AU2021212558A1

AU2021212558A1 - Fusion protein comprising anti-TAA antibody, anti-PD-L1 antibody, and IL-2, and uses thereof

Info

Publication number: AU2021212558A1
Application number: AU2021212558A
Authority: AU
Inventors: Eun Ju Shin; Young Chul Sung; Jin Won Youn
Original assignee: Genexine Inc
Current assignee: Genexine Inc
Priority date: 2020-01-31
Filing date: 2021-01-27
Publication date: 2022-07-21
Also published as: KR102559355B1; WO2021153979A1; KR20210098148A

Abstract

The present invention relates to a fusion protein comprising an anti-TAA antibody, an anti-PD-L1 antibody, and IL-2, and uses thereof, wherein the fusion protein: has excellent binding affinity to CEA, which is a tumor-associated antigen, and to PD-L1, which is an immune gateway; can specifically bind to tumor cells expressing CEA and/or PD-L1; can induce proliferation of T cells and NK cells and activate immune cells; and can exhibit excellent anticancer effects when administered to cancer-induced mice, and thus can be advantageously used in the treatment of cancer diseases.

Description

[DESCRIPTION]

[Invention Title]

FUSION PROTEIN COMPRISING ANTI-TAA ANTIBODY, ANTI-PD-LI

ANTIBODY AND IL-2, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean Patent

Application No. 10-2020-0011984, filed on January 31, 2020, the disclosure of which

is incorporated herein by reference in its entirety.

[Technical Field]

The present invention relates to a fusion protein including an anti-TAA

antibody, an anti-PD-Li antibody and IL-2, and uses thereof. More specifically, the

present invention relates to a fusion protein including an anti-CEA antibody, an anti

PD-Li antibody and IL-2, and uses thereof.

[Background Art]

The anticancer drug market occupies the largest share of the overall drug

market, and since the growth potential (CACR) is also the highest at 11.6%, it is

expected to expand to a scale of 153 trillion Korean won in 2020. Among them,

immune-oncology drugs have a mechanism in which the immune system attacks

cancer cells by stimulating the body's immune system, and recently, it is known in the

field of anticancer drugs that the effect is greater and more persistent in metastatic

cancer than the second-generation targeted anticancer drugs.

In this regard, tumor cells expressing tumor-specific antigens may be removed

by the subject's immune system at an early stage of development. However, actively

proliferating tumor cells express immune checkpoint proteins such as PD-LI, in order

to avoid immune surveillance. Such immune checkpoint proteins are known as

mechanisms for preventing the immune response of tumor cells by interfering with the

anticancer immune surveillance system.

Among immune checkpoint proteins, a mechanism is known in which PD-I

expressed on T cells and PD-Li expressed on cancer cells bind to each other to inhibit

the function of T cells. Immune checkpoint inhibitors that are released by such a

mechanism for inhibiting the binding of PD-i and PD-L include pembrolizumab

(Keytruda, pembrolizumab, anti-PD-1: Merck), nivolumab (Opdivo, nivolumab, anti

PD-1: BMS), atezolizumab (Tecentriq, atezolizumab, anti-PD-L i: Roche) and the like.

Ipilimumab (Yervoy, Ipilimumab, anti-CTLA4: BMS), which is an immune

checkpoint inhibitor that was first approved by the FDA before an immune checkpoint

inhibitor for PD-iand PD-L1, binds to CTLA-4 on the surface of T cells to prevent T

cells from being inactivated, and it was first approved as a therapeutic agent for

melanoma patients as a mechanism for helping the proliferation of T cells.

Such an immune checkpoint inhibitor showed clinical trial results that are

superior to conventional anticancer drugs, which only extended their survival time, by

improving the overall survival of patients and progression free survival (PFS) without

worsening the disease and the like. In addition, the immune checkpoint inhibitor

showed great potential in anticancer treatment due to fewer side effects and tolerance

problems of conventional anticancer drugs, such as emesis, alopecia and a decrease in

leukocytes. In addition, the clinical results of the immune checkpoint inhibitor

showed an almost complete long-term survival of end-stage cancer patients, which surprised the world, but only about 20% to 40% of patients responded among various types of cancer such as lung cancer, skin cancer and breast cancer. The remaining

60% to 80% of patients, particularly, colorectal cancer patients had a limitation in

terms of low or no therapeutic efficacy.

In the case of a group of patients showing low or no responsiveness to immune

checkpoint inhibitors, the number of T cells in tumor tissue was remarkably

insufficient. That is, the immune status of a patient is one of the important factors

for determining the responsiveness to immune checkpoint inhibitors. It is speculated

that many cancer patients are in 'an immune cold or immune desert state' because the

immune system is disturbed by anticancer agents, radiation therapy and the like, and

this may be a cause of low responsiveness to immune checkpoint inhibitors.

Therefore, the situation is that there is a need to develop a substance for activating the

immune system of a patient and to study new therapies using the substance in

combination with existing immune checkpoint inhibitors.

Meanwhile, cytokines are substances that regulate various immune responses,

and are important factors that determine the development, proliferation, maintenance,

activity and killing of immune cells according to the type and characteristics thereof.

Various cytokine therapeutic agents have been developed for the purpose of activating

the immune system of patients. In particular, the development of IL-2-based

cytokines (IL-2, IL-7, IL-15, IL-21) that are important for the homeostasis of T cells

and NK cells has been actively conducted. However, cytokine therapeutic agents

have a limitation in that they cannot be confirmed to have clear therapeutic efficacy

due to their short half-life.

[Disclosure]

[Technical Problem]

Accordingly, the inventors of the present invention have researched to develop

an effective immune anticancer agent capable of inhibiting immune checkpoint

proteins while activating the immune system, and as a result, the inventors of the

present invention completed the present invention by confirming that a fusion protein

including an anti-CEA antibody, an anti-PD-Li antibody and IL-2 exhibits an excellent

anticancer effect.

[Technical Solution]

In order to achieve the above object, one aspect of the present invention

provides a fusion protein of Structural Formula 1 below.

[Structural Formula 1]

N'-A-Li-B-L 2-C-L 3 -D-C'

Another aspect of the present invention provides a fusion protein dimer in

which two of the fusion protein are bound.

Still another aspect of the present invention provides a pharmaceutical

composition for preventing or treating cancer, including the fusion protein or the fusion

protein dimer as an active ingredient.

[Advantageous Effects]

The fusion protein of the present invention has excellent binding affinity to

CEA, which is a tumor-associated antigen, and to PD-LI, which is an immune gateway

protein, and can specifically bind to tumor cells expressing CEA and/or PD-Li. In addition, the fusion protein of the present invention can induce the proliferation of T cells and NK cells and activate immune cells, and can exhibit excellent anticancer effects when administered to cancer-induced mice. Therefore, the fusion protein of the present invention can be advantageously used in the treatment of cancer diseases.

[Description of Drawings]

FIG. 1 is a diagram showing the results of analysis by SDS-PAGE after

purifying the aCEA-aPDL1-hyFc-IL2v fusion protein dimer, which is an exemplary

embodiment of the present invention.

FIG. 2 is a diagram showing the results of analysis by SE-HPLC after

formulating the aCEA-aPDL1-hyFc-IL2v fusion protein dimer, which is an exemplary

embodiment of the present invention.

FIG. 3 is a diagram measuring the binding force between the aCEA-aPDL1

hyFc-IL2v fusion protein dimer, which is an exemplary embodiment of the present

invention, and cells expressing CEA.

FIG. 4 is a diagram measuring the binding force between the aCEA-aPDL1

hyFc-IL2v fusion protein, which is an exemplary embodiment of the present invention,

and cells expressing PD-Li.

FIG. 5 is a diagram measuring the binding force between the aCEA-aPDL1

hyFc-IL2v fusion protein dimer, which is an exemplary embodiment of the present

invention, and cells expressing CEA and PD-Li.

FIG. 6 is a diagram confirming the NFAT-Luc signal changes by the binding

inhibition of PD-i/PD-Li, after treating cells expressing PD-i and cells expressing

PD-Li withthe aCEA-aPDLi-hyFc-IL2v fusionprotein dimer, which is an exemplary

embodiment of the present invention

FIG. 7 is a diagram confirming the proliferation rate of immune cells such as

T cells and NK cells, after human-derived peripheral blood mononuclear cells were

treated with the aCEA-aPDL1-hyFc-IL2v fusion protein dimer, which is an exemplary

embodiment of the present invention.

FIG. 8 is a diagram comparing the degree of apoptosis of tumor cells, after

tumor cells were treated with human-derived peripheral blood mononuclear cells that

had been treated with the aCEA-aPDL1-hyFc-IL2v fusion protein dimer, which is an

exemplary embodiment of the present invention, or recombinant IL-2.

FIG. 9 is a diagram measuring the half-life of the aCEA-aPDL-hyFc-IL2v

fusion protein, which is an exemplary embodiment of the present invention, in the

body.

FIG. 10 is a diagram showing the amount of accumulation of the aCEA

aPDL1-hyFc-IL2v fusion protein dimer, which is an exemplary embodiment of the

present invention, in the body by each tissue.

FIG. 11 is a diagram showing the distribution of CD8+ T cells, NK cells and

Treg cells according to time, after administering the aCEA-aPDL1-hyFc-IL2v fusion

protein dimer, which is an exemplary embodiment of the present invention, to solid

cancer-induced mice.

FIG. 12 is a diagram measuring the tumor size and absolute lymphocyte count,

after administering the aCEA-aPDL1-hyFc-IL2v fusion protein dimer, which is an

exemplary embodiment of the present invention, to solid cancer-induced mice.

[Best Mode]

Hereinafter, the present invention will be described in detail.

Fusion protein including anti-CEA antibody, anti-PD-Li antibody and

IL-2

One aspect of the present invention provides a fusion protein of Structural

Formula 1 below:

[Structural Formula 1]

N'-A-L 1 -B-L 2-C-L 3 -D-C'

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

wherein 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 programmed

death-ligand 1 (PD-Li),

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

variant thereof,

wherein D is IL-2, a fragment thereof or a variant thereof, and

wherein Li, L 2 and L3 are each a peptide linker.

A is a tumor-associated antigen, wherein the tumor-associated antigen is not

cancer-specific among antigens expressed according to cell carcinoma, but 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 carcinoembryonic antigen

(CEA), a-fetoprotein (AFP) and mucin 1 (MUC1). Specifically, A may be an scFv

of an antibody that specifically binds to CEA.

The CEA is a tumor-associated antigen present in the blood found in cancers

such as colon cancer, breast cancer, lung cancer, liver cancer, noncancerous diseases

or smokers, and it may be used to check the progress of cancer treatment and whether or not it recurs.

The AFP is a tumor-associated antigen detected in the blood found in patients

with carcinomas such as liver cancer, gastric cancer, ovarian fetal cancer, hepatitis or

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 MUCI is a protein found in specific epithelial cells, and it is measured

higher in patients with breast cancer, ovarian cancer, lung cancer and prostate cancer

than in normal subjects. By measuring the expression level of MUC1 in the blood, it

is possible to predict the treatment and recurrence of cancer.

The A may consist 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 may have a modified binding force with an Fc receptor and/or

complement such that antibody-dependent cellular cytotoxicity (ADCC) or

complement-dependent cytotoxicity (CDC) may be weakened. The modified

immunoglobulin may be selected from the group consisting of IgGI, IgG2, IgG3, IgG4,

IgAl, 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 (CLI). It may further include 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 maybe derived from IgGI, IgG2, IgG3, IgG4, IgAl, IgA2, IgD orIgE. ModifiedFc 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 1 4 4 th amino acid and/or the 1 4 5 th aminoacid of SEQ ID NO: 9 may

be modified. Preferably, it may be a variant in which K, which is the 1 4 4th amino

acid of SEQ ID NO: 9, is substituted with G or S, and E, which is the 1 4 5th amino acid,

is substituted with G or S.

In addition, the Fc fragment of the present invention may be in the form of 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 Clto Clq 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 may include 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). 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 an exemplary embodiment of the present invention, for the C, a modified

immunoglobulin Fc domain (hyFcM1), which is a variant of the Fc domain of the

immunoglobulin was used, and the C may consist of the amino acid sequence

represented by SEQ ID NO: 5.

D may be IL-2 or a variant thereof.

As used herein, the term "IL-2", unless otherwise stated, refers to any wild

type IL-2 obtained from any vertebrate source, including mammals, for examples,

primates (e.g., humans) and rodents (e.g., mice and rats). The term includes untreated

IL-2 and any form of IL-2 obtained from treatment in cells. In addition, the term also

includes wild-type variants of IL-2, for example, splice variants or allelic variants.

Specifically, human IL-2 may consist of the amino acid sequence represented by SEQ

ID NO: 7.

As used herein, the term "variant of IL-2" includes a truncated form of IL-2

and a form in which IL-2 is linked to another molecule by fusion or chemical

conjugation, and it may include any mutant form in various forms. In addition, the

variant of IL-2 may be a mutant obtained by substituting some amino acids of wild

type IL-2. In this case, the variant of IL-2 may be modified to have low toxicity in

vivo. In this case, in vivo toxicity may mean a side effect caused by binding of IL-2

to the alpha chain of the IL-2 receptor. In order to attenuate the side effect of IL-2,

various IL-2 variants have been developed, and these IL-2 variants are the same as

disclosed in US Patent No. 5,229,109 and Korean Patent No. 1667096.

In the variant of IL-2, the 3 8th or 4 2 nd amino acid of SEQ ID NO: 7 may be substituted. Specifically, one embodiment of the variant of IL-2 may be one in which the wild-type 3 8th arginine is substituted with alanine (R38A). In addition, one embodiment of the variant of IL-2 may be a form in which the wild-type 42d phenylalanine is substituted with another amino acid, and specifically, the variant of

IL-2 may be one in which the 4 2 "d amino acid of the sequence of SEQ ID NO: 7 is

substituted as F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R and F42K.

In addition, the variant of IL-2 may be a form in which the 3 8th and 4 2nd amino

acids are substituted in wild-type IL-2. For example, the variant of IL-2 may include

at least one amino acid substitution selected from R38A, F42A and a combination

thereof in the amino acid sequence represented by SEQ ID NO: 7. The variant of IL

2 may have the amino acid sequence of SEQ ID NO: 8.

The A and B may be coupled via 1 to 50 peptide linkers (first linker, Li).

Specifically, the peptide linker may consist of 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. In an embodiment, Li may consist of 20

amino acids. The A and B may be coupled via a peptide linker (first linker, Li)

consisting of the amino acid sequence represented by SEQ ID NO: 3.

The B and C may be coupled via 1 to 50 peptide linkers (second linker, L 2 ).

The Li may consist of 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 amino

acids. In an embodiment, the L 2 may consist of 30 amino acids. In addition, the 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 an embodiment, the L 2 may consist of the amino acid sequence represented by SEQ

ID NO: 16.

The C and D may be coupled via 1 to 50 peptide linkers (third linker, L3 ).

Specifically, the peptide linker may consist of 1 to 50 contiguous amino acids. In an

embodiment, L3 may consist of 4 amino acids. The C and D may be coupled via a

peptide linker (third linker, L 3 ) consisting of the amino acid sequence represented by

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 of the fusion protein including the anti-CEA antibody, anti-PD-Li antibody

and IL-2 are bound. The fusion protein including the anti-CEA antibody, anti-PD-Li

antibody and IL-2 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 identical or may be

different fusion proteins. Preferably, the dimer may be a homodimer.

Polynucleotide encoding the fusion protein

Still another aspect of the present invention provides a polynucleotide

encoding a fusion protein including an anti-CEA antibody, an anti-PD-Li antibody and

IL-2. Specifically, the polynucleotide may be a nucleotide sequence encoding the

amino acid sequence represented by SEQ ID NO: 15. The fusion protein including

the anti-CEA antibody, anti-PD-Li antibody and IL-2 is as described above. Aslong

as the polynucleotide encodes 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 migration of a protein through the endoplasmic reticulum (ER) membrane.

The characteristics of a signal sequence are well known in the art, and it

typically includes 16 to 30 amino acid residues, but may include more or fewer amino

acid residues. A typical signal peptide consists of three regions of a basic N-terminal

region, a central hydrophobic region and a more polar C-terminal region. The central

hydrophobic region includes 4 to 12 hydrophobic residues that anchor the signal

sequence through the membrane lipid bilayer during the migration of an 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 signal

sequence of the 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

Still another aspect of the present invention provides a vector including the

polynucleotide.

The vector can be introduced into a host cell and recombined and inserted into

the host cell genome. Alternatively, the vector is understood as a nucleic acid means

including a polynucleotide sequence that is 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 and the like, and it

may be commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.),

E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus

subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13,

YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4,Xgt10,

Xgt11, XZAP, etc.), animal virus vectors (retroviruses, adenovirus, vaccinia virus, etc.)

or 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 that is most suitable for the purpose.

As used herein, the term "gene expression" or "expression" of a target protein

is understood to mean the transcription of a DNA sequence, the translation of an

mRNA transcript and the 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 may include a human cytomegalovirus (CMV) promoter to

promote the 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.

Transformed cells expressing the fusion protein

Still 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 prokaryotic cells, eukaryotic

cells, mammalian cells, plant cells, insect cells, fungal cells or cellular origin cells, but is not limited thereto. 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 the present invention is not limited thereto, and any cell that can

be used as a mammalian host cell known to those skilled in the art may be used.

In addition, when introducing an expression vector into a host cell, the CaCl2

precipitation method, the Hanahan method in which a reducing substance called

dimethyl sulfoxide (DMSO) is used in the CaCl2 precipitation method to increase the

efficiency, the electroporation method, the calcium phosphate precipitation method,

the protoplast fusion method, the agitation method using silicon carbide fiber, the

Agrobacterium-mediated transformation method, the transformation method using

PEG, dextran sulfate, the lipofectamine and drying/inhibition-mediated transformation

method may 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-associated gene

possessed by the host cell may be manipulated through a method known to those

skilled in the art in order to adjust the sugar chain pattern of the fusion protein (e.g.,

sialic acid, fucosylation, glycation).

Production method of the fusion protein

Still another aspect of the present invention provides a method for producing

a fusion protein including an anti-CEA antibody, an anti-PD-Li antibody and IL-2,

including culturing the transformed cell. Specifically, the production method may

include the steps of i) culturing the transformed cells to obtain a culture product; and ii) recovering the fusion protein from the culture product.

The method of culturing the transformed cells may be performed by using a

method well known in the art. Specifically, the culture may be continuously cultured

in a batch process, injection batch or repeated fed batch process (fed batch or repeated

fed batch process).

Use of the fusion protein or dimer thereof

Still another aspect of the present invention provides a pharmaceutical

composition for preventing or treating cancer, including a fusion protein including an

anti-CEA antibody, an anti-PD-Li antibody and IL-2 or a fusion protein dimer in

which two of the fusion protein are bound as an active ingredient.

The fusion protein including the anti-CEA antibody, anti-PD-Li antibody and

IL-2 or the fusion protein dimer in which two of the fusion protein are bound are as

described above.

The fusion protein or dimer thereof of the present invention may specifically

bind to CEA, which is 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 may block the immune

evasion mechanism of tumor cells by including an scFv of an antibody that specifically

binds to PD-L1, thereby binding to PD-Li expressed in tumor cells and inhibiting PD

Li/PD-i binding. In addition, the fusion protein of the present invention or a dimer

thereof may activate immune cells by including IL-2, a fragment thereof or variant

thereof. That is, the fusion protein of the present invention or a dimer thereof may

specifically bind to tumor cells, block the immune evasion mechanism of tumor cells,

and activate immune cells around tumor cells, and thus, it may be advantageously used in the prevention or treatment of cancer diseases.

The cancer may be selected from the group consisting of 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.

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 treating or preventing cancer or

infectious disease according to the present invention, the active ingredient may be

included in an arbitrary amount (effective amount) depending on the use, formulation,

purpose of formulation and the like, as long as it can exhibit anticancer activity or

exhibit a therapeutic effect on infectious disease, 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 those skilled in the art.

As used herein, the term "treatment" may be used to mean both therapeutic

and prophylactic treatment. Herein, prophylaxis may be used to mean that a

pathological condition or disease of an individual is alleviated or mitigated. In an

embodiment, the term "treatment" includes both application or any form of

administration for treating a disease in a mammal, including a human. In addition,

the term includes inhibiting or slowing down a disease or disease progression; and includes meanings of restoring or repairing impaired or lost function such that a disease is partially or completely alleviated; stimulating inefficient processes; or alleviating a serious disease.

As used herein, the term "efficacy" refers to capacity that can be determined

by one or parameters, for example, survival or disease-free survival over a certain

period of time such as one year, five years or ten years. In addition, the parameter

may include inhibition of size of at least one tumor in an individual.

Pharmacokinetic parameters such as bioavailability and underlying

parameters such as clearance rate may also affect efficacy. Thus, "enhanced efficacy"

(e.g., improvement in efficacy) may be due to enhanced pharmacokinetic parameters

and improved efficacy, which may be measured by comparing clearance rate and tumor

growth in test animals or human subjects, or by comparing parameters such as survival,

recurrence, or disease-free survival.

As used herein, the term "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 does

not cause adverse effects. A level of the effective amount may be determined

depending on factors including the patient's health condition, type and severity of

disease, activity of drug, the patient's sensitivity to drug, mode of administration, time

of administration, route of administration and excretion rate, duration of treatment,

formulation or simultaneously used drugs, and other factors well known in the medical

field. In an embodiment, the therapeutically effective amount means an amount of

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 carrier as long as the carrier is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax and inert solid may be contained as the carrier. A pharmaceutically acceptable adjuvant (buffer, dispersant) may also be contained in the pharmaceutical composition.

Specifically, by including a pharmaceutically acceptable carrier in addition to

the active ingredient, the pharmaceutical composition may be prepared into a

parenteral formulation depending on its route of administration using conventional

methods known in the art. Herein, the term "pharmaceutically acceptable" means

that the carrier does not have more toxicity than the subject to be applied (prescribed)

can adapt while not inhibiting activity of the active ingredient.

When the pharmaceutical composition is prepared into a parenteral

formulation, it may be made into preparations in the form of injections, transdermal

patches, nasal inhalants or suppositories with suitable carriers according to methods

knownintheart. Ina case of being made into injections, sterile water, ethanol, polyol

such as glycerol or propylene glycol, or a mixture thereof may be used as a suitable

carrier; and an isotonic solution, such as Ringer's solution, phosphate buffered saline

(PBS) containing triethanol amine or sterile water for injection and 5% dextrose or the

like may preferably be used. Formulation of pharmaceutical compositions is known

in the art, and reference may specifically be made to the literature [Remington's

PharmaceuticalSciences (19th ed., 1995)] and the like. This literature is considered

as part of the present specification.

A preferred dose of the pharmaceutical composition may range from 0.01

pg/kg to 10 g/kg or 0.01 mg/kg to 1 g/kg per day, depending on the patient's condition,

body weight, sex, age, severity of the patient and route of administration. The dose may be administered once a day or may be divided into several times a day. Such a dose should not be construed as limiting the scope of the present invention in any aspect.

Subjects to which the pharmaceutical composition can be applied (prescribed)

are mammals and humans, with humans being particularly preferred. In addition to

the active ingredient, the pharmaceutical composition of the present application may

further contain any compound or natural extract, which has already been validated for

safety and is known to have anticancer activity or a therapeutic effect on an infectious

disease, so as to boost or reinforce anticancer activity.

The 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 may be

appropriately selected by those skilled in the art 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 for the disease to be treated, or formulated in the form of a

combination preparation with other drugs.

Still another aspect of the present invention provides a method for preventing

or treating cancer, including administering a fusion protein including an anti-CEA

antibody, an anti-PD-Li antibody and IL-2 or a fusion protein dimer in which two of

the fusion protein are bound as an active ingredient.

It is preferable to apply the therapeutically effective amount differently

according to various factors including the specific composition including the type and

degree of the reaction to be achieved, whether other agents are used in some cases, the subject's age, weight, general health status, gender and diet, administration time, administration route and the secretion rate of the composition, the treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the field of medicine. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the foregoing description.

The subject is applicable to any mammal, and the mammal includes not only

humans and primates, but also domestic animals such as cattle, pigs, sheep, horses,

dogs and cats.

[Modes of the Invention]

Hereinafter, the present invention will be described in more detail by 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 aCEA-aPDL1-hyFc-IL2v gene construct

In order to prepare a fusion protein in which the scFv of an anti-CEA antibody

(aCEA), the scFv of an anti-PD-Li antibody (aPDL1) and the variant of IL-2 (IL2v)

are combined, the aCEA-aPDL1-hyFc-IL2v gene construct in the form where aCEA,

a first linker, aPDL1, a second linker, the Fc domain (hyFc), a third linker and IL2v

were combined in order in the direction from the N-terminus to the C-terminus was

prepared.

Specifically, first, by using the amino acid sequence of hMN-14, which is a

known amino acid sequence (Accession number: DB05097), the scFv of an anti-CEA

antibody consisting of the amino acid sequence represented by SEQ ID NO: 1 was prepared. In addition, the scFv of an anti-PD-Li antibody consisting of the amino acid sequence represented by SEQ ID NO: 2 was prepared by using the amino acid sequence of the anti-PD-Li antibody. The constructed scFv of the anti-CEA antibody and the scFv of the anti-PD-Li antibody were combined with a peptide linker (first linker) consisting of the amino acid sequence represented by SEQ ID NO: 3, which was combined with a peptide linker (second linker) consisting of the amino acid sequence represented by SEQ ID NO: 16 on the N-terminal side of the FC domain.

In addition, the variant of IL-2 was constructed to include two mutants, which

are expected to increase the therapeutic efficacy through selective binding to NK cells

while decreasing the binding force to IL-2 receptor a. The variant of IL-2 has amino

acid substitutions of R38A and F42A in human IL-2 (SEQ ID NO: 7), and the one

consisting of the amino acid sequence represented by SEQ ID NO: 8 was used. The

variant of IL-2 was coupled to the C-terminal side of the FC domain with a peptide

linker (third linker) consisting of the amino acid sequence represented by SEQ ID NO:

6.

Afterwards, genes encoding each amino acid sequence were synthesized and

prepared as sub-vectors. In order to prepare the four synthesized genes into one gene

construct, the Golden Gateway assembly was performed to obtain an expression vector

using pBispec vector.

In the same manner as for the preparation of the aCEA-aPDL-hyFc-IL2v

gene construct, an expression vector including the aPDL1-aCEA-hyFc-IL2v gene

construct in the form where aPDLI, a first linker, aCEA, a second linker, hyFc, a third

linker and IL2v were combined in order in the direction from the N-terminus to the C

terminus was prepared.

Further, in the same manner as for the preparation of the aCEA-aPDLI-hyFc

IL2v gene construct, an expression vector including the aCEA-hyFc gene construct in

the form where aCEA, a second linker and hyFc were combined in order in the

direction from the N-terminus to the C-terminus was prepared. Furthermore, an

expression vector including the aCEA-hyFc-IL2v gene construct in the form where

aCEA, a second linker, hyFc, a third linker and IL2v were combined in order in the

direction from the N-terminus to the C-terminus was prepared. In addition, an

expression vector including the aPDL1-aCEA-hyFc gene construct in the form where

aPDL1, a first linker, aCEA, a second linker and hyFc were combined in order in the

direction from the N-terminus to the C-terminus was prepared. Furthermore, an

expression vector including the hyFc-IL2v gene construct in the form where hyFc, a

third linker and IL2v were combined in order in the direction from the N-terminus to

the C-terminus was prepared.

Example 2. Production of aCEA-aPDL1-hyFc-IL2v fusion protein dimer

In order to produce the aCEA-aPDL1-hyFc-IL2v fusion protein dimer,

suspension-adapted CHO cells were seeded at 6x106 cells/mL, and then, these cells

were transformed by treating 200 mL of the expression vector including the aCEA

aPDL1-hyFc-IL2v gene construct prepared in Example 1 and ExpiFectamineT M CHO

reagent. In order to obtain a large amount of the aCEA-aPDL1-hyFc-IL2v fusion

protein dimer, ExpiFectamine T M CHO enhancer and ExpiCHO TM feed were added the

day after transformation, and the culture temperature was shifted to 32°C. After 7

days, the culture medium was collected and centrifuged at 3,600 rpm for 15 minutes

to obtain a supernatant. Afterwards, the supernatant was filtered using a 0.22 m

filter.

In order to purify the aCEA-aPDL1-hyFc-IL2v fusion protein dimer from the supernatant, the aCEA-aPDL1-hyFc-IL2v fusion protein dimer including an Fc domain was purified by using AKTA Prime (GE Healthcare Life sciences) and Protein

A resin. 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 fractions with high purity were

isolated and formulated in 50 mM hepes and 250 mM sucrose at pH 5.0. As a result

of checking the formulated aCEA-aPDL1-hyFc-IL2v fusion protein dimer through

SDS-PAGE, it was confirmed at about 250 kDa under non-reducing conditions and at

about 100 kDa under reducing conditions (FIG. 1, non-reducing: fusion protein dimer,

reducing: fusion protein monomer). In addition, as a result of determining the purity

through SE-HPLC analysis, a purity of 90.22% was confirmed (FIG. 2).

By using the expression vector including the aPDL1-aCEA-hyFc-IL2v gene

construct, the expression vector including the aCEA-hyFc gene construct, the

expression vector including the aCEA-hyFc-IL2v gene construct, the expression

vector including the aPDL1-aCEA-hyFc and the expression vector including the hyFc

IL2v gene construct prepared in Example 1 above, the aPDL1-aCEA-hyFc-IL2v

fusion protein dimer, the aCEA-hyFc fusion protein dimer, the aCEA-hyFc-IL2v

fusion protein dimer, the aPDL1-aCEA-hyFc fusion protein dimer and the hyFc-IL2v

fusion protein dimer were produced in the same manner as in the production method

of the aCEA-aPDL1-hyFc-IL2v fusion protein, respectively

Example 3. Confirmation of binding of CEA-expressing cells to aCEA

aPDL1-hyFc-IL2v fusion protein dimer: in vitro

In order to determine whether the aCEA-aPDL1-hyFc-IL2v fusion protein dimer and the aPDL1-aCEA-hyFc-IL2v fusion protein dimer produced in Example 2 bind to CEA, which is a tumor antigen, CEA-expressing MKN45 cells (Korean Cell

Line Bank (KCLB), Cat. No. 80103) were used to confirm the degree of binding.

Specifically, after aliquoting the MKN45 cells to a number of 3x105 cells,

each fusion protein dimer was diluted to a concentration of 300 g to 0 g by

concentration and cultured at4°C for 30 minutes. APBS buffer (Welgene, Cat. No.

LB004-02-500 mL) containing 1% FBS (Hyclone, Cat. No. SH30084.03) was

prepared for use as a FACS assay buffer. Thereafter, after washing with the FACS

assay buffer, 1.5 L of PE-anti-human IgG antibody (Biolegend, Cat. No. 490304),

which is a secondary buffer, was treated to each sample in 100 L of the FACS assay

buffer, and it was cultured 4°C for 30 minutes. After washing with the FACS assay

buffer, it was transferred to a FACS tube for FACS analysis.

As a result, it was confirmed that the EC50 value of the MKN45 cell line and

the aCEA-aPDL1-hyFc-IL2v fusion protein dimer was about 15.8 nM. Inaddition,

it was confirmed that the EC50 value of the MKN45 cell line and the aPDL1-aCEA

hyFc-IL2v fusion protein dimer was about 104.7 nM (FIG. 3).

Example 4. Confirmation of binding of cells expressing PD-L1 to aCEA

aPDL1-hyFc-IL2v fusion protein dimer: in vitro

In order to determine whether the aCEA-aPDL1-hyFc-IL2v fusion protein

dimer and the aPDL1-aCEA-hyFc-IL2v fusion protein dimer produced in Example 2

bind to PD-Li, which is an immune checkpoint protein, MDA-MB-231 cell lines

(Korean Cell Line Bank (KCLB), Cat. No. 30026), which express PD-Li, were used

to confirm the degree of binding.

Specifically, after aliquoting the MDA-MB-231 cell line to a number of5x105 cells, each fusion protein dimer was treated by diluting by concentration while diluting

2 times at 12,000 ng/mL, and cultured at 4°C for 30 minutes. Thereafter, after

washing with the FACS assay buffer, 1.5 L of PE-anti-human IgG antibody

(Biolegend, Cat. No. 490304), which is a secondary buffer, was treated to each sample

in 100 L of the FACS assay buffer, and it was cultured 4°C for 30 minutes. After

washing with the FACS assay buffer, it was transferred to a FACS tube for FACS

analysis. Asa result, it was confirmed that the EC50 value of the MDA-MB-231 cell

line and the aCEA-aPDL1-hyFc-IL2v fusion protein dimer was about 4.98 nM. In

addition, it was confirmed that the EC50 value of the MDA-MB-231 cell line and the

aPDL1-aCEA-hyFc-IL2v fusion protein dimer was about 1.16 nM (FIG. 4).

Example 5. Confirmation of binding of cells expressing PD-L1 and CEA

to aCEA-aPDL1-hyFc-IL2v fusion protein dimer: in vitro

In order to determine whether the aCEA-aPDL1-hyFc-IL2v fusion protein

dimer and the aPDL1-aCEA-hyFc-IL2v fusion protein dimer produced in Example 2

bind well to CEA, which is a tumor antigen, and PD-Li, which is an immune

checkpoint protein, mouse-derived MC38 cell lines (Kerafast, Cat. No. ENH204-FP)

were used to confirm the degree of binding. A mouse-derived MC38 cell line

expressing human-derived CEA and PD-Liwas prepared, and this cell line was named

"C4 cell line."

For the C4 cell line, a gene encoding the amino acid sequence represented by

SEQ ID NO: 17 was inserted into Lenti-X TM vector (Clontech, Cat. No. 632164) by

using XhoI and XbaI restriction enzymes to construct a lentivirus. Thereafter, the

lentivirus was transfected into the MC-38-CEA cell line (Kerafast, Cat. No. ENH202).

Specifically, after reacting 1.5 mL of the transfection mixture (PLUSTM

Reagent, Invitrogen, Cat. No. 11514015) with 1.5 mL of a plasmid DNA complex, the

corresponding solution was treated to the Lenti-XTM 293T cell line (Clontech, Cat. No.

632180) and cultured for 6 hours, and then, the existing medium was removed, and the

culture medium was added and cultured for 48 hours. In this case, the plasmid DNA

complex was a plasmid in which the gene encoding the amino acid sequence

represented by SEQ ID NO: 17 was inserted into the Lenti-XTM vector (Clontech, Cat.

No. 632164) by using XhoI and XbaI restriction enzymes, and Lenti-X Packaging

Single Shots plasmid (Clontech, Cat. No. 631275). Afterwards, only the supernatant

of the Lenti-XTM293T cell line that produced the lentivirus was obtained and filtered

through a 0.45 m filter.

The filtered supernatant was treated with MC-38-CEA and transfected.

After culturing for 48 hours, in order to determine whether hPD-L1 was expressed, the

expression level was confirmed by using APC-anti human PD-Li antibody (BD

Bioscience, Cat. No. 563741). In addition, cell lines that expressed hPD-L1 well

were selected and stored through single cell selection.

In this case, the aCEA-hyFc fusion protein dimer, the aCEA-hyFc-IL2v

fusion protein dimer and the aPDL1-hyFc-IL2v fusion protein dimer were used as

comparison groups.

Specifically, after aliquoting the C4 cell line to 5x105 cells, each fusion protein

dimer was treated by diluting by concentration while diluting 2 times at 12,000 ng/mL,

and cultured at 4°C for 30 minutes. Thereafter, after washing with the FACS assay

buffer, 1.5 L of PE-anti-human IgG antibody (Biolegend, Cat. No. 490304), which is

a secondary buffer, was treated to each sample in 100 L of the FACS assay buffer,

and it was cultured 4°C for 30 minutes. After washing with the FACS assay buffer,

it was transferred to a FACS tube for FACS analysis.

As a result, it was confirmed that although the protein molecular weight was

about 250 kDa, the aCEA-aPDLI-hyFc-IL2v fusion protein dimer had a high binding

force with the EC50 value of about 2.6 nM (FIG. 5).

Example 6. Confirmation of PD-1/PD-L1 binding inhibitory effect of

aCEA-aPDL1-hyFc-IL2v fusion protein dimer: in vivo

It was determined whether the aCEA-aPDL1-hyFc-IL2v fusion protein dimer

inhibits PD-i/PD-Li binding. In this case, the aCEA-hyFc fusion protein dimer, the

aCEA-hyFc-IL2v fusion protein dimer and the aPDL1-hyFc-IL2v fusion protein

dimer were used as comparison groups.

In order to determine the PD-I/PD-L binding inhibitory effect of the aCEA

aPDL-hyFc-IL2v fusion protein dimer, PD-i Effector Cells (Promega, Cat. No.

Ji15A) and PD-Li-aAPC/CHO- Ki cells (Promega, Cat. No. J109A) were co-cultured.

In this case, the TCR signal is blocked by the binding of PD-i/PD-Li expressed by

bothcells. After the two cells were treated with the aCEA-aPDLi-hyFc-IL2v fusion

protein dimer, the NFAT-Luc fluorescence signal was detected using a fluorescence

microscope. When the two cells are treated with the aCEA-aPDL-hyFc-IL2v

fusion protein dimer, the PD-i/PD-Li binding is inhibited and the TCR signal is

transmitted, thereby activating the lower NFAT-Luc signal.

As a result of determining through this method, it was confirmed that the EC50

value of the aCEA-aPDL-hyFc-IL2v fusion protein dimer was about 4.5 nM (FIG.

6).

Example 7. Confirmation of immune cell proliferation effect aCEA

aPDL1-hyFc-IL2v fusion protein dimer: in vitro

In order to determine whether the aCEA-aPDL1-hyFc-IL2v fusion protein

dimer proliferates in immune cells, the immune cells were treated with the aCEA

aPDL1-hyFc-IL2v fusion protein dimer, and then, the proliferation rate of each

immune cell was compared. In this case, a group treated with recombinant IL-2

(rhIL-2, Proleukin@ Novartis, Cat. No. 653601261) was set as a comparison group.

Specifically, human-derived peripheral blood mononuclear cells (PBMC)

were aliquoted to a number of 2x105 cells, and then stained using Cell TraceTM violet

at a concentration of 2.5 mM. Afterwards, the aCEA-aPDL1-hyFc-IL2v fusion

protein dimer was diluted three times from a concentration of 105.26 nM together with

anti-CD3 antibody stimulation, and then cultured for 5 days. Thereafter, the analysis

was performed by staining with a FACS antibody capable of identifying CD4+ T cells,

CD8+ T cells, NK cells and regulatory T cells.

Specifically, after washing the cells cultured for 5 days with the FACS assay

buffer, the cells were reacted with the FACS antibodies (PerCP/Cyanine5.5 anti-human

CD3 Antibody (Biolegend, Cat. No. 300430), Brilliant Violet 650TM anti-human CD4

Antibody (Biolegend, Cat.No. 300536), Brilliant Violet 605TM anti-human CD8a

Antibody (Biolegend, Cat.No. 301040), BV786 Mouse Anti -Human CD56 (BD

Biosciences, Cat. No. 564058), (1) PE anti-mouse/rat/human FOXP3 Antibody

(Biolegend, Cat. No. 320008)), which are capable of identifying CD4+ T cells, CD8+

T cells, NK cells and regulatory T cells, at 4°C for 30 minutes, and then, 1.5 L of PE

anti-human IgG antibody (Biolegend, Cat. No. 490304), which is a secondary buffer,

was treated to each sample in 100 L of the FACS assay buffer, and it was cultured

4°C for 30 minutes. After washing with the FACS assay buffer, it was transferred to

a FACS tube for FACS analysis.

As a result, it was confirmed that the proliferation rates of NK cells and CD8+

T cells by the aCEA-aPDL1-hyFc-IL2v fusion protein dimer increased in a

concentration-dependent manner. In addition, it was confirmed that the proliferation

rate of regulatory T cells (Treg) was significantly reduced in the aCEA-aPDL1-hyFc

IL2v fusion protein dimer compared to recombinant IL-2 (FIG. 7). Through this, it

was confirmed that the aCEA-aPDL1-hyFc-IL2v fusion protein dimer increases the

proliferation of NK cells and CD8+ T cells, which are important for tumor killing, to

a level similar to that of recombinant IL-2, and in the case of Treg, by inducing

significantly lower proliferation, the anticancer efficacy was superior to that of

recombinant IL-2.

Example 8. Confirmation of immune cell activation effect aCEA-aPDL1

hyFc-IL2v fusion protein dimer: in vitro

In order to determine the immune cell activation effect of the aCEA-aPDL1

hyFcRn-IL2v fusion protein dimer, the amount of tumor cells killed by immune cells

after treating immune cells with the aCEA-aPDL1-hyFcRn-IL2v fusion protein dimer

was confirmed. The killed tumor cells were measured by checking the 7AAD+ cells

that were stained by intervening in the DNA of the dead cells and measuring the

amount of LDH released from the dead cells. Human-derived PBMCs were treated

with the aCEA-aPDL1-hyFcRn-IL2v fusion protein dimer for each concentration and

activated, and then cultured with tumor cells at a ratio of 20:1. In this case, a group

treated with recombinant IL-2 (rhIL-2) was set as a comparison group.

First, in order to identify 7-AAD+ cells, human PBMC (Effector cell) and

tumor cells (Target cell) were mixed at ratios of 5:1, 20:1, and 80:1 (E:T), respectively,

and cultured for 4 hours at 37°C and 5% C02 conditions. Thereafter, after washing

using the FACS assay buffer, a 7-AAD (1000X stock) solution was diluted 1:1000 to

30 minutes before FACS analysis, and it was treated to the cells, and FACS analysis

was performed.

Further, in order to measure the amount of LDH released from dead cells,

human PBMC (Effector cell) and tumor cells (Target cell) were mixed at ratios of 5:1,

20:1, and 80:1 (E:T), respectively, and cultured for 4 hours at 37°C and 5% C02

conditions. Afterwards, the supernatant was obtained from the cell culture solution,

and 100 L of the LDH measure reaction mixture (250 L catalyst, 11.25 mL dye) was

treated in each well, and it was cultured at room temperature for 30 minutes while

blocking light. Thereafter, the absorbance was measured at wavelengths of 490 nm

to 492 nm and 620 nm by using a microplate reader.

As a result of determining the degree of apoptosis of tumor cells, it was

confirmed that the degree of apoptosis of tumor cells in the group treated with the

aCEA-aPDL1-hyFcRn-IL2v fusion protein dimer increased in proportion to the

concentration, and it showed an immune cell activation effect corresponding to

recombinant IL-2 (FIG. 8).

Example 9. Confirmation of in vivo half-life of CEA-PDL1-hyFc-IL2v

fusion protein dimer

In order to determine the pattern of in vivo absorption, distribution,

metabolism and excretion of the aCEA-aPDL1-hyFcRn-IL2v fusion protein dimer,

PK analysis was performed.

Specifically, 1 mg/kg of the aCEA-aPDL1-hyFc-IL2v fusion protein dimer

was subcutaneously administered to 8-week-old male SD rats, and blood was collected

by hour to determine the concentration of the aCEA-aPDL1-hyFc-IL2v fusion protein

in the blood.

Anti-human IgG Fc antibody was coated on an ELISA plate at 4°C overnight

or at 25°C for 2 hours, then treated with a blocking buffer (2.5% skim milk, 1% BSA

in PBS), and the blocking process was carried out at 25°C for 1 hour. Afterwards, a

calibration standard diluted to an appropriate concentration, QC and rat serum samples

were loaded into each well and cultured at 37°C for 1 hour.

Anti-human IgG Fc-HRP antibody (Abcam, ab97225) was treated, and it was

reacted at 25°C for 30 minutes. Afterwards, a TMB substrate was added to cause a

coloring reaction, and the reaction was stopped by treating with a 0.2 NH 2 SO4 solution,

and absorbance was measured at a wavelength of 450 nm. In this case, the

absorbance for the concentration of the calibration standard was measured by

obtaining a graph with the 4-parameter logistics regression equation to calculate the

concentration of each sample.

As a result, it was confirmed that the half-life of the aCEA-aPDL1-hyFc-IL2v

fusion protein dimer was 7.2 hours, which was significantly increased compared to 7

minutes, which is the half-life of recombinant IL-2 (FIG. 9).

Example 10. Confirmation of in vivo accumulation of aCEA-aPDL1

hyFc-IL2v fusion protein dimer

The reason for constructing a triple-specific fusion protein compared to a

single- or bi-specific antibody is to secure the disadvantages of a single- or bi-specific

antibody and safely use a drug. In order to confirm this, after administration of the

aCEA-aPDL-hyFc-IL2v fusion protein dimer in a solid cancer animal model, it was

confirmed in which tissue it was accumulated. In this case, the hyFc-IL2v fusion

protein dimer and the hyFc-IL2wt fusion protein dimer were used as comparison

groups.

First, in order to label each fusion protein dimer with a dye, each fusion protein

dimer was prepared in a buffer containing no amine-containing chemical, treated with

CFTM750 dye dissolved in DMSO, and then reacted at room temperature for about 1

hour while shaking slowly. Afterwards, unbound dye was removed by centrifugation,

and each dye-labeled fusion protein was used by diluting in PBS to a desired

concentration. In this case, the yield of the reaction was determined by measuring

the absorbance at a wavelength of 280 nm and a wavelength of 755 nm, which is the

maximum absorption wavelength of CFTM750.

Specifically, solid cancer-induced mice were prepared by transplanting the C4

cell line into 8-week-old male SD rats. Solid cancer-induced mice were stained with

the hyFc-IL2v fusion protein dimer, the hyFc-IL2wt fusion protein dimer or the aCEA

aPDL1-hyFc-IL2v fusion protein dimer, and then subcutaneously administered once

at 1 mg/kg. Two days later, the solid cancer-induced mice were sacrificed, and the

amount of the fusion protein dimers accumulated in each tissue was compared.

As a result, as shown in FIG. 10, the aCEA-aPDL1-hyFc-IL2v fusion protein

dimer was accumulated in the tumor tissue (C4), and the degrees of accumulation in

the lung, liver, kidney and spleen were lower than those of the comparison groups.

(FIG. 10). Through this, it was confirmed that the aCEA-aPDL1-hyFc-IL2v fusion

protein dimer can lower toxicity that may be caused by accumulation in the lung, liver

and spleen.

Example 11. Confirmation of in vivo immune cell activation by aCEA

aPDL1-hyFc-IL2v fusion protein dimer

C57BL/6 mice were transplanted with the C4 cell line expressing human

derived CEA and PD-Li prepared in Example 5 to prepare solid cancer-induced mice.

The aCEA-aPDL-hyFc-IL2v fusion protein dimer was subcutaneously administered

to the solid cancer-induced mice at 5 mg/kg. On days 0, 1, 3, 5, 7 and 9, each tissue

of the solid tumor-induced mouse was isolated, and immune cells in the tissue were

analyzed by flow cytometry.

As a result, NK cells showed the greatest increase in the blood and spleen

about 5 days after injection of aCEA-aPDL1-hyFc-IL2v fusion protein dimer, and then

decreased, and they showed the greatest increase in the tumor tissue on day 3,

continued until day 5 and then decreased. On the other hand, CD8+ T cells showed

the greatest increase on day 5 in the blood and spleen, and then decreased slowly

thereafter, and it was confirmed that the increased state continued until day 9 in the

tumor tissue. Treg also showed an increase on day 3, but it was not a significant

result (FIG. 11).

Example 12. Confirmation of anticancer effect ofaCEA-PDL1-hyFc

IL2v fusion protein dimer using solid cancer-induced mice

The anticancer effect of the aCEA-aPDL1-hyFc-IL2v fusion protein dimer

was determined by using solid cancer-induced mice. Specifically, after transplanting

the C4 cell line expressing human-derived CEA and PD-Li prepared in Example 5

with a number of 5x105 cells into the right flank of mice, the tumor size and absolute

lymphocyte count over time were measured in the group subcutaneously injected once

at 5 mg/kg of the aCEA-aPDL1-hyFc-IL2v fusion protein dimer and in the group

repeated twice for once per a week. In this case, the group administered with PBS

was set as a negative control group.

As a result, compared to the negative control group, the tumor size of the

group administered with the aCEA-aPDL-hyFc-IL2v fusion protein dimer was reduced, and particularly, the tumor size of the group administered repeatedly was significantly reduced. In addition, it was confirmed that the absolute lymphocyte count of the group administered with the aCEA-aPDL-hyFc-IL2v fusion protein dimer was increased compared to that of the negative control group (FIG. 12).

Through this, it was confirmed that the aCEA-aPDL1-hyFc-IL2v fusion protein dimer

exhibits an excellent anticancer effect.

Claims

[CLAIMS]

[Claim 1]

A fusion protein of Structural Formula 1 below,

[Structural Formula 1]

N'-A-L 1 -B-L 2-C-L 3 -D-C'

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

wherein 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 programmed

death-ligand 1 (PD-Li),

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

variant thereof,

wherein D is IL-2, a fragment thereof or a variant thereof, and

wherein Li, L 2 and L3 are each a peptide linker.
[Claim 2]

The fusion protein of claim 1, wherein A is an scFv of an antibody that

specifically binds to any one tumor-associated antigen selected from the group

consisting of CEA, AFP and MUC1.
[Claim 3]

The fusion protein of claim 1, wherein A is an scFv of an antibody that

specifically binds to CEA.
[Claim 4]

The fusion protein of claim 1, wherein A consists of the amino acid sequence

represented by SEQ ID NO: 1.
[Claim 5]

The fusion protein of claim 1, wherein B consists of the amino acid sequence

represented by SEQ ID NO: 2.
[Claim6]

The fusion protein of claim 1, wherein C consists of the amino acid sequence

represented by SEQ ID NO: 5.
[Claim 7]

The fusion protein of claim 1, wherein the variant of IL-2 comprises at least one

amino acid substitution selected from the group consisting of R38A, F42A and a

combination thereof in the amino acid sequence represented by SEQ ID NO: 7.
[Claim 8]

The fusion protein of claim 1, wherein D consists of the amino acid sequence

represented by SEQ ID NO: 8.
[Claim 9]

The fusion protein of claim 1, wherein LI, L 2 and L3 are each a peptide linker consisting of 1 to 50 amino acids.
[Claim 10]

The fusion protein of claim 1, wherein Li consists of the amino acid sequence

represented by SEQ ID NO: 3.
[Claim 11]

The fusion protein of claim 1, wherein L 2 consists of the amino acid sequence

represented by SEQ ID NO: 16.
[Claim 12]

The fusion protein of claim 1, wherein L 3 consists of the amino acid sequence

represented by SEQ ID NO: 6.
[Claim 13]

The fusion protein of claim 1, wherein the fusion protein consists of the amino

acid sequence represented by SESQ ID NO: 15.
[Claim 14]

A fusion protein dimer in which two of the fusion protein according to any

one of claims I to 13 are bound.
[Claim 15]

A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein according to any one of claims 1 to 13 or the fusion protein dimer of claim 14 as an active ingredient.
[Claim 16]

The pharmaceutical composition of claim 15, wherein the cancer is any one

selected from the group consisting of 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.

【DRAWINGS】

【FIG. 1】

1/12

【FIG. 2】

2/12

【FIG. 3】

3/12

【FIG. 4】

4/12

【FIG. 5】

5/12

【FIG. 6】

6/12

【FIG. 7】

7/12

【FIG. 8】

8/12

【FIG. 9】

9/12

【FIG. 10】

10/12

【FIG. 11】

11/12

【FIG. 12】

12/12

<110> Genexine, Inc.

<120> FUSION PROTEIN COMPRISING ANTI‐TUMOR‐ASSOCIATED ANTIGEN ANTIBODY, ANTI‐PD‐L1 ANTIBODY AND IL‐2 AND USES THEREOF

<130> PCT5040722

<150> KR 10‐2020‐0011984 <151> 2020‐01‐31

<160> 17

<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> 4 <212> PRT

<213> Artificial Sequence

<220> <223> linker (L3)

<400> 6 Gly Gly Gly Ser 1

<210> 7 <211> 133 <212> PRT <213> Artificial Sequence

<220> <223> IL‐2

<400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125

Ile Ser Thr Leu Thr 130

<210> 8 <211> 133 <212> PRT <213> Artificial Sequence

<220> <223> IL‐2v

<400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30

Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125

Ile Ser Thr Leu Thr 130

<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 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> 883 <212> PRT

<213> Artificial Sequence

<220> <223> anti‐CEA‐anti‐PDL1‐hyFc‐IL2v 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 Ser Ala Pro 740 745 750

Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu 755 760 765

Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro 770 775 780

Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala 785 790 795 800

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu 805 810 815

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro 820 825 830

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly 835 840 845

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile 850 855 860

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser 865 870 875 880

Thr Leu Thr

<210> 16 <211> 30 <212> PRT <213> Artificial Sequence

<220> <223> linker (L2)

<400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 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> 266 <212> PRT <213> Artificial Sequence

<220> <223> TPA signal peptide‐PD‐L1 Ectodomain‐Transmembrane

<400> 17 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Phe Thr Val Thr Val Pro Lys 20 25 30

Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys 35 40 45

Phe Pro Val Glu Lys Gln Leu Asp Leu Ala Ala Leu Ile Val Tyr Trp 50 55 60

Glu Met Glu Asp Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu Asp

65 70 75 80

Leu Lys Val Gln His Ser Ser Tyr Arg Gln Arg Ala Arg Leu Leu Lys 85 90 95

Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu Gln Ile Thr Asp Val Lys 100 105 110

Leu Gln Asp Ala Gly Val Tyr Arg Cys Met Ile Ser Tyr Gly Gly Ala 115 120 125

Asp Tyr Lys Arg Ile Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile 130 135 140

Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu 145 150 155 160

Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser 165 170 175

Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys 180 185 190

Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu Arg Ile Asn Thr 195 200 205

Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg Leu Asp Pro Glu 210 215 220

Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu Pro Leu Ala His 225 230 235 240

Pro Pro Asn Glu Arg Thr His Leu Val Ile Leu Gly Ala Ile Leu Leu 245 250 255

Cys Leu Gly Val Ala Leu Thr Phe Ile Phe 260 265