Multifunctional protein

Abstract

A multifunctional polypeptide chain or a protein. A polypeptide chain X, comprising an antigen 1 binding domain R1, an auxiliary peptide chain linking domain R2 and an antigen 2 binding domain R3. The auxiliary peptide chain linking domain R2 is a cytokine or a cytokine binding domain in a cytokine receptor. A protein, which is a heterodimer composed of the polypeptide chain X as a main peptide chain and an auxiliary peptide chain Y. The auxiliary peptide chain Y comprises an antigen 3 binding domain R4 and a main peptide chain X linking domain R5, or the auxiliary peptide chain Y is the main peptide chain linking domain R5. The multifunctional protein mediates specific cell killing by binding to different tumor antigens with the two antigen binding domains of tumor-associated antigens therein. The multifunctional protein can function as a cytokine by introducing a cytokine or a cytokine receptor complex.

Claims

1. A protein, comprising a main peptide chain and a co-peptide chain to constitute a heterodimer; the main peptide chain comprises an antigen 1 binding domain R1, a co-peptide chain linkage domain R2 and an antigen 2 binding domain R3; the co-peptide chain comprises an antigen 3 binding domain R4 and a main peptide chain linkage domain R5; the main peptide chain linkage domain R5 binds each other to the co-peptide chain linkage domain R2; the antigen 1 binding domain R1 is anti-CD19 ScFv, the co-peptide chain linkage domain R2 is IL15Rαsushi, the antigen 2 binding domain R3 is anti-CD3 ScFv, the antigen 3 binding domain R4 is the extracellular domain of PD1, the main peptide chain linkage domain R5 is IL15; and the main peptide chain comprises the amino acid sequence of SEQ ID NO: 8, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO: 9.

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the protein of claim 1.

3. An immune cell, wherein: the surface of the immune cell is bound with the protein according to claim 1.

Description

FIGURES

(1) FIG. 1. A schematic diagram showing the molecular structure of a multifunctional protein. A: The multifunctional protein molecule is composed of a main peptide chain X and an auxiliary peptide chain Y, wherein the main peptide chain X includes an antigen binding domain R1, a co-peptide chain linkage domain R2, an antigen binding domain R3, and a co-peptide chain Y includes an antigen binding domain R4, the main peptide chain linkage domain R5; B: The multifunctional protein molecule is composed of a main peptide chain X and a co-peptide chain Y, wherein the main peptide chain X includes an antigen-binding domain R1, a co-peptide chain linkage domain R2, and an antigen-binding domain R3. The peptide chain Y comprises a main peptide chain linkage domain R5; C: The multifunctional protein molecule consists only of the main peptide chain X, wherein the main peptide chain X comprises an antigen binding domain R1, a co-peptide chain linkage domain R2, and an antigen binding domain R3.

(2) FIG. 2. The gene expression framework of the multifunctional protein molecule.

(3) FIG. 3. The expressed and purified multifunctional protein molecules on SDS-PAGE. Lane 1: TiTE-1, main peptide chain about 65 KD, co-peptide chain about 30 KD; Lane 2: TiTE-6, main peptide chain about 65 KD, co-peptide chain about 30 KD; Lane 3: Protein marker, the molecular weight from top to bottom respectively as 160 KD, 120 KD, 100 KD, 70 KD, 50 KD, 40 KD, 30 KD, 25 KD.

(4) FIG. 4. The killing results of the multifunctional protein molecule TiTE-1, 15, 16, and 5: A, The negative control of TiTE-6 protein for killing malme-3M-CD19-luc; B, TiTE-1 protein for killing malme-3M-CD19-luc; C, TiTE-15 protein for killing malme-3M-CD19-luc; D, TiTE-16 protein for killing malme-3M-CD19-luc; E, TiTE-5 protein for killing malme-3M-CD19-luc; F, TiTE-5 protein for killing malme-3M-CD22-luc. It is demonstrated that the multi-functional proteins TiTE-1, 15, 16, and 5 provided by the present invention can kill tumor cells in vitro at a very low concentration, and it shows the best result when the concentration is used as 0.5-5 ng/10{circumflex over ( )}6 cells.

(5) FIG. 5. The killing results of the multifunctional protein molecule TiTE-6, 8, 9, 10, 11, 12, 13, and 14 respectively: A, the negative control of TiTE-2 protein for killing BV173-luc; B, TiTE-6 protein for killing BV173-luc C, TiTE-8, 9, 10, 11, 12, 13, 14 proteins for killing BV173-luc. It was demonstrated that the multifunctional proteins TiTE-6, 8, 9, 10, 11, 12, 13, and 14 provided by the present invention shows killing ability for WT1-positive tumor cells.

(6) FIG. 6. The killing results of multifunctional protein molecules TiTE-2, 3, 4: A, the negative control TiTE-6 protein for killing malme-3M-luc; B, TiTE-2 protein for killing malme-3M-luc; C, TiTE-3 protein for killing malme-3M-luc; D, TiTE-4 protein for killing malme-3M-luc. It is demonstrated that the multifunctional proteins TiTE-2, 3, and 4 provided by the present invention can kill tumor cells expressing the intracellular antigen in vitro.

(7) FIG. 7. The results of the stimulation of NK cells by multifunctional protein molecules. Almost all cells died after 5 days when the NK cell expansion was stimulated without any interleukin; NK cell expansion were obtained by the stimulation of the multi-functional proteins provided by the present invention, and cells were amplified about 140 times in 18 days.

(8) FIG. 8. FACS analysis with multifunctional protein molecules TiTE-1, 6, 8, 9, 10, 11, 12: A, The negative control with T cell alone; B, The experimental group of T cells with TiTE-1; C, The negative control with BV173 alone; The experimental group of TiTE-1 on BV173; E, The negative control with BV173; F, The experimental group of TiTE-6 on BV173; G, The experimental group of TiTE-8 on BV173; H, The experimental group of TiTE-9 on BV173; I, The experimental group of TiTE-10 on BV173; J, The experimental group of TiTE-11 on BV173; K, The experimental group of TiTE-12 on BV173; L, T cell negative control; M, The experimental group of TiTE-6 on T cell; N, The experimental group of TiTE-8 on T cell; 0, The experimental group of TiTE-9 on T cell; P, The experimental group of TiTE-10 on T Cell; Q, The experimental group of TiTE-11 on T cell; R, The experimental group of TiTE-12 on T cell. The experiments demonstrated that the multifunctional protein molecule TiTE-1 binds well to CD3 antigen and CD19 antigen respectively; the AntiMHC/WT1 and antiCD3 of TiTE-6, 8, 9, 10, 11, 12 have ability to bind to both intracellular antigen WT1 and CD3 antigen, respectively.

(9) FIG. 9. FACS results of multifunctional protein molecules TiTE-2, 3, and 4: A, malme-3M negative control; B, The experimental group of TiTE-2 on malme-3M; C, The experimental group of TiTE-2 on malme-3M; D, The experimental group of TiTE-4 one malme-3M; E, T cell negative control; F, The experimental group of TiTE-2 on T cell; G, The experimental group of TiTE-3 on T cell; H, The experimental group of TiTE-4 on T cell. It can be seen from the figure that the multifunctional protein molecules TiTE-2, 3 bind well to the MHC/GP100 antigen and CD3 antigen, respectively, and TiTE-4 binds well to the MHC/Mart1 antigen and CD3 antigen.

(10) FIG. 10. FACS results of multifunctional protein molecules TiTE-15, 16: A, BV173 negative control; B, The experimental group of TiTE-15 on BV173; C, the experimental group of TiTE-16 on BV173; D, T cell negative control; E, The experimental group of TiTE-15 on T cell; F, The experimental group of TiTE-16 on T cell. It can be seen from the figure that the multifunctional protein molecules TiTE-15, 16 bind well to the CD19 antigen and the CD3 antigen, respectively.

(11) FIG. 11. FACS results of multifunctional protein molecule TiTE-5: A; malme-3M-CD19-Luc negative control; B, The experimental group of TiTE-5 on malme-3M-CD19-Luc; C, Negative control with malme-3M-CD22-Luc; D, The experimental group of TiTE-5 on malme-3M-CD22-Luc; E, T cell negative control; F, The experimental group of TiTE-5 on T cell; It can be seen from the figure that the multifunctional protein molecule TiTE-5 binds well to the CD19 antigen, CD20 antigen and CD3 antigen, respectively.

EXAMPLES

(12) The experimental methods used in the following examples are conventional methods unless otherwise specified.

(13) The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1. Construction of the Vector Expressing a Multifunctional Protein Molecule

(14) 1. Construction of the Novel Multifunctional Protein TiTE-1 Targeting to CD19-Positive Tumor Cells

(15) A novel multifunctional protein TiTE-1 targeting to CD19-positive tumor cells, which was fused by the main peptide chain X1 with the co-peptide chain Y1 to obtain a protein (FIG. 1).

(16) The main peptide chain X1 included an antigen 1 binding domain R1, a cytokine or the cytokine binding domain of a cytokine receptor R2 and an antigen 2 binding domain R3; The co-peptide chain Y1 included an antigen 3 binding domain R4 and a main peptide chain X linkage domain R5.

(17) The antigen-binding domain (R1) of the main peptide chain (X1) was selected from antiCD19-ScFv (SEQ ID NO. 1), the co-peptide chain linkage domain (R2) was selected from IL15Rαsushi (SEQ ID NO. 2), and the antigen-binding domain (R3) is selected from antiCD3-ScFv (SEQ ID NO. 3); The extracellular domain of the receptor PD1 of PDL1 and PDL2 (SEQ ID NO. 4) was selected as the antigen binding domain (R4) of the co-peptide chain (Y1), and IL15 (SEQ ID NO. 5) was selected as the primary peptide chain domain (R5).

(18) 2. The Signal Peptide (Amino Acid Sequence:

(19) MALPVTALLLPLALLLHAARP (SEQ ID NO: 51)), HindIII restriction site was added to the 5′ end of the main peptide chain, and the linker peptide between the co-peptide chain domain of the main peptide chain (R2: L15Rαsushi) and the antigen binding domain (R3: antiCD3-ScFv) contained a BamHI restriction site; a P2A peptide was added between the 3′ end of the main peptide chain and the 5′ end of the co-peptide chain (amino acid sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 52)); Xba I of restriction site was added to the 3′ end of the co-peptide chain.

(20) 3. The antiCD19-IL15Rαsushi fragment, antiCD3 fragment and P2A-PD1-IL15 fragment were PCR amplified and run on nucleic acid gel electrophoresis; overlapping PCR amplified antiCD3-P2A-PD1-IL15 fragment on nucleic acid gel electrophoresis. The antiCD19-IL15Rαsushi fragment was cleaved using HindIII and BamHI. AntiCD3-P2A-PD1-IL15 was cut using BamHI and Xba I; the vector PCDNA3.1 (Invitrogen) was cleaved using HindIII and Xba I.

(21) 4. The target fragments were recovered by gel electrophoresis, and three fragments recovered were ligated and transformed, and the clones were selected and sequenced, and finally the target plasmid PCDNA3.1-TiTE-1 was obtained.

(22) The recombinant vector PCDNA3.1-TiTE-1 is an expression cassette for the expression of the multifunctional protein TiTE-1 targeting CD19-positive tumor cells (the nucleotide sequence of the expression cassette is composed of the nucleotide sequence encoding the main peptide chain X1 (SEQ ID NO. 6) and the nucleotide sequence (SEQ ID NO. 7) encoding the co-peptide chain Y1, and the last nucleotide of the SEQ ID NO. 6 is immediately adjacent to the first nucleotide of the SEQ ID NO. 7) replaces the fragment between HindIII and XbaI of PCDNA3.1 vector (Invitrogen, USA), the resulting recombinant vector was obtained to express a functional multifunctional protein TiTE-1 consisting of a main peptide chain X1 (SEQ ID NO. 8) and a co-peptide chain Y1 (SEQ ID NO. 9).

(23) 5. According to the above steps, the expression vectors were constructed for the multifunctional proteins TiTE-2, TiTE-3, TiTE-4, TiTE-5, TiTE-6, TiTE-7, TiTE-8, TiTE-9, TiTE-10, TiTE-11, TiTE-12; The expression vectors of TiTE-13, TiTE-14, TiTE-15, and TiTE-16 were constructed in a similar manner, and the structures thereof are shown in Table 1 below, and the expression framework is shown in FIG. 2.

(24) TABLE-US-00001 TABLE 1 the structure of multifunctional proteins Main peptide Antigen Co-peptide Antigen Antigen chain binding linkage binding binding linkage domain R1 domain R2 domain R3 domain R4 domain R5 TiTE-1  AntiCD19- IL15Rαsushi AntiCD3- Extracellular IL15 ScFv ScFv region of PD1 TiTE-2  AntiMHC/ IL15Rαsushi AntiCD3- Extracellular IL15 GP100-VHH ScFv region of PD1 TiTE-3  AntiMHC/ IL15Rαsushi AntiCD3- AntiMHC/ IL15 GP100-VHH ScFv GP100-VHH TiTE-4  AntiMHC/ IL15Rαsushi AntiCD3- Extracellular IL15 Mart 1-VHH ScFv region of PD1 TiTE-5  AntiCD19- IL15Rαsushi AntiCD3- AntiCD22- IL15 ScFv ScFv ScFv TiTE-6  AntiMHC/ IL15Rαsushi AntiCD3- Extracellular IL15 WT1-VH ScFv region of PD1 TiTE-7  AntiMHC/ IL15Rαsushi AntiCD16- Extracellular IL15 WT1-VH ScFv region of PD1 TiTE-8  AntiMHC/ IL4Rα-N- AntiCD3- Extracellular IL4  WT1-VH FN3 ScFv region of PD1 TiTE-9  AntiMHC/ IL15Rαsushi AntiCD3- Extracellular IL15 WT1-VH ScFv region of PD1 TiTE-10 Extracellular IL15Rαsushi AntiCD3- AntiMHC/ IL15 region of PD1 ScFv WT1-VH TiTE-11 AntiCD3- IL15Rαsushi AntiMHC/ Extracellular IL15 ScFv WT1-VH region of PD1 TiTE-12 AntiMHC/ IL15 AntiCD3- Extracellular IL15 WT1-VH ScFv region of PD1 Rαsushi TiTE-13 AntiMHC/ IL15Rαsushi AntiCD3- — — WT1-VH ScFv TiTE-14 AntiMHC/ IL15Rαsushi AntiCD3- — IL15 WT1-VH ScFv TiTE-15 AntiCD19- IL15Rαsushi AntiCD3- — — ScFv ScFv TiTE-16 AntiCD19- IL15Rαsushi AntiCD3- — IL15 ScFv ScFv

(25) Wherein, the AntiMHC/GP100-VHH comprises the amino acid sequence of SEQ ID NO. 10.

(26) The AntiMHC/Mart1-VHH comprises the amino acid sequence of SEQ ID NO. 11.

(27) The AntiMHC/WT1-VH comprises the amino acid sequence of SEQ ID NO. 12.

(28) The IL4Rα-N-FN3 comprises the amino acid sequence of SEQ ID NO. 13.

(29) The AntiCD16-ScFv comprises the amino acid sequence of SEQ ID NO. 14.

(30) The AntiCD22-ScFv comprises the amino acid sequence of SEQ ID NO. 15.

(31) The IL4 comprises the amino acid sequence of SEQ ID NO. 16.

(32) The main peptide chain of TiTE-2 comprises the amino acid sequence of SEQ ID NO. 17, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 9.

(33) The main peptide chain of TiTE-3 comprises the amino acid sequence of SEQ ID NO. 17, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 18.

(34) The main peptide chain of TiTE-4 comprises the amino acid sequence of SEQ ID NO. 19, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 9.

(35) The main peptide chain of TiTE-5 comprises the amino acid sequence of SEQ ID NO. 8, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 20.

(36) The main peptide chain of TiTE-6 comprises the amino acid sequence of SEQ ID NO. 21, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 9.

(37) The main peptide chain of TiTE-7 comprises the amino acid sequence of SEQ ID NO. 22, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 9.

(38) The main peptide chain of TiTE-8 comprises the amino acid sequence of SEQ ID NO. 23, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 24.

(39) The main peptide chain of TiTE-9 comprises the amino acid sequence of SEQ ID NO. 25, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 26.

(40) The main peptide chain of TiTE-10 comprises the amino acid sequence of SEQ ID NO. 27, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 28.

(41) The main peptide chain of TiTE-11 comprises the amino acid sequence of SEQ ID NO. 29, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 9.

(42) The main peptide chain of TiTE-12 comprises the amino acid sequence of SEQ ID NO. 30, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 31.

(43) TiTE-13 is the main peptide chain, and the main peptide chain comprises the amino acid sequence of SEQ ID NO. 21.

(44) The main peptide chain of TiTE-14 comprises the amino acid sequence of SEQ ID NO. 21, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 5.

(45) TiTE-15 is a main peptide chain, and it comprises the amino acid sequence of SEQ ID NO. 8.

(46) The main peptide chain of TiTE-16 comprises the amino acid sequence of SEQ ID NO. 8, and the co-peptide chain comprises the amino acid sequence of SEQ ID NO. 5.

(47) The encoding nucleic acid sequence expressing TiTE-2 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence. wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 35, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 7.

(48) The encoding nucleic acid sequence expressing TiTE-3 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence. wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 35, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 36.

(49) The encoding nucleic acid sequence expressing TiTE-4 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last peptide of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence. wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 37, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 7.

(50) The encoding nucleic acid sequence expressing TiTE-5 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 6, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 38;

(51) The encoding nucleic acid sequence expressing TiTE-6 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 39, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 7.

(52) The encoding nucleic acid sequence expressing TiTE-7 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last peptide of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 40, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 7.

(53) The encoding nucleic acid sequence expressing TiTE-8 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 41, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 42.

(54) The encoding nucleic acid sequence expressing TiTE-9 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 43, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 44.

(55) The encoding nucleic acid sequence expressing TiTE-10 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 45, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 46.

(56) The encoding nucleic acid sequence expressing TiTE-11 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last peptide of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 47, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 7.

(57) The encoding nucleic acid sequence expressing TiTE-12 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 48, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 49.

(58) The coding nucleic acid sequence expressing TiTE-13 is SEQ ID NO. 39.

(59) The encoding nucleic acid sequence expressing TiTE-14 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last base of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 39, and the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 50.

(60) The nucleic acid sequence encoding TiTE-15 is SEQ ID NO. 6.

(61) The encoding nucleic acid sequence expressing TiTE-16 is composed of a main peptide chain encoding nucleic acid sequence and a co-peptide chain encoding nucleic acid sequence, and the last peptide of the 3′ end of the main peptide chain encoding nucleic acid sequence is next to the 1st base of the 5′ end of the co-peptide chain encoding nucleic acid sequence; wherein the main peptide chain encoding nucleic acid sequence is SEQ ID NO. 6, the co-peptide chain encoding nucleic acid sequence is SEQ ID NO. 50.

(62) To make recombinant vectors from PCDNA3.1-TiTE-2 to PCDNA3.1-TiTE-16 PCDNA3.1 vector is cut by HindIII and XbaI and the corresponding nucleic acid sequence from the nucleic acid sequence expressing TiTE-2 to TiTE-16 is inserted into PCDNA3.1 respectively.

Example 2: The Expression and Purification of Multi-Targeting Functional Proteins

(63) 1. 293F (in vitrogen) was cultured at 37° C., 8% CO2, 120 rpm until the cell density reached to 1×10{circumflex over ( )}6 cell/ml. 2. The vector PCDNA3.1-TiTE-1 constructed in Example 1 was transfected into the cells of the above 1 using PEI and the concentration of plasmid used was 1 mg/L, and PEI concentration was 3 mg/L. The cells were incubated for 5-6 days at 37° C., 8% CO2, 120 rpm.

(64) 3. The culture media of the above 2 was centrifuged at 4000 rpm, and the supernatant was collected, and the protein was bound to Protein/cap to L beads and eluted with 500 μL of 0.1Mof Gly-HCl, pH 2.6-3.0, and finally the eluate was collected.

(65) 4. The proteins were detected on SDS-PAGE (FIG. 3). It can be seen that the target proteins of about 65 KD and 30 KD were obtained, representing the main peptide chain X and the co-peptide chain Y of the multifunctional protein molecule TiTE-1.

(66) 5. The same method was used to express and purify TiTE-2, TiTE-3, TiTE-4, TiTE-5, TiTE-7, TiTE-8, TiTE-9, TiTE-10, TiTE-11, TiTE-12, etc. multi-functional proteins.

Example 3: Multi-Functional Proteins TiTE-1, 15, 16 Mediated T Cell Killing CD19+Target Cells In Vitro

(67) 1. 1×10{circumflex over ( )}4 of target cells, malme-3M-CD19-luc obtained by transfecting CD19 antigen gene (the nucleic acid sequence is SEQ ID NO. 32) and Luc gene (the nucleic acid sequence is SEQ ID NO. 33) into the Malme-3M purchased from ATCC to express CD19 antigen and the Luc protein, in 50 μL were plated in a 96-well plate, and cultured at 37° C., 5% CO2 for 18-20 h.

(68) 2. After the cells attached to the wall, the medium was aspirated and discarded, and 50 μL of fresh medium was added and the cells were cultured at 37° C., 5% CO2 for 1-3 h.

(69) 3, The target protein TiTE-1 obtained in Example 2 was stepwise diluted to different concentrations of 50, 5, 0.5, 0.05, 0.005 ng/μL respectively.

(70) On experimental group: 50 μL of 1×10{circumflex over ( )}5 of T cells, which were derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation, and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days, were added to 50, 5 0.5, 0.05, and 0.005 ng of TiTe-1, the target protein obtained in Example 2 and were incubated further for 1-2 h at 37° C. to obtain T cells incubated with the antibody.

(71) On negative control group: 50, 5, 0.5, 0.05, 0.005 ng of a bispecific control antibody (TiTE-6) with no killing effect on the target cells were added to 50 μL of 1×10{circumflex over ( )}5 T cells, respectively, and incubate at 37° C. for 1-2 h.

(72) 4. 50 μL of T cells incubated with the antibody were added to a 96-well plate with added target cells, and incubate at 37° C., 5% CO2 for 22-24 h.

(73) 5, 100 μL of 1% Triton lysate was added onto each well, repeatedly blew cells, and stood for 3-5 min, the cells were completely lysed; 50 μL of lysate was added into a black 96-well plate, 50 μL substrate (300 m/mL Luc and 2 mg/mL ATP was mixed in a volume ratio of 3:1) was added and the fluorescence value on each well was quickly measured.

(74) 6. The killing efficiency was calculated as follows: the killing efficiency={(negative control fluorescence value−experimental group fluorescence value)/negative control fluorescence value}×100%.

(75) The result is shown in FIG. 4B. It can be seen that the multifunction protein TiTE-1 by the present invention can kill CD19-positive tumor cells in vitro at a very low concentration compared with the control group in FIG. 4A. The optimal killing effect in vitro can be obtained at a concentration of 0.5-5 ng/10{circumflex over ( )}6 cells.

(76) 7. In the same way, TiTE-15 and TiTE-16 killing experiments were carried out to verify that the tumor cells were killed by using a very low concentration of a multifunctional protein.

(77) The results are shown in FIGS. 4C and 4D. It can be seen that multifunctional proteins TiTE-15 and 16 by the present invention could kill tumor cells in vitro at a very low concentration, and the optimal concentration was 0.5-5 ng/10{circumflex over ( )}6 cells.

(78) 8. In the same manner, TiTE-5 killing experiment was carried out malme-3M-CD19-luc and malme-3M-CD22-luc by transfecting a CD22 antigen gene (SEQ ID NO. 34) and a Luc gene (SEQ ID NO. 33) into the genome of ATCC-purchased Malme-3M were used as the target cells. The results are shown in FIGS. 4E and 4F. It can be seen that the multifunctional protein TiTE-5 provided by the present invention has killing ability on CD19 and CD22 positive cells. The tumor cells can be killed in vitro at a very low concentration, and the optimal killing effect was obtained when the concentration is 0.5-5 ng/10{circumflex over ( )}6 cells.

Example 4: Multi-Functional Protein Molecule TiTE-6, 8, 9, 10, 11, 12, 13, 14 Mediate T Cell Killing of WT1 Positive Target Cells In Vitro

(79) 1. 1×10{circumflex over ( )}4 of target cell BV173 (ATCC purchased BV173 transfected by Luc gene (SEQ ID NO. 33)) in 50 μL was plated in a 96-well plate, and cultured at 37° C., 5% CO2 for 1-2 h.

(80) 2, The target protein TiTE-6 obtained in Example 2 was stepwise diluted to different concentrations of 50, 5, 0.5, 0.05 ng/μL.

(81) On experimental group: 50 uL of 1×10{circumflex over ( )}5 of T cells, which were derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation, and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days, were added to 50, 5 0.5, 0.05, and 0.005 ng of TiTe-6, the target protein obtained in Example 2 and were incubated further for 1-2 h at 37° C. to obtain T cells incubated with the antibody.

(82) On negative control group: 50, 5, 0.5, 0.05, 0.005 ng of a bispecific control antibody (TiTE-2) with no killing effect on the target cells were added to 50 μL of 1×10{circumflex over ( )}5 T cells, respectively, and incubate at 37° C. for 1-2 h.

(83) 3. 50 μL of T cells incubated with the antibody was added to a 96-well plate with target cells, and incubated at 37° C., 5% CO2 for 22-24 h.

(84) 4. 100 μL of 1% Triton lysate was added onto each well, repeatedly blew cells, and stood for 3-5 min, the cells were completely lysed; 50 μL of lysate was added into a black 96-well plate, 50 μuL substrate (300 m/mL Luc and 2 mg/mL ATP was mixed in a volume ratio of 3:1) was added and the fluorescence value on each well was quickly measured.

(85) 5. The killing efficiency was calculated as follows: the killing efficiency={(negative control fluorescence value−experimental group fluorescence value)/negative control fluorescence value}×100%.

(86) The result is shown in FIG. 5B. It can be seen that the multifunctional protein TiTE-6 provided by the present invention can kill tumor cells at a very low concentration.

(87) 6. The killing experiments of TiTE-8, 9, 10, 11, 12, 13, and 14 were carried out in the same manner, and the effective target ratio was 10:1, and the antibody concentration was 5 ng of the corresponding antibody in each case. The experimental results are shown in FIG. 5C. It can be seen that WT1 positive tumor cells could be killed by multi-functional proteins provided by the present invention are killed.

Example 5: Multi-Functional Protein TiTE-2, 3, 4 Mediate T Cell Killing for the Target Cells In Vitro

(88) 1. 50 μL of 1×10{circumflex over ( )}4 target cell malme-3M-luc (obtained by transfection of Luc gene into malme-3M purchased by ATCC) was plated in a 96-well plate and cultured at 37° C., 5% CO2 for 18-20 h.2. After the cells attached to the wall, the medium was aspirated and discarded, and 50 μL of fresh medium was added and incubated at 37° C., 5% CO2 for 1-3 h.

(89) 3, TiTE-2, 3, 4 obtained in Example 2 were stepwise diluted to different concentrations of 50, 5, 0.5, 0.05, 0.005 ng/μL.

(90) On experimental group: 50 μL of 1×10{circumflex over ( )}5 of T cells, which were derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation, and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days, were added to 50, 5 0.5, 0.05, and 0.005 ng of TiTe-2, 3, 4, the target proteins obtained in Example 2. The cells with the proteins were incubated further for 1-2 h at 37° C. to obtain T cells incubated with the antibody.

(91) On negative control group: 50, 5, 0.5, 0.05, 0.005 ng of a bispecific control antibody (TiTE-6) with no killing effect on the target cells were added to 50 μL of 1×10{circumflex over ( )}5 T cells, respectively, and incubate at 37° C. for 1-2 h.

(92) 4. 50 μL of T cells incubated with the antibody was added to a 96-well plate plated with target cells, and incubate at 37° C., 5% CO2 for 22-24 h.

(93) 5, 100 μL of 1% Triton lysate was added onto each well, repeatedly blew cells, and stood for 3-5 min, the cells were completely lysed; 50 μL of lysate was added into a black 96-well plate, 50 μL substrate (300 m/mL Luc and 2 mg/mL ATP was mixed in a volume ratio of 3:1) was added and the fluorescence value on each well was quickly measured.

(94) 6. The killing efficiency was calculated as follows: the killing efficiency={(negative control fluorescence value−experimental group fluorescence value)/negative control fluorescence value}×100%.

(95) The result is shown in FIG. 6. It can be seen that multi-functional proteins TiTE-2, 3, and 4 provided by the present invention could kill tumor cells in vitro at a very low concentration, and the optimal concentration for the best killing effect is 0.5-5 ng/10{circumflex over ( )}6 cells.

Example 6. Multifunctional Protein TiTE-1 Stimulates the Expansion of NK Cell

(96) 1. 6×10{circumflex over ( )}5 of NK92 cells (China Type Culture Collection) were culture in 2 mL medium (Alpha basal medium, 12.5% horse serum, 12.5% FBS, 0.2 mM inositol, 0.1 mM mercaptoethanol, 0.02 mM folic acid) with 40 ng/mL of multifunctional protein TiTE-1 obtained in Example 2 at 37° C., 5% CO2.

(97) 2. After 2-3 days of culture, the total number of cells was counted and cultured continuously for 18 days, the cell density was adjusted to 3×10{circumflex over ( )}5 cells/mL for each passage, and 40 ng/mL of multifunctional protein TiTE-1 was maintained.

(98) The cell growth curve is shown in FIG. 7. It can be seen that the multifunctional protein TiTE-1 provided by the present invention can stimulate NK cell expansion and has the function of IL15/IL15Rαsushi.

(99) The IL15/IL15Rαsushi domains of TiTE2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, and 15 are identical to TiTE-1, and their functions are not significantly different.

Example 7

(100) FACS verification of CD19 and CD3 antigen binding for multifunctional protein TiTE-1 and antiMHC/WT1 and antiCD3 of TiTE-6, 8, 9, 10, 11, 12 for binding to intracellular antigen WT1 and CD3, respectively.

(101) 1. T cell experimental group and BV173 experimental group: 5 μg each of multifunctional protein TiTE-1, 6, 8, 9, 10, 11, 12 were added to the mixture of BV173 cells and 3×10{circumflex over ( )}5 T cells which were derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation, and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days, and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were re-suspended in 200 μL of PBS. 2 μL APC labelled Mouse anti-Human CD279 (BD, Cat. No. 558694) was added and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were re-suspended in 200 μL of PBS.

(102) T cell negative control group and BV173 cell negative control group: BV173 cells (ATCC) were mixed with 3×10{circumflex over ( )}5 of T cells derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days and 2 μL of APC labelled Mouse anti-Human CD279 (BD, Cat. No. 558694) was added and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re-suspended in 200 μL of PBS as a negative control.

(103) 2. The results of flow cytometry shown in FIG. 8. It can be seen from the figure that the multifunctional protein TiTE-1 bound well to CD19 antigen and CD3 antigen, respectively; antiMHC/WT1 and anti-CD3 of TiTE-6, 8, 9, 10, 11, and 12 bound well to intracellular antigen antiCD3 function well with WT1 and CD3 antigen, respectively.

Example 8

(104) FACS verification of the binding function of TiTE-2, 3 for MHC/GP100 and CD3 antigens and TiTE-4 for MHC/Mart1 and CD3 antigens respectively.

(105) 1. T cell and malme-3M-Luc (by transfecting luc gene into Malme-3M purchased by ATCC) experimental group: 5 μg of multifunctional protein TiTE-2, 3, 4 each was added to 3×10{circumflex over ( )}5 T cells and malme-3M-Luc cells, respectively. and the cells were incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 μL of PBS. 2 μL of PE conjugated anti-hIL-15 (R&D, IC2471P) was added and kepton ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 μL of PBS.

(106) T cell group and malme-3M-Luc cell negative control group: 2 μL APC Mouse anti-Human CD279 (BD, No. 558694) was added to 3×10{circumflex over ( )}5 T cells and malme-3M-Luc, respectively, and the cells with the antibody were incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re-suspended in 200 μL of PBS as a negative control.

(107) 2. The results of flow cytometry analysis shown in FIG. 9. It can be seen from the figure that the multifunctional proteins TiTE-2 and 3 bind well to MHC/GP100 antigen and CD3 antigen, respectively, TiTE-4 binds to MHC/Mart1 antigen and CD3 antigen well.

Example 9

(108) FACS verification of the binding function of antiCD19 and antiCD3 of the multifunctional protein TiTE-15, 16 to CD19 antigen and CD3 antigen, respectively 1. T cell and BV173 cell experimental group: 5 μg of multi-function proteins TiTE-15, 16 were added to BV173 cells and 3×10{circumflex over ( )}5 T cells derived from mononuclear cell-rich white membrane layer of normal human peripheral blood by density gradient centrifugation, and stimulated by OKT3 (50 ng/mL) and IL2 (300 IU/mL) for 14 days, and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were re-suspended in 200 μL of PBS. 2 μL of FITC-Labeled recombinant Protein L (ACRO Biosystem, RPL-PF141) was added and incubated on ice for 30 min. The supernatant was removed by centrifugation, washed twice in 500 μL PBS, and re-suspended in 200 μL of PBS.

(109) T cell and BV173 cell negative control group: 3*10E5 T cells respectively (PBMC stimulated with 50 ng/mL OKT3, 300 IU/mL IL2) were mixed with BV173 cells (ATCC) first, and 2 μL APC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to the cells and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re-suspended in 200 μL of PBS as a negative control.

(110) 2. The results of flow cytometry analysis shown in FIG. 10. It can be seen from the figure that the multifunctional proteins TiTE-15 and 16 bind well to the CD19 antigen and the CD3 antigen, respectively.

Example 10

(111) FACS verification of the binding function of antiCD19, antiCD20 and antiCD3 of multifunctional protein TiTE-5 to respective antigens.1. T cell and malme-3M-CD19-Luc/malme-3M-CD22-Luc experimental groups. Cell experimental group: 5 μg of multi-function protein TiTE-5 was added to 3×10{circumflex over ( )}5 T cells and malme-3M-CD19-Luc cells (by transfecting CD19 antigen gene and Luc gene into Malme-3M purchased by ATCC) and malme-3M-CD22-Luc (by transfecting CD22 antigen gene and Luc gene into Malme-3M purchased by ATCC) respectively, and incubated on ice for 30 min. The supernatant was re-suspended in 200 μL of PBS. 2 μL of PE conjugated anti-hIL-15 (R&D, IC2471P) was added and incubated on ice for 30 min. The supernatant was centrifuged and the cells were re-suspended in 200 μL of PBS.

(112) T cell and malme-3M-CD19-Luc, malme-3M-CD22-Luc cell negative control groups: 3×10{circumflex over ( )}5 T cells were mixed with malme-3M-CD19-Luc, malme-3M-CD22-Luc, respectively. 2 μL PE conjugated anti-hIL-15 (R&D, article number IC2471P) was added to each groups and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re-suspended in 200 μL of PBS as a negative control.

(113) 2. The results of flow cytometry analysis shown in FIG. 11. It can be seen from the figure that the multifunctional protein TiTE-5 binds well to the CD19 antigen, CD20 antigen and CD3 antigen, respectively.

INDUSTRIAL APPLICATION

(114) The experiments of the present invention demonstrate that the multifunctional protein of the present invention can bind to different tumor antigens through two antigen binding domains that could bind to tumor-associated antigens, mediate specific cell killing, and improve the accuracy of targeting. It can block the immunosuppressive signal and improve the ability to kill tumor if one of the antigen binding domains is an immune check-point related antigen; the multifunctional protein of the present invention can play a role of a cytokine since it contains a cytokine and cytokine receptor complex.