WO2024117781A1 - Fusion protein comprising cd137 extracellular domain, immune cells expressing same, and use thereof - Google Patents

Abstract

cBBR T cells in which a 4-1BB/CD3ζ (cBBR) fusion protein or a variant thereof is overexpressed in T cells specifically kill cancer cells in which 4-1BBL is overexpressed. Compared to cBBR T cells comprising a native form 4-1BB extracellular domain, the T cells, in which cBBR is overexpressed, comprising a 4-1BB extracellular domain variant have lower binding to 4-1BBL and cytotoxicity. If stimulated by Urelumab, the cBBR T cells, in which cBBR is overexpressed, comprising the variant have remarkably better activity than normal T cells. Furthermore, if reacted with an anti-BCMA/ anti-4-1BB bispecific antibody, the T cells show remarkably better killing activity, on cancer cells in which BCMA is expressed, than normal T cells. Therefore, T cells in which the cBBR fusion protein is overexpressed can be used as an anticancer agent. In addition, since it has been identified that NK cells in which a cBBR fusion protein is overexpressed are effectively activated by an anti-4-1BB antibody, it has been identified that the NK cells in which a cBBR fusion protein is overexpressed can be used as an anticancer agent.

Description

Fusion protein containing the CD137 extracellular domain, immune cells expressing the same, and uses thereof

The present invention relates to a fusion protein containing the CD137 extracellular domain or a fragment thereof, immune cells expressing the fusion protein, and uses thereof.

Our immune system is being fine-tuned to effectively eliminate invading pathogens while maintaining tolerance to self-antigens. Among these, T cells expressing CD3 are known as effector cells that can eliminate tumor cells. Immune checkpoints are co-signaling molecules that play a central role in T cell activation and regulate the signal of the T cell receptor (TCR) to be suppressed or elevated (Immunological reviews, 2017, 276:52- 65).

In addition, natural killer cells (NK cells) are known to exhibit anticancer activity by eliminating cancer cells. The activity of NK cells is regulated by a balance of various activating and inhibitory receptor signaling. It is known that the anticancer activity of NK cells is also achieved by distinguishing cancer cells through various immune receptors present on their surface. NK cells can eliminate not only cancer cells but also cancer stem cells, so they are in the spotlight as a source of development for treatments that can not only suppress the occurrence, proliferation, and metastasis of cancer, but also reduce the recurrence of cancer after cure.

Meanwhile, 4-1BB is one of the TNF-receptor superfamily (TNFRSF), a costimulatory molecule that is expressed upon activation of immune cells, both innate and adaptive immune cells, and regulates the activation of various immune cells. . The 4-1BB agonist enhances immune cell proliferation, survival, cytokine secretion, and CD8+ T cell lytic activity. This action has shown anticancer effects in several mouse tumor models and is attracting attention as a target for anticancer drugs. However, to this day, new methods that can enhance the anticancer activity of immune cells against cancer cells through 4-1BB activation are being studied.

Accordingly, the present inventors studied a new method that can enhance the anti-cancer activity of immune cells against cancer cells, and as a result, a fusion protein containing the extracellular domain and intracellular signaling domain of CD137 (or 4-1BB) was introduced into immune cells. When overexpressed, it was confirmed that cytotoxicity against cancer cells increased compared to normal immune cells. In particular, it was confirmed that when the extracellular domain of CD137 (or 4-1BB) is overexpressed in NK cells and stimulated with urelumab, INF-γ (interferon gamma) is released 5 times more than normal NK cells. The invention was completed.

In order to solve the above problem, one aspect of the present invention provides a fusion protein comprising the following (i) to (iii): (i) the extracellular domain of CD137 (4-1BB) or a fragment thereof; (ii) Transmembrane domain; and (iii) an intracellular signaling domain.

Another aspect of the present invention provides a polynucleotide encoding the fusion protein, a vector and a virus containing the polynucleotide.

Another aspect of the present invention provides immune cells expressing the fusion protein.

Another aspect of the present invention includes the steps of i) preparing the virus; and ii) processing the virus into immune cells.

Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer comprising the immune cells as an active ingredient; and pharmaceutical compositions and kits for cancer treatment comprising the immune cells and bispecific antibodies.

Another aspect of the invention provides CD137 extracellular domain variants or fragments thereof.

cBBR T cells overexpressing the 4-1BB/CD3ζ fusion protein (cBBR) or a variant thereof specifically killed cancer cells overexpressing 4-1BBL. In the case of T cells overexpressing cBBR containing the 4-1BB extracellular domain variant, it was confirmed that the binding affinity and cytotoxicity to 4-1BBL were reduced compared to cBBR T cells containing the native 4-1BB extracellular domain. did. When cBBR T cells overexpressing cBBR containing the above mutant were stimulated with Urelumab, their activity significantly increased compared to normal T cells. Furthermore, when reacted with anti-BCMA/anti-4-1BB bispecific antibodies, it showed a significantly superior killing effect on BCMA-expressing cancer cells compared to normal T cells. Therefore, T cells overexpressing cBBR can be used as an anticancer agent.

Figure 1a is a schematic diagram of a nucleic acid cassette encoding a fusion protein combining 4-1BB and CD3ζ intracellular domains. At this time, SP represents the signal peptide, CRD represents the cysteine-rich domain, TM represents the transmembrane domain, and ICD represents the intracellular domain of 4-1BB. Additionally, CD3ζ represents the intracellular domain of CD3ζ.

Figure 1b is a schematic diagram of a nucleic acid cassette encoding a fusion protein combining 4-1BB and CD3ζ intracellular domains. Specifically, the domain structure of a fusion protein expressing only CRD1 and CDR1/CRD2 among CRD1, CRD2, CRD3, and CRD4 of the CD137 extracellular domain is schematized.

Figure 2 is a diagram showing that a fusion protein combining 4-1BB and CD3ζ intracellular domains is stably expressed in transformed NK92MI cells.

Figure 3 shows the level of expression of 4-1BB after treating 1.0 x 10 ⁶ NK92MI cells with Urelumab at different concentrations.

Figure 4 shows the expression level of 4-1BB quantified through FACS analysis after treatment with urelumab at different concentrations.

Figure 5 shows the measurement of the expression level of INF-γ secreted by transformed NK92MI cells and normal NK cells cultured in the presence of urelumab. At this time, the group in which NK92MI cells and normal NK cells were cultured together with K562 cells was used as the control group.

Figure 6 shows a schedule for an experiment in which PBMC-derived NK cells were cultured, transfected with CD137 mRNA, and then co-cultured with Urelumab.

Figure 7 shows the expression level of 4-1BB over time using flow cytometry.

Figure 8 shows the amount of IFN-γ secreted through ELISA.

Figure 9 shows that, despite the presence of only CRD1 and CRD1/CRD2 of the CD137 extracellular domain, it was confirmed through flow cytometry that Urelumab and the 4B4-1 antibody bind well.

Figure 10 is a diagram showing a schematic diagram of T (CER T, cBBR T) cells overexpressing chimeric engager receptor (cBBR) as an embodiment of the present invention.

Figure 11 is a graph showing the results of confirmation of cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R) overexpressed in Jurkat T cells and 4-1BBL overexpressed in Nalm6 cells through flow cytometry.

Figure 12 is a graph showing the results of confirming the expression of cBBR or its variants (I64R, V71R) overexpressed in blood-derived T cells through flow cytometry.

Figure 13 is a graph showing the results of confirming the binding rate of cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R) overexpressed in Jurkat T cells and 4-1BBL overexpressed in Nalm6 cells through flow cytometry.

Figure 14 shows the killing ability of T cells (cBBR T cells) overexpressing cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R, I64R/V71R) against cancer cells overexpressing 4-1BBL by CFSE fluorescence staining and This is a graph showing the results measured by flow cytometry.

Figure 15 is a graph showing the level of activation of cBBR T cells overexpressing cBBR variants (I64R, V71R, I64R/V71R) by urelumab, confirmed by flow cytometry after staining with anti-CD8a antibody and anti-CD107a antibody. am.

Figure 16 is a graph showing the results of confirming the degree of activation of cBBR T cells overexpressing cBBR variants (I64R, V71R, I64R/V71R) by urelumab by measuring the concentration of IFN-γ.

Figure 17 shows cBBR T cells or Ramos cells overexpressing cBBR variants (I64R, V71R) treated with a bispecific antibody (anti-BCMA/anti-4-1BB), and then cBBR T cells or Ramos cells and the bispecific antibody. This is a graph showing the results of confirming the degree of binding through flow cytometry. BiTE: anti-BCMA/anti-4-1BB bispecific antibody, 19F2: anti-BCMA antibody, 4B4-1: anti-4-1BB antibody

Figure 18 is a graph showing the results of confirming the cancer cell killing ability of cBBR T cells by co-culturing cBBR T cells overexpressing cBBR variants (I64R, V71R) with cancer cells expressing dual specific antibodies and BCMA.

Figure 19 confirms that tumor suppression occurred when transformed T cells and AB-101 antibody were administered.

Fusion protein containing CD137 extracellular domain

One aspect of the present invention provides a fusion protein comprising (i) to (iii) below:

(i) CD137 (4-1BB) extracellular domain or fragment thereof;

(ii) Transmembrane domain; and

(iii) Intracellular signaling domain.

As used herein, “CD137” is a membrane protein belonging to the tumor necrosis factor receptor family. CD137 is referred to as tumor necrosis factor receptor superfamily member 9 or 4-1BB. Specifically, CD137 is a tumor necrosis factor receptor and mainly acts as a co-stimulatory molecule in T cells. The CD137 extracellular domain contains four cysteine-rich pseudo repeat (CRD) domains. The CD137 protein may preferably be human CD137 protein, but is not limited thereto.

The "CD137 extracellular domain fragment" refers to a fragment in which part of the N-terminus and/or C-terminus of the native CD137 extracellular domain protein is truncated, and is identical to or similar to the native CD137 extracellular domain 4- This may indicate 1BBL binding force. The “wild type” includes all proteins found in nature or nucleic acids encoding them and can be used interchangeably with wild type.

In the present invention, the CD137 extracellular domain or fragment thereof includes CRD1 comprising the amino acid sequence of SEQ ID NO: 2; CRD2 comprising the amino acid sequence of SEQ ID NO: 3; CRD3 comprising the amino acid sequence of SEQ ID NO: 6; CRD4 comprising the amino acid sequence of SEQ ID NO: 7; And it may include any one selected from the group consisting of combinations thereof.

In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD2. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2 and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2 and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD3 and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD3, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2, CRD3, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, CRD3, and CRD4.

Preferably, the CD137 extracellular domain or fragment thereof may include or consist of the amino acid sequence of CRD1. Preferably, the CD137 extracellular domain or fragment thereof may include or consist of the amino acid sequences of CRD1 and CRD2. Preferably, the extracellular domain of CD137 or a fragment thereof may include or consist of the amino acid sequences of CRD1, CRD2, CRD3, and CRD4.

In one embodiment of the present invention, the CD137 extracellular domain or fragment thereof may include the amino acid sequence of SEQ ID NO: 15.

In addition, if it has the same activity as the extracellular domain of CD137 or a fragment thereof, or the location of the gene encoding the extracellular domain of CD137 on the chromosome is the same, the protein consists of a sequence in which one or several amino acids are added, deleted, or substituted. It can be. The CD137 extracellular domain has about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% amino acid identity with the amino acid sequence of SEQ ID NO: 15. It may contain or consist of sequences.

The CD137 extracellular domain or fragment thereof may further include mutations.

As used herein, “mutation” refers to a form in which some amino acids of the above-described CD137 extracellular domain or fragment thereof are substituted. That is, the variant of the CD137 extracellular domain or fragment thereof may have a different sequence from the native CD137 extracellular domain or fragment thereof. In the present invention, the mutant may have reduced binding ability to 4-1BBL (CD137L) compared to native CD137. Here, the binding ability with 4-1BBL can be measured using methods known to those skilled in the art.

The mutation may be included in any one selected from the group consisting of CRD1, CRD2, CRD3, CRD4, and combinations thereof.

In one embodiment, the mutation may be included in CRD1. In one embodiment, the mutation may be included in CRD2. In one embodiment, the mutation may be included in CRD3. In one embodiment, the mutation may be included in CRD4. In one embodiment, the mutation may be included in CRD1 and CRD2. In one embodiment, the mutation may be included in CRD1 and CRD3. In one embodiment, the mutation may be included in CRD1 and CRD4. In one embodiment, the mutation may be included in CRD2 and CRD3. In one embodiment, the mutation may be included in CRD2 and CRD4. In one embodiment, the mutation may be included in CRD3 and CRD4. In one embodiment, the mutation may be included in CRD1, CRD2, and CRD3. In one embodiment, the mutation may be included in CRD1, CRD2, and CRD4. In one embodiment, the mutation may be included in CRD1, CRD3, and CRD4. In one embodiment, the mutation may be included in CRD2, CRD3, and CRD4. In one embodiment, the mutation may be included in CRD1, CRD2, CRD3, and CRD4.

Specifically, the mutation may be included in CRD2 and/or CRD3.

At this time, the mutation of CRD2 is any one amino acid selected from the group consisting of P3, S6, Q13, T15, C16, D17, I18, Q21, K23, V25, F26, and combinations thereof in the amino acid sequence of SEQ ID NO: 3. It may have been replaced. Additionally, the mutation of CRD3 may be a substitution of any one amino acid selected from the group consisting of S14, M15, C16, and combinations thereof in the amino acid sequence of SEQ ID NO: 6.

The "amino acids" introduced by the substitution include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, Methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan ( It may be any one selected from the group consisting of tryptophan, histidine, and proline.

More specifically, the mutation of CRD2 may be a substitution of any one amino acid selected from the group consisting of I18R, V25R, and combinations thereof in the amino acid sequence of SEQ ID NO: 3.

In one embodiment, the mutation in CRD2 may be one in which isoleucine (I), the 18th amino acid of SEQ ID NO: 3, is substituted with arginine (R) (I18R). As an example, a mutation in CRD2 may be a substitution of valine (V), the 25th amino acid of SEQ ID NO: 3, with arginine (R) (V25R). In one embodiment, the mutation in CRD2 may be one in which isoleucine (I), the 18th amino acid of SEQ ID NO: 3, and valine (V), the 25th amino acid, are each substituted with arginine (R) (I18R, V25R).

In one embodiment, CRD2 containing the mutation may include or be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 26.

Additionally, in one embodiment of the present invention, the CD137 extracellular domain containing the above mutation may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 27.

In the present invention, when the CD137 extracellular domain or a fragment thereof contains a mutation, the binding ability of the CD137 extracellular domain to 4-1BBL (CD137L) may be reduced compared to the native type.

The term “transmembrane domain” used in the present invention refers to a portion of the protein structure located in the cell membrane that connects the extracellular domain and the intracellular signaling domain and penetrates the cell membrane. The fusion protein according to the present invention can be fixed to the cell membrane through the transmembrane domain. The transmembrane domain includes T cell receptor (TCR), CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, It may be derived from any one molecule selected from the group consisting of CD137, CD152, and CD154. Specifically, the transmembrane domain may be derived from CD137.

In one embodiment of the present invention, the transmembrane domain may be derived from CD137. Specifically, the transmembrane domain may include the amino acid sequence of SEQ ID NO: 8.

In addition, as long as it has the same activity as the transmembrane domain or the location of the gene encoding the transmembrane domain derived from CD137 on the chromosome is the same, the protein may be composed of a sequence in which one or several amino acids are added, deleted, or substituted. there is. The transmembrane domain derived from CD137 may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 8.

In the present invention, the (i) CD137 extracellular domain and (ii) transmembrane domain may be connected through a spacer. The spacer refers to a linker and may be a protein or peptide. Additionally, it may be composed of about 1 to about 1,000 amino acids, and may include about 10 to about 300 amino acids, about 15 to about 100 amino acids, about 15 to about 60 amino acids, or about 15 to about 45 amino acids. Additionally, the linker may be a fragment of a protein in the human body, such as an Fc region. In addition, the linker consists of CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 and CD154. It may be derived from any one molecule selected from the group.

In one embodiment of the present invention, the spacer may be derived from CD137. Specifically, the spacer may include or be composed of the amino acid sequence of SEQ ID NO: 11.

As used in the present invention, the term "intracellular signaling domain" refers to the case where an extracellular binding domain present on the cell surface binds to a biological target molecule (e.g., binding of CD137 extracellular domain and 4-1BBL). ), refers to a site that transmits signals into the cell to induce reactions such as cell activation, release of cytotoxic factors, cytokine production, and cell proliferation.

In the present invention, the intracellular signaling domain may include a co-stimulatory signaling domain and/or a primary signaling domain.

As used herein, the term “co-stimulatory domain” refers to the intracellular signaling domain of a co-stimulatory molecule. The “co-stimulatory molecule” is a cell surface molecule other than the Fc receptor that provides a secondary signal that induces efficient activation and function of immune cells when the extracellular binding site binds to the target molecule. The costimulatory domain may be derived from any one molecule selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and CD137. .

In one embodiment, the costimulatory domain may be derived from CD137. Specifically, the co-stimulatory domain may include or consist of the amino acid sequence of SEQ ID NO: 9.

As used herein, the term “primary signaling domain” refers to a protein that constitutes the CD3 complex of T cells and refers to a signaling domain that regulates the primary activation of T cells in a stimulatory or inhibitory manner. The primary signaling domain that stimulates activation of the T cell may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM (Immunoreceptor tyrosine-based activation motif). ITAM containing the primary signaling domain may be derived from any one molecule selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

In one embodiment of the present invention, the primary signaling domain may be derived from CD3ζ. The term “CD3ζ” used herein refers to a protein that constitutes the CD3 complex. The CD3 complex is a protein complex and T cell co-receptor involved in activating both cytotoxic T cells and T helper cells. The CD3 complex contains the CD3α chain, CD3β chain, CD3γ chain, CD3δ chain, CD3ε chain, and CD3ζ. Specifically, the primary signaling domain may include or consist of the amino acid sequence of SEQ ID NO: 10.

As used herein, “CD28” is one of the proteins expressed on T cells that provide costimulatory signals necessary for T cell activation and survival. At this time, the intracellular domain of CD28 may have the amino acid sequence of SEQ ID NO: 34.

As used herein, “OX40” refers to tumor necrosis factor receptor superfamily, member 4, also known as CD134. At this time, the intracellular domain of OX40 may have the amino acid sequence of SEQ ID NO: 35.

In addition, as long as it has the same activity as each of the co-stimulatory domains and the primary signaling domain or has the same coding gene location on the chromosome, each of the domains consists of a sequence in which one or several amino acids are added, deleted, or substituted. It can be. Specifically, the co-stimulatory domain may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 9. Specifically, the primary signaling domain may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10. there is.

The intracellular signaling domain of the present invention may be an appropriate combination of the costimulatory domain and the primary signaling domain. In one embodiment, the intracellular signaling domain may include CD137 and CD3ζ. At this time, the co-stimulatory domain and the primary signaling domain may be connected through a linker.

Structure of fusion protein

Specifically, the fusion protein of the present invention may contain the following structural formula (I).

<Structural Formula (I)>

N'-A-(L1)n-TM-(L2)m-B-(L3)l-C-C' (I)

At this time, in the structural formula (I),

Wherein N' is the N terminus of each polypeptide,

The C' is the C terminus of each polypeptide,

where - means bonding,

wherein A is the extracellular domain of CD137 or a fragment thereof;

The TM is a transmembrane domain;

B and C are intracellular signaling domains, each independently a co-stimulatory domain and a primary signaling domain;

Wherein L1, L2 and L3 are each peptide linkers,

The n, m and l are each independently 0 or 1.

At this time, the extracellular domain or fragment thereof, transmembrane domain, and intracellular signaling domain of CD137 are the same as described above.

The L1 may be composed of about 1 to about 100 amino acids, and may include about 10 to about 80 amino acids, about 15 to about 70 amino acids, about 15 to about 60 amino acids, or about 15 to about 45 amino acids. The L2 and L3 may each include about 1 to about 30 amino acids. Specifically, L2 and L3 may each include about 5 to about 25 amino acids, about 10 to about 20 amino acids, or about 15 amino acids. More specifically, the L2 and L3 are each about 2 to about 30, about 2 to about 28, about 2 to about 26, about 2 to about 24, about 2 to about 22, and about 2 to about 20. , about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 14 amino acids, about 2 to about 12 amino acids, or about 2 to about 10 amino acids. In one embodiment, the L1 may be a peptide derived from CD137, and may include or consist of the amino acid of SEQ ID NO: 11.

Specific examples of fusion proteins

Includes CD137 intracellular domain and CD3z

1) N'-CRD1 (SEQ ID NO: 2)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-CD3z (SEQ ID NO: 10)-C'

2) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD3z (SEQ ID NO: 10) - C'

3) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD3z (SEQ ID NO: 10)-C'

4) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-CD3z (SEQ ID NO: 10)-C'

5) N'-CRD1 (SEQ ID NO: 2) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD3z (SEQ ID NO: 10) - C'

6) N'-CRD4 (SEQ ID NO: 7)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-CD3z (SEQ ID NO: 10)-C'

Includes CD137 intracellular domain and CD28

7) N'-CRD1 (SEQ ID NO: 2)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-CD28 (SEQ ID NO: 34)-C'

8) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34) - C'

9) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34)-C'

10) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-CD28 (SEQ ID NO: 34)-C'

11) N'-CRD1 (SEQ ID NO: 2) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34) - C'

12) N'-CRD4 (SEQ ID NO: 7)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-CD28 (SEQ ID NO: 34)-C'

Includes CD137 intracellular domain and OX40

13) N'-CRD1 (SEQ ID NO: 2)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'

14) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35) - C'

15) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35)-C'

16) N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'

17) N'-CRD1 (SEQ ID NO: 2) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35) - C'

18) N'-CRD4 (SEQ ID NO: 7)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'

At this time, N' refers to the N terminus of the fusion protein, and C' refers to the C terminus of the fusion protein.

Polynucleotide encoding fusion protein

Another aspect of the present invention provides a polynucleotide encoding the fusion protein. The fusion protein is the same as described above. Specifically, the polynucleotide encoding the fusion protein may include or be composed of any one nucleotide selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 23 and SEQ ID NO: 28.

Additionally, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When preparing a polynucleotide sequence by chemical synthesis, a synthesis method well known in the art, for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used. and triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.

Specifically, the polynucleotide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, and at least about the base sequences of SEQ ID NO: 21 to SEQ ID NO: 23 and SEQ ID NO: 28, respectively. 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about It may include a nucleic acid sequence having an identity of 97%, at least about 98%, at least about 99%, or at least about 100%.

The polynucleotide may additionally include a nucleic acid encoding a signal sequence or leader sequence. The term “signal sequence” used herein 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 proteins through the ER (Endoplasmic reticulum) membrane.

The signal sequence has well-known characteristics in the art, and usually contains 16 to 30 amino acid residues, but may contain 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 throughout the membrane lipid bilayer while the immature polypeptide moves. After initiation, the signal sequence is cleaved within the lumen of the ER by a cellular enzyme commonly known as signal peptidase. At this time, the signal sequence may be a secretion signal sequence of tPa (Tissue Plasminogen Activation), HSV gDs (Signal sequence of Herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells, including mammals, can be used. In addition, the signal sequence can be used as a wild-type signal sequence, or by replacing it with a codon that is frequently expressed in host cells. In one specific example, the signal sequence may be the signal sequence of CD137. In one embodiment of the present invention, the signal sequence may include or consist of the amino acid sequence of SEQ ID NO: 1.

Vector loaded with polynucleotide

Another aspect of the present invention provides a vector loaded with the polynucleotide. The vector may contain or consist of a polynucleotide encoding a fusion protein of structural formula (I). The fusion protein is the same as described above.

As used herein, the term “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 vehicle containing a nucleotide sequence capable of spontaneous replication as an episome. The vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, mini-chromosomes 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 (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.) , Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), It may be an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.) or an insect virus vector (baculovirus, etc.). Since the expression level and modification of the protein of the vector varies depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

Preferably, the vector may be a viral vector, and the viral vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, baculovirus, papillomavirus, and parvovirus. You can. In one embodiment of the present invention, the vector may be a lentiviral vector.

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 variants thereof. The expression vector contains a human CMV (cytomegalovirus) promoter to promote continuous transcription of the target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of RNA after transcription. can do.

Viruses containing polynucleotides

Another aspect of the present invention provides a virus containing the polynucleotide. At this time, the polynucleotide is the same as described above. The virus may be a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, baculovirus, papillomavirus or parvovirus.

Immune cells expressing fusion protein

Another aspect of the present invention provides immune cells expressing the fusion protein. The fusion protein is the same as described above.

At this time, the extracellular domain of CD137 may include at least one domain selected from the group consisting of CRD1 (cysteine-rich pseudo repeat 1), CRD2, CRD3, CRD4, and combinations thereof. At this time, the extracellular domain of CD137 overexpressed in immune cells may have any of the following structures:

i) N'-CRD1-C'; ii) N'-CRD1-CRD2-C'; iii) N'-CRD1-CRD2-CRD3-C'; or iv) N'-CRD1-CRD2-CRD3-CRD4-C'.

At this time, N' refers to the N terminus of the fusion protein, and C' refers to the C terminus of the fusion protein. Also, at this time, CRD1, CRD2, CRD3, and CRD4 may include mutations. At this time, the mutation is as described above.

In addition, the extracellular domain of CD137 overexpressed in immune cells may include the intracellular domain of CD137 or a fragment thereof; The intracellular domain of CD3ζ or a fragment thereof; The intracellular domain of CD28 or a fragment thereof; and OX40 intracellular domain or fragment thereof; it may further include at least one intracellular domain selected from the group consisting of. At this time, a specific example of the intracellular domain of CD137 overexpressed in immune cells is as follows:

i) N'-Intracellular domain of CD137 or fragment-C' thereof;

ii) the intracellular domain of N'-CD3ζ or its fragment -C';

iii) N'-intracellular domain of CD28 or fragment-C' thereof;

iv) N'-OX40 intracellular domain or fragment thereof-C'';

v) N'-intracellular domain of CD137 or fragment thereof-intracellular domain of CD3ζ or fragment thereof-C';

vi) N'-Intracellular domain of CD137 or fragment thereof-Intracellular domain of CD28 or fragment thereof-C''

vii) N'-CD137 intracellular domain or fragment thereof-OX40 intracellular domain or fragment thereof-C';

viii) N'-intracellular domain of CD3ζ or fragment thereof-intracellular domain of CD28 or fragment thereof-C';

ix) N'-CD3ζ intracellular domain or fragment thereof-OX40 intracellular domain or fragment thereof-C'; or

x) N'-CD28 intracellular domain or fragment thereof-OX40 intracellular domain or fragment thereof-C'.

At this time, N' refers to the N terminus of the fusion protein, and C' refers to the C terminus of the fusion protein.

In addition, the intracellular domain of CD137, the intracellular domain of CD3ζ, the intracellular domain of CD28, and the intracellular domain of OX40 are the same as described above.

The immune cells may be natural killer cells, T cells, or macrophages. The immune cell expressing the fusion protein is a cell transformed by introducing the polynucleotide, and can be transformed using a vector loaded with the polynucleotide or a virus containing the polynucleotide. The polynucleotide, vector and virus are the same as described above.

As used herein, the term "natural killer cell (NK cell)" refers to a cytotoxic lymphocyte that constitutes a major component of the innate immune system, and is defined as a large granular lymphocyte (LGL) and a lymphoid progenitor cell. (Common lymphoid progenitor, CLP) constitutes a third cell differentiated from B and T cells. It is a lymphocyte cell that plays a part in the immune response, exists in approximately 10-15% of normal blood, and has a high killing ability when reacting with non-self. In the case of cells infected with various viruses, bacterial invasion, or the creation of abnormal cells, natural killer cells can react immediately and non-specifically to remove foreign substances. In this specification, natural killer cells are also referred to as NK cells.

As used herein, the term “T cell” refers to a type of lymphocyte that hosts antigen-specific adaptive immunity, including immature T cells that have not come into contact with an antigen and effector T cells that have matured after contact with an antigen (helper T cells, cytotoxic T cells). cells, natural killer T cells) and memory T cells.

Activation of T cells occurs when a naïve T cell antigen receptor (TCR) binds to the MHC:antigen complex and activates the CD3 complexes of T cells: CD3 gamma (γ), delta (δ), epsilon (ε), and zeta ( ζ) is induced by phosphorylating the ITAM of the chain. The ITAM phosphorylation induces phosphorylation of ZAP-70 protein, LAT protein, and SLP-76 protein, thereby inducing a signal response that activates transcription factors such as NFκB, AP-1, and NFAT. Transcription factors such as NFκB, AP-1, and NFAT induce T cell division and differentiation by promoting the expression of interleukin-2 (IL-2).

As used herein, the term “macrophage” is also referred to as a phagocytic cell and is a major cell responsible for innate immunity. The macrophages may exist as settlers in tissues, such as Kupffer cells, Langerhans cells, and microglial cells, or may exist in the form of monocytes in the blood. At this time, the monocytes may differentiate into dendritic cells or macrophages. Macrophages control inflammatory responses through phagocytosis and secretion of cytokines, which absorb and digest foreign proteins such as cell tissue, foreign substances, microorganisms, and cancer cells. In addition, after removing the antigen, it acts as an antigen-presenting cell that delivers the antigen to lymphocytes, thereby inducing an immune response.

In the present invention, the immune cells can be obtained from an individual or from various tissues. Additionally, the immune cells include unmodified immune cells obtained from an individual and may include immature immune cells as well as mature immune cells. The immune cells can be obtained by separating them from tissues, hematopoietic cells, hematopoietic stem cells, or hematopoietic progenitor cells. Specifically, the immune cells may include, but are not limited to, hematopoietic cells, hematopoietic stems or precursors from placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc. The immune cells may be autologous or foreign cells. Additionally, the immune cells may be derived from mammals such as rats, mice, livestock, and humans. Preferably, the immune cells may be obtained from an individual seeking treatment. At this time, the immune cells can be directly isolated from the blood of the individual and used, or they can be used by differentiating immature undifferentiated (or immature) immune cells or stem cells obtained from the individual. In addition, the immune cells may be differentiated from iPSCs (Induced pluripotent stem cells).

Methods for introducing the vector or virus into immune cells can be methods known in the art, such as transient transfection, microinjection, transduction, cell fusion, and calcium phosphate precipitation. Method, Liposome-mediated transfection, DEAE Dextran-mediated transfection, Polybrene-mediated transfection, electroporation , can be introduced into cells by a gene gun or other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988). However, it is not limited to this.

Transduced or transfected immune cells can be propagated ex vivo following vector or viral infection. In one embodiment, the transfected immune cells are allowed to proliferate for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, It can be cultured for 13, 14, 15, 16, 17, 18, 19 or 20 days, preferably for 13 to 15 days.

Methods for confirming whether the vector has been successfully introduced into the immune cells include, for example, molecular biological assays (e.g., Southern and Northern blotting, RT-PCR and PCR) well known to those skilled in the art; It may include detection of the presence or absence of a particular peptide by biochemical assays (e.g., immunological methods (e.g., ELISAs, Western blots, flow cytometry)).

Method for producing immune cells expressing fusion proteins

Another aspect of the present invention includes the steps of i) preparing a virus containing the polynucleotide; and ii) processing the virus into immune cells. It provides a method for producing immune cells expressing the fusion protein. At this time, the polynucleotide, virus containing the polynucleotide, immune cell, and fusion protein are the same as described above.

Methods for producing the virus may use methods known in the art. Additionally, immune cells transfected with the virus can be proliferated by further culturing after infection.

The term "culture" used in the present invention refers to growing cells in appropriately controlled environmental conditions, and the culture process of the present invention can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted by a person skilled in the art depending on the cells selected. The medium refers to a known medium used for culturing cells, and includes all known media for cell culture or modified media thereof.

Specifically, immune cells transfected with the above virus persist for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 days after infection. , may be cultured for 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days, preferably for 13 to 15 days.

In addition, the production of immune cells expressing the fusion protein can be confirmed by confirming the expression of the fusion protein through methods such as molecular biological assays and biochemical assays well known to those skilled in the art.

Pharmaceutical composition containing immune cells expressing fusion protein

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising immune cells expressing the fusion protein as an active ingredient. At this time, the pharmaceutical composition may further include an anti-CD137 antibody or a bispecific antibody. The fusion protein and immune cells are the same as described above.

In addition, the above cancers include stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, and salivary gland cancer. , lymphoma, kidney cancer, melanoma, multiple myeloma, brain cancer, osteosarcoma, glioblastoma, IgG-opsonized tumor, lymphoma, neuroma, mesothelioma, and esophageal cancer.

As used herein, the term “antibody” refers to an immunoglobulin molecule that reacts immunologically with a specific antigen, and refers to a protein molecule that specifically recognizes the antigen. The heavy and light chains of immunoglobulins may each include a constant region and a variable region. The light and heavy chain variable regions of immunoglobulins include three variable regions called complementarity determining regions (CDRs) and four framework regions (FRs). The CDR is an antigen binding site that mainly binds to the epitope of the antigen.

The antibody may refer to any one selected from IgG, IgE, IgM, IgD, and IgA, and may be a subclass of IgG, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Additionally, the antibody may be an agonistic antibody or an antagonistic antibody. In addition, the antibodies include whole antibodies, monoclonal antibodies, polyclonal antibodies, single domain antibodies, single chain antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs, and Fab fragments. , F(ab') fragment, Fv(sdFv) linked by a disulfide bond, and any of the above epitope binding fragments, and is not limited thereto, as long as it specifically binds to the cancer antigen.

As used herein, the term “dual specific antibody” refers to a substance that binds to at least one target or antigen. In the present invention, the bispecific antibody includes a first domain that specifically binds to a tumor antigen; and a second domain that specifically binds to the extracellular domain of CD137.

One specific example of the bispecific antibody may be a fragment of an anti-CD137 antibody, for example, scFv, bound to an antibody that specifically binds to an antigen. At this time, the scFv may be bound to the C terminus of an antibody that specifically binds to an antigen, and at this time, the antibody and scFv may be bound through a peptide linker. At this time, the peptide linker may be (G4S)n. Also, at this time, the linker of the peptide may be (G4S) ₄ .

Another specific example of the bispecific antibody may be an engager. Specifically, the bispecific antibody may be in the form of BiTE (Bi-specific T-cell engager). Specifically, the first domain may be an antibody or scFv that specifically binds to a cancer antigen, and the second domain may be an scFv that specifically binds to CD137. Additionally, the first domain and the second domain may be linked through a peptide linker.

At this time, the cancer antigen is alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a. , CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, glypican -3(GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY- ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survi It may be any one selected from the group consisting of bin, TAG72, TEMs, VEGFR2, and WT-1.

Additionally, the extracellular domain binding site of CD137, which is the second domain of the bispecific antibody, can serve as an agonistic antibody. Therefore, the bispecific antibody can activate CD137 by specifically binding to the extracellular domain of CD137.

Therefore, in the present invention, the bispecific antibody specifically binds to the cancer antigen overexpressed on the tumor surface and specifically binds to the CD137 extracellular domain of the fusion protein of the present invention, thereby inhibiting tumor cells and the fusion protein of the present invention. It can be used as a cell engager to bind the expressed immune cells. By using the bispecific antibody as a cell binder, it can target cancer and exhibit safer and superior anticancer activity by activating immune cells specifically for cancer cells.

In one embodiment of the present invention, when T cells expressing the fusion protein of the present invention are treated in combination with anti-BCMA/anti-4-1BB bispecific antibodies, it is confirmed that cytotoxicity increases compared to wild type T cells. (Figure 17).

As used herein, the term “prevention” refers to all actions that suppress or delay the onset of a disease by administering the pharmaceutical composition. The term “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by administering the pharmaceutical composition.

In the pharmaceutical composition of the present invention, immune cells expressing the fusion protein may be included in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as they can show activity. The pharmaceutical composition of the present invention can be used to kill about 1×10 ² cells to about 1×10 ¹⁶ cells, about 1×10 ² cells to about 1×10 ¹⁵ cells, about 1×10 ² cells to about 1×10 ¹⁴ cells, or about 1 cell. It may include immune cells expressing the fusion protein of ×10 ² cells to about 1 × 10 ¹³ cells. Preferably, the pharmaceutical composition may include about 2×10 ² to about 2×10 ¹¹ cells of immune cells expressing the fusion protein.

Here, “effective amount” refers to the amount of active ingredient that can induce an anti-aging effect. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

Compositions of the present invention can be used unfrozen or frozen for later use. If freezing is required, use standard cryopreservation agents (e.g. DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D- Sorbitol, i-inositol, D-lactose, choline chloride, and Epilife ^® cell freezing medium (Cascade biologics) can be added to the cells before freezing.

The pharmaceutical composition of the present invention may be formulated to further include a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not significantly irritate living organisms and does not inhibit the biological activity and properties of the administered ingredient. The pharmaceutical composition of the present invention can be applied as a cell therapy agent, and pharmaceutically acceptable carriers that can be included as a cell therapy agent include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc. If it is known in the industry, it can be used without restrictions. The pharmaceutical composition of the present invention can be prepared according to commonly used techniques in the form of various dosage forms.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and can be administered through any route as long as it can induce movement to the diseased area. Here, “administration” means introducing a predetermined substance into an individual by an appropriate method. In some cases, loading the pharmaceutical composition of the present invention into a vehicle equipped with a means to direct it to the lesion may be considered. Preferably, it may be parenteral administration. At this time, parenteral administration may include subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid administration.

The preferred administration method and formulation of the pharmaceutical composition of the present invention is injection. Injections include aqueous solvents such as physiological saline solution, Ringer's solution, Hank's solution, or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol, or glycerin. It can be manufactured using, etc., and for mucosal penetration, impermeable agents known in the art suitable for the barrier to pass through can be used, and as a stabilizer to prevent deterioration, ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA It may additionally contain pharmaceutical carriers such as emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol. Regarding the formulation of pharmaceutical compositions, it is known in the art, and specifically, references can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The above documents are considered part of this specification.

As used herein, the term “pharmaceutically effective amount” refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Factors including gender, age, weight, health status, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, drugs combined or used simultaneously, and other factors. It can be readily determined by a person skilled in the art according to factors well known in the medical field.

The preferred dosage of the pharmaceutical composition of the present invention is about 1×10 cells/kg to about 1×10 ¹² cells/kg per day, based on cells, depending on the patient's condition, weight, gender, age, patient's severity, and route of administration. It may be about 1×10 cells/kg to about 1×10 ¹¹ cells/kg or about 1×10 cells/kg to about 1×10 ¹⁰ cells/kg. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The number of administrations can be once or twice or more within the range of clinically acceptable side effects. The pharmaceutical composition of the present invention may be administered 1 to 3 times/day, 1 to 3 times/week, or 1 to 10 times/month. For example, the pharmaceutical composition of the present invention can be used 1 to 9 times/month, 1 to 8 times/month, 1 to 7 times/month, 1 to 6 times/month, 1 to 5 times/month, 1 It may be administered once to four times/month, once to three times/month, or once to twice per month. Regarding the administration site, it can be administered at one or two or more locations. For animals other than humans, the dosage per kg or per individual is the same as that for humans, or, for example, the above-mentioned administration is based on the volume ratio (e.g., average value) of the organs (e.g., heart, etc.) between the target animal and human. The converted dose can be administered. These dosages should not be construed as limiting the scope of the invention in any respect.

The term "individual" used herein refers to a subject who has or may develop a disease to which the composition of the present invention can be applied (prescribed), and may be a mammal such as a rat, mouse, or livestock, including humans. . Preferably, it may be a human, but is not limited thereto.

If necessary, the pharmaceutical composition of the present invention may additionally contain known substances that exhibit a preventive or therapeutic effect on the above diseases.

The pharmaceutical composition of the present invention may further include a substance that increases the activity of immune cells expressing the fusion protein. The pharmaceutical composition of the present invention can be administered in combination with conventionally known substances for preventing or treating the above diseases. At this time, the immune cells expressing the fusion protein, substances showing a preventive or therapeutic effect for the disease, and/or substances that increase the activity of the immune cells can be administered simultaneously or sequentially.

Another aspect of the present invention provides the use of immune cells expressing the fusion protein or a pharmaceutical composition containing the same for preventing or treating cancer.

Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to an individual an immune cell expressing the fusion protein or a pharmaceutical composition containing the same. Immune cells expressing the fusion protein, pharmaceutical composition, cancer, prevention, treatment, subject, and administration are the same as described above.

Combination of immune cells expressing fusion proteins and dual-specific antibodies

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an immune cell expressing the fusion protein and a dual-specific antibody as active ingredients.

Another aspect of the present invention provides a kit for preventing or treating cancer, including an immune cell expressing the fusion protein and a dual-specific antibody.

Immune cells and dual-specific antibodies expressing the fusion protein contained in the pharmaceutical composition and kit may be included in one formulation and may be administered in combination. At this time, the immune cells expressing the fusion protein and the dual-specific antibody may be administered simultaneously or sequentially.

Immune cells expressing the fusion protein, bispecific antibodies, cancer, prevention and treatment are the same as described above.

CD137 extracellular domain, variants thereof or fragments thereof

Another aspect of the invention provides CD137 extracellular domain variants or fragments thereof. At this time, the variant may include a mutation in any one selected from the group consisting of CRD1, CRD2, CRD3, CRD4, and combinations thereof.

Specifically, the variant may include mutations in CRD2 and/or CRD3. The mutations of CRD2 and CRD3 are the same as described above.

More specifically, the CD137 extracellular domain variant or fragment thereof may include or consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 27.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

Preparation example 1. Antibody information used

Antibody information used in the following experimental examples is shown in Table 1 below.

Antibodies	Source	Cat No.
CD3 Monoclonal Antibody (UCHT1), FITC	bioscience	11-0038-42
CD56 (NCAM) Monoclonal Antibody, PE	bioscience	12-0567-42
CD56 (NCAM) Monoclonal Antibody, FITC	bioscience	11-0566-42
CD16 Monoclonal Antibody, APC	bioscience	17-0168-42
Brilliant Violet 605™ anti-human CD226 (DNAM-1) Antibody	Biolegend	338324
PE anti-human CD335 (NKp46) Antibody	Biolegend	331908
APC anti-human CD336 (NKp44) Antibody	Biolegend	325110
Brilliant Violet 421™ anti-human CD314 (NKG2D) Antibody	Biolegend	320822
BV711 Mouse Anti-Human CD337 (NKp30)	BD Bioscience	563383
Urelumab	MedChemExpress	HY-99055
Biotin Antibody, PE, REAfinity	MiltenyiBiotec	130-110-951
PE Mouse Anti-Human CD137	BD Pharmingen	555956

Experimental Method 1. Cell analysis using flow cytometry

Fluorescently labeled antibodies of each FITC, PE, APC, BV421, BV605, and BV711 wavelength were used to stain NK cells. Specifically, 1.0 x 10 ⁵ NK cells were harvested and washed once with Flow Cytometry Staining Buffer (eBioscience, 00-4222-26). Reaction was performed in a refrigerator at 4°C, shielded from light for 30 minutes, and analyzed using flow cytometry.

Example 1. Preparation of transformed PB-NK cells

Example 1.1. Genetic construction manufacturing

The transgene containing the 4-1BB and CD3ζ domains was inserted into the pVAX1 vector using EcoRI and XhoI. The vector was converted into a linear template strand using XbaI (NEB, R0145S) for mRNA production. Afterwards, in vitro transcription was performed using the mMESSAGE mMACHINE ^TM T7 ULTRA Transcription Kit (Invitrogen, AM1345). For detailed information, refer to the manufacturer's instructions. The structure of the specific fusion protein is shown in Figures 1A and 1B. In addition, the fusion protein containing the extracellular domain of CD137 and the intracellular domain of CD3ζ was collectively named BBz. Specific amino acid sequences and nucleic acid sequences are shown in Table 2.

sequence number	designation		order
One	Signal sequence (SP)		MGNSCYNIVATLLVLNFERTRS
2	CRD1			LQDPCSNCPAGTFCDNNRNQIC
3	CRD2			SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC
6	CRD3			DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK
7	CRD4			DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG
16	4-1BB extracellular domain			LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG
11	linker			PSPADLSPGASSVTPPAPAREPGHSPQ
8	Transmembrane domain (TM)		IISFFLALTSTALLFLLFFLTLRFSVV
9	Intracellular domain (ICD) of CD137			KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
10	Intracellular domain (ICD) of CD3z			RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
18	Intracellular domain (ICD) of BBz		MGNSCYNIVATLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQITCDRCRQCKGVFRTRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
30	nucleotide coding amino acid of BBz		atgggcaaca gctgctacaa catcgtggcc acactgctgc tggtgctgaa cttcgagaga accagaagcc tgcaggaccc ctgcagcaat tgtcctgccg gcaccttctg cgacaacaac cggaatcaga tctgcagccc ctgtcctcct aacagcttta gctctgccgg cggacagaga acctgcgaca t ctgcagaca gtgcaagggc cggttccgga ccagaaaaga gtgcagcagc acctccaacg ccgagtgcga ttgcacacct ggcttccatt gtctcggagc cggctgttct atgtgcgagc aggattgcaa gcagggccaa gagctgacca agaagggctg caaggattgc tgcttcggca cc agaagcgg ggcatctgtc ggccctggac aaattgctct ctggacggca agagcgtgct ggtcaacggc accaaagaac gggatgtcgt gtgcggacct tctccagccg atttgtctcc tggcgcctct agcgttacac ctcctgctcc agctagagag cctggccact ctcctcagat catctcattc tttctggccc tgacctctac agccctgctg tttctgctgt tcttcctgac actgcggttc agcgtggtca agagaggccg gaagaagctg ctgtacatct tcaagttcatg cgg cccgtgcaga ccacacaaga ggaagatggc tgctcctgca gattccccga ggaagaagaa ggcggctgcg aacttagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac g tggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa
34	Intracellular domain (ICD) of CD28			RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
35	Intracellular domain (ICD) of OX40		ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

Example 1.2. Isolation and proliferation of PB-NK cells

The process was carried out by referring to Therabest's SOP (Standard Operating Procedure). Specifically, PBMC (Peripheral Blood Mononuclear Cell) was isolated using ficoll-Paque PLUS (Cytiva, 17144002). To obtain pure NK cells, Miltenyi Biotec's MACS (Magnetically activated cell sorting) system was used. The isolated NK cells were co-cultured with X-ray irradiated GE-K562 cells in a 1:2 ratio. On day 0, ALyS505N-0 (CSTI, 1020P10), 4% human AB serum (GeminiBio, 100-512), 1% L-Glutamine (ThermoFisher, 25030149), 500 IU/mL IL-2 (Hileukin), 3 ng/ NK using medium containing mL IL-12 (BD Bioscience, 554613), 50 ng/mL IL-15 (R&D systems, 247ILB-500CF), and 30 ng/mL IL-18 (MBL, B003-2). Cells were activated. Afterwards, NK cells were cultured in medium from which cytokines other than IL-2 were removed.

Example 1.3. Transformation of PB-NK cells

NK cells were electroporated using the Neon ^® Transfection system (ThermoFisher) and MaxCyte's GTx. First, NK cells were suspended at 5.0x10 ⁶ cells/100 μL with Buffer T (Invitrogen, MPK10096). 2.5 μg of mRNA was added to the cell suspension, and electroporation was performed at 1850 V, 20 ms, 1 purse. GTx followed the manufacturer's instructions, and electroporation was performed with the code set to 'Expanded NK-5'. Afterwards, a cell line expressing 4-1BB was isolated using anti-PE MicroBeads from Miltenyi Biotec (MiltenyiBiotec, 130-048-801).

Example 2. Preparation of cBBR immune cells

Example 2.1. Lentiviral vector preparation

Example 2.1.1 Genetic Construction Preparation

Signal sequence (SEQ ID NO: 1), extracellular domain of CD137 (4-1BB) (SEQ ID NO: 15) or a variant thereof (SEQ ID NO: 16, 17 or 27), linker (SEQ ID NO: 11), transmembrane domain (SEQ ID NO: 8 ), a polynucleotide (SEQ ID NO: 12, 13, 14) encoding a fusion protein (SEQ ID NO: 18, 19, 20 or 31) containing a co-stimulatory domain (SEQ ID NO: 9) and a primary signaling domain (SEQ ID NO: 10) or 30) was synthesized and loaded into the pCDH-EF1-MCS-IRES-Puro vector (SBI, CD532A-2). The amino acid sequences of the fusion proteins are shown in Tables 3 to 6. The fusion protein according to the present invention was named “cBBR (Chimeric 4-1BB receptor)”.

Fusion protein	domain	amino acid sequence	sequence number
cBBR (SEQ ID NO: 18)	Signal peptide	MGNSCYNIVATLLVLNFERTRS	One
	CRD1
	LQDPCSNCPAGTFCDNNRNQICS	2
	CRD2
	PCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC	3
	CRD3		DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK	6
	CRD4		DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG	7
	linker		PSPADLSPGASSVTPPAPAREPGHSPQ	11
	Transmembrane (TM)	IISFFLALTSTALLFLLFFLTLRFSVV	8
co-stimulatory domain		KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL	9
CD3z signal domain		RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR	10

Fusion protein	domain	amino acid sequence	sequence number
cBBR_I64R (SEQ ID NO: 19)	Signal peptide	MGNSCYNIVATLLVLNFERTRS	One
	CRD1
	LQDPCSNCPAGTFCDNNRNQICS	2
	CRD2 mutant	PCPPNSFSSAGGQRTCD R CRQCKGVFRTRKECSSTSNAEC	4
	CRD3		DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK	6
	CRD4		DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG	7
	linker		PSPADLSPGASSVTPPAPAREPGHSPQ	11
	Transmembrane (TM)	IISFFLALTSTALLFLLFFLTLRFSVV	8
	co-stimulatory domain		KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL	9
CD3z signal domain		RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR	10

Fusion protein	domain	amino acid sequence	sequence number
cBBR_V71R (SEQ ID NO: 20)	Signal peptide	MGNSCYNIVATLLVLNFERTRS	One
	CRD1
	LQDPCSNCPAGTFCDNNRNQICS	2
	CRD2 mutant	PCPPNSFSSAGGQRTCDICRQCKG R FRTRKECSSTSNAEC	5
	CRD3		DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK	6
	CRD4		DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG	7
	linker		PSPADLSPGASSVTPPAPAREPGHSPQ	11
	Transmembrane (TM)	IISFFLALTSTALLFLLFFLTLRFSVV	8
	co-stimulatory domain		KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL	9
CD3z signal domain		RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR	10

Fusion protein	domain	amino acid sequence	sequence number
cBBR_ I64R/V71R (SEQ ID NO: 31)	Signal peptide	MGNSCYNIVATLLVLNFERTRS	One
	CRD1
	LQDPCSNCPAGTFCDNNRNQICS	2
	CRD2 mutant	PCPPNSFSSAGGQRTCD R CRQCKG R FRTRKECSSTSNAEC	26
	CRD3		DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK	6
	CRD4		DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG	7
	linker		PSPADLSPGASSVTPPAPAREPGHSPQ	11
	Transmembrane (TM)	IISFFLALTSTALLFLLFFLTLRFSVV	8
	co-stimulatory domain		KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL	9
CD3z signal domain		RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR	10

The 4-1BBL expression vector was constructed by synthesizing the polynucleotide of SEQ ID NO: 25 and loading it into the pCDH-EF1-MCS-IRES-Puro vector (SBI, CD532A-2).

Example 2.2. Lentivirus production

pCDH-EF1-MCS-IRES-Puro vector loaded with polynucleotides encoding VSV-G, Rev, and cBBR or variants thereof (SEQ ID NO: 12, 13, 14 or 30) or a polynucleotide encoding 4-1BBL ( The pCDH-EF1-MCS-IRES-Puro vector loaded with SEQ ID NO: 25) was transduced into Lenti-X 293T cells (Clontech, 632180) using Lipofectamine 2000 (Thermo Fisher, 11668019). 72 hours after transfection, each supernatant was harvested, mixed with Lenti-X Concentrator (Clontech, 631232) in a 1:3 ratio, and stored in the refrigerator for more than 1 hour. Each of the above mixed solutions was centrifuged at 1,500g at 4°C, and the pellet was collected and used as virus particles capable of expressing cBBR or a variant thereof and virus particles expressing the 4-1BBL gene, respectively.

Example 2.3. cBBR T cell production

Example 2.3.1. Production of cell lines expressing cBBR

K562 cells and Nalm6 cells were purchased from the European collection of authenticated cell cultures (ECACC), and Jurkat T cells and Ramos cells were purchased from the Korean Cell Line Bank. Various cBBR Jurkat T cells and 4-1BBL Nalm6 cells were transduced using the lentivirus prepared in Example 2.2 above. All cells were cultured using RPMI-1640 (Hyclone, SH30027.01) medium containing 10% FBS (Gibco, 10100147).

Example 2.3.2. Production of blood-derived T cells expressing cBBR

First, to isolate T cells in the blood, PBMC (Peripheral Blood Mononuclear Cell) in the blood was isolated using Ficoll Paque PLUS (Cytiva, 17144002). T cells in PBMC were isolated using CD4+ T cell isolation kit, human (Miltenyi biotec, 130-096-533) and CD8+ T cell isolation kit, human (Miltenyi biotec, 130-096-495). T cells isolated as above were activated by adding T Cell TransAct, human (Miltenyi biotec, 130-111-160). Activated T cells were cultured using RPMI-1640 medium containing 10% FBS and 300 U/mL Proleukin IL-2 (NOVARTIS). The day after inducing activation, cBBR or its variants (I64R, V71R) were transduced into T cells using the lentivirus prepared in Example 2.2, and cultured for up to 14 days. The cells prepared as above are hereinafter referred to as “cBBR T cells.”

Example 2.4. cBBR NK cell production

Example 2.4.1. Preparation of NK cells

Lymphoblast patient-derived NK cells NK92MI (ATCC, CRL-2408) were supplemented with 12.5% FBS (fetal bovine serum, Gibco, 10100147), 12.5% horse serum (Gibco, 16050122), and 1% penicillin/streptomycin ( Gibco, 15140-122), alpha MEM (Cytiva, containing 0.2mM inositol (DAEJUNG, 5008-4105), 0.1mM 2-mercaptoethanol (Gibco, 21985023), and 0.02mM folic acid (Sigma Aldrich, F8758). SH30265.01) was cultured in a CO ₂ incubator using medium.

Example 2.4.2. Construction of NK cell lines expressing cBBR

An NK cell line expressing cBBR was created using the NK cells obtained in Example 2.4.1 in the same manner as in Example 2.3. The cells prepared as above are hereinafter referred to as “cBBR NK92MI cells” or “4-1BB expressing NK92MI cells.”

Experimental Example 1. Confirmation of changes in activity of transformed NK cells

Experimental Example 1.1. Confirmation of changes in the activity of NK cells expressing BBz by urelumab

A cell line was developed to determine whether NK92MI cells expressing 4-1BB are activated by the monoclonal antibody urelumab (Figure 2). Titration evaluation was performed to confirm the appropriate concentration of urelumab (Figures 3 and 4). Then, in order to confirm the IFN-γ produced by NK cells, the concentration of IFN-γ was measured in the NK cell culture medium using the Human IFN-gamma Quantikine ELISA kit (R&D systems, DIF50C). As a result, unlike NT NK92MI, it was confirmed that when 10 μg/mL of urelumab (MedChemExpress, HY-P99055) was treated in BBzNK92MI, the amount of IFN-γ increased by more than 9-fold (Figure 5).

Experimental Example 1.2. Confirmation of changes in PB-NK cell activity caused by urelumab

As with cell lines, cloning was performed to determine whether the same results were observed in peripheral blood (PB) NK cells and whether the structure without the intracellular domain (ICD) of 4-1BB showed stronger efficacy (Figure 1a). Electroporation (hereinafter referred to as EP) was performed on PB-NK using mRNA produced through IVT ( in vitro transcription). Immediately after EP, PB-NK was cultured on a plate coated with urelumab for 24 hours, and the produced IFN-γ was confirmed. In both BBz and tBBzNK cells, IFN-γ increased by approximately 3 to 4 times. However, it was confirmed that there was no change in the experimental group treated with soluble urelumab (Figure 8).

From these results, it was confirmed that the effect of urelumab alone on activating PB-NK was low, but that when one side of urelumab was fixed, the effect of urelumab on activating PB-NK increased. In other words, it can be seen that agonist 4-1BB antibody, such as urelumab, is not activated by binding to CD137, but that urelumab can activate NK with overexpressed CD137 only when immunological synapse is formed. Also, interestingly, BBz expressed 1.3 times more IFN-γ than tBBzNK cells, although the expression gradually decreased after 4 hours.

Experimental Example 1.3. Domain mapping for isolation of 4-1BB expressing cells

The genetic recombination efficiency of iPSCs is relatively low. To compensate for these shortcomings, enrichment is an essential process. Cloning was performed to find the minimal domain of 4-1BB that binds to the antibody of MiltenyiBiotec's GMP 4B4-1 clone. 4B4-1 (PE Mouse Anti-Human CD137 (BD Biosciences, 555956)) is reported to bind to CRD (Cystein-Rich Domain) 2 of 4-1BB, and urelumumab is reported to bind to CRD1, so cloning was performed based on this ( Figure 1b). However, unlike the reference, it was confirmed that not only 12BBz but also 1BBz K562 binds to 4B4-1 (FIG. 9).

Experimental Example 2. Analysis of transduced cells using flow cytometry

Cell surface protein expression of the cell line expressing cBBR or a variant thereof, or 4-1BBL, produced by the method of Example 2, and blood-derived T cells (cBBR T cells) expressing cBBR or a variant thereof was measured using FITC, PE. and was confirmed using a fluorescently labeled antibody of APC wavelength.

Specifically, each cBBR Jurkat T cell, 4-1BBL Nalm6 cell, and cBBR T cell (1.0×10 ⁵ cells) were washed once with Flow Cytometry Staining Buffer (eBioscience, 00-4222-26), and then incubated with each antibody. was treated and reacted at 4°C, shielded from light for 30 minutes. After the reaction was completed, the stained cells were analyzed using flow cytometry. At this time, the antibodies used are shown in Table 7.

antibody	manufacturing company	catalog number
CD3 Monoclonal Antibody (OKT3), FITC	bioscience	11-0037-42
CD8a Monoclonal Antibody (SK1), APC	bioscience	17-0087-42
PE Mouse Anti-Human CD137	BD Biosciences	555956
PE Mouse Anti-Human CD137 Ligand	BD Biosciences	559446
Goat Anti-Human IgG H&L (DyLight®488)	Abcam	ab96907
PE anti-human CD269 (BCMA)	Biolegend	357504
PE Mouse Anti-Human CD107a	BD Biosciences	555801

As a result, as shown in Figures 11 and 12, high CD137 expression was observed in cBBR Jurkat T cells and cBBR T cells compared to the cBBR control group (cells that did not overexpress cBBR) regardless of amino acid substitution. In addition, in the case of Nalm6 cells that overexpressed 4-1BBL (4-1BBL Nalm6 cells), it was confirmed that the expression rate of 4-1BBL was high compared to cells that did not overexpress 4-1BBL.

Experimental Example 3. Confirmation of binding ability of cBBR and 4-1BBL

The binding ability of cBBR or its variant, and 4-1BBL was confirmed using cBBR JurKat T cells and 4-1BBL Nalm6 cell line produced by the method of Example 2.

Specifically, cBBR Jurkat T cells were stained with 1 μM CellTracker Deep Red Dye (Invitrogen, C34565), and 4-1BBL Nalm6 cell line was stained with 5 μM CellTrace CFSE Cell Proliferation Kit (Invitrogen, C34554). The two types of stained cells were co-cultured for 30 minutes and then fixed by adding 4% paraformaldehyde solution (Biosesang, PC2031-100-00). The fixed cells were analyzed using flow cytometry (% of CFSE+ 7-AAD+ cells).

As a result, as shown in Figure 13, it was confirmed that the binding affinity of cBBR overexpressed in T cells to 4-1BBL overexpressed in Nalm6 cells was reduced due to amino acid substitution (mutant).

Experimental Example 4. Confirmation of cancer cell killing ability of cBBR T cells

To confirm the killing ability of cBBR T cells produced by the method of Example 2 against cancer cells, cBBR T cells and 4-1BBL Nalm6 cells were co-cultured to measure the cytotoxicity of cBBR T cells against cancer cells.

Specifically, the toxicity of cBBR T cells was performed using the Cytotoxicity Assay Kit (Abcam, ab270780). 4-1BBL Nalm6 cells were stained with the CFSE reagent included in the kit, mixed with cBBR T cells at a ratio of 0.5:1, 1:1, and 2:1, respectively, and co-cultured for 24 hours. At this time, Nalm6, in which 4-1BBL was not transduced, was used as a control. After co-culture, the cells were stained with 7-AAD and dead cells were confirmed through flow cytometry (% of CFSE+ 7-AAD+ cells).

As a result, as shown in Figure 14, cBBR T cells (including mutants) exhibited cytotoxicity against 4-1BBL Nalm6 cells due to the binding of the 4-1BB extracellular domain and 4-1BBL. In particular, cytotoxicity was reduced in cBBR T cells (I64R, V71R, I64R/V71R) in which some amino acids of cBBR were substituted (mutants). The above results are believed to be due to a decrease in binding force between the CD137 (4-1BB) extracellular domain of the cBBR variant and 4-1BBL.

Experimental Example 5. Confirmation of activation of cBBR T cells by urelumab

Experimental Example 5.1. Confirmation of cBBR T cell activation by urelumab through flow cytometry

A 96-well plate was coated with urelumab (MedChemExpress, HY-P99055) (1 μg/mL, 10 μg/mL), and then cBBR T cells (I64R, V71R, I64R/V71R) prepared by the method of Example 2 above were added. were inoculated at a concentration of 5×10 ⁵ cell/mL and reacted for 24 hours. After the reaction was completed, the cells were stained with anti-CD8a antibody (APC) and anti-CD107a antibody (PE), respectively, and analyzed using flow cytometry. As a result, as shown in Figure 15, the binding affinity of cBBR T cells with amino acid substitutions (mutants) to 4-1BBL decreased. However, it was confirmed that the activity caused by Urelumab, an anti-CD137 antibody (agonist), was not reduced.

Experimental Example 5.2. Confirmation of the IFN-γ secretion ability of cBBR T cells by urelumab

cBBR T cells (I64R, V71R, I64R/V71R) produced by the method of Example 2 were treated with urelumab at a concentration of 1 μg/mL or 10 μg/mL, and then incubated for 24 hours to induce activation. After the reaction, the concentration of IFN-γ secreted from activated cBBR T cells into the culture medium was measured using the Human IFN-gamma Quantikine ELISA Kit (R&D systems, SIF50C). As a result, as shown in Figure 16, the binding affinity of cBBR T cells with amino acid substitutions (mutants) to 4-1BBL decreased. However, it was confirmed that the activity caused by Urelumab, an anti-CD137 antibody (agonist), was not reduced.

Example 3. Production of bispecific antibody

The bispecific antibody used in the present invention was produced by Genescript Probio. The amino acid sequence of the bispecific antibody is shown in Table 8 below. At this time, the bispecific antibody is an anti-BCMA antibody, and an scFv that binds to 4-1BB is bound to the C terminus.

designation	amino acid sequence	sequence number
Heavy chain region	MGWSCIILFLVATATGVHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGLSWVRQAPGKGLEWVSLIDSSGSSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEHGLFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSG SSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGS GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	32
light chain region	MGWSCIILFLVATATGVHSEIVLTQSPGTLSLSPGERATLSCKASQDIDDAINWYQQKPGQAPRLLIYDASLRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSLRTPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC	33

Experimental Example 6. Activation of cBBR T cells by bispecific antibodies

Experimental Example 6.1. Confirmation of binding ability of cBBR T cells with cBBR and bispecific antibodies

The binding ability of cBBR T cells (I64R, V71R) produced by the method of Example 2 and the bispecific antibody (BiTE) produced by the method of Example 3 was confirmed.

Specifically, each cBBR T cell (Control, I64R, V71R) and Ramos cell (1.0 × 10 ⁵ cells) were washed once with Flow cytometry staining buffer (eBioscience, 00-4222-26), and then washed in Example 3 above. The bispecific antibody prepared by the method was treated and reacted at 4°C for 30 minutes, shielded from light. After the reaction was completed, the stained cells were analyzed using flow cytometry.

As a result, as shown in Figure 17, it was confirmed that the CD137 binding site (anti-CD137 Fab site) of the bispecific antibody and the CD137 extracellular domain of cBBR T cells bound. In addition, it can be confirmed that the BCMA binding site (anti-BCMA Fab site) of the bispecific antibody binds to the BCMA protein of Ramos cells.

Experimental Example 6.2. Confirmation of cancer cell killing ability of cBBR T cells by double binding

In order to confirm the killing ability of cBBR T cells whose activation was induced by a bispecific antibody (BiTE) against cancer cells, cBBR T cells (I64R, V71R) were treated with a bispecific antibody, and then cancer cells were killed using the method of Experimental Example 3. Cytotoxicity was measured. At this time, the bispecific antibody was treated at a concentration of 1 or 10 μg/mL for 24 hours.

As a result, as shown in Figure 18, when cBBR T cells (I64R, V71R) with amino acid substitutions (mutants) and Ramos cells were co-cultured and then treated with anti-BCMA/anti-4-1BB bispecific antibody, Cytotoxicity increased depending on the treatment concentration of the bispecific antibody. Through the above results, it was confirmed that an immunological synapse was formed between cBBR T cells (Effector cells) and Ramos cells (Target cells) by the bispecific antibody.

Experimental Example 7. Confirmation of activity of transformed T cells

One day before administering the transfected T cells, 3x10 ⁶ Ramos cells (ATCC, CRL-1596) were intraperitoneally administered to NSG mice (IP Injection). Before administering the transformed T cells, 3 mg of luciferin (Perkin elmer, cat.122799) was administered intraperitoneally and the fluorescence value of Ramos cells was measured using IVIS ^® fluorescence equipment. Afterwards, 1x10 ⁷ cBBR T cells and 200 ug of AB-101 bispecific antibody were administered intraperitoneally. Four days after administration of the transformed T cells, 3 mg of luciferin was administered intraperitoneally and the fluorescence value of Ramos cells was measured using an IVIS ^® fluorescence equipment. The measured fluorescence values were analyzed using the CleVue analysis program. The results are shown in Figure 19.

Claims (41)

1. Fusion protein comprising the following (i) to (iii):

   (i) CD137 (4-1BB) extracellular domain or fragment thereof;

   (ii) Transmembrane domain; and

   (iii) Primary signaling domain.
2. According to paragraph 1,

   The CD137 extracellular domain or fragment thereof is

   CRD1 (Cystein-rich pseudo repeat 1) containing the amino acid sequence of SEQ ID NO: 2; CRD2 comprising the amino acid sequence of SEQ ID NO: 3; CRD3 comprising the amino acid sequence of SEQ ID NO: 6; CRD4 comprising the amino acid sequence of SEQ ID NO: 7; And a fusion protein comprising any one selected from the group consisting of combinations thereof.
3. According to paragraph 2,

   The CD137 extracellular domain includes CRD1, CRD2, CRD3, and CRD4.
4. According to paragraph 2,

   A fusion protein in which the CD137 extracellular domain further includes mutations.
5. According to clause 4,

   The fusion protein is one in which the mutation is selected from the group consisting of CRD2, CRD3, and combinations thereof.
6. According to clause 5,

   The mutation of CRD2 is a substitution of any one amino acid selected from the group consisting of P3, S6, Q13, T15, C16, D17, I18, Q21, K23, V25, F26, and combinations thereof in the amino acid sequence of SEQ ID NO: 3. It's a fusion protein.
7. According to clause 5,

   The mutation of CRD3 is a fusion protein in which any one amino acid selected from the group consisting of S14, M15, C16, and combinations thereof is substituted in the amino acid sequence of SEQ ID NO: 6.
8. According to clause 6,

   The mutation of CRD2 is a fusion protein in which any one amino acid selected from the group consisting of I18R, V25R, and combinations thereof is substituted in the amino acid sequence of SEQ ID NO: 3.
9. According to clause 8,

   The mutation of CRD2 is a fusion protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 26.
10. According to paragraph 1,

    The transmembrane domain is T cell receptor (TCR), CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, A fusion protein selected from the group consisting of CD137, CD152, and CD154.
11. According to clause 10,

    A fusion protein in which the transmembrane domain is derived from CD137.
12. According to clause 11,

    A fusion protein in which the transmembrane domain includes the amino acid sequence of SEQ ID NO: 8.
13. According to paragraph 1,

    A fusion protein, wherein the intracellular signaling domain includes a co-stimulatory domain and an activation domain.
14. According to clause 13,

    A fusion protein, wherein the constant costimulatory domain is any one selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and CD137.
15. According to clause 14,

    A fusion protein in which the costimulatory domain is derived from CD137.
16. According to clause 15,

    A fusion protein in which the co-stimulatory domain consists of the amino acid sequence represented by SEQ ID NO: 9.
17. According to clause 13,

    A fusion protein, wherein the primary signaling domain is any one selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.
18. According to clause 17,

    A fusion protein in which the primary signaling domain is derived from CD3ζ.
19. According to clause 18,

    A fusion protein wherein the primary signaling domain includes the amino acid sequence of SEQ ID NO: 10.
20. According to paragraph 1,

    (i) CD137 extracellular domain or fragment thereof; and (ii) a fusion protein further comprising a linker between the transmembrane domains.
21. According to paragraph 1,

    The fusion protein contains the following structural formula (I):

    N'-A-(L1)n-TM-(L2)m-B-(L3)l-C-C' (I)

    At this time, in the structural formula (I),

    Wherein N' is the N terminus of each polypeptide,

    The C' is the C terminus of each polypeptide,

    where - means bonding,

    wherein A is the CD137 extracellular domain or fragment thereof;

    The TM is a transmembrane domain;

    B and C are intracellular signaling domains, each independently a co-stimulatory domain and a primary signaling domain;

    Wherein L1, L2 and L3 are each peptide linkers,

    The n, m and l are each independently 0 or 1.
22. A polynucleotide encoding the fusion protein of any one of claims 1 to 21.
23. A vector loaded with the polynucleotide of claim 22.
24. A virus containing the polynucleotide of claim 22.
25. Immune cells expressing the fusion protein of claim 1.
26. According to clause 25,

    The immune cells are natural killer cells, T cells, or macrophages.
27. According to clause 25,

    An immune cell in which the cell expressing the fusion protein is transformed by introducing the polynucleotide of claim 22.
28. A pharmaceutical composition for preventing or treating cancer comprising the immune cell of claim 25 as an active ingredient.
29. According to clause 28,

    A pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition further comprises a bispecific antibody.
30. According to clause 29,

    The bispecific antibody includes a first domain that specifically binds to a cancer antigen; And a pharmaceutical composition for preventing or treating cancer, comprising a second domain that specifically binds to CD137.
31. According to clause 30,

    The cancer antigens include alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b. , CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO- 1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, A pharmaceutical composition for preventing or treating cancer, which is any one selected from the group consisting of TAG72, TEMs, VEGFR2 and WT-1.
32. According to clause 28,

    The above cancers include stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. , a pharmaceutical composition for preventing or treating cancer, which is any one selected from the group consisting of kidney cancer, melanoma, multiple myeloma, brain cancer, osteosarcoma, glioblastoma, IgG-opsonized tumor, lymphoma, neuroma, mesothelioma, and esophageal cancer.
33. A kit for cancer treatment comprising the immune cell of item 25 and the dual specific antibody of item 29.
34. i) preparing the virus of paragraph 24; and

    ii) Processing the virus into immune cells; A method of producing immune cells expressing the fusion protein of claim 1, comprising:
35. CD137 extracellular domain variant or fragment thereof.
36. According to clause 35,

    The variant is a variant or fragment thereof that includes a mutation in any one selected from the group consisting of CRD1, CRD2, CRD3, CRD4, and combinations thereof.
37. According to clause 36,

    The variant is a variant or fragment thereof that includes a mutation in any one selected from the group consisting of CRD2, CRD3, and combinations thereof.
38. According to clause 37,

    The mutation of CRD2 is a substitution of any one amino acid selected from the group consisting of P3, S6, Q13, T15, C16, D17, I18, Q21, K23, V25, F26, and combinations thereof in the amino acid sequence of SEQ ID NO: 3. A variant or fragment thereof.
39. According to clause 37,

    The mutation of CRD3 is a variant or fragment thereof in which any one amino acid selected from the group consisting of S14, M15, C16, and combinations thereof is substituted in the amino acid sequence of SEQ ID NO: 6.
40. According to clause 38,

    The mutation of CRD2 is a variant or fragment thereof in which any one amino acid selected from the group consisting of I18R, V25R, and a combination thereof is substituted in the amino acid sequence of SEQ ID NO: 3.
41. According to clause 40,

    The CRD2 mutation is a variant or fragment thereof comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 26.

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