KR20190141511A - New peptide, chemeric antigen receptor and immune cell expressing the same - Google Patents

Abstract

The present invention relates to a novel peptide and a chimeric antigen receptor comprising the same. The present invention relates to a chimeric antigen receptor (CAR) which has an intracellular signaling domain including a part or the entirety of the novel peptide and thus exhibits an excellent effect of augmenting the anticancer activity of immune cells; and to immune cells expressing the same.

Description

Novel peptides, chimeric antigen receptors comprising the same and immune cells expressing the same {NEW PEPTIDE, CHEMERIC ANTIGEN RECEPTOR AND IMMUNE CELL EXPRESSING THE SAME}

The present invention relates to novel peptides, chimeric antigen receptors comprising the same and immune cells expressing the same.

Human natural killer cells (NK cells) are responsible for innate immunity and can remove virus-infected cells or tumor cells, making them safe and effective immune cell therapies.

Recently, chimeric antigen receptors are expressed on T cells to increase the recognition and activation of specific antigens of tumor cells, resulting in dramatic therapeutic effects and approved as an autoimmune cell therapy. Unlike T cells, NK cells can be applied not only to autologous but also to taga, and to study CAR-NK cell therapeutics that can overcome side effects such as GVHD and Cytokine release syndrome (CRS) of CAR-T transgenic. Lots of things are going on.

In order to enhance the efficacy of immune cells into which the chimeric antigen receptor has been introduced, various supplemental methods such as CD28, 4-1BB (CD137), OX40 (CD134), DAP10, DAP12, or ICOS are used as intracellular signaling domains constituting the chimeric antigen receptor Application of stimulus domains is possible.

4-1BB (CD137) is a member of the Tumor necrosis factor receptor (TNFR) family, expressed in T cells, B cells, natural killer cells, and dendritic cells, and acts as a co-stimulatory molecule in T cells to prevent activation induced cell death (AICD). It is known to play an important role in the activation of T cells by preventing and increasing the cytotoxicity and secretion of type 1 cytokines (IL-2, IFNg, TNFa). In addition, the introduction of 4-1BB (CD137) as a co-stimulatory factor in CAR-T therapy increased the survival of T cells, prolonged survival in the body, and suppressed exhaustion, thereby inducing CD28 as a co-stimulatory factor. In vivo efficacy has been reported.

However, in mouse spontaneous killer cells, the combination of CD137 and CD137 ligand acts as an activation signal such as increased proliferation and IFNg secretion, whereas in human spontaneous killer cells, IFNg secretion and cytotoxicity have been reported. Not known

Korean Laid-Open Patent No. 2016-0062760

An object of the present invention is to provide a novel peptide.

An object of the present invention is to provide a chimeric antigen receptor for enhancing the anticancer immune cell treatment efficiency using immune cells.

1. In the amino acid sequence of SEQ ID NO: 39, (a) substitution of residues 231-235 with TTGAA; (b) substitution of the residues 237-239 for AAA; And (c) a variant amino acid sequence for SEQ ID NO: 39 comprising one or more variations selected from the group consisting of substitution of residue 245 with C.

2. The peptide according to the above 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

3. A nucleic acid molecule encoding the peptide of the above 1 or 2.

4. The nucleic acid molecule according to the above 3, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6 to 10.

5. A chimeric antigen receptor (chimeric antigen receptor) comprising all or part of the peptide of the above 1 as an intracellular signaling domain.

6. The method according to the above 5, further comprising a whole or a portion of the peptide as a first intracellular signaling domain, further comprising a second intracellular signaling domain comprising all or a portion of the CD3-zeta, the first And the second intracellular signaling domain is located in order in the intracellular direction at the cell membrane.

7. In the above 5,

A transmembrane domain linked to the intracellular signaling domain;

A spacer domain linked to the transmembrane domain; And

Extracellular domain linked to the spacer domain

Further comprising, chimeric antigen receptor.

8. The chimeric antigen receptor according to the above 7, further comprising a signal sequence connected to the extracellular domain.

9. The chimeric antigen receptor according to the above 8, wherein the signal sequence comprises all or part of CD16 or CD8α.

10. The method according to the above 7, wherein the transmembrane domain comprises all or part of CD8α, chimeric antigen receptor.

11. The method according to the above 7, wherein the spacer domain comprises all or part of CD8α, chimeric antigen receptor.

12. The chimeric antigen receptor according to the above 7, wherein the extracellular domain comprises all or part of CD16V or NKG2D.

13. Immune cells expressing chimeric antigen receptor of any one of 5 to 12 above.

14. The immune cell according to 13 above, wherein the immune cell is a natural killer cell (NK cell).

15. A pharmaceutical composition for treating tumors comprising the immune cells of 14 above.

16. A nucleic acid molecule encoding the chimeric antigen receptor of any one of 5 to 12 above.

17. The method according to the above 16, wherein the nucleic acid molecule encodes at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 29, 35, and 37 or a variant thereof having at least 80% sequence identity, Nucleic Acid Molecules.

18. The nucleic acid molecule of above 17, wherein the nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 34, and 36 or variants thereof having 80% or more sequence identity, Nucleic Acid Molecules.

The chimeric antigen receptor according to the present invention is excellent in natural killer cell activation efficiency.

The chimeric antigen receptor according to the invention can be used with various cancer target antibodies depending on the type of cancer targeted.

The chimeric antigen receptor according to the invention can be applied to a variety of carcinomas by applying various antigen recognition sites.

Natural killer cells expressing the chimeric antigen receptor according to the present invention have excellent cytotoxicity against cancer cells.

Natural killer cells expressing the chimeric antigen receptor according to the present invention can be usefully used for the treatment of immune cells.

1A illustrates a CD16V-BBZ CAR, CD16V-BB (231-235) Z CAR, CD16V-BB (237-239) Z CAR or CD16V-BB (231-235, 237-239) Z according to an embodiment of the invention. The expression level of each CAR in NK92MI cells transduced with CAR is shown.
1B shows a CD16V-BBZ CAR, CD16V-BB (231-235) Z CAR, CD16V-BB (237-239) Z CAR or CD16V-BB (231-235, 237-239) Z according to an embodiment of the invention. Evaluation of natural killer cell killing ability in combination with antibody to Ramos cells of NK92MI cells transduced with CAR.
Figure 2A shows the expression level of each CAR in NK92MI cells transduced with CD16V-BBZ CAR, CD16V-BB (F245C) Z CAR or CD16V-BB (231-235, F245C) Z CAR according to an embodiment of the present invention. Indicates.
2B shows antibody combination against Ramos cells in NK92MI cells transduced with CD16V-BBZ CAR, CD16V-BB (F245C) Z CAR or CD16V-BB (231-235, F245C) Z CAR according to an embodiment of the present invention Shows the natural killer cell killing ability of the city.
Figure 3A shows the expression level of each CAR in NK92MI cells transduced with NKG2D-BBZ CAR, NKG2D-BB (F245C) Z CAR or NKG2D-BB (231-235, F245C) Z CAR according to an embodiment of the present invention. Indicates.
3B shows natural killing of MCF cells in NK92MI cells transduced with NKG2D-BBZ CAR, NKG2D-BB (F245C) Z CAR or NKG2D-BB (231-235, F245C) Z CAR according to an embodiment of the present invention. Cell killer evaluation is shown.

Hereinafter, the present invention will be described in detail.

The present invention relates to novel peptides.

Peptides of the invention in the amino acid sequence of SEQ ID NO: 39, (a) the substitution of residues 231-235 with TTGAA; (b) substitution of the residues 237-239 for AAA; And (c) a variant amino acid sequence for SEQ ID NO: 39 comprising one or more variations selected from the group consisting of substitution of residue 245 with C.

This sequence is a variant of the amino acid sequence corresponding to the intracellular signaling domain (901-1026nt, GenBank NM 001561.5, 4-1BB) derived from CD137.

Since 41BB has no intrinsic kinase activity, signaling is induced by interaction with its associated molecule, TNF receptor-associated factor (TRAF) 1-3. TRAF2 is known to bind to human 41BB and transmit an activation signal, while TRAF3 has a negative impact.

The present inventors devised the present invention by discovering that when the specific sequence of the intracellular signaling domain of human 41BB is replaced, the anticancer activity of the immune cell into which the chimeric antigen receptor containing the same is significantly increased is devised.

Specifically, the peptides of the present invention have one or more of three mutations in the wild type 4-1BB intracellular signaling domain, and BB (231-235) is a PVQTT sequence which is a TRAF binding region of the TNFR family CD30 and CD40. Is substituted with TTGAA, BB (237-239) is the substitution of EED237-239 with AAA among the two acidic residues (EED237-239, EEE248-250), BB (F245C) is CRFP sequence of human 41BB Is replaced by.

To give a specific example, the peptide of the present invention may be one comprising the amino acid sequence of SEQ ID NO: 1 to 5.

The present invention also relates to a nucleic acid molecule encoding the peptide.

The nucleic acid molecule of the present invention is not limited as long as it can encode the peptide, and for example, may include a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-10.

The present invention also relates to a chimeric antigen receptor comprising the peptide.

The chimeric antigen receptor of the present invention may comprise the peptide as an intracellular signaling domain.

According to one embodiment of the invention, the chimeric antigen receptor comprises a transmembrane domain connected to the intracellular signaling domain; A spacer domain linked to the transmembrane domain; And an extracellular domain linked to the spacer domain. In addition, according to an embodiment of the present invention, the chimeric antigen receptor may further include a signal sequence connected to a terminal which is not connected to the spacer domain of the extracellular domain. According to one embodiment of the invention, each of the above domains may be directly connected to each other or may be connected by a linker.

According to one embodiment of the invention, the signal sequence may be such that the extracellular domain can be located outside the cell membrane of immune cells (eg, natural killer cells) when the chimeric antigen receptor is expressed. For example, the signal sequence may include all or a part of CD16 or CD8α.

According to one embodiment of the invention, the extracellular domain is a domain for binding specifically to the antibody or specifically recognize the antigen, for example Fc receptor (Fc receptor), single-chain variable fragment (single-chain variable fragment) , An antigen-binding fragment of an antibody such as ScFv), a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1. Thus, the term "extracellular domain" of the present invention is used in the same sense as "antigen recognition site", "antigen binding fragment" or "antibody binding site".

The chimeric antigen receptor according to one embodiment of the present invention can be used with various antibodies depending on the cell type of the cancer to be treated by including the Fc receptor as an extracellular domain. According to one embodiment, the Fc receptor may be any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89. According to a more specific embodiment, the Fc receptor may comprise all or part of the CD16 V158 variant (CD16V).

According to another embodiment, the chimeric antigen receptor of the present invention may include as an extracellular domain an antigen binding fragment of an antibody that directly recognizes the antigen as an extracellular domain without co-administration with the antibody. According to one embodiment, the antigen-binding fragment may be a Fab fragment, F (ab ') fragment, F (ab') 2 fragment or Fv fragment. According to an embodiment of the present invention, the antibody may be any one of various kinds of antibodies capable of antigen-specific binding. For example, an antibody may be one light chain and one heavy chain combined, and two light chains and two heavy chains combined. For example, when two light chains and two heavy chains are combined, the first unit in which the first light chain and the first heavy chain are combined, and the second unit in which the second light chain and the second heavy chain are combined may be combined with each other. Herein, the bond may be, but is not limited to, a disulfide bond. According to an embodiment of the present invention, the two units may be the same as or different from each other. For example, the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other. As described above, an antibody prepared such that the first unit and the second unit can recognize two different antigens is commonly referred to in the art as a bispecific antibody. In addition, for example, the antibody may be a combination of three or more of the above units. The antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but is not limited thereto.

According to another embodiment of the present invention, the extracellular domain used in the present invention may be a natural cytotoxicity receptor. According to a specific embodiment, the natural killer receptors include, but are not limited to, NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.

According to an embodiment of the present invention, the transmembrane domain is located through the cell membrane, and any one can be located through the cell membrane without interfering with the function of the extracellular domain and the intracellular signaling domain. Any use is available. For example, the transmembrane domain may comprise all or part of CD8α.

According to an embodiment of the present invention, the extracellular domain and the transmembrane domain may be connected by a spacer domain. For example, the spacer domain can be a hinge domain. According to a specific embodiment, the spacer domain may include all or part of the CD8α.

According to an embodiment of the present invention, the intracellular signaling domain is a part which is located inside the cell membrane of the natural killer cell, that is, the cytoplasm, when the antibody bound to the extracellular domain of the present invention binds to the target antigen, It may include a sequence capable of transmitting a signal for activating natural killer cells.

According to one embodiment of the invention, the chimeric antigen receptor may comprise one or more intracellular signaling domains. In the case of including two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other. For example, in the case of including three intracellular signaling domains, one end of the first intracellular signaling domain is connected to an end which is not connected to the spacer domain of the transmembrane domain, and the first intracellular signaling domain is connected. One end of a second intracellular signaling domain is linked to a terminus that is not linked to the transmembrane domain of and a third to a terminus that is not linked to the first intracellular signaling domain of the second intracellular signaling domain. One end of the intracellular signaling domain may be linked. That is, the first, second and third intracellular signaling domains may be located in order in the cell membrane in the intracellular direction. Even if two, four or more intracellular signaling domains can be linked to one another in the same way. According to one embodiment of the invention, each of the above domains may be directly connected to each other or may be connected by a linker.

According to one embodiment of the invention the chimeric antigen receptor may be one comprising two intracellular signaling domains. For example, it may include a first intracellular signaling domain connected to the transmembrane domain and a second intracellular signaling domain connected to a terminal not connected to the transmembrane domain of the first intracellular signaling domain. According to a more specific embodiment, the first intracellular signaling domain may include all or a part of any one selected from the group consisting of the peptides of SEQ ID NOs: 1 to 5, the second intracellular signal The delivery domain may be one that includes all or part of CD3-zeta.

According to one specific embodiment, the chimeric antigen receptor comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 29, 35 and 37 or variants thereof having 80% or more sequence identity. Can be.

The present invention further provides immune cells (eg, natural killer cells (NK cells)) expressing the chimeric antigen receptor according to the invention described above.

The immune cells of the present invention may be toxic to tumor cells. Since the chimeric antigen receptor according to the present invention is determined which tumor cell is specifically toxic depending on which antibody the extracellular domain binds to, the immune cells expressing the chimeric antigen receptor according to the present invention are toxic. Tumor cells that can be expressed are not particularly limited. According to one embodiment, when the immune cells of the present invention (eg, natural killer cells) are used with rituximab, they may be toxic to malignant lymphoma cells. For example, the malignant lymphoma cells may be expressing CD20. Also for example, the malignant lymphoma may be B-cell lymphoma.

The present invention further provides that the number of immune cells (eg, natural killer cells) expressing chimeric antigen receptors according to the invention described above is comprised between two and 7.5 times the number of tumor cells (eg, malignant lymphoma cells) in the subject of treatment, It provides a pharmaceutical composition for the prevention or treatment of a tumor or tumor metastasis.

According to an embodiment of the present invention, the pharmaceutical composition of the present invention comprises from 0.75 times the number of the immune cells (eg, natural killer cells) in one dose to the number of the tumor cells (eg, malignant lymphoma cells) in the treatment target. It may be included 10 times. For example, the number of immune cells (eg, natural killer cells) in a single dose may be comprised between 2 and 7.5 times the number of the tumor cells (eg, malignant lymphoma cells) in the treatment subject.

The present invention also provides a nucleic acid sequence encoding the chimeric antigen receptor according to the invention described above.

According to one embodiment of the invention, the nucleic acid sequence comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 34 and 36 or variants thereof having at least 80% sequence identity. It may be.

The present invention also provides a vector comprising the nucleic acid sequence according to the present invention described above.

The present invention also provides a method for treating a tumor comprising administering the above-described immune cells to a subject.

The present invention also provides a method for preventing tumor metastasis, comprising administering the above-described immune cells to a subject.

The subject may be a mammal with a tumor, specifically, a human, but is not limited thereto.

Administration can be in an amount of 2 to 7.5 times the number of immune cells (eg, natural killer cells) expressing the chimeric antigen receptor according to the invention, of the number of tumor cells (eg, malignant lymphoma cells) in the treatment subject.

The method of administration is not particularly limited and may be administered via conventional oral or parenteral routes.

The tumor is not particularly limited, and may be, for example, malignant lymphoma, leukemia, breast cancer, lung cancer, or the like, and more specifically, B-cell lymphoma.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, but the scope of the present invention is not limited to those illustrated in these examples.

Example

1. Experimental Methods and Reagents

Cell line

Human B-family cell line Ramos, human erythroleukemic cell line K562, human breast cancer cell line MCF-7 and human natural killer cell line NK-92MI were supplied from ATCC (American Type Culture Collection, Manassas, VA, USA). Human embryonic kidney fibroblast 293T was used from KTCC.

K562 is maintained at RPMI-1640 (Gibco, Grand Island, NY, USA) containing 10% FBS, and Ramos is RPMI-1640 containing 10% FBS (fetal bovine serum; Gibco, Grand Island, NY, USA) (ATCC) (Manassas, VA). MCF-7 was maintained in EMEM (ATCC) medium containing 10% FBS (Gibco). NK92MI cells transduced with NK92MI were maintained in CellGro® (Cellgenix) serum-free medium containing 1% human plasma. 293T cells were maintained in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco, Grand Island, NY, USA).

Plasmid

Signal sequence and extracellular domain of FCRG3A V158 variant (CD16V); NKG2D extracellular domain; Signal sequence of CD8αa, hinge and transmembrane domain of CD8αa; Hinge and transmembrane domain, intracellular signaling domain of CD28; Various mutants of 4-1BB and 41BB and intracellular signaling domains of CD3ζ (CD3z) were artificially synthesized, respectively. They were assembled in various combinations using SOE-PCR (splicing by overlapping extension by PCR). PCR results were confirmed by direct sequencing. PCR products were digested with Nhe1 and EcoRI and used to prepare MSCV-EF1α-GFP vector or EF1a-MCS vector (NKG2D CAR-NK), a third generation self-inactivating lentiviral expression vector (Ligation) at the Nhe1 and EcoRI sites of the isolated vector).

The chimeric antigen receptor (CAR) according to an embodiment of the present invention is summarized in Table 1. Domains of all CARs according to an embodiment of the present invention are connected in tandem with each other, and also in frame.

CAR
Kinds
(Generation) Abbreviation signal
order ECD Hinge TM Signal-1 Signal-2 Signal-3 2nd CD16V-BBZ CD16 CD16V CD8α CD8α 4-1BB CD3ζ CD16V-BB (△ F245C) Z CD16 CD16V CD8α CD8α 4-1BB
(△ F245C) CD3ζ CD16V-BB (△ 231-235) Z CD16 CD16V CD8α CD8α 4-1BB
(△ 231-235) CD3ζ CD16V-BB (△ 231-235, ΔF245C) Z CD16 CD16V CD8α CD8α 4-1BB (△ 231-235, △ F245C) CD3ζ CD16V-BB (△ 237-239) Z CD16 CD16V CD8α CD8α 4-1BB
(△ 237-239) CD3ζ CD16V-BB (△ 231-235, △ 237-239) Z CD16 CD16V CD8α CD8α 4-1BB
(△ 231-235, △ 237-239) CD3ζ

CD16V-BBZ CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

The CD16V-BB (ΔF245C) Z CAR (2nd generation) contains the signal sequence domain (34-84 nucleotides, GenBank Accession No. X52645) of CD16; Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (ΔF245C) (901-1026 nucleotides, mutations of 994-996 nucleotides in GenBank NM 001561.5, C mutations of amino acids 245); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

The CD16V-BB (Δ231-235) Z CAR (2nd generation) contains the signal sequence domain (34-84 nucleotides, GenBank Accession No. X52645) of CD16; Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (Δ231-235) (901-1026 nucleotides, mutations of nucleotides 952-966 in GenBank NM 001561.5, TTGAA mutations of amino acids 231-235); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

CD16V-BB (Δ231-235, ΔF245C) Z CAR (2nd generation) were found in the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domain from CD137 (Δ231-235, ΔF245C) (901-1026 nucleotide, 952-966, 994-996 nucleotides of GenBank NM 001561.5, TTGAA mutation of amino acids 231-235, 245 C mutation of amino acid 1); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

The CD16V-BB (Δ237-239) Z CAR (2nd generation) contains the signal sequence domain (34-84 nucleotides, GenBank Accession No. X52645) of CD16; Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (Δ237-239) (901-1026 nucleotides, mutations of 970-978 nucleotides in GenBank NM 001561.5, AAA mutations of amino acids 237-239); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

CD16V-BB (Δ231-235, Δ237-239) Z CAR (2nd generation) are the signal sequence domains of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8a (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domain derived from CD137 (Δ231-235, Δ237-239) (901-1026 nucleotide, 952-966, 970-978 nucleotides of GenBank NM 001561.5, TTGAA mutations of amino acids 231-235 , AAA mutation of amino acids 237-239); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

CAR
Kinds
(Generation) Abbreviation signal
order ECD Hinge TM Signal-1 Signal-2 Signal-3 2nd NKG2D-BBZ CD8α NKG2D CD8α CD8α 4-1BB CD3ζ NKG2D-BB (△ F245C) Z CD8α NKG2D CD8α CD8α 4-1BB
(△ F245C) CD3ζ NKG2D-BB (△ 231-235, △ F245C) Z CD8α NKG2D CD8α CD8α 4-1BB
(△ 231-235, △ F245C) CD3ζ

NKG2D-BBZ CAR (2nd generation) comprises the signal sequence domain of CD8α (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8α (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3ζ.

NKG2D-BB (ΔF245C) Z CAR (2nd generation) comprises the signal sequence domain of CD8α (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8α (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (ΔF245C) (901-1026 nucleotides, mutations of 994-996 nucleotides in GenBank NM 001561.5, C mutations of amino acids 245); And a stop codon TGA linked to an intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

NKG2D-BB (Δ231-235, ΔF245C) Z CAR (2nd generation) were found in the signal sequence domain (890-952 nucleotide, GenBank NM 001768.6) of CD8α; Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8α (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domain from CD137 (Δ231-235, ΔF245C) (901-1026 nucleotide, 952-966, 994-996 nucleotides of GenBank NM 001561.5, TTGAA mutation of amino acids 231-235, 245 C mutation of amino acid 1); And the stop codon TGA and the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3z.

Table 3 shows a sequence listing of chimeric antigen receptors (CARs) according to an embodiment of the present invention and the domains used for the preparation thereof.

SEQ ID NO: Sequence name Description of the sequence One 4-1BB (△ 231-235 mutant) amino acid Δ231-235 mutant amino acid sequence of intracellular signaling domain (GenBank NM 001561.5) from 4-1BB (CD137) 2 4-1BB (△ 237-239 mutant) amino acid Δ237-239 mutant amino acid sequence of intracellular signaling domain (GenBank NM 001561.5) from 4-1BB (CD137) 3 4-1BB (△ 231-235, △ 237-239 mutant) amino acid Δ231-235, Δ237-239 mutant amino acid sequences of the intracellular signaling domain (GenBank NM 001561.5) from 4-1BB (CD137) 4 4-1BB (△ F245C_mutant) amino acid ΔF245C mutant amino acid sequence of the intracellular signaling domain (GenBank NM 001561.5) from 4-1BB (CD137) 5 4-1BB (△ 231-235, △ F245C mutant) amino acid Δ231-235, ΔF245C mutant amino acid sequences of the intracellular signaling domain (GenBank NM 001561.5) from 4-1BB (CD137) 6 4-1BB (△ 231-235 mutant) nucleotide Nucleotide sequence corresponding to SEQ ID NO: 1 7 4-1BB (△ 237-239 mutant) nucleotide Nucleotide sequence corresponding to SEQ ID NO: 2 8 4-1BB (△ 231-235, △ 237-239 mutant) nucleotide Nucleotide sequence corresponding to SEQ ID NO: 3 9 4-1BB (△ F245C_mutant) nucleotide Nucleotide sequence corresponding to SEQ ID NO: 4 10 4-1BB (231-235, ΔF245C mutant) nucleotide Nucleotide sequence corresponding to SEQ ID NO: 5 11 CD16 nucleotide Signal sequence and extracellular domain of CD16 (34-84 nt, GenBank Accession No. X52645) 12 CD16 amino acid Amino acid sequence corresponding to SEQ ID NO: 11 13 CD16V nucleotide Codon optimized sequence of the G mutation of 559 nucleotides in the signal sequence and extracellular domain of CD16V (85-651nt, GenBank Accession No. X52645) 14 CD16V amino acid Amino acid sequence corresponding to SEQ ID NO: 13 15 CD8a nucleotide Codon optimized sequence of hinge and transmembrane domain (1292-1507nt, GenBank NM 001768.6) derived from human CD8a 16 CD8a amino acid Amino acid sequence corresponding to SEQ ID NO: 15 17 CD3z nucleotide Codon-Optimized Sequences of the Intracellular Signaling Domain from CD3z (299-634 nt, GenBank NM000734.3) 18 CD3z amino acid Amino acid sequence corresponding to SEQ ID NO: 17 19 CD16V-BB (△ 231-235) Z nucleotide Codon Optimized Nucleotide Sequences of CD16V-BB (Δ231-235) Z CAR 20 CD16V-BB (△ 231-235) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 19 21 CD16V-BB (△ 237-239) Z nucleotide Codon Optimized Nucleotide Sequences of CD16V-BB (Δ237-239) Z 22 CD16V-BB (△ 237-239) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 21 23 CD16V-BB (△ 231-235, △ 237-239) Z nucleotide Codon Optimized Nucleotide Sequences of CD16V-BB (Δ231-235, Δ237-239) Z 24 CD16V-BB (△ 231-235, △ 237-239) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 23 25 CD16V-BB (△ F245C) Z nucleotide Codon Optimized Nucleotide Sequences of CD16V-BB (ΔF245C) Z 26 CD16V-BB (△ F245C) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 25 27 CD16V-BB (△ 231-235, ΔF245C) Z nucleotide Codon Optimized Nucleotide Sequences of CD16V-BB (Δ231-235, ΔF245C) Z 28 CD16V-BB (△ 231-235, ΔF245C) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 27 29 CD8a nucleotide Signal sequence from human CD8a (1-63nt, GenBank NM 001768.6) 30 CD8a amino acid Amino acid sequence corresponding to SEQ ID NO: 29 31 NKG2D nucleotide Extracellular domain derived from human NKG2D (788-1192nt, GenBank ID: AF461811.1) 32 NKG2D amino acid Amino acid sequence corresponding to SEQ ID NO: 31 33 NKG2D-BB (△ F245C) Z nucleotide Codon Optimized Nucleotide Sequence of NKG2D-BB (ΔF245C) Z 34 NKG2D-BB (△ F245C) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 33 35 NKG2D-BB (231-235, ΔF245C) Z nucleotide Codon Optimized Nucleotide Sequences of NKG2D-BB (231-235, ΔF245C) Z 36 NKG2D-BB (231-235, ΔF245C) Z amino acid Amino acid sequence corresponding to SEQ ID NO: 35 37 4-1BB (CD137) nucleotide Codon optimized sequence of intracellular signaling domain (901-1026nt, GenBank NM 001561.5) from 4-1BB (CD137) 38 4-1BB (CD137) amino acid Amino acid sequence corresponding to SEQ ID NO: 37 39 4-1BB (CD137) nucleotide Natural sequence of intracellular signaling domain (901-1026nt, GenBank NM 001561.5) derived from 4-1BB (CD137)

Virus Production and Gene Transfer

To prepare VSVG-pseudotyped lentiviral, various types of PCDH1-MSCV-CD16-construct-EF1-copGFP vectors, or PCDH1-MSCV-EF1, were applied to 293T cells cultured in DMEM medium containing 10% FBS. -copGFP control vector (for mok infection virus production using empty vector) and EF1a-NKG2D-construct vector or EF1a-GFP control vector (for moq infection viral production using empty vector); And HIV-based pPACKH1 lentiviral package kit (System Biosciences) were co-transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.). Various types of CD16V-41BB mutant construct expression vectors or control plasmids with about 80% 293T cells in a 75T flask; And pPACKH1 lentiviral packaging plasmids were transfected and replaced with DMEM medium containing 10% FBS and Sodium butyrate (Sigma Aldrich, USA) 6 hours later. 48 hours after transfection, the conditioned medium containing the lentiviral was removed and filtered using a 0.45 μm filter unit (Milliopore, Billerica, Mass., USA) to remove cell debris. The supernatant containing lentiviral was concentrated about 50-fold by centrifugation at 3000 rpm for 40 minutes at 4 ° C using Amicon Filter (Millipore). The concentrated virus was stored at -80 ° C.

For lentiviral infection, NK92MI cells in logarithmic phase are adjusted to 1 x 10 ⁶ cells / ml using Cellgro (Cellgenix) containing 1% human plasma. 50 MOI of lentiviral supernatant and 8 μg / ml polybrene were added to the cells and centrifuged at 1800 g for 90 minutes at 32 ° C. After centrifugation, the cells are placed in a humidified incubator at 37 ° C. and 5% CO 2 for 48 hours. Cells are then washed twice with RPMI-1640 medium containing 10% FBS and placed in RPMI-1640 medium containing 10% FBS until used in the experiment. Control cells were transduced using the vector only.

CD16V  or NKG2D - 41BB  Mutation Construct  Including receptor expression detection

NK92MI cells transduced with each CD16V-41BB mutant chimeric antibody receptor, control vector-transduced NK92MI (NK92MI-mock), or NK-92MI parent cells were washed twice with FACS buffer and then 7-AAD ( Beckman coulter), anti-CD3, anti-CD56 and anti-CD16 (BD Biosciences) mAbs. Expression ratio and mean fluorescence intensity (MFI) of the stained cells were measured using BD LSRFortessa.

NK92MI cells transduced with NKG2D-41BB mutant chimeric antibody receptor were washed twice with FACS buffer, followed by 7-AAD (Beckman coulter), anti-CD3, anti-CD56 and anti-NKG2D (R & D systems) mAbs. By staining. Expression ratio and mean fluorescence intensity (MFI) of the stained cells were measured using BD LSRFortessa.

NK92MI cells were first gated for singlets and then gated for 7AAD- and CD3-CD56 +. Transformation efficiency of the CD16V-41BB mutant construct and the control vector was measured by flow cytometry analysis of GFP and CD16 expressing cells among CD3-CD56 + cells. Transformation efficiency of the NKG2D-41BB mutant construct and the control vector was measured by flow cytometry of NKG2D expressing cells among CD3-CD56 + cells.

Calcein  Release Cytotoxic Essay

Target cells were diluted in RPMI-1640 medium containing 10% FBS at 1 × 10 ⁶ cells / mL and at 37 ° C. using 30 μM Calcein acetoxymethyl ester (Calcein-AM; Molecular probes). Labeled for 1 hour. After washing twice with RPMI-1640 medium containing 10% FBS, labeled target cells were placed in 96-well plates at 1 × 10 ⁴ cells / well. Each CD16V-41BB mutant CAR-transduced NK92MI cell, control vector-transduced NK92MI (NK92MI-mock), or NK-92MI parental cells were harvested and washed and then subjected to various effector-to- target) and added with or without rituximab. As a control, anti-human antibody (Sigma aldrich) independent of rituximab was used. After 2 hours, the plates were centrifuged at 2000 rpm at 4 ° C. for 3 minutes, and 100 μL of supernatant was collected and calcein emission was measured at 485 nm excitation wavelength and 535 nm emission wavelength with a fluorescent microplate reader (Victor3, PerkinElmer). The specific calcein release was calculated by the following equation: percent specific lysis = (test release-spontaneous release) x 100 / (maximal release-spontaneous release). Spontaneous release was used as the supernatant containing only the target cells stained with calcein and 1% Triton X-100 was used for maximal lysis.

2. Results

(1) various 4- 1BB  Containing mutations chimera  Expressing antigen receptor (CAR) NK92MI  CD20-positive of cells Lymphoma  Cytotoxicity Assessment on Cells

-Various 4- 1BB  Mutation is a costimulatory motif With CD16V  Connected chimera  Transduction and Expression of Antigen Receptors

The V158 variant of CD16 is an Fc receptor with high affinity with the Fc portion of the antibody, which can produce a good therapeutic effect in combination with antibodies and immune cells expressing CD16V. The CD16V and CD8a hinge and transmembrane domains, the intracellular signaling domain of the costimulatory molecule 4-1BB, and the CD3z, a T cell stimulating molecule, were combined.

We have different sequences in the intracellular domains of human and mouse 4-1BB and 231-T1, which is the site of binding of TNF receptor-associated factor (TRAF) 1, 2, 3 in the Tumor necrosis factor receptor (TNFR) such as CD30, 40. The amino acid sequence PVQTT 235 was replaced with TTGAA mouse 229-233 to prepare a CD16V-BB (Δ231-235) Z chimeric antigen receptor containing CD137 (Δ231-235) as an intracellular signaling domain. In addition, CD137 (Δ237-239) is included as an intracellular signaling domain by replacing AED 237-239 amino acid sequence EED, which is the site where TRAF (TNF receptor-associated factor) 1, 2, 3 binds to 4-1BB. CD16V-BB (Δ237-239) Z chimeric antigen receptor was constructed, and CD16V-BB (Δ231-235, which replaced both amino acid sequences 231-235 and amino acids 237-239 in the 4-1BB intracellular domain Δ237-239) Z chimeric antibody receptor was constructed.

Thus prepared chimeric antibody receptors (CD16V-BBZ, CD16V-BB (Δ231-235) Z, CD16V-BB (Δ237-239) Z, CD16V-BB (Δ231-235, Δ237-239) Z) The lentiviral vector containing the MSCV promoter was expressed in NK92MI cells using 50 multiples of infection (MOI). Expression of chimeric antibody receptors in transduced NK92MI cells was confirmed by detecting human CD16 using monoclonal mouse anti-human CD16 antibodies. As a result of flow cytometry, it was confirmed that the chimeric antibody receptors were transduced at an efficiency of 90% or more in NK92MI cells (FIG. 1A).

4- 1BB  With mutations CD16V chimera  Expressing antibody receptors NK92MI  Cellular Rituximab Antibodies  CD20-positive according to combination Lymphoma  Assessing apoptosis of cells

To determine whether NK92MI cells expressing chimeric antibody receptors have increased cancer cell killing capacity, CD16V chimeric antibody receptors (CD16V-BBZ, CD16V-BB) containing 4-1BB mutations and NK92MI cells transfected with GFP-only vectors were introduced. CD20- in combination with rituximab antibody in NK92MI cells expressing (Δ231-235) Z, CD16V-BB (Δ237-239) Z, CD16V-BB (Δ231-235, Δ237-239) Z Cytotoxicity against positive lymphoma cell cells (Ramos cells) was assessed through a Calcein-AM release assay.

After culturing the transduced NK92MI cells and the CD20-positive lymphoma cell line Ramos stained with Calcein-AM and antibody, the degree of lysis of cancer cells was evaluated by measuring the amount of Calcein-AM released (FIG. 1B). In FIG. 1B, 10: 1, 5: 1, and 2.5: 1 represent ratios of the number of transfected NK92MI cells, which are effector cells, and the number of Ramos cells, which are target cells. Since NK92MI cells do not express CD16, the presence of anti-CD20 antibody rituximab does not increase the death of Ramos cells.

As shown in FIG. 1B, when Rituximab is not present, a control group (Mock) transfected with a GFP-expressing vector and NK92MI cells expressing a chimeric antibody receptor containing a 4-1BB or 4-1BB mutation are Ramos cells. Almost no cell killing ability was observed. The previously produced 4-1BB is a chimeric antibody receptor (CD16V-BBZ) included as an intracellular signaling domain and a chimeric antibody receptor (CD16V-BB (△ 231-235) Z and CD16V-BB containing a 4-1BB mutation) NK92MI cells expressing (Δ237-239) Z, CD16V-BB (Δ231-235, Δ237-239) Z are 10: 1, 5: 1, 2.5: 1 effector cells in the presence of rituximab. Higher cell killing in cells expressing CD16V-BB (Δ231-235) Z chimeric antibody receptor, including 4-1BB (Δ231-235) mutations, showing killing ability against Ramos cells at target cell ratio Ability revealed.

- 4- 1BB (△ F245C A) containing mutations CD16V chimera  Evaluation of efficacy of antibody receptor

To increase the cytotoxicity of the chimeric receptor containing the 4-1BB mutation, LCK, one of the tyrosine kinases, which differs in sequence between humans and mice in the intracellular domain of 4-1BB and plays an important role in signaling in immune cells (Lymphocyte-specific protein tyrosine kinase, p56lck) is located in the position of the CXCP motif to bind amino acid F 245 is replaced by C 4-1BB (ΔF245C) CD16V-BB (△ containing the intracellular signaling domain (△) F245C) Z chimeric antigen receptor was constructed. In addition, the CD16V-BB (Δ231-235, ΔF245C) Z chimeric antibody receptor was prepared by adding the ΔF245C mutation to CD16V-BB (Δ231-235) Z, which had improved cell death ability.

To compare cytotoxicity, CD16V-BBZ, CD16V-BB (ΔF245C) Z, CD16V-BB (Δ231-235), and ΔF245C) Z chimeric antibody receptors were transformed into NK92MI cells via lentiviruses and at high levels. It was confirmed that it is expressed as (Fig. 2A).

As shown in FIG. 2B, low cell killing ability was observed for Ramos cells in the absence of rituximab, but CD16V-BBZ, CD16V-BB (ΔF245C) Z, CD16V-BB (Δ231-) in the presence of rituximab. 235), all of the ΔF245C) Z chimeric antibody receptors showed cell killing ability against Ramos cells, and all of the chimeric antibody receptors containing the 4-1BB (ΔF245C) mutation showed high cell killing ability.

(2) 4- 1BB  (△ F245C Containing NKG2D chimera  Cytotoxicity Evaluation of NK92MI Cells Expressing Antibody Receptors on Human Breast Cancer Cells

- 4- 1BB (△ F245C ) Mutations as co-stimulatory motifs With NKG2D  Connected chimera  Transduction and Expression of Antigen Receptors

Human NKG2D extracellular domain, CD8a hinge and transmembrane domain, costimulatory molecule 4-1BB intracellular signaling domain and T cell stimulating molecule CD3z were coupled.

In order to increase the cytotoxicity of the NKG2D chimeric antibody receptor, we included a 4-1BB mutant (ΔF245C) replacing amino acid F 245 of the costimulatory motif 4-1BB with C as an intracellular signaling domain. BB (ΔF245C) Z chimeric antigen receptor was constructed. In addition, 4-1BB 31-235 amino acid sequence PVQTT with improved cell killing ability in the CD16V chimeric antibody receptor was replaced with TTGAA mouse 229-233 and 4-1BB mutation with amino acid F replaced with C (△ 231) NKG2D-BB (Δ231-235, ΔF245C) Z chimeric antibody receptor containing -235, ΔF245C) was prepared (Table 2).

The NKG2D chimeric antibody receptor thus produced was expressed in NK92MI cells via lentivirus. Expression of chimeric antibody receptors in transduced NK92MI cells was confirmed by detecting human NKG2D using monoclonal mouse anti-human NKG2D antibodies. As a result of flow cytometry, it was confirmed that the chimeric antibody receptors were transfected with high efficiency over 90% in NK92MI cells (FIG. 3A).

- 4- 1BB (△ F245C A) containing mutations NKG2D chimera  Increasing Apoptosis by Antibody Receptors

To determine whether NK92MI cells expressing chimeric antibody receptors have increased cancer cell killing capacity, NK92MI cells transfected with GFP-only vectors and NKG2D chimeric antibody receptors (NKG2D-BBZ, NKG2D-) containing 4-1BB or mutation Cytotoxicity against breast cancer cell line MCF-7 expressing NKG2D ligand of NK92MI expressing BB (Δ231-235) Z, NKG2D-BB (Δ231-235, ΔF245C) Z) was analyzed through Calcein-AM release assay. Evaluated.

The degree of lysis of cancer cells after co-culture with NK92MI cells transfected with NKG2D chimeric antibody receptor containing 4-1BB mutation and MCF-7 breast cancer cells stained with Calcein-AM was evaluated by measuring the amount of Calcein-AM released. (FIG. 3B). In Fig. 3B, 5: 1, 2.5: 1 and 1: 1 represent the ratio of the number of transfected NK92MI cells, which are effector cells, and the number of MCF-7 cells, which are target cells. NKG2D-BBZ, NKG2D-BB (Δ231-235) Z, NKG2D-BB (Δ231-235, ΔF245C) Z chimeric antibody receptors all showed cell killing ability against MCF-7 cells in a 2-hour in vitro cytotoxicity assay. Seemed. In particular, the chimeric antibody receptor NKG2D-BB (Δ231-235) Z and NKG2D-BB (Δ231-235, ΔF245C) Z containing the 4-1BB (ΔF245C) mutations were found to further increase cell death ability ( 3B).

<110> GREEN CROSS LABCELL <120> NEW PEPTIDE, CHEMERIC ANTIGEN RECEPTOR AND IMMUNE CELL EXPRESSING          THE SAME <130> 03014 <160> 39 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> PRT <213> Artificial Sequence <220> 4-1BB (231-235 mutant) amino acid <400> 1 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Thr Thr Gly Ala Ala Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 2 <211> 42 <212> PRT <213> Artificial Sequence <220> 4-1BB (237-239 mutant) amino acid <400> 2 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Pro Val Gln Thr Thr Gln Ala Ala Ala Gly Cys Ser Cys Arg Phe              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 3 <211> 42 <212> PRT <213> Artificial Sequence <220> 4-1BB (231-235, 237-239 mutant) amino acid <400> 3 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Thr Thr Gly Ala Ala Gln Ala Ala Ala Gly Cys Ser Cys Arg Phe              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 4 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB (F245C_mutant) amino acid <400> 4 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Cys              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 5 <211> 42 <212> PRT <213> Artificial Sequence <220> 4-1BB (231-235, F245C mutant) amino acid <400> 5 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Thr Thr Gly Ala Ala Gln Glu Glu Asp Gly Cys Ser Cys Arg Cys              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 6 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (231-235 mutant) nucleotide <400> 6 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gaccaccgga 60 gctgctcagg aggaagacgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120 gagctg 126 <210> 7 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (237-239 mutant) nucleotide <400> 7 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gcctgtgcag 60 accacacagg cagcggctgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120 gagctg 126 <210> 8 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (231-235, 237-239 mutant) nucleotides <400> 8 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gaccaccgga 60 gctgctcagg cagcggctgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120 gagctg 126 <210> 9 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (F245C_mutant) nucleotide <400> 9 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gcctgtgcag 60 accacacagg aggaagacgg ctgctcctgc cggtgccccg aggaagagga aggcgggtgc 120 gagctg 126 <210> 10 <211> 126 <212> DNA <213> Artificial Sequence <220> 4-1BB (231-235, F245C mutant) nucleotide <400> 10 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gaccaccgga 60 gctgctcagg aggaagacgg ctgctcctgc cggtgccccg aggaagagga aggcgggtgc 120 gagctg 126 <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> CD16 nucleotide <400> 11 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg g 51 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD16 amino acid <400> 12 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly     <210> 13 <211> 567 <212> DNA <213> Artificial Sequence <220> <223> CD16V nucleotide <400> 13 atgcgaaccg aggatctgcc taaagccgtg gtcttcctgg agcctcagtg gtacagagtg 60 ctggagaagg actctgtgac actgaaatgc cagggcgctt attcaccaga ggataacagc 120 actcagtggt tccacaatga atccctgatc agctcccagg catctagtta ctttattgac 180 gccgctacag tggacgattc tggagagtat cgatgccaga ctaacctgag caccctgtcc 240 gatcccgtgc agctggaagt ccacatcgga tggctgctgc tccaggcacc aagatgggtc 300 ttcaaggagg aagaccccat tcacctgcgc tgtcatagct ggaagaatac cgctctgcat 360 aaagtgacat acctccagaa cggaaagggc cgaaaatact tccaccataa ttccgacttt 420 tatatcccca aggcaaccct gaaagatagt gggtcatatt tttgtcgggg gctggtggga 480 agtaaaaacg tctcaagcga gactgtgaat atcaccatta cacagggcct ggctgtcagc 540 accatctcct ctttctttcc ccctggg 567 <210> 14 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> CD16V amino acid <400> 14 Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln   1 5 10 15 Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly              20 25 30 Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser          35 40 45 Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val      50 55 60 Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser  65 70 75 80 Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala                  85 90 95 Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His             100 105 110 Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly         115 120 125 Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys     130 135 140 Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly 145 150 155 160 Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly                 165 170 175 Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly             180 185 <210> 15 <211> 216 <212> DNA <213> Artificial Sequence <220> <223> CD8a nucleotide <400> 15 gccaaaccta ccacaactcc tgctccaaga ccacccacac ccgctcctac tattgcatct 60 cagccactga gtctgcgacc agaggcctgc cggcccgccg ccggcggggc cgtgcatacc 120 aggggcctgg acttcgcctg tgatatctac atttgggctc cactggctgg gacttgcggc 180 gtgctgctgc tgtctctggt cattactctg tattgt 216 <210> 16 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> CD8a amino acid <400> 16 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro   1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro              20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp          35 40 45 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu      50 55 60 Ser Leu Val Ile Thr Leu Tyr Cys  65 70 <210> 17 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3z nucleotide <400> 17 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 60 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 120 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 180 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 240 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 300 tatgatgcac tgcacatgca ggccctgccc cctcgg 336 <210> 18 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3z amino acid <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly   1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr              20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys          35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys      50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg  65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                  85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             100 105 110 <210> 19 <211> 1299 <212> DNA <213> Artificial Sequence <220> <223> CD16V-BB (231-235) Z nucleotide <400> 19 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60 gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120 gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180 ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240 gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300 cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360 gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420 tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480 aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540 gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600 tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660 gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720 ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780 ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840 ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggaccac cggagctgct 900 caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260 tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299 <210> 20 <211> 432 <212> PRT <213> Artificial Sequence <220> <223> CD16V-BB (231-235) Z amino acid <400> 20 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys         195 200 205 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile     210 215 220 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 225 230 235 240 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr                 245 250 255 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu             260 265 270 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile         275 280 285 Phe Lys Gln Pro Phe Met Arg Thr Thr Gly Ala Ala Gln Glu Glu Asp     290 295 300 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 305 310 315 320 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly                 325 330 335 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr             340 345 350 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys         355 360 365 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys     370 375 380 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 385 390 395 400 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                 405 410 415 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             420 425 430 <210> 21 <211> 1299 <212> DNA <213> Artificial Sequence <220> CD16V-BB (237-239) Z nucleotide <400> 21 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60 gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120 gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180 ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240 gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300 cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360 gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420 tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480 aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540 gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600 tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660 gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720 ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780 ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840 ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900 caggcagcgg ctggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260 tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299 <210> 22 <211> 432 <212> PRT <213> Artificial Sequence <220> CD16V-BB (237-239) Z amino acid <400> 22 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys         195 200 205 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile     210 215 220 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 225 230 235 240 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr                 245 250 255 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu             260 265 270 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile         275 280 285 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Ala Ala Ala     290 295 300 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 305 310 315 320 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly                 325 330 335 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr             340 345 350 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys         355 360 365 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys     370 375 380 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 385 390 395 400 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                 405 410 415 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             420 425 430 <210> 23 <211> 1299 <212> DNA <213> Artificial Sequence <220> CD16V-BB (231-235, 237-239) Z nucleotide <400> 23 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60 gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120 gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180 ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240 gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300 cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360 gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420 tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480 aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540 gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600 tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660 gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720 ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780 ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840 ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggaccac cggagctgct 900 caggcagcgg ctggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260 tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299 <210> 24 <211> 432 <212> PRT <213> Artificial Sequence <220> CD16V-BB (231-235, 237-239) Z amino acid <400> 24 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys         195 200 205 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile     210 215 220 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 225 230 235 240 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr                 245 250 255 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu             260 265 270 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile         275 280 285 Phe Lys Gln Pro Phe Met Arg Thr Thr Gly Ala Ala Gln Ala Ala Ala     290 295 300 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 305 310 315 320 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly                 325 330 335 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr             340 345 350 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys         355 360 365 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys     370 375 380 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 385 390 395 400 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                 405 410 415 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             420 425 430 <210> 25 <211> 1299 <212> DNA <213> Artificial Sequence <220> CD16V-BB (F245C) Z nucleotide <400> 25 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60 gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120 gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180 ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240 gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300 cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360 gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420 tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480 aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540 gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600 tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660 gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720 ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780 ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840 ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900 caggaggaag acggctgctc ctgccggtgc cccgaggaag aggaaggcgg gtgcgagctg 960 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260 tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299 <210> 26 <211> 432 <212> PRT <213> Artificial Sequence <220> <223> CD16V-BB (F245C) Z amino acid <400> 26 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys         195 200 205 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile     210 215 220 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 225 230 235 240 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr                 245 250 255 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu             260 265 270 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile         275 280 285 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp     290 295 300 Gly Cys Ser Cys Arg Cys Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 305 310 315 320 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly                 325 330 335 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr             340 345 350 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys         355 360 365 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys     370 375 380 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 385 390 395 400 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                 405 410 415 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             420 425 430 <210> 27 <211> 1299 <212> DNA <213> Artificial Sequence <220> CD16V-BB (231-235, F245C) Z nucleotide <400> 27 atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60 gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120 gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180 ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240 gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300 cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360 gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420 tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480 aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540 gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600 tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660 gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720 ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780 ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840 ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggaccac cggagctgct 900 caggaggaag acggctgctc ctgccggtgc cccgaggaag aggaaggcgg gtgcgagctg 960 cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020 tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080 cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140 gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200 aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260 tatgatgcac tgcacatgca ggccctgccc cctcggtga 1299 <210> 28 <211> 432 <212> PRT <213> Artificial Sequence <220> CD16V-BB (231-235, F245C) Z amino acid <400> 28 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys         195 200 205 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile     210 215 220 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 225 230 235 240 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr                 245 250 255 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu             260 265 270 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile         275 280 285 Phe Lys Gln Pro Phe Met Arg Thr Thr Gly Ala Ala Gln Glu Glu Asp     290 295 300 Gly Cys Ser Cys Arg Cys Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 305 310 315 320 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly                 325 330 335 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr             340 345 350 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys         355 360 365 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys     370 375 380 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 385 390 395 400 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala                 405 410 415 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg             420 425 430 <210> 29 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> CD8a nucleotide <400> 29 atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60 cct 63 <210> 30 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CD8a amino acid <400> 30 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro              20 <210> 31 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> NKG2D nucleotide <400> 31 ctgttcaacc aggaggtcca gatcccactg accgaaagtt actgcggacc atgtcccaag 60 aactggatct gctacaagaa caactgttac cagttctttg acgagtctaa gaactggtat 120 gaatctcagg ccagttgcat gtcacagaat gcttcactgc tgaaggtgta cagcaaagag 180 gaccaggatc tgctgaagct ggtgaaatcc tatcactgga tgggcctggt ccatatccca 240 accaacgggt cttggcagtg ggaggacgga agcattctgt cccccaatct gctgacaatc 300 attgaaatgc agaagggcga ttgtgctctg tacgcaagct ccttcaaagg gtatatcgag 360 aactgctcca cccccaatac atacatttgt atgcagagga cagtg 405 <210> 32 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> NKG2D amino acid <400> 32 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly   1 5 10 15 Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe              20 25 30 Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser          35 40 45 Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu      50 55 60 Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro  65 70 75 80 Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn                  85 90 95 Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala             100 105 110 Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr         115 120 125 Ile Cys Met Gln Arg Thr Val     130 135 <210> 33 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> NKG2D-BB (F245C) Z nucleotide <400> 33 atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60 cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120 aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180 tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240 gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300 ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360 atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420 gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480 acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540 ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600 tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660 tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720 cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggtgc 780 cccgaggaag aggaaggcgg gtgcgagctg cgagtgaagt tcagcaggtc cgccgacgct 840 cctgcatacc agcagggaca gaaccagctg tataacgagc tgaatctggg ccggagagag 900 gaatacgacg tgctggacaa aaggcggggc cgggaccccg aaatgggagg gaagccacga 960 cggaaaaacc cccaggaggg cctgtacaat gagctgcaaa aggacaaaat ggccgaggct 1020 tattctgaaa tcgggatgaa gggagagaga aggcgcggaa aaggccacga tggcctgtac 1080 caggggctga gcaccgctac aaaggacacc tatgatgcac tgcacatgca ggccctgccc 1140 cctcggtga 1149 <210> 34 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> NKG2D-BB (F245C) Z amino acid <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr              20 25 30 Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn          35 40 45 Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln      50 55 60 Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys  65 70 75 80 Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly                  85 90 95 Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser             100 105 110 Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp         115 120 125 Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser     130 135 140 Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr 145 150 155 160 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser                 165 170 175 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly             180 185 190 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp         195 200 205 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile     210 215 220 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 225 230 235 240 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys                 245 250 255 Ser Cys Arg Cys Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val             260 265 270 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn         275 280 285 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val     290 295 300 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 305 310 315 320 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys                 325 330 335 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg             340 345 350 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys         355 360 365 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg     370 375 380 <210> 35 <211> 1149 <212> DNA <213> Artificial Sequence <220> NKG2D-BB (231-235, F245C) Z nucleotide <400> 35 atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60 cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120 aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180 tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240 gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300 ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360 atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420 gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480 acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540 ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600 tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660 tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720 cagcccttta tgaggaccac cggagctgct caggaggaag acggctgctc ctgccggtgc 780 cccgaggaag aggaaggcgg gtgcgagctg cgagtgaagt tcagcaggtc cgccgacgct 840 cctgcatacc agcagggaca gaaccagctg tataacgagc tgaatctggg ccggagagag 900 gaatacgacg tgctggacaa aaggcggggc cgggaccccg aaatgggagg gaagccacga 960 cggaaaaacc cccaggaggg cctgtacaat gagctgcaaa aggacaaaat ggccgaggct 1020 tattctgaaa tcgggatgaa gggagagaga aggcgcggaa aaggccacga tggcctgtac 1080 caggggctga gcaccgctac aaaggacacc tatgatgcac tgcacatgca ggccctgccc 1140 cctcggtga 1149 <210> 36 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> NKG2D-BB (231-235, F245C) Z amino acid <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu   1 5 10 15 His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr              20 25 30 Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn          35 40 45 Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln      50 55 60 Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys  65 70 75 80 Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly                  85 90 95 Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser             100 105 110 Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp         115 120 125 Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser     130 135 140 Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr 145 150 155 160 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser                 165 170 175 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly             180 185 190 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp         195 200 205 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile     210 215 220 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 225 230 235 240 Gln Pro Phe Met Arg Thr Thr Gly Ala Ala Gln Glu Glu Asp Gly Cys                 245 250 255 Ser Cys Arg Cys Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val             260 265 270 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn         275 280 285 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val     290 295 300 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 305 310 315 320 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys                 325 330 335 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg             340 345 350 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys         355 360 365 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg     370 375 380 <210> 37 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (CD137) nucleotide <400> 37 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 38 <211> 42 <212> PRT <213> Artificial Sequence <220> 4-1BB (CD137) amino acid <400> 38 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met   1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe              20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu          35 40 <210> 39 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB (CD137) nucleotide <400> 39 aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gcctgtgcag 60 accacacagg aggaagacgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120 gagctg 126

Claims (18)

In the amino acid sequence of SEQ ID 39, (a) substitution of TTGAA for residues 231-235; (b) substitution of the residues 237-239 for AAA; And (c) a variant amino acid sequence for SEQ ID NO: 39 comprising one or more variations selected from the group consisting of substitution of residue 245 with C.
The peptide of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5.
A nucleic acid molecule encoding the peptide of claim 1 or 2.
The nucleic acid molecule of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-10.
A chimeric antigen receptor comprising all or part of the peptide of claim 1 as an intracellular signaling domain.
The method according to claim 5, wherein all or part of the peptide as a first intracellular signaling domain, and further comprises a second intracellular signaling domain comprising all or a portion of CD3-zeta, wherein the first and second 2 The intracellular signaling domains are located in the cell membrane in order in the intracellular direction, chimeric antigen receptor.
The method according to claim 5,
A transmembrane domain linked to the intracellular signaling domain;
A spacer domain linked to the transmembrane domain; And
Extracellular domain linked to the spacer domain
Further comprising, chimeric antigen receptor.
The chimeric antigen receptor of claim 7, further comprising a signal sequence linked to the extracellular domain.
The chimeric antigen receptor of claim 8, wherein the signal sequence comprises all or part of a CD16 or CD8α.
The chimeric antigen receptor of claim 7, wherein the transmembrane domain comprises all or a portion of CD8α.
The chimeric antigen receptor of claim 7, wherein the spacer domain comprises all or part of CD8α.
The chimeric antigen receptor of claim 7, wherein the extracellular domain comprises all or a portion of CD16V or NKG2D.
An immune cell expressing the chimeric antigen receptor of any one of claims 5-12.
The immune cell of claim 13, wherein the immune cell is a natural killer cell (NK cell).
Pharmaceutical composition for the treatment of tumor comprising immune cells of claim 14.
A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 5-12.
The nucleic acid molecule of claim 16, wherein the nucleic acid molecule encodes at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 29, 35, and 37 or a variant thereof having at least 80% sequence identity. .
The nucleic acid molecule of claim 17, wherein the nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 34, and 36 or variants thereof having 80% or more sequence identity. .

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