KR20210098147A - Fusion protein comprising anti-tumor-associated antigen antibody, anti-pd-l1 antibody and 4-1bbl and uses thereof - Google Patents

Abstract

The present invention relates to a fusion protein comprising an anti-tumor-associated antigen (TAA) antibody, an anti-PD-L1 antibody and 4-1BBL, and uses thereof. More specifically, the present invention relates to a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and 4-1BBL, and uses thereof. The fusion protein of the present invention has excellent binding ability between CEA, which is a tumor-associated antigen, and PD-L1, which is an immune checkpoint protein, and can specifically bind to tumor cells expressing CEA and/or PD-L1. In addition, the fusion protein of the present invention can activate T cells without showing toxicity seen in a conventional 4-1BBL therapeutic agent. Thus, the fusion protein of the present invention can be advantageously used to treat cancer diseases.

Description

FUSION PROTEIN COMPRISING ANTI-TUMOR-ASSOCIATED ANTIGEN ANTIBODY, ANTI-PD-L1 ANTIBODY AND 4-1BBL AND USES THEREOF}

The present invention relates to a fusion protein comprising an anti-TAA antibody, an anti-PD-L1 antibody and 4-1BBL and uses thereof. More specifically, the present invention relates to a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and 4-1BBL and uses thereof.

The anticancer drug market occupies the largest portion of the overall drug market and has the highest growth potential (CACR) of 11.6%, which is expected to expand to 153 trillion won in 2020. Among them, immuno-oncology drugs have a mechanism in which the immune system attacks cancer cells by stimulating the body's immune system.

In this regard, tumor cells expressing tumor-specific antigens can be eliminated by an individual's immune system at an early stage of development. However, actively proliferating tumor cells express immune checkpoint proteins such as PD-L1 to evade immune surveillance. These immune checkpoint proteins are known as a mechanism to evade the immune response of tumor cells by interfering with the anticancer immune surveillance system.

Among immune checkpoint proteins, the mechanism by which PD-1 expressed on T cells and PD-L1 expressed on cancer cells bind to suppress the function of T cells is known. Immune checkpoint inhibitors released with the mechanism of inhibiting the binding of PD-1 and PD-L1 include pembrolizumab (Keytruda, pembrolizumab, anti-PD-1: Merck), nivolumab (Opdivo, nivolumab, anti-PD- 1: BMS), and atezolizumab (Tecentriq, atezolizumab, anti-PD-L1: Roche). Ipilimumab (Yervoy, Ipilimumab, anti-CTLA4: BMS), the world's first FDA-approved immune checkpoint inhibitor prior to the immune checkpoint inhibitor for PD-1 and PD-L1, binds to T-cell surface CTLA-4 It was approved for the first time as a treatment for melanoma patients as a mechanism to prevent cells from being incapacitated and to help the proliferation of T cells.

These immuno-oncology drugs showed superior clinical trial results than conventional anti-cancer drugs, which merely extended the survival period, such as improving the overall survival and progression free survival (PFS) of patients without aggravation of the disease. In addition, there are few side effects and resistance problems such as vomiting, hair loss, and leukocyte reduction of existing anticancer drugs, so immunotherapy showed great potential in anticancer treatment. In addition, the clinical results of the immune checkpoint inhibitor surprised the world by showing a long-term survival close to a complete cure for terminal cancer patients, but only about 20% to 40% of patients responded in various cancer types such as lung cancer, skin cancer, and breast cancer. . The remaining 60% to 80% of patients and, in particular, in the case of colorectal cancer patients, have a low or no therapeutic efficacy.

In the case of the patient group showing low or no response to the immune checkpoint inhibitor, the number of T cells in the tumor tissue was markedly insufficient. In other words, the patient's immune status is one of the important factors that determine the response to the immune checkpoint inhibitor. In the case of many cancer patients, the immune system is disturbed by chemotherapy and radiation therapy, resulting in 'immune cold or immune desert', which is speculated to be the cause of low reactivity to immune checkpoint inhibitors. Therefore, there is a need to develop a substance that activates the patient's immune system and research on a new treatment method that uses it in combination with an existing immune checkpoint inhibitor.

On the other hand, cytokines are substances that regulate various immune responses, and are important factors that determine the development, proliferation, maintenance, activity, and death of immune cells according to their types and characteristics. Various cytokine therapeutics are being developed for the purpose of activating the immune system of patients. In particular, the development of IL-2 cytokines (IL-2, IL-7, IL-15, IL-21) that are important for homeostasis of T cells and NK cells are being actively developed. However, cytokine therapeutics have a limitation in that they cannot confirm distinct therapeutic efficacy due to their short half-life.

Accordingly, the present inventors studied to develop an effective immuno-oncology agent capable of inhibiting the immune checkpoint protein while activating the immune system. As a result, the fusion protein including the anti-CEA antibody, anti-PD-L1 antibody and 4-1BBL is excellent The present invention was completed by confirming that it exhibits an anticancer effect.

In order to achieve the above object, one aspect of the present invention provides a fusion protein of the following Structural Formula 1 or Structural Formula 2.

[Structural Formula 1]

N′-AL ₁ -BL ₂ -CL ₃ -DC′

[Structural Formula 2]

N′-BL ₁ -AL _{2 -CL 3} -DC′

Another aspect of the present invention provides a fusion protein dimer in which the two fusion proteins are bound.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the fusion protein or the fusion protein dimer as an active ingredient.

The fusion protein of the present invention has excellent binding ability between CEA, a tumor-associated antigen, and PD-L1, an immune checkpoint protein, and can specifically bind to tumor cells expressing CEA and/or PD-L1. In addition, the fusion protein of the present invention can activate T cells without showing the toxicity seen in the existing 4-1BBL therapeutic agent. Therefore, the fusion protein of the present invention can be usefully used to treat cancer diseases.

1 is a view showing the results of analysis by SDS-PAGE after purifying the αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer according to an embodiment of the present invention.
2 is a view showing the results of SE-HPLC analysis after formulating the αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer, which is an embodiment of the present invention.
3 is a view showing the results of analysis by SDS-PAGE after purifying the αPDL1-αCEA-hyFc-4-1BBL fusion protein dimer according to an embodiment of the present invention.
4 is a view showing the results of SE-HPLC analysis after formulating the αPDL1-αCEA-hyFc-4-1BBL fusion protein dimer, which is an embodiment of the present invention.
5 is a hyFc-4-1BBLM fusion protein dimer containing 4-1BBLM (trimer), αCEA-hyFc containing a fragment (monomer) of a CEA-hyFc-4-1BBLM fusion protein dimer and a 4-1BBL variant. A diagram comparing the 4-1BBL activity of the -4-1BBL fusion protein dimer with that of urelzumab.
6 is a view confirming that T cells are specifically activated only in cancer cells expressing CEA or PD-L1 of the αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer according to an embodiment of the present invention.
7 is a diagram schematically showing that the αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer, which is an embodiment of the present invention, specifically activates T cells only in cancer cells expressing CEA or PD-L1.

Hereinafter, the present invention will be described in detail.

Fusion protein comprising anti-CEA antibody, anti-PD-L1 antibody and 4-1BBL

One aspect of the present invention, as a fusion protein of the following Structural Formula 1 or Structural Formula 2,

[Structural Formula 1]

N′-AL ₁ -BL ₂ -CL ₃ -DC′

[Structural Formula 2]

N′-BL ₁ -AL _{2 -CL 3} -DC′

The N 'is the N-terminus of the fusion protein,

The C 'is the C-terminus of the fusion protein,

wherein A is a single-chain variable fragment (scFv) of an antibody that specifically binds to a tumor-associated antigen,

wherein B is an scFv of an antibody that specifically binds to PD-L1 (Programmed death-ligand 1),

wherein C is an Fc domain of an immunoglobulin, a fragment thereof, or a variant thereof,

wherein D is 4-1BBL, a fragment thereof, or a variant thereof,

The L ₁ , L ₂ and L ₃ provide a fusion protein that is a peptide linker, respectively.

A is a tumor-associated antigen, wherein the tumor-associated antigen is not cancer-specific among antigens expressed according to cell carcinoma, and refers to an antigen present in a trace amount even in the corresponding normal cells. Specifically, A may be an scFv of an antibody that specifically binds to any one tumor-associated antigen selected from the group consisting of CEA (Carcinoembryonic antigen), AFP (α-fetoprotein), and MUC1 (Mucin 1). Specifically, A may be an scFv of an antibody that specifically binds to CEA.

The CEA is a tumor-related antigen present in the blood found in cancers such as colon cancer, breast cancer, lung cancer, liver cancer, noncancerous diseases, or smokers, and can be used to check the progress of cancer treatment and whether or not it recurs.

The AFP is a tumor-associated antigen detected in blood found in patients with carcinomas such as liver cancer, gastric cancer, ovarian fetal cancer, hepatitis, and cirrhosis. In general, AFP is a fetal serum protein that is produced during gestation and begins to decline after birth to levels observed in adults after 18 months.

The MUC1 is a protein found in specific epithelial cells, and is measured higher in breast cancer, ovarian cancer, lung cancer, and prostate cancer patients than in normal subjects. By measuring the expression level of MUC1 in the blood, treatment and recurrence of cancer can be predicted.

A may be composed of the amino acid sequence represented by SEQ ID NO: 1.

The B may consist of the amino acid sequence represented by SEQ ID NO: 2.

The C may be an Fc domain of an immunoglobulin, a fragment thereof, or a variant thereof. The variant of the Fc domain may be an Fc domain of a modified immunoglobulin or a fragment thereof. In this case, the Fc domain of the modified immunoglobulin has a modified binding ability with an Fc receptor and/or complement, thereby causing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This may be weakened. The modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof.

As used herein, the term "Fc domain", "Fc fragment" or "Fc" includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, but includes the variable regions of its heavy and light chains. and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. In addition, the immunoglobulin Fc domain fragment may include the CH2 and CH3 domains of an antibody. The CH2 and CH3 domains may be derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE. Modified Fc or modified Fc fragments are also referred to herein as “hFc” or “hyFc”.

As used herein, the term “Fc domain variant” refers to one in which some amino acids in the Fc domain are substituted or prepared by combining different types of Fc domains. The Fc domain variant can prevent cleavage at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 may be modified. Preferably, it may be a variant in which K, which is the 144th amino acid of SEQ ID NO: 9, is substituted with G or S, and E, which is the 145th amino acid, is substituted with G or S.

In addition, the Fc fragment of the present invention may be a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed. Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc fragment sharply reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of ADCC or CDC, thereby not inducing an unnecessary immune response in vivo. In this regard, the immunoglobulin Fc fragment in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier. As used herein, the term “deglycosylation” refers to enzymatic removal of sugars from an Fc fragment. In addition, the term "aglycosylation" means that the Fc fragment is produced in an unglycosylated form by prokaryotes, preferably E. coli.

In addition, the Fc domain of the modified immunoglobulin comprises the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 5 (hyFcM1), SEQ ID NO: 12 (hyFcM2), SEQ ID NO: 13 (hyFcM3) or SEQ ID NO: 14 (hyFcM4) may be doing In addition, the Fc domain of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 5 (non-lytic mouse Fc).

In one embodiment of the present invention, C used is a modified immunoglobulin Fc domain (hyFcM1), which is a variant of the immunoglobulin Fc domain, and C may consist of the amino acid sequence shown in SEQ ID NO: 5.

D may be 4-1BBL, a fragment thereof, or a variant thereof.

The 4-1BBL variant or fragment thereof may be one in which the amino acid corresponding to positions 146/241, 232/244 or 140/199 in the amino acid sequence shown in SEQ ID NO: 7 is substituted with cysteine.

As used herein, the term “4-1BB ligand” refers to a membrane protein belonging to the tumor necrosis factor superfamily called 4-1BBL or CD137L. 4-1BBL is found in antigen presenting cells. In addition, 4-1BBL can specifically bind to 4-1BB. In this case, the wild-type 4-1BBL may be a natural 4-1BBL found in mammals. In one embodiment, the 4-1BBL may consist of the amino acid sequence represented by SEQ ID NO: 7, which is 4-1BBL derived from humans.

Human-derived 4-1BBL consists of an intracellular domain (amino acids 1 to 28), a transmembrane protein (amino acids 29 to 49), and an extracellular domain (amino acids 50 to 254). there is. The extracellular region was formed as a secondary structure comprising a total of 10 beta strands (PDB 2X29). The first beta strand starts at amino acid 83, and the last beta strand consists of amino acids 239. That is, in the case of a fragment including amino acids 83 to 239, it is known that there is activity because it contains all secondary structures.

In one embodiment of the 4-1BBL variant, the 146th and 241th amino acids (146/241) in the amino acid sequence shown in SEQ ID NO: 7 may be substituted with cysteine. That is, the 4-1BBL mutant may be Q146C and T241C.

Another embodiment of the 4-1BBL variant may be one in which the 232th and 244th amino acids (232/244) in the amino acid sequence shown in SEQ ID NO: 7 are substituted with cysteine. That is, the 4-1BBL variant may be A232C and I244C.

Another embodiment of the 4-1BBL variant may be one in which the 140th and 199th amino acids (140/199) in the amino acid sequence shown in SEQ ID NO: 7 are substituted with cysteine. That is, the 4-1BBL mutant may be V140C and F199C.

In this case, the fragment of the 4-1BBL mutant may be a mutated extracellular domain (ECD) of 4-1BBL. As used herein, "extracellular region" refers to a region exposed outside the cell as a part of a membrane protein. In this case, it may refer to a portion excluding the transmembrane domain and the intracellular region. One specific example of the extracellular region of 4-1BBL may be a polypeptide (SEQ ID NO: 17) comprising the 50th amino acid to the 254th amino acid of human 4-1BBL, specifically, the 83rd amino acid to the 239th amino acid It may mean a polypeptide comprising (SEQ ID NO: 18). In this case, the mutated extracellular region of 4-1BBL may consist of an amino acid sequence of any one of SEQ ID NOs: 22 to 27.

In addition, the fragment of the 4-1BBL mutant refers to a part of the extracellular region of 4-1BBL including the mutations of 146/241, 232/244 or 140/199 described above. Specifically, in the fragment of the 4-1BBL variant, positions 146/241, 232/244 or 140/199 in the amino acid sequence of SEQ ID NO: 17 are substituted with cysteine, and starting from the amino acid at position 1 of SEQ ID NO: 17, N - from terminal to C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33 amino acid residues may be deleted. In addition, starting from the amino acid at position 205 of SEQ ID NO: 17, 1, 2, 3, 4, 5, 6, 7, 8, 9 from the C-terminus to the N-terminus , 10, 11, 12, 13, 14 or 15 amino acid residues may be deleted.

Deletion of the amino acid residue may occur in either or both of the "N-terminal to C-terminal direction" and "C-terminal to N-terminal direction" described above. Specifically, the extracellular region fragment of 4-1BBL in which the amino acid residue is deleted may consist of the amino acid sequence of SEQ ID NO: 18. More specifically, the mutated extracellular region fragment of 4-1BBL in which the amino acid residue is deleted may consist of the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27.

One embodiment of such a fragment of the 4-1BBL variant may be represented by a polypeptide represented by the following Structural Formula 3:

[Structural Formula 3]

N-terminal extension domain-core domain-C-terminal extension domain (I)

In the above formula (I),

the core domain is a polypeptide having the amino acid sequence of SEQ ID NO: 28, 29 or 30;

The N-terminal extension domain is a polypeptide having the amino acid sequence of SEQ ID NO: 31, in which 1 to 33 amino acids are consecutively deleted from the N-terminus to the C-terminus starting from the amino acid at position 1 of SEQ ID NO: 31. can;

The C-terminal extension domain is a polypeptide having the amino acid sequence of SEQ ID NO: 32, in which 1 to 15 amino acids are consecutively deleted from the C-terminus to the N-terminus starting from the amino acid at position 16 of SEQ ID NO: 32. can

In this case, the N-terminal extension domain may be one in which 30 amino acids are consecutively deleted from the N-terminus to the C-terminus starting from the amino acid at position 1 of SEQ ID NO: 31. In addition, the N-terminal extension domain may be one in which 22 amino acids are consecutively deleted from the N-terminus to the C-terminus starting from the amino acid at position 1 of SEQ ID NO: 31. In addition, the N-terminal extension domain may be one in which 17 amino acids are consecutively deleted from the N-terminus to the C-terminus starting from the amino acid at position 1 of SEQ ID NO: 31. In addition, the C-terminal extension domain may be one in which 7 amino acids are consecutively deleted from the C-terminus to the N-terminus starting from the amino acid at position 16 of SEQ ID NO: 32.

One embodiment of the fragment of the 4-1BBL mutant may consist of the amino acid sequence of any one of SEQ ID NOs: 28 to 30. In another embodiment, the fragment of the 4-1BBL mutant may consist of any one amino acid sequence selected from among the amino acid sequences of SEQ ID NO: 33 to SEQ ID NO: 41. In another embodiment, the fragment of the 4-1BBL mutant may consist of any one amino acid sequence selected from SEQ ID NO: 42 to SEQ ID NO: 44. In another embodiment, the fragment of the 4-1BBL mutant may consist of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 26.

That is, D may consist of any one amino acid sequence selected from SEQ ID NO: 22 to SEQ ID NO: 44.

The A and B may be coupled through ₁ to 50 peptide linkers (first linker, L 1 ). Specifically, the peptide linker comprises 1 to 50 contiguous amino acids, 5 to 45 contiguous amino acids, 10 to 40 contiguous amino acids, or 15 to 35 contiguous amino acids, or 20 to 30 contiguous amino acids. It may consist of amino acids. In one embodiment, L ₁ may consist of 20 amino acids. The A and B may be coupled through _{a peptide linker (first linker, L 1} ) consisting of the amino acid sequence shown in SEQ ID NO: 3.

The B and C may be coupled through 1 to 50 peptide linkers (second linker, L _{2 ).} wherein L ₁ is 1 to 50 consecutive amino acids, 5 to 45 consecutive amino acids, 10 to 40 consecutive amino acids, or 15 to 35 consecutive amino acids, or 20 to 30 amino acids can In one embodiment, L ₂ may consist of 30 amino acids. In addition, L ₂ may include at least one cysteine. Specifically, L ₂ may include one, two, or three cysteines. In addition, the L ₂ may be derived from the hinge of the immunoglobulin. In one embodiment, L ₂ may be composed of the amino acid sequence represented by SEQ ID NO: 16.

The C and D may be coupled through 1 to 50 peptide linkers (third linker, L _{3 ).} Specifically, the peptide linker may consist of 1 to 50 consecutive amino acids. In one embodiment, L ₃ may consist of 5 amino acids. The C and D may be coupled through _{a peptide linker (third linker, L 3} ) consisting of the amino acid sequence shown in SEQ ID NO: 6.

The fusion protein may consist of the amino acid sequence represented by SEQ ID NO: 15.

Another aspect of the present invention provides a fusion protein dimer in which two fusion proteins comprising the anti-CEA antibody, the anti-PD-L1 antibody and 4-1BBL are bound. The fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and 4-1BBL is as described above.

In this case, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond by a cysteine present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same, but may be different fusion proteins. Preferably, the dimer may be a homodimer.

Polynucleotide encoding the fusion protein

Another aspect of the present invention provides a polynucleotide encoding a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and 4-1BBL. Specifically, the polynucleotide may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 15. The fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and 4-1BBL is as described above. As long as the polynucleotides encode the same amino acid sequence, one or more bases may be substituted.

The polynucleotide may further include a nucleic acid encoding a signal sequence. As used herein, the term “signal sequence” refers to a signal peptide that directs secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates the movement of the protein through the ER (endoplasmic reticulum) membrane.

The signal sequence is well known in the art, and typically contains 16 to 30 amino acid residues, but may include more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.

After initiation, the signal sequence is cleaved in the lumen of the ER by cellular enzymes commonly known as signal peptidases. In this case, the signal sequence may be a secretion signal sequence of tissue Plasminogen Activation (tPa), a signal sequence of Herpes simplex virus glycoprotein D (HSV gDs), or a growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells including mammals and the like may be used.

Vector loaded with polynucleotide encoding the fusion protein

Another aspect of the present invention provides a vector comprising the polynucleotide.

The vector can be introduced into a host cell, recombination and insertion into the host cell genome. or said vector is understood as a nucleic acid means comprising a polynucleotide sequence capable of spontaneous replication as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

Specifically, the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retroviruses) ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the vector exhibits different protein expression levels and modifications depending on the host cell, it is preferable to select and use a host cell most suitable for the purpose.

As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector includes a human cytomegalovirus (CMV) promoter to promote continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the stable-state level of RNA after transcription. can do.

Transformed cells expressing the fusion protein

Another aspect of the present invention provides a transformed cell into which the vector is introduced.

The host cell of the transformed cell may include, but is not limited to, a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin. As an example of the prokaryotic cell, E. coli may be used. In addition, yeast may be used as an example of eukaryotic cells. In addition, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells or HEK293T cells may be used as the mammalian cells. , but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art is available.

In addition, when introducing an expression vector into a host cell, the CaCl ₂ precipitation method, the CaCl ₂ precipitation method using a reducing material called DMSO (dimethyl sulfoxide), which increases the efficiency by using the Hanahan method, electroporation method, calcium phosphate precipitation method , protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition mediated transformation method can be used.

As described above, in order to optimize the properties of the fusion protein as a therapeutic agent or for other purposes, the glycosylation-related gene possessed by the host cell is manipulated through a method known to those skilled in the art, and the sugar chain pattern of the fusion protein (e.g., sialic acid, fucosylation, glycation) can be adjusted.

Fusion protein production method

Another aspect of the present invention provides a method for producing a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody, and 4-1BBL, comprising culturing the transformed cell. Specifically, the production method comprises the steps of i) culturing the transformed cells to obtain a culture; and ii) recovering the fusion protein from the culture.

The method of culturing the transformed cells may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or injection batch or repeated fed batch process (fed batch or repeated fed batch process).

Use of a fusion protein or a dimer thereof

Another aspect of the present invention is a fusion protein comprising po, an anti-CEA antibody, an anti-PD-L1 antibody and 4-1BBL or a fusion protein dimer to which the two fusion proteins are bound as an active ingredient. Prevention of cancer Or it provides a pharmaceutical composition for treatment.

The fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and 4-1BBL or the dimer of the fusion protein to which the two fusion proteins are bound is as described above.

The fusion protein or dimer thereof of the present invention may specifically bind to CEA, a tumor-associated antigen expressed in tumor cells, by including an scFv of an antibody that specifically binds to CEA. In addition, the fusion protein of the present invention or a dimer thereof includes an antibody scFv that specifically binds to PD-L1, thereby binding to PD-L1 expressed in tumor cells and inhibiting PD-L1/PD-1 binding. Thus, it is possible to block the immune evasion mechanism of tumor cells. In addition, the fusion protein or a dimer thereof of the present invention can activate immune cells by including 4-1BBL or a fragment thereof or a variant thereof. That is, the fusion protein of the present invention or a dimer thereof specifically binds to tumor cells, can block the immune evasion mechanism of tumor cells, and can activate immune cells around tumor cells, thereby preventing or treating cancer diseases. can be usefully used for

The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. may be selected from the group.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. In the pharmaceutical composition for treatment or prevention of cancer or infectious disease of the present invention, the active ingredient exhibits anticancer activity or an arbitrary amount (effective amount) depending on the use, formulation, purpose of formulation, etc. as long as it can exhibit a therapeutic effect on infectious disease may be included, and a typical effective amount will be determined within the range of 0.001 wt % to 20.0 wt % based on the total weight of the composition. Herein, the term “effective amount” refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

As used herein, the term “treatment” may be used to include both therapeutic treatment and prophylactic treatment. In this case, prevention may be used in the sense of alleviating or reducing a pathological condition or disease of an individual. In one embodiment, the term “treatment” includes any form of administration or application for treating a disease in a mammal, including a human. The term also includes inhibiting or slowing the disease or its progression; restoring or repairing damaged or missing function, partially or completely relieving the disease; or stimulate inefficient processes; It includes the meaning of alleviating serious diseases.

As used herein, the term “efficacy” refers to one or more parameters, such as survival or disease-free survival over a period of time, such as 1 year, 5 years, or 10 years. can be determined by In addition, the parameter may include suppressing the size of at least one tumor in the subject.

Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Thus, "enhanced efficacy" (e.g., improvement in efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, comparing clearance rates and tumor growth in test animals or human subjects, or whether survival, recurrence rates, or disease It can be measured by comparing parameters such as survival in the absence.

Herein, "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition effective to prevent or treat a target disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and It means an amount that does not cause side effects. The level of the effective amount may be determined by the patient's health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or factors including drugs used concurrently and other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of a drug effective to treat cancer.

In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond which the application (prescription) target is adaptable.

When the pharmaceutical composition is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, a suitable carrier may be sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterilized for injection. Water, an isotonic solution such as 5% dextrose, etc. may be used. Formulation of pharmaceutical compositions is known in the art, and specifically, reference may be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)] and the like. This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, patient's severity, and administration route. can be Administration may be performed once or divided into several times a day. Such dosages should not be construed as limiting the scope of the present invention in any respect.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract known to have an anticancer activity or therapeutic effect on an infectious disease, and safety has already been verified for the increase/reinforcement of anticancer activity.

The administration route, dosage, and frequency of administration of the fusion protein or fusion protein dimer may be administered to a subject in various ways and amounts depending on the patient's condition and presence or absence of side effects, and the optimal administration method, dosage and frequency of administration are A person skilled in the art can select within an appropriate range. In addition, the fusion protein or fusion protein dimer may be administered in combination with other drugs or physiologically active substances with known therapeutic effects on the disease to be treated, or formulated in the form of a combination preparation with other drugs.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1. Preparation of αCEA-αPDL1-hyFc-4-1BBL gene construct

In order to prepare a fusion protein in which the scFv (αCEA) of the anti-CEA antibody, the scFv (αPDL1) of the anti-PD-L1 antibody, and the fragment of the 4-1BBL mutant are combined, αCEA, αCEA, The αCEA-αPDL1-hyFc-4-1BBL gene construct in which linker 1, αPDL1, second linker, Fc domain (hyFc), third linker, and fragments of the 4-1BBL mutant are combined in order was prepared.

Specifically, first, using the amino acid sequence of hMN-14, which is a known amino acid sequence (Accession number: DB05097), an anti-CEA antibody scFv having the amino acid sequence shown in SEQ ID NO: 1 was prepared. In addition, an anti-PD-L1 antibody scFv having the amino acid sequence shown in SEQ ID NO: 2 was prepared using the amino acid sequence of the anti-PD-L1 antibody. The prepared scFv of the anti-CEA antibody and the scFv of the anti-PD-L1 antibody were combined with a peptide linker (first linker) consisting of the amino acid sequence shown in SEQ ID NO: 3, which was combined with SEQ ID NO: on the N-terminal side of the FC domain. It was bound with a peptide linker (second linker) consisting of the amino acid sequence represented by 16.

In addition, the fragment of the 4-1BBL mutant contains a mutation introducing a disulfide bond, so that the amino acids corresponding to positions 146/241, 232/244 or 140/199 of wild-type 4-1BBL are converted to cysteine so that the monomer is well trimerized. The extracellular region of the substituted 4-1BBL mutant was prepared as a fragment, and the one consisting of the amino acid sequence shown in SEQ ID NO: 33 was used. In addition, a trimer (4-1BBLM) of the fragment of the mutant 4-1BBL was prepared by linking three fragments of the mutant 4-1BBL by a linker. The fragment of the 4-1BBL mutant or 4-1BBLM was bound to the C-terminal side of the FC domain with a peptide linker (third linker) consisting of the amino acid sequence shown in SEQ ID NO: 6.

Then, a gene encoding each amino acid sequence was synthesized and prepared as a sub-vector. To prepare the four synthesized genes into one gene construct, Golden GATE way assembly was performed to obtain an expression vector using pBispec vector.

In the same manner as for the preparation of the αCEA-αPDL1-hyFc-4-1BBL gene construct, αPDL1, the first linker, αCEA, the second linker, hyFc, the third linker and 4 An expression vector containing the αPDL1-αCEA-hyFc-4-1BBL gene construct bound in the -1BBL sequence was prepared.

In addition, in the same method as the preparation method of the αCEA-αPDL1-hyFc-4-1BBL gene construct, as a comparative example, hyFc, the third linker, and 4-1BBLM were linked in the order from the N-terminus to the C-terminus. An expression vector containing the hyFc-4-1BBLM gene construct in the form was prepared. In addition, an expression vector containing the αCEA-hyFc-4-1BBLM gene construct in the form of αCEA, the second linker, hyFc, the third linker, and 4-1BBLM combined in the order from the N-terminus to the C-terminus was prepared did. Furthermore, an expression vector containing the αCEA-hyFc-4-1BBL gene construct in the form of αCEA, the second linker, hyFc, the third linker, and 4-1BBL combined in the order from the N-terminus to the C-terminus was prepared did.

Example 2. αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer production

To produce an αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer, suspension-adapted CHO cells were seeded at 6×10 ⁶ cells/ml, and then αCEA-αPDL1-hyFc prepared in Example 1 Transformation was achieved by treatment with 200 ml of an expression vector containing the -4-1BBL gene construct and ExpiFectamine ^{TM CHO reagent.} In order to secure a large amount of αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer, ExpiFectamine ^TM CHO enhancer and ExpiCHO ^TM feed were added the day after transformation, and the incubation temperature was increased to 32°C. shifted. After 7 days, the culture medium was collected and centrifuged at 3,600 rpm for 15 minutes to obtain a supernatant. Thereafter, the supernatant was filtered using a 0.22 μm filter. At this time, the produced αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer was named “IO-129”.

To purify the αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer from the supernatant, αCEA-αPDL1-hyFc-4-1BBL containing an Fc domain using AKTA Prime (GE Healthcare Life sciences) and Protein A resin The fusion protein dimer was purified. As the elution buffer, 100 mM Glycine-HCL, 50 mM L-Arginine and 5% Glycerol (pH 3.0) were used.

As a result, a fusion protein dimer with a purity of 90% or more was obtained only by purification using Protein A resin. Only a fraction with high purity was isolated and formulated in 50 mM hepes, 250 mM sucrose, pH 5.0. As a result of confirming the formulated αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer through SDS-PAGE, it was confirmed at about 250 kDa under non-reducing conditions, and about 250 kDa under reducing conditions. It was confirmed at 100 kDa ( FIG. 1 , non-reducing: fusion protein dimer, reduced: fusion protein monomer). In addition, as a result of confirming the purity through SE-HPLC analysis, a purity of 99.8% was confirmed (FIG. 2).

In addition, prepared in Example 1 The αPDL1-αCEA-hyFc-4-1BBL fusion protein was prepared in the same manner as for the production of the αCEA-αPDL1-hyFc-4-1BBL fusion protein using an expression vector containing the αPDL1-αCEA-hyFc-4-1BBL gene construct. produced. At this time, the αPDL1-αCEA-hyFc-4-1BBL fusion protein was named “IO-130”.

The αPDL1-αCEA-hyFc-4-1BBL fusion protein was purified from the supernatant, and only a high-purity fraction was isolated and formulated in 50 mM hepes, 250 mM sucrose, pH 5.0. As a result of confirming the formulated αPDL1-αCEA-hyFc-4-1BBL fusion protein through SDS-PAGE, it was confirmed at about 250 kDa under non-reducing conditions and at about 100 kDa under reducing conditions (FIG. 3). In addition, as a result of confirming the purity through SE-HPLC analysis, a purity of 91.4% was confirmed (FIG. 4).

In addition, prepared in Example 1 Using the expression vector containing the hyFc-4-1BBLM gene construct, the expression vector containing the αCEA-hyFc-4-1BBLM gene construct, and the expression vector containing the αCEA-hyFc-4-1BBL gene construct hyFc-4-1BBLM fusion protein (SEQ ID NO: 19) dimer, αCEA-hyFc-4-1BBLM fusion protein (SEQ ID NO: 20) dimer and A dimer of αCEA-hyFc-4-1BBLM fusion protein (SEQ ID NO: 21) was produced.

Example 3. Comparison of activity between 4-1BBLM (trimer) and 4-1BBL (monomer)

4-1BBL basically forms a trimer structure, and the activity of the trimer is greater than that of the monomer. However, the activity of 4-1BBL increases exponentially as it forms a trimer, a hexamer, or a dodecer, and this has a side effect that toxicity appears when used as a monotherapy as in the case of urelumab.

Therefore, in order to compare the activity of a fusion protein dimer including a fragment (monomer) or 4-1BBLM (trimer) of a 4-1BBL variant, Promega, 4-1BB Bioassay kit was used. Recombinant 4-1BBL (R&D systems, Cat no. 2295-4L-025/CF) was cross-linked with an anti-His tag Ab (Biolegend, Cat no. 652502, B2223517) with a control and hyFc-4-1BBLM fusion protein , αCEA-hyFc-4-1BBLM fusion protein dimer, αCEA-hyFc-4-1BBL fusion protein dimer, and urelumab, which showed toxicity in clinical practice due to high activity, were used to measure the activity. Specifically, NFκB-Luc2/4-1BB Jurkat cells (Promega, Cat.No. J235A) were dispensed on each plate, and each fusion protein was diluted 3 times from 441 μg and treated, followed by 37°C 5% CO ₂ condition. Incubated for 6 hours and the luminescence (luminescence) was measured.

As a result, compared to the control group and the αCEA-hyFc-4-1BBL fusion protein dimer, the activity of the hyFc-4-1BBLM fusion protein and the αCEA-hyFc-4-1BBLM fusion protein dimer surpassed that of urelumab, 4 - In the case of the fusion protein dimer containing -1BBLM (trimer), it was confirmed that there is a possibility that toxicity may appear (FIG. 5).

Through this, αCEA-αPDL1-hyFc-4-1BBL fusion protein dimers and αPDL1-αCEA-hyFc-4-1BBL fusion protein dimers including fragments (monomers) of the 4-1BBL variant were finally selected.

Example 4. Confirmation of tumor antigen-specific activation of αCEA-αPDL1-hyFc-4-1BBL fusion protein dimer

Based on the results obtained in Example 3, a fusion protein comprising a fragment (monomer) of a 4-1BBL mutant is specifically bound to cancer cells expressing CEA, a tumor antigen, or cancer cells expressing PD-L1, an immune checkpoint protein. We tried to determine whether the activity of 4-1BBL can be revealed only in tumor tissue. Specifically, CEA-expressing MKN45 cancer cells (Korea Cell Line Bank (KCLB), Cat. No. 80103) and NFκB-Luc2/4-1BB Jurkat cells were co-cultured. In addition, MDA-MB-231 cancer cells expressing PD-L1 (Korea Cell Line Bank (KCLB), Cat. No. 30026) and NFκB-Luc2/4-1BB Jurkat cells were co-cultured. The co-cultured cells and NFκB-Luc2/4-1BB Jurkat cells were treated with urelumab, αCEA-αPDL1-hyFc-h4-1BBL fusion protein dimer or αPDL1-αCEA-hyFc-h4-1BBL fusion protein dimer, respectively. Then, it was confirmed by using the 4-1BB Bioassay kit (Promega, Cat. No. J235A) whether T cell activation was induced by the fusion protein dimer.

As a result, in the case of urelumab, the degree of T cell activation was high even in the absence of CEA or PD-L1 expressing cancer cells. confirmed that. On the other hand, in the case of αCEA-αPDL1-hyFc-h4-1BBL fusion protein dimer and αPDL1-αCEA-hyFc-h4-1BBL fusion protein dimer, T cells are activated only when co-cultured with cancer cells expressing CEA or PD-L1. and T cell activity was higher when the degree of activation was co-cultured with cancer cells expressing PD-L1 (FIG. 6).

Through this, the fusion protein dimer including the fragment (monomer) of the 4-1BBL mutant binds specifically to CEA or PD-L1, thereby activating T cells, and cancer cells expressing CEA or PD-L1 It was confirmed that by accumulating a fragment (monomer) of the 4-1BBL mutant in the tumor tissue, the activity of 4-1BBL was exhibited (FIG. 7).

<110> Genexine, Inc. <120> FUSION PROTEIN COMPRISING ANTI-TUMOR-ASSOCIATED ANTIGEN ANTIBODY, ANTI-PD-L1 ANTIBODY AND 4-1BBL AND USES THEREOF <130> FPD/201911-0091 <160> 44 <170> KoPatentIn 3.0 <210> 1 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> anti-CEA antibody of scFv <400> 1 Glu 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 Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Tyr Ser Leu Tyr Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 240 <210> 2 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 antibody of scFv <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Ser 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 Val Tyr Ile Cys 85 90 95 Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Thr Pro Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ser Thr Ser Ser Arg Tyr Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 His Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> linker 1 (L1) <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> IgG1 hinge <400> 4 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 5 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> hyFcM1 <400> 5 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker (L3) <400> 6 Gly Gly Gly Gly Ser 1 5 <210> 7 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> wild type human 4-1BBL <400> 7 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 <210> 8 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> wild type mouse 4-1BBL <400> 8 Met Asp Gln His Thr Leu Asp Val Glu Asp Thr Ala Asp Ala Arg His 1 5 10 15 Pro Ala Gly Thr Ser Cys Pro Ser Asp Ala Ala Leu Leu Arg Asp Thr 20 25 30 Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr 35 40 45 Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg 50 55 60 Asp Arg Glu Ala Ala Trp Pro Ala Leu Asn Phe Cys Ser Arg His 65 70 75 80 Pro Lys Leu Tyr Gly Leu Val Ala Leu Val Leu Leu Leu Leu Leu Ile Ala 85 90 95 Ala Cys Val Pro Ile Phe Thr Arg Thr Glu Pro Arg Pro Ala Leu Thr 100 105 110 Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln 115 120 125 Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly 130 135 140 Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys 145 150 155 160 Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr 165 170 175 Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val 180 185 190 Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu Ser Pro 195 200 205 Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val 210 215 220 Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr 225 230 235 240 Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser 245 250 255 Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly 260 265 270 Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg Asp Trp Glu 275 280 285 Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu Val Lys Pro 290 295 300 Asp Asn Pro Trp Glu 305 <210> 9 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> hyFc <400> 9 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 10 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> human IgD constant region (Genbank accession No. P01880) <400> 10 Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg 1 5 10 15 His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30 Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45 Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55 60 Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly 65 70 75 80 Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95 Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110 Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125 Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu 145 150 155 160 Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170 175 Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190 Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205 Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215 220 Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu 225 230 235 240 Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255 Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270 Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285 Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300 Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe 305 310 315 320 Ala Pro Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala 325 330 335 Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr 340 345 350 Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365 Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys 370 375 380 <210> 11 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Partial human IgG4 constant region (Genbank accession No. AH25985) <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> hyFcM2 <400> 12 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 13 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> hyFcM3 <400> 13 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Gly Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 14 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> hyFcM4 <400> 14 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 15 <211> 926 <212> PRT <213> Artificial Sequence <220> <223> anti-CEA-anti-PDL1-hyFc-4-1BBL fusion protein <400> 15 Glu 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 Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Tyr Ser Leu Tyr Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 260 265 270 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 275 280 285 Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser 290 295 300 Leu Glu Trp Val Ala Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr 305 310 315 320 Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 325 330 335 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 340 345 350 Val Tyr Ile Cys Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr 355 360 365 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 370 375 380 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 385 390 395 400 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 405 410 415 Cys Lys Ala Ser Gln Asp Val Thr Pro Ala Val Ala Trp Tyr Gln Gln 420 425 430 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr Ser Ser Arg 435 440 445 Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 450 455 460 Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 465 470 475 480 Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu Thr Phe Gly Gln Gly Thr 485 490 495 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 500 505 510 Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 515 520 525 Pro Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro 530 535 540 Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 545 550 555 560 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 565 570 575 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 580 585 590 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 595 600 605 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 610 615 620 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 625 630 635 640 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 645 650 655 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 660 665 670 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 675 680 685 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 690 695 700 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 705 710 715 720 Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr 725 730 735 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Asp 740 745 750 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 755 760 765 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 770 775 780 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 785 790 795 800 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 805 810 815 Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 820 825 830 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 835 840 845 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Ala Ser Ser Glu Ala 850 855 860 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 865 870 875 880 Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 885 890 895 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 900 905 910 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 915 920 925 <210> 16 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> linker (L2) <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 1 5 10 15 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 20 25 30 <210> 17 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL extracellular domain <400> 17 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1 5 10 15 Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala 35 40 45 Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro 50 55 60 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 65 70 75 80 Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe 85 90 95 Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 100 105 110 Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 115 120 125 Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 130 135 140 Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly 145 150 155 160 Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala 165 170 175 Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr 180 185 190 Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 195 200 205 <210> 18 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL extracellular core domain <400> 18 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 1 5 10 15 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 20 25 30 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 35 40 45 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln 50 55 60 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 65 70 75 80 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 85 90 95 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 100 105 110 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 115 120 125 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 130 135 140 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 145 150 155 <210> 19 <211> 847 <212> PRT <213> Artificial Sequence <220> <223> hyFc-4-1BBLM <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 1 5 10 15 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys Ser Pro Lys Ala Gln Ala Gly Gly Gly Gly Ser 245 250 255 Ala Gln Pro Gln Ala Glu Gly Ser Leu Gly Gly Gly Gly Ser Ala Lys 260 265 270 Ala Ser Ala Pro Ala Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu Leu 275 280 285 Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu 290 295 300 Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly 305 310 315 320 Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu 325 330 335 Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys Leu Glu Leu 340 345 350 Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu 355 360 365 His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu 370 375 380 Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe 385 390 395 400 Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly 405 410 415 Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr 420 425 430 Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys Pro Glu Ile Pro 435 440 445 Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Ser Gly Gly Ser Gly 450 455 460 Gly Ser Gly Gly Ser Gly Gly Ser Glu Gln Glu Glu Arg Asp Pro Ala 465 470 475 480 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 485 490 495 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 500 505 510 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 515 520 525 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys 530 535 540 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 545 550 555 560 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 565 570 575 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 580 585 590 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 595 600 605 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 610 615 620 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys Pro 625 630 635 640 Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Ser Gly 645 650 655 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Gln Glu Glu Arg 660 665 670 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 675 680 685 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 690 695 700 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 705 710 715 720 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 725 730 735 Phe Phe Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 740 745 750 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 755 760 765 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 770 775 780 Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 785 790 795 800 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 805 810 815 His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 820 825 830 Val Cys Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 835 840 845 <210> 20 <211> 1088 <212> PRT <213> Artificial Sequence <220> <223> anti-CEA-hyFc-4-1BBLM <400> 20 Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr 20 25 30 Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser 50 55 60 Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe 85 90 95 Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala 145 150 155 160 Ser Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His Thr Gly 180 185 190 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe 195 200 205 Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Tyr Ser Leu Tyr Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser 260 265 270 His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 275 280 285 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 290 295 300 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 305 310 315 320 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 325 330 335 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 340 345 350 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 355 360 365 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 370 375 380 Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn 385 390 395 400 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 405 410 415 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 420 425 430 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 435 440 445 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 450 455 460 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 465 470 475 480 Ser Leu Ser Leu Gly Lys Ser Pro Lys Ala Gln Ala Gly Gly Gly Gly 485 490 495 Ser Ala Gln Pro Gln Ala Glu Gly Ser Leu Gly Gly Gly Gly Gly Ser Ala 500 505 510 Lys Ala Ser Ala Pro Ala Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu 515 520 525 Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val 530 535 540 Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala 545 550 555 560 Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 565 570 575 Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys Leu Glu 580 585 590 Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala 595 600 605 Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala 610 615 620 Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala 625 630 635 640 Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu 645 650 655 Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu 660 665 670 Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys Pro Glu Ile 675 680 685 Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Ser Gly Gly Ser 690 695 700 Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Gln Glu Glu Arg Asp Pro 705 710 715 720 Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala 725 730 735 Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro 740 745 750 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 755 760 765 Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe 770 775 780 Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 785 790 795 800 Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 805 810 815 Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 820 825 830 Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly 835 840 845 Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala 850 855 860 Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys 865 870 875 880 Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Ser 885 890 895 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Gln Glu Glu 900 905 910 Arg Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln 915 920 925 Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr 930 935 940 Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr 945 950 955 960 Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr 965 970 975 Val Phe Phe Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser 980 985 990 Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala 995 1000 1005 Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser 1010 1015 1020 Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu 1025 1030 1035 1040 Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala 1045 1050 1055 Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe 1060 1065 1070 Arg Val Cys Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 1075 1080 1085 <210> 21 <211> 666 <212> PRT <213> Artificial Sequence <220> <223> anti-CEA-hyFc-4-1BBL <400> 21 Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr 20 25 30 Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser 50 55 60 Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe 85 90 95 Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala 145 150 155 160 Ser Gln Asp Val Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His Thr Gly 180 185 190 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe 195 200 205 Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Tyr Ser Leu Tyr Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser 260 265 270 His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 275 280 285 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 290 295 300 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 305 310 315 320 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 325 330 335 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 340 345 350 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 355 360 365 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 370 375 380 Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn 385 390 395 400 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 405 410 415 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 420 425 430 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 435 440 445 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 450 455 460 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 465 470 475 480 Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu 485 490 495 Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val 500 505 510 Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala 515 520 525 Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 530 535 540 Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu 545 550 555 560 Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala 565 570 575 Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala 580 585 590 Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala 595 600 605 Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu 610 615 620 Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu 625 630 635 640 Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile 645 650 655 Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 660 665 <210> 22 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) extracellular domain <400> 22 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1 5 10 15 Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala 35 40 45 Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro 50 55 60 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 65 70 75 80 Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe 85 90 95 Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 100 105 110 Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 115 120 125 Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 130 135 140 Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly 145 150 155 160 Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala 165 170 175 Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys 180 185 190 Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 195 200 205 <210> 23 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) extracellular core domain <400> 23 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 1 5 10 15 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 20 25 30 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 35 40 45 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys 50 55 60 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 65 70 75 80 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 85 90 95 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 100 105 110 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 115 120 125 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 130 135 140 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 145 150 155 <210> 24 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) extracellular domain <400> 24 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1 5 10 15 Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala 35 40 45 Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro 50 55 60 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 65 70 75 80 Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe 85 90 95 Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 100 105 110 Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 115 120 125 Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 130 135 140 Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly 145 150 155 160 Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala 165 170 175 Trp Gln Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe Arg Val Thr 180 185 190 Pro Glu Cys Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 195 200 205 <210> 25 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) extracellular core domain <400> 25 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 1 5 10 15 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 20 25 30 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 35 40 45 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln 50 55 60 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 65 70 75 80 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 85 90 95 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 100 105 110 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 115 120 125 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 130 135 140 Gln Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe Arg 145 150 155 <210> 26 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) extracellular domain <400> 26 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1 5 10 15 Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala 35 40 45 Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro 50 55 60 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 65 70 75 80 Thr Lys Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val Phe Phe 85 90 95 Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 100 105 110 Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 115 120 125 Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 130 135 140 Asn Ser Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser Ala Gly 145 150 155 160 Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala 165 170 175 Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr 180 185 190 Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 195 200 205 <210> 27 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) extracellular core domain <400> 27 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 1 5 10 15 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 20 25 30 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 35 40 45 Lys Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val Phe Phe Gln 50 55 60 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 65 70 75 80 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 85 90 95 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 100 105 110 Ser Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 115 120 125 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 130 135 140 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 145 150 155 <210> 28 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) extracellular core domain (deletion of N,C-terminal amino acid residues) <400> 28 Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn 1 5 10 15 Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu 20 25 30 Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys 35 40 45 Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys Leu 50 55 60 Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu 65 70 75 80 Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu 85 90 95 Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser 100 105 110 Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg 115 120 125 Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln 130 135 140 Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe 145 150 155 <210> 29 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) extracellular core domain (deletion of N,C-terminal amino acid residues) <400> 29 Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn 1 5 10 15 Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu 20 25 30 Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys 35 40 45 Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu 50 55 60 Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu 65 70 75 80 Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu 85 90 95 Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser 100 105 110 Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg 115 120 125 Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln 130 135 140 Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe 145 150 155 <210> 30 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) extracellular core domain (deletion of N,C-terminal amino acid residues) <400> 30 Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn 1 5 10 15 Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu 20 25 30 Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys 35 40 45 Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val Phe Phe Gln Leu 50 55 60 Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu 65 70 75 80 Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu 85 90 95 Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser 100 105 110 Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg 115 120 125 Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln 130 135 140 Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe 145 150 155 <210> 31 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL N-terminal extension domian <400> 31 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1 5 10 15 Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Ala Gly <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL C-terminal extension domian <400> 32 Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 1 5 10 15 <210> 33 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) fragment (deletion of N-termianl) amino acid residues) <400> 33 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 1 5 10 15 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 20 25 30 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 35 40 45 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 50 55 60 Phe Phe Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 65 70 75 80 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 85 90 95 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 100 105 110 Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 115 120 125 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 130 135 140 His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 145 150 155 160 Val Cys Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 165 170 175 <210> 34 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) fragment (deletion of N-termianl) amino acid residues) <400> 34 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 1 5 10 15 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 20 25 30 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 35 40 45 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 50 55 60 Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 65 70 75 80 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 85 90 95 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 100 105 110 Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 115 120 125 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 130 135 140 His Ala Trp Gln Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe Arg 145 150 155 160 Val Thr Pro Glu Cys Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 165 170 175 <210> 35 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) fragment (deletion of N-termianl) amino acid residues) <400> 35 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 1 5 10 15 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 20 25 30 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 35 40 45 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val 50 55 60 Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 65 70 75 80 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 85 90 95 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 100 105 110 Ala Arg Asn Ser Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser 115 120 125 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 130 135 140 His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 145 150 155 160 Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 165 170 175 <210> 36 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) fragment (deletion of N-termianl) amino acid residues) <400> 36 Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg 1 5 10 15 Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp 20 25 30 Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu 35 40 45 Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala 50 55 60 Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys Leu Glu Leu Arg Arg Val 65 70 75 80 Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln 85 90 95 Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp 100 105 110 Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln 115 120 125 Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu 130 135 140 His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala 145 150 155 160 Thr Val Leu Gly Leu Phe Arg Val Cys Pro Glu Ile Pro Ala Gly Leu 165 170 175 Pro Ser Pro Arg Ser Glu 180 <210> 37 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) fragment (deletion of N-termianl) amino acid residues) <400> 37 Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg 1 5 10 15 Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp 20 25 30 Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu 35 40 45 Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala 50 55 60 Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val 65 70 75 80 Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln 85 90 95 Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp 100 105 110 Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln 115 120 125 Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu 130 135 140 His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Cys 145 150 155 160 Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Cys Pro Ala Gly Leu 165 170 175 Pro Ser Pro Arg Ser Glu 180 <210> 38 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) fragment (deletion of N-termianl) amino acid residues) <400> 38 Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg 1 5 10 15 Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp 20 25 30 Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu 35 40 45 Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala 50 55 60 Lys Ala Gly Cys Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val 65 70 75 80 Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln 85 90 95 Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp 100 105 110 Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Cys Gln 115 120 125 Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu 130 135 140 His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala 145 150 155 160 Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu 165 170 175 Pro Ser Pro Arg Ser Glu 180 <210> 39 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) fragment (deletion of N-termianl) amino acid residues) <400> 39 Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala 1 5 10 15 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 20 25 30 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 35 40 45 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 50 55 60 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Cys 65 70 75 80 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 85 90 95 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 100 105 110 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 115 120 125 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 130 135 140 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 145 150 155 160 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Cys Pro 165 170 175 Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 180 185 <210> 40 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) fragment (deletion of N-termianl) amino acid residues) <400> 40 Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala 1 5 10 15 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 20 25 30 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 35 40 45 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 50 55 60 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln 65 70 75 80 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 85 90 95 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 100 105 110 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 115 120 125 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 130 135 140 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 145 150 155 160 Gln Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe Arg Val Thr Pro 165 170 175 Glu Cys Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 180 185 <210> 41 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) fragment (deletion of N-termianl) amino acid residues) <400> 41 Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala 1 5 10 15 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln 20 25 30 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly 35 40 45 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr 50 55 60 Lys Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val Phe Phe Gln 65 70 75 80 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser 85 90 95 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala 100 105 110 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn 115 120 125 Ser Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln 130 135 140 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 145 150 155 160 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro 165 170 175 Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 180 185 <210> 42 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (Q146C/T241C) fragment (deletion of N,C-termianl amino acid residues) <400> 42 Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp 1 5 10 15 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 20 25 30 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 35 40 45 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 50 55 60 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 65 70 75 80 Phe Phe Cys Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 85 90 95 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 100 105 110 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 115 120 125 Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 130 135 140 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 145 150 155 160 His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 165 170 175 Val Cys Pro Glu Ile Pro Ala Gly 180 <210> 43 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (A232C/I244C) fragment (deletion of N,C-termianl amino acid residues) <400> 43 Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp 1 5 10 15 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 20 25 30 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 35 40 45 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 50 55 60 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 65 70 75 80 Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 85 90 95 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 100 105 110 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 115 120 125 Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 130 135 140 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 145 150 155 160 His Ala Trp Gln Leu Thr Gln Gly Cys Thr Val Leu Gly Leu Phe Arg 165 170 175 Val Thr Pro Glu Cys Pro Ala Gly 180 <210> 44 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> human 4-1BBL mutein (V140C/F199C) fragment (deletion of N,C-termianl amino acid residues) <400> 44 Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp 1 5 10 15 Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 20 25 30 Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 35 40 45 Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 50 55 60 Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Cys Tyr Tyr Val 65 70 75 80 Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 85 90 95 Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 100 105 110 Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 115 120 125 Ala Arg Asn Ser Ala Phe Gly Cys Gln Gly Arg Leu Leu His Leu Ser 130 135 140 Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 145 150 155 160 His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 165 170 175 Val Thr Pro Glu Ile Pro Ala Gly 180

Claims (17)

As a fusion protein of the following Structural Formula 1 or 2,
[Structural Formula 1]
N′-AL ₁ -BL ₂ -CL ₃ -DC′
[Structural Formula 2]
N′-BL ₁ -AL _{2 -CL 3} -DC′
The N 'is the N-terminus of the fusion protein,
The C 'is the C-terminus of the fusion protein,
wherein A is a single-chain variable fragment (scFv) of an antibody that specifically binds to a tumor-associated antigen,
wherein B is an scFv of an antibody that specifically binds to PD-L1 (Programmed death-ligand 1),
wherein C is an Fc domain of an immunoglobulin, a fragment thereof, or a variant thereof,
wherein D is 4-1BBL, a fragment thereof, or a variant thereof,
The L ₁ , L ₂ and L ₃ are each a peptide linker fusion protein. According to claim 1,
Wherein A is CEA, AFP and MUC1 The scFv of an antibody that specifically binds to any one tumor-associated antigen selected from the group consisting of these, the fusion protein. According to claim 1,
The A is the scFv of the antibody that specifically binds to CEA, the fusion protein. According to claim 1,
Wherein A is the amino acid sequence represented by SEQ ID NO: 1, the fusion protein. According to claim 1,
Wherein B is the amino acid sequence represented by SEQ ID NO: 2, the fusion protein. According to claim 1,
Wherein C is the amino acid sequence represented by SEQ ID NO: 5, the fusion protein. According to claim 1,
The 4-1BBL variant or fragment thereof is a fusion protein in which the amino acids corresponding to positions 146/241, 232/244 or 140/199 in the amino acid sequence shown in SEQ ID NO: 7 are substituted with cysteine. According to claim 1,
The fragment of the 4-1BBL mutant is a mutated extracellular region of 4-1BBL, the fusion protein. According to claim 1,
Wherein D is a fusion protein consisting of any one amino acid sequence selected from SEQ ID NOs: 22 to 44. According to claim 1,
The L ₁ , L ₂ and L ₃ are each a peptide linker consisting of 1 to 50 amino acids, a fusion protein. According to claim 1,
The L ₁ is a fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 3. According to claim 1,
The L ₂ is a fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 16. According to claim 1,
The L ₃ is a fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 6. According to claim 1,
The fusion protein is a fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 15. A fusion protein dimer in which two fusion proteins of any one of claims 1 to 14 are bound. A pharmaceutical composition for preventing or treating cancer comprising the fusion protein of any one of claims 1 to 14 or the dimer of the fusion protein of claim 15 as an active ingredient. 17. The method of claim 16,
The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Any one selected from the group, pharmaceutical composition.

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