combination to sequence positive clones. The protein sequences of the selected clones are shown in table 1.

The corresponding genes of these proteins were expressed by HEK293 following fusion with the C-terminal of the IgG1Fc domain and further confirmed by TIGIT/CD155 blockade reporter analysis.

Example 2

Transient transfection

HEK293 cells were plated at 1.5X 10 ⁵ Cells were cultured overnight in 24-well tissue culture dishes in 1mL complete medium per well. Transfection with 500ng DNA was performed with Lipofectamine 3000 (Life Technologies, cat. # L3000015) according to the manufacturer's instructions. After 3-5 days, the supernatant was collected and its binding activity was measured by ELISA.

HEK293 cells were cultured with 5% ultra-low IgG fetal bovine serum (Life technologies, cat. # 16250-078) using Lipofectamine 3000 according to manufacturer's instructions and transfected on a large scale to produce purified material.

Example 3

TIGIT/CD155 Block reporter assay

TIGIT/CD155 blocking bioassay (a-e) (Cat. # J2201, J2205) is a cell-based bioluminescence 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 comprises two genetically engineered cell lines:

tigit effector cell line: jurkat T cells are designed to express human TIGIT with a luciferase reporter gene, the expression of which is driven by its own promoter, which responds to TCR activation and co-stimulation of CD 226; and

CD155aAPC/CHO-K1 cell line: CHO-K1 cells were designed to express human CD155, and the genetically engineered cell surface proteins were designed to activate the T Cell Receptor (TCR) complex in an antigen-independent manner.

The TIGIT effector cell line and the CD155aAPC/CHO-K1 cell line are provided in a mode of instant use and fusion, and the cells stored in a freezing way can be directly subjected to fusion, plating and application in the measuring process without cell culture and expansion. TIGIT inhibited CD226 activity and promoter-mediated fluorescence when both cells were co-cultured. The addition of an anti-TIGIT antibody prevents TIGIT from interacting with CD155 or inhibits TIGIT from acting on the CD266 homodimer, thereby generating promoter-mediated fluorescence. The TIGIT/CD155 blocking assay includes fusion, plating and assaying of the cells using essential media and serum. Using Bio-Glo ^TM Luciferase assay systems and standardised luminometers e.g.

The Discover System quantitatively detects bioluminescent signals.

The TIGIT/CD155 blockade reporter assay is used to study the single domain antibodies as a cell-based functional assay. The single domain antibody was cloned and fused to the C-terminus of IgG1 Fc. The fusion molecule is transiently expressed and purified. Fig. 1 shows TIGIT reporter assay results. The different curves compare the following single domain antibodies by reflecting the correlation between fluorescence values (relative photometric values) and concentrations:

a)41.1 D11

b)41.2 D1

c)41.3 B4

d)42.2 D6

e) 41.1B4 mutants

f) 42.3D6 mutant

Each of the fusion molecules was D11, D1, B4, D6, the mutant B4 and the mutant D6 clones were fused to the C-terminus of Fc (SEQ ID NO: 7) via a linker as shown in SEQ ID NO:8, respectively. Anti-human TIGIT antibodies (Catalog # MAB 7898) purchased from R & D Systems were used as positive controls based on cell function assays.

Example 4

Humanization

The D1VHH homology model was constructed using pdb 3H6 as a structural model and its humanization design was confirmed again using another structural model, pdb 4p 59. The humanized D1 sequence (SEQ ID NOS: 34-47) was fused to the N-and C-termini of IgG1Fc, and the molecule was cloned, expressed and purified. The affinity of the human D1-Fc fusion molecule to human TIGIT was examined using a ForteBio instrument, and the results are shown in FIG. 2. The data indicate that the human D1 molecule has a strong affinity for human TIGIT with KD values below 3nM. Specifically, all human D1 molecules have strong affinity for human TIGIT with KD values below 3nM.

Finally, it should be understood that the embodiments disclosed are merely illustrative of the principles of the subject matter disclosed herein, and that no limitation on the scope of the invention is intended by the claims. Thus, it is to be understood that the disclosed subject matter is in no way limited to the specific methods, procedures, and/or reagents, etc., described herein, unless otherwise indicated. In addition, it should be recognized that certain modifications, variations, or alterations to those techniques which are conventional in the art may be made in light of the teachings herein without departing from the spirit of this specification. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined only by the appended claims, and therefore the invention is not limited to the exact details shown or described above.

Table 1: protein sequences of Single Domain antibodies

Table 2: additional sequences

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Sequence listing

<110> Oumei pharmaceuticals, inc

<120> human TIGIT specific single domain antibody and application thereof

<130> AG3-021WO

<140> 63012221

<141> 2020-04-19

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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 sequences

<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 ectodomain fragment comprising VEGF receptor II second and third IgC 2-like (immunoglobulin-like) domains

<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> the second IgC 2-like (immunoglobulin-like) domain comprising VEGF receptor I and the VEGF receptor II

VEGF-binding portion of third IgC 2-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-beta receptor II (TGF-beta RII)

<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-beta 1 binding moieties comprising TGF-beta receptor II ectodomain fragments

<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-beta 1 binding moieties comprising TGF-beta receptor II ectodomain fragments

<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-beta 1 binding moieties comprising two TGF-beta receptor II ectodomain 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 Nivolumab of PD-1 antibody

<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 light chain sequence

<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> antibody Pembrolizumab heavy chain sequence

<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 light chain

<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> PD-L1 antibody ASKB1296 heavy chain

<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> 24

<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> 24

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 antibody 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 antibody 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> Iplilimumab light chain amino acid sequence

<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> Iplilimumab heavy chain amino acid sequence

<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> humanized 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> humanized 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)..(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> 379D 1-IgG 1Fc fusion molecule amino acid sequence, version 1

<220>

<221> MISC_FEATURE

<222> (3)..(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> 379D 1-IgG 1Fc fusion molecule amino acid sequence, version 2

<220>

<221> MISC_FEATURE

<222> (3)..(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> 379D 1-IgG 1Fc fusion molecule amino acid sequence with knob mutation, version 3

<220>

<221> MISC_FEATURE

<222> (3)..(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> 379D 1-IgG 1Fc fusion molecule amino acid sequence with knob mutation, version 4

<220>

<221> MISC_FEATURE

<222> (3)..(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> Iplilimumab heavy chain amino acid sequence having 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> Iplilimumab heavy chain amino acid sequence having a 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 (35)

1. A single domain antibody that binds to human TIGIT protein, said single domain antibody comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 1-5 and 6.

2. The single domain antibody according to claim 1, wherein the CDR domain of said single domain antibody has one, two, three, four or five amino acid mutations, deletions or additions.

3. A single domain antibody comprising CDR1, CDR2 and CDR3 identical to any one of the sequences derived from SEQ ID NOs 1-5 and 6.

4. The single domain antibody of claim 1, wherein said single domain antibody is humanized.

5. The single domain antibody of claim 1, wherein said single domain antibody is mature and has enhanced binding capacity for human TIGIT relative to the parent antibody.

6. The single domain antibody of claim 1, wherein said single domain antibody is humanized and mature.

7. A humanized single domain antibody that binds to human TIGIT protein, comprising an amino acid sequence selected from the group consisting of SEQ ID NO 34,35,36,37 and 38.

8. A humanized single domain antibody that binds to human TIGIT protein comprising an amino acid sequence having at least 90%, at least 95%, or at least 98% homology to SEQ ID NO 34,35,36,37 or 38.

9. The single domain antibody of any one of claims 1 to 8, wherein said single domain antibody is fused to the N-terminus or C-terminus of the light or heavy chain of a second antibody, optionally via a peptide linker.

10. The single domain antibody of any one of claims 1-8, wherein said single domain antibody is fused, optionally via a peptide linker, to the N-terminus or C-terminus of the light or heavy chain of a second antibody, wherein said second antibody binds to an antigen expressed by a tumor cell or an immune cell.

11. The single domain antibody of any one of claims 1 to 8, wherein said single domain antibody is fused to the N-terminus or C-terminus of an Fc polypeptide or albumin molecule, optionally via a peptide linker.

12. The single domain antibody of any one of claims 1 to 8, wherein said single domain antibody and a second single domain antibody are fused to form an antibody-antibody fusion molecule.

13. The single domain antibody-antibody fusion molecule of claim 10, wherein said second antibody binds to an antigen selected from the group consisting of human PD-1, human PD-L1, CTLA4, HER2, EGFR, VEGFR2, VEGF, claudin18.2, FAP, CD20, mesothelin, CMET, and 5T4.

14. The single domain antibody-antibody fusion molecule of claim 10, wherein the second antibody binds to PD-L1 and comprises a light chain having the amino acid sequence of SEQ ID NO 19 or 22 and a heavy chain having the amino acid sequence of SEQ ID NO 20,21 or 23.

15. The single domain antibody-antibody fusion molecule of claim 10, wherein the second antibody binds to PD-1 and comprises a light chain having the amino acid sequence of SEQ ID NO 15 or 17 and a heavy chain having the amino acid sequence of SEQ ID NO 16 or 18.

16. The single domain antibody-antibody fusion molecule of claim 10, wherein the second antibody binds to CTLA4 and comprises a light chain having the amino acid sequence of SEQ ID No. 32 and a heavy chain having the amino acid sequence of SEQ ID No. 33.

17. The single domain antibody-antibody fusion molecule of any one of claims 13-16, wherein said fusion molecule further comprises a TGF β receptor II extracellular domain or a functional analog thereof.

18. The single domain antibody-antibody fusion molecule of claim 17, wherein the TGF β receptor II extracellular domain comprises the amino acid sequence of SEQ ID NO 11,12,13 or 14.

19. The fusion molecule of claim 11, further comprising a TGF β receptor II extracellular domain or a functional analog thereof.

20. The fusion molecule of claim 19 wherein the extracellular domain of TGF β receptor II comprises the amino acid sequence set forth in SEQ ID NO 11,12,13 or 14.

21. A fusion molecule comprising a single domain antibody according to any one of claims 1 to 8, an antigen binding portion that binds human PD-1 or PD-L1, and a TGF β receptor II extracellular domain or a functional analog thereof.

22. A fusion molecule comprising the single domain antibody of any one of claims 1-8, optionally comprising an antigen binding portion that binds human PD-1 or PD-L1, and a cytokine selected from the group consisting of: IL-2, IL-2 functional analogs, IL-15 functional analogs, IL-12 functional analogs, IL-21 functional analogs, interferon alpha, functional analogs of interferon alpha, interferon gamma, functional analogs of interferon gamma, TNF alpha, functional analogs of TNF alpha.

23. The fusion molecule of claim 22, wherein the cytokine moiety is masked and wherein the mask is fused to the other portion of the fusion molecule by a cleavable or non-cleavable peptide linker.

24. A fusion molecule comprising a single domain antibody according to any one of claims 1 to 7 and a bifunctional molecule capable of binding to PD-L1 and TGF- β,

wherein the bifunctional molecule comprises two identical light chains and two identical heavy chain polypeptide chains of an antibody,

wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO 19 and the heavy chain polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO 24.

25. A fusion molecule comprising a single domain antibody according to any one of claims 1-10 and a bifunctional molecule that binds to PD-L1 and TGF- β,

wherein said bifunctional molecule comprises an antibody of two identical light chains and two identical heavy chain polypeptide chains,

wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO. 15 and the heavy chain polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO. 25,26,27,28,29,30 or 31.

26. The fusion molecule of claim 11, which binds to PD-L1 and TIGIT, wherein the fusion molecule comprises two identical light chains having the amino acid sequence of SEQ ID No. 19 and two identical heavy chain polypeptide chains having at least 95%, at least 99% or 100% homology to amino acid SEQ ID No. 39.

27. The fusion molecule of claim 11 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 40,41,42 and 43.

28. The fusion molecule of claim 11, wherein the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 42 and 43,

wherein the fusion molecule further comprises a heavy chain having the amino acid sequence of SEQ ID NO. 44 and a light chain having the amino acid sequence of SEQ ID NO. 32.

29. A polynucleotide encoding a single domain antibody according to any one of claims 1 to 8.

30. A polynucleotide encoding the fusion molecule of claim 10.

31. An expression vector comprising the polynucleotide of any one of claims 29 or 30.

32. A host cell comprising the vector of claim 31.

33. A method of producing a single domain antibody according to any one of claims 1 to 8 or a fusion molecule according to claim 10, comprising culturing a host cell according to claim 32 under conditions which allow expression of said single domain antibody or said fusion molecule, and isolating said single domain antibody or fusion molecule.

34. A pharmaceutical composition comprising a single domain antibody according to any one of claims 1 to 8 or a fusion molecule according to claim 10, and a pharmaceutically acceptable excipient.

35. A method of treating cancer or an infectious disease or stimulating the immune system of a patient in need thereof, comprising the use of the pharmaceutical composition of claim 34.

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