WO2023204290A1 - Nanoparticule multispécifique - Google Patents

Nanoparticule multispécifique Download PDF

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WO2023204290A1
WO2023204290A1 PCT/JP2023/015857 JP2023015857W WO2023204290A1 WO 2023204290 A1 WO2023204290 A1 WO 2023204290A1 JP 2023015857 W JP2023015857 W JP 2023015857W WO 2023204290 A1 WO2023204290 A1 WO 2023204290A1
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chain variable
variable region
amino acid
seq
cdr2
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PCT/JP2023/015857
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Japanese (ja)
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勇紀 籠谷
雄介 伊藤
誠一 太田
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愛知県
国立大学法人東京大学
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Definitions

  • the present invention relates to multispecific nanoparticles, methods for producing multispecific nanoparticles, and the like.
  • CAR-T cell therapy involves introducing chimeric antigen receptors (CARs) into T cells collected from peripheral blood to create CAR-T cells that can attack cancer cells, and use them for treatment. This is the method used (Non-Patent Document 1).
  • CARs chimeric antigen receptors
  • Chimeric antigen receptors are artificial antigen receptors that combine an extracellular domain containing an antibody variable region that recognizes cancer antigens and a signaling domain of costimulatory molecules involved in T cell activation. be.
  • CARs Chimeric antigen receptors
  • first generation CAR whose signal transduction domain consists only of CD3 ⁇
  • second and third generation CARs containing different structures and types of signal transduction domains have been developed.
  • Ta. CAR-T cell therapy is a technological technology that recognizes tumor cells and makes it possible to efficiently kill tumor cells by activating CAR-expressing T cells (CAR-T cells) themselves. It is a cancer treatment.
  • CAR-T cell therapy Treatment methods that target the CD19 molecule have been approved in Europe, the United States, and Japan for B-cell leukemia and lymphoma. The response rate is extremely high, and it has been reported that over 80% of remissions can be achieved even in refractory cases. Furthermore, CAR-T cell therapy targeting other antigens and cancer types is being developed, and is expected to be a breakthrough cancer treatment technology following immune checkpoint inhibition therapy.
  • CAR-T cell therapy Another problem with CAR-T cell therapy is that the therapeutic effect cannot be stably obtained.
  • CAR-T cell therapy CAR-T cells are generated using T cells collected from a patient. Therefore, it is known that the quality of CAR-T cells can vary widely depending on the patient's condition. In particular, it has been reported that in patients who have undergone repeated chemotherapy, etc., the quality of collected T cells decreases during the culture stage, and CAR-T cells disappear early after infusion (Non-patent Document 2). ).
  • An object of the present invention is to provide a new non-cell preparation that does not need to be individually prepared for each patient and can stably exhibit a high killing effect on tumor cells at low cost.
  • CAR-T cell therapy requires the step of collecting the subject's own T cells, introducing the CAR gene, and then returning them to the patient.
  • a series of operations to individually prepare CAR-T cells is a necessary step to create cancer cell-specific cells, but it is a factor that requires high treatment costs and This is also a factor that can cause the quality of treatment to vary widely from patient to patient.
  • the present inventors developed nanoparticles with multiple immunoregulatory molecules mounted on the surface layer.
  • T cells existing in the living body can be activated "on the spot.”
  • this activation is specifically induced in the presence of tumor cells. Therefore, specificity equivalent to that of CAR-T cells was ensured, and cost reduction and stabilization of efficacy were simultaneously achieved.
  • the nanoparticles of the present invention can be additionally loaded with immune regulating molecules such as costimulatory molecules and cytokine molecules, thereby making it possible to freely adjust and enhance the effects.
  • immune regulating molecules such as costimulatory molecules and cytokine molecules
  • the present invention is based on the above research results and provides the following.
  • the target binding region is The multispecific nanoparticle according to (1), comprising (a) a T cell receptor complex binding region, a NK cell surface antigen binding region, or a macrophage surface antigen binding region, and (b) a cancer antigen binding region.
  • the T cell receptor complex binding region is selected from the group consisting of an anti-CD3 antibody or a fragment thereof, an anti-T cell receptor (TCR) antibody or a fragment thereof, and an HLA/peptide fusion molecule; ) Multispecific nanoparticles as described in ).
  • the anti-CD3 antibody is [1] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 72 to 74, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively.
  • chain variable region [2] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 80 to 82, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively.
  • chain variable region [3] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 88 to 90, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively.
  • chain variable region [4] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 96 to 98, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 99 to 101, respectively.
  • chain variable region [5] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 106, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively.
  • chain variable region [6] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 112 to 114, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively.
  • chain variable region [7] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 120 to 122, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively.
  • chain variable region [8] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 128 to 130, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively.
  • chain variable region [9] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 136 to 138, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively.
  • chain variable region [10] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 144 to 146, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively.
  • chain variable region [11] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 152 to 153, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively.
  • chain variable region [12] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 160 to 162, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively.
  • chain variable region [13] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 168 to 170, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 171 to 173, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NO: 345 to 347, respectively, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 348, and consisting of the amino acid sequence GAS (SEQ ID NO: 349)
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 350
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 356 to 358, respectively.
  • chain variable region [17] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 362 to 364, respectively.
  • chain variable region [18] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 370 to 372, respectively.
  • chain variable region [19] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 375 to 377, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380, respectively.
  • chain variable region [20] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 383 to 385, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively.
  • chain variable region [21] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 391 to 393, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively.
  • chain variable region [22] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 399 to 401, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 402 to 404, respectively.
  • a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 438 to 440, respectively.
  • chain variable region [25] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 446 to 448, respectively.
  • chain variable region [26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 451 to 453, and CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 454, and consisting of the amino acid sequence AAS (SEQ ID NO: 455).
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 456, [27] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 459 to 461, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 462, and consisting of the amino acid sequence AAS (SEQ ID NO: 463) A light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 464, [28] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 467 to 492, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 494 to 518, [29] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 519 to 521, and CDR1 consisting of the amino
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 524, [30] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 532, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 566 to 568, respectively, and CDR1 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively;
  • the anti-TCR antibody comprises a light chain variable region comprising CDR2 and CDR3, or
  • the multispecific nanoparticle according to (3) comprising a chain variable region.
  • the NK cell surface antigen binding region is an anti-CD16 antibody or a fragment thereof, an anti-NKG2D antibody or a fragment thereof, an anti-Nkp30 antibody or a fragment thereof, an anti-Nkp44 antibody or a fragment thereof, an anti-Nkp46 antibody or a fragment thereof, an anti-2B4 antibody or a fragment thereof, an IgG Fc region or a fragment thereof, a MICA protein or a fragment thereof, a MICB protein or a fragment thereof, a B7H6 protein or a fragment thereof, and an influenza virus-derived hemagglutinin or a fragment thereof, according to (2). multispecific nanoparticles.
  • the anti-CD16 antibody is [34] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 300 to 302, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 303 to 305, respectively.
  • the anti-NKG2D antibody is [35] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 308 to 310, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 311 to 313, respectively.
  • the anti-NKp46 antibody comprises a chain variable region, or [36] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 316 to 318, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 319 to 321, respectively.
  • the IgG Fc region comprises a chain variable region, [37]
  • the macrophage surface antigen-binding region is an anti-FC ⁇ RI (CD64) antibody or a fragment thereof, an anti-FC ⁇ RIIA (CD32) antibody or a fragment thereof, an anti-FC ⁇ RIIIA (CD16A) antibody or a fragment thereof, an anti-CD40 antibody or a fragment thereof, an IgG Fc
  • the multispecific nanoparticle according to (2) selected from the group consisting of a region or a fragment thereof, a CD40L protein or a fragment thereof, an anti-CD47 antibody or a fragment thereof, and a TLR4a ligand.
  • the anti-CD40 antibody is [39] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 324 to 326, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 327 to 329, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 332 to 334, respectively, and CDR1, CDR2 consisting of the amino acid sequences shown in SEQ ID NOs: 335 to 337, respectively; and a light chain variable region comprising CDR3;
  • the IgG Fc region is [41] A human IgG1 Fc region consisting of the amino acid sequence shown in SEQ ID NO: 30, or [42] A human IgG3 Fc region consisting of the amino acid sequence shown in SEQ ID NO: 61,
  • the anti-CD47 antibody or fragment thereof is [43] A heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 590 to 592, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 593 to 595, respectively, or [ 44] comprises
  • the cancer antigen is selected from the group consisting of CD19, BCMA, mesothelin, GD2, CD20, CD22, EGFR, CD33, CD123, ERBB2, CD133, CEA, CEACAM5, MUC1, and PSMA; (2) The multispecific nanoparticle according to any one of (8) to (8). (10) One or more selected from the group consisting of costimulatory molecules, cytokine molecules, cytokine receptors, migration molecules, immune checkpoint inhibitory molecules, and immune checkpoint molecules on the surface and/or inside of the nanoparticles.
  • the costimulatory molecule is selected from the group consisting of CD80 protein, CD86 protein, 4-1BB ligand protein, ICOS ligand protein, CD40 protein, CD70 protein, OX40 ligand protein, GITR ligand protein, and LIGHT protein.
  • the cytokine molecule is IL-7 protein, IL-15 protein, IL-21 protein, IL-2 protein, IL-12 protein, IL-4 protein, IL-10 protein, IL-18 protein, GM-CSF protein, one or more selected from the group consisting of IFN- ⁇ protein, TNF- ⁇ protein, and TGF- ⁇ protein
  • the cytokine receptor is a TGF- ⁇ receptor, an IL-6 receptor, an IL-1 receptor, a TNF receptor, an IL-4 receptor, an IL-10 receptor, an IL-13 receptor, and a CSF-1 receptor.
  • the migration molecule is one or more selected from the group consisting of CXCR4 protein, ITGA4 protein, ITGB1 protein, ICAM-1 protein, VCAM-1 protein, and LFA3 protein
  • the immune checkpoint inhibitor molecule may be a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a Lag-3 inhibitor, a Tim-3 inhibitor, a TIGIT inhibitor, a B7-H3 inhibitor, or a VISTA inhibitor.
  • the immune checkpoint molecule is one or more selected from the group consisting of PD1 protein, Lag-3 protein, Tim-3 protein, TIGIT protein, ICOS protein, CD47 protein, and BTLA protein.
  • Specificity nanoparticles (12) The multispecific nanoparticle according to any one of (1) to (11), wherein the lipid membrane is composed of a cell membrane, or a cell membrane and a liposome.
  • a pharmaceutical composition comprising the multispecific nanoparticle according to any one of (1) to (14).
  • a fusion protein comprising a membrane binding region, and a T cell receptor complex binding region and a cancer antigen binding region on its N-terminal side or C-terminal side, The fusion protein, wherein the T cell receptor complex binding region is selected from the group consisting of an anti-CD3 antibody or a fragment thereof, an anti-T cell receptor (TCR) antibody or a fragment thereof, and an HLA/peptide fusion molecule.
  • the anti-CD3 antibody is [1] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 72 to 74, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively. chain variable region, [2] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 80 to 82, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively.
  • chain variable region [3] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 88 to 90, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively.
  • chain variable region [4] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 96 to 98, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 99 to 101, respectively.
  • chain variable region [5] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 106, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively.
  • chain variable region [6] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 112 to 114, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively.
  • chain variable region [7] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 120 to 122, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively.
  • chain variable region [8] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 128 to 130, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively.
  • chain variable region [9] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 136 to 138, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively.
  • chain variable region [10] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 144 to 146, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively.
  • chain variable region [11] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 152 to 153, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively.
  • chain variable region [12] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 160 to 162, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively.
  • chain variable region [13] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 168 to 170, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 171 to 173, respectively.
  • chain variable region [14] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 176 to 178, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 179 to 181, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NO: 345 to 347, respectively, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 348, and consisting of the amino acid sequence GAS (SEQ ID NO: 349)
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 350
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 356 to 358, respectively.
  • chain variable region [17] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 362 to 364, respectively.
  • chain variable region [18] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 370 to 372, respectively.
  • chain variable region [19] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 375 to 377, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380, respectively.
  • chain variable region [20] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 383 to 385, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively.
  • chain variable region [21] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 391 to 393, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively.
  • chain variable region [22] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 399 to 401, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 402 to 404, respectively.
  • a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 438 to 440, respectively.
  • chain variable region [25] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 446 to 448, respectively.
  • chain variable region [26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 451 to 453, and CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 454, and consisting of the amino acid sequence AAS (SEQ ID NO: 455).
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 456, [27] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 459 to 461, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 462, and consisting of the amino acid sequence AAS (SEQ ID NO: 463) A light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 464, [28] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 467 to 492, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 494 to 518, [29] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 519 to 521, and CDR1 consisting of the amino
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 524, [30] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 532, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 566 to 568, respectively, and CDR1 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively; comprising a light chain variable region including CDR2 and CDR3;
  • the anti-TCR antibody is [33] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 184 to 186, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 187 to 189, respectively.
  • the anti-CD3 antibody is [1] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 72 to 74, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively. chain variable region, [2] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 80 to 82, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively.
  • chain variable region [3] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 88 to 90, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively.
  • chain variable region [4] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 96 to 98, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 99 to 101, respectively.
  • chain variable region [5] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 106, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively.
  • chain variable region [6] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 112 to 114, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively.
  • chain variable region [7] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 120 to 122, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively.
  • chain variable region [8] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 128 to 130, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively.
  • chain variable region [9] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 136 to 138, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively.
  • chain variable region [10] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 144 to 146, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively.
  • chain variable region [11] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 152 to 153, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively.
  • chain variable region [12] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 160 to 162, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively.
  • chain variable region [13] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 168 to 170, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 171 to 173, respectively.
  • chain variable region [14] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 176 to 178, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 179 to 181, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NO: 345 to 347, respectively, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 348, and consisting of the amino acid sequence GAS (SEQ ID NO: 349)
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 350
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 356 to 358, respectively.
  • chain variable region [17] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 362 to 364, respectively.
  • chain variable region [18] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 370 to 372, respectively.
  • chain variable region [19] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 375 to 377, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380, respectively.
  • chain variable region [20] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 383 to 385, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively.
  • chain variable region [21] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 391 to 393, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively.
  • chain variable region [22] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 399 to 401, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 402 to 404, respectively.
  • a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 438 to 440, respectively.
  • chain variable region [25] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 446 to 448, respectively.
  • chain variable region [26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 451 to 453, and CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 454, and consisting of the amino acid sequence AAS (SEQ ID NO: 455).
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 456, [27] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 459 to 461, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 462, and consisting of the amino acid sequence AAS (SEQ ID NO: 463) A light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 464, [28] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 467 to 492, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 494 to 518, [29] A heavy chain variable region comprising CDR1, CDR2, and CDR3 each consisting of the amino acid sequence shown in SEQ ID NO: 519 to 521, and CDR1 consisting of the amino
  • a light chain variable region comprising CDR2 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 524, [30] A heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 532, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 527 to 529, respectively, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively.
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 566 to 568, respectively, and CDR1 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively;
  • the anti-TCR antibody comprises a light chain variable region comprising CDR2 and CDR3, or
  • a method for producing multispecific nanoparticles capable of binding to two or more target cells comprising: (i) two or more fusion proteins containing a membrane-binding region and a target-binding region; or (ii) a cell expressing on its cell membrane a fusion protein containing a membrane-binding region and two or more target-binding regions; a cell membrane fragmentation step that fragments the
  • the method includes a nanoparticle forming step of forming nanoparticles by reconstituting the cell membrane fragments after the fragmentation step, and a recovery step of collecting the nanoparticles.
  • the target binding region is The method according to (24), comprising (a) a T cell receptor complex binding region, a NK cell surface antigen binding region, or a macrophage surface antigen binding region, and (b) a cancer antigen binding region.
  • the reconstitution in the nanoparticle forming step mixes the cell membrane fragment and the liposome and/or the nanoparticle core, and the lipid membrane in which the cell membrane fragment and the liposome are fused forms a nanoparticle that forms the surface layer of the lipid membrane.
  • a fusion protein comprising a membrane-binding region, and an NK cell surface antigen-binding region and a cancer antigen-binding region on its N-terminal side or C-terminal side
  • the NK cell surface antigen-binding region is an anti-CD16 antibody or a fragment thereof, an anti-NKG2D antibody or a fragment thereof, an anti-Nkp30 antibody or a fragment thereof, an anti-Nkp44 antibody or a fragment thereof, an anti-Nkp46 antibody or a fragment thereof, an anti-2B4 antibody or a fragment thereof , IgG Fc region or a fragment thereof, MICA protein or a fragment thereof, MICB protein or a fragment thereof, B7H6 protein or a fragment thereof, and influenza virus-derived hemagglutinin or a fragment thereof.
  • a fusion protein comprising a membrane-binding region, and a macrophage surface antigen-binding region and a cancer antigen-binding region on its N-terminal side or C-terminal side
  • the macrophage surface antigen-binding region is an anti-FC ⁇ RI (CD64) antibody or a fragment thereof, an anti-FC ⁇ RIIA (CD32) antibody or a fragment thereof, an anti-FC ⁇ RIIIA (CD16A) antibody or a fragment thereof, an anti-CD40 antibody or a fragment thereof, an IgG Fc region or its fragment.
  • CD40L protein or fragment thereof, anti-CD47 antibody or fragment thereof, and TLR4a ligand is an anti-FC ⁇ RI (CD64) antibody or a fragment thereof, an anti-FC ⁇ RIIA (CD32) antibody or a fragment thereof, an anti-FC ⁇ RIIIA (CD16A) antibody or a fragment thereof, an anti-CD40 antibody or a fragment thereof, an IgG Fc region or its fragment.
  • a new non-cell preparation that does not need to be individually prepared for each patient and can stably exhibit a high killing effect on tumor cells at low cost.
  • FIG. 2 is a diagram showing nanoparticles in one embodiment of the present invention.
  • Figure 1A schematically shows the effects of the present invention on two target cells (T cells, NK cells, or macrophages, and cancer cells) based on nanoparticles having a fusion protein containing two or more target binding regions on the surface layer. show.
  • FIG. 1B shows the effect of the present invention based on nanoparticles having two or more fusion proteins containing target binding regions on the surface layer.
  • 1 is a diagram illustrating an overview of a method for producing nanoparticles in an embodiment of the present invention.
  • FIG. FIG. 2 is a diagram showing the CD3/CD19 binding protein and K562-mOKT3/FMC63 cells produced in Example 1.
  • Figure 3A shows the structure of the CD3/CD19 binding protein.
  • FIG. 3 is a diagram showing a nanoparticle core produced in Example 2.
  • Figure 4A shows the appearance of the PLGA nanoparticle core dispersion.
  • Figure 4B shows the results of observing the spherical shape of the PLGA nanoparticle core using a transmission electron microscope.
  • Figure 4C (left) shows the results of measuring the particle size and concentration of PLGA nanoparticle cores immediately after synthesis.
  • FIG. 4C shows the results of measuring the particle size and concentration of PLGA nanoparticle cores after cryopreservation/thawing.
  • FIG. 3 is a diagram showing the process of producing nanoparticles in Example 2.
  • FIG. 5A shows the results of detecting the expression of CD80 protein and 41BBL protein on the cell surface by flow cytometry.
  • Figure 5B shows each step of preparing nanoparticles.
  • 3 is a diagram showing the results of analyzing nanoparticles produced in Example 2.
  • FIG. FIG. 6A shows the results of observing nanoparticles using a transmission electron microscope (TEM). PLGA nanoparticle cores are shown before (-) and after (+) cell membrane reconstitution.
  • TEM transmission electron microscope
  • FIG. 6B shows the results of Western blotting analysis of components contained in the protein fractions obtained in each step of nanoparticle preparation.
  • FIG. 7 is a diagram showing the effector effect of nanoparticles in Example 3. The figure shows the survival rate of tumor cells after co-culture. “NP/T cell” indicates the ratio of the number of mOKT3/FMC63 nanoparticles (NP) to the number of T cells.
  • FIG. 4 is a diagram showing the results of measuring T cell activation based on nanoparticles in Example 4.
  • Figure 8A shows the results of detecting CD25 protein expression on the cell surface by flow cytometry.
  • FIG. 8B shows the measurement results of CD25 expression level under each condition.
  • FIG. 4 is a diagram showing the results of measuring T cell activation based on nanoparticles in Example 4.
  • FIG. 7 is a diagram showing the effect of nanoparticles into which costimulatory molecules CD80 protein and 41BBL protein were additionally introduced in Example 5.
  • Figure 10A shows the cell doubling rate of T cells.
  • “NP/T cell” indicates the ratio of the number of nanoparticles (NP) to the number of T cells.
  • Figure 10B shows the survival rate of tumor cells after co-culture. The results for mOKT3/FMC63 nanoparticles (CD80/41BBL (-)) and mOKT3/FMC63/CD80/41BBL nanoparticles (CD80/41BBL (+)) are shown. The results of measuring the percentage of cytokine-producing cells in CD8-positive T cells in Example 5 are shown.
  • FIG. 7 is a diagram showing the results of comparing tandem nanoparticles and separated nanoparticles in Example 6.
  • FIG. 13A schematically shows the configurations of tandem nanoparticles and separated nanoparticles.
  • FIG. 13B shows the results of measuring T cell activation based on tandem nanoparticles or isolated nanoparticles.
  • Figure 13C shows the survival rate of tumor cells after co-culture with tandem nanoparticles and isolated nanoparticles and T cells.
  • FIG. 7 is a diagram showing the effect of nanoparticles into which cytokine molecules IL7 protein and IL15 protein were additionally introduced in Example 7.
  • FIG. 14A shows the results of detecting the expression of each protein by flow cytometry in K562 cells additionally introduced with IL7 protein and IL15 protein.
  • FIG. 14B shows the cell doubling rate of T cells by mOKT3/FMC63/CD80/41BBL nanoparticles (-IL7/IL15) and mOKT3/FMC63/CD80/41BBL/IL7/IL15 nanoparticles (+IL7/IL15).
  • NP/T cell indicates the ratio of the number of nanoparticles (NP) to the number of T cells.
  • FIG. 7 is a diagram showing the results of evaluating T cell memory traits in Example 7.
  • FIG. 15A shows the results of evaluating the expression levels of marker proteins CCR7, CD45RA, CD62L, CD28, FSC, and CD27 by flow cytometry.
  • FIG. 15B shows undifferentiated memory traits when mOKT3/FMC63/CD80/41BBL nanoparticles (-IL7/IL15) and mOKT3/FMC63/CD80/41BBL/IL7/IL15 nanoparticles (+IL7/IL15) were administered.
  • “NP/T cell” indicates the ratio of the number of nanoparticles (NP) to the number of T cells.
  • FIG. 7 is a diagram showing the results of evaluating phosphorylated STAT5 in Example 7.
  • Figure 16A shows the results of measuring phosphorylated STAT5 (pSTAT5) levels by flow cytometry.
  • FIG. 16B shows the results of quantifying phosphorylated STAT5 (pSTAT5) fluorescence intensity.
  • FIG. 3 is a diagram showing the CD3/BCMA binding protein and mOKT3/BCMA98/CD80/41BBL nanoparticles produced in Example 8.
  • Figure 17A shows the structure of CD3/BCMA binding protein. In the figure, SP indicates a signal peptide.
  • Figure 17B shows the results of detecting CD25 protein expression on the cell surface by flow cytometry.
  • FIG. 7 is a diagram showing the results of evaluating T cell proliferation and effector effect induction in Example 8.
  • Figure 18A shows the cell doubling rate of T cells.
  • Figure 18B shows the survival rate of tumor cells after co-culture. The results of measuring the percentage of cytokine-producing cells in Example 8 are shown.
  • Figure 19A shows a representative flow cytometry plot.
  • FIG. 19B shows the IL2 positive rate (CD4 + IL2 + ) in CD4 positive T cells and the IFN ⁇ positive rate (CD8 + IFN ⁇ + ) in CD8 positive T cells.
  • FIG. 7 is a diagram showing the results of evaluating the effect of mOKT3/ss1/CD80/41BBL/IL7/IL15 nanoparticles containing mesothelin binding protein in Example 9.
  • FIG. 20A shows the results of evaluating the expression level of CD25 protein by flow cytometry. T cell activation by mOKT3/ss1/CD80/41BBL/IL7/IL15 nanoparticles (ss1) and mOKT3/FMC63/CD80/41BBL/IL7/IL15 nanoparticles (FMC63) is shown.
  • FIG. 20B shows the results of quantifying the expression level of CD25 protein.
  • Figure 20C shows the survival rate of K562-CD19/Mesothelin cells after co-culture.
  • FIG. 7 is a diagram showing the sustained release effect of molecules encapsulated in PLGA nanoparticles in Example 10.
  • Figure 21A shows the fluorescence spectrum of FITC-BSA encapsulated PLGA nanoparticles.
  • FIG. 21B shows the results of measuring the time change in the concentration of FITC-BSA released to the outside of the dialysis membrane using a fluorometer.
  • FIG. 7 is a diagram showing the in vivo antitumor effect of nanoparticles in Example 11.
  • FIG. 22A shows the dosing schedule for NALM6-GL cells, T cells, and nanoparticles.
  • FIG. 22B shows the imaging results of NALM6-GL cells at 2 to 5 weeks after NALM6-GL cell transplantation in the No NP group, NP ⁇ 3 group, and NP ⁇ 6 group.
  • FIG. 22C shows logarithmically transformed luminescence intensity.
  • FIG. 7 is a diagram showing the analysis results of mice to which nanoparticles were administered in Example 11.
  • Figure 23A shows the results of measuring the number of CD8-positive T cells in peripheral blood.
  • Figure 23B shows the results of survival analysis by Kaplan-Meier method. The results of evaluating T cell memory traits in Example 11 are shown.
  • Figure 24A shows a representative flow cytometry diagram.
  • FIG. 24B shows the percentage of memory stem cell (TSCM) and effector memory (TEM) fractions.
  • FIG. 7 is a diagram showing the effect of nanoparticles into which bone marrow homing molecules have been introduced in Example 12.
  • FIG. 25A shows the results of flow cytometry detection of the expression of CXCR4 protein, ITGA4 protein, and ITGB1 protein that were additionally introduced into K562 cells.
  • Figure 25B shows the dosing schedule for NALM6 cells, T cells, and nanoparticles.
  • FIG. 25C shows mOKT3/FMC63/CD80/41BBL/CXCR4/ITGA4/ITGB1 nanoparticles (“CXCR4+VLA4”, (-)) or mOKT3/FMC63/CD80/41BBL nanoparticles (“CXCR4+VLA4”) containing bone marrow homing molecules. ”, (+)) shows the percentage of T cells in the bone marrow or spleen of mice administered.
  • FIG. 7 is a diagram showing the results of producing nanoparticles with different particle diameters and testing their effects in Example 13.
  • Figure 26A shows the particle size distribution of PLGA nanoparticle cores made from 1 mg/mL or 20 mg/mL PLGA.
  • Figure 26B shows the cell doubling rate of T cells.
  • NP size indicates the particle size of nanoparticles.
  • FIG. 26C shows the results of quantifying the expression level of CD25 protein. Five fusion proteins designed as immune regulatory molecules are shown. The effect of membrane vesicles (MVs) loaded with cytokine molecules on their surface layer is shown.
  • Figure 28A shows the absence of membrane vesicles (MV:-) under conditions of not co-culturing with A20 cells (in the figure, A20 cells:-) or under conditions of co-culturing with A20 cells (in the figure, A20 cells:+).
  • MV:WT mouse CD8 cultured in the presence of MV (2C11/1D3/41BBL)
  • MV:IL12 IL12 fusion protein introduced into the surface layer
  • Figure 28B shows the results in the absence of membrane vesicles (MV:-), in the presence of MV(2C11/1D3/41BBL) (MV:WT), and in the presence of MV(2C11/1D3/41BBL/IL12) (MV:IL12).
  • mice CD8 cultured in the presence of MV (2C11/1D3/41BBL/IL18) (MV:IL18), or in the presence of MV (2C11/1D3/41BBL/IL12/IL18) (MV:IL12+IL18)
  • MV:IL18 mouse CD8 cultured in the presence of MV (2C11/1D3/41BBL/IL12/IL18)
  • MV:IL12+IL18 mouse CD8 cultured in the presence of MV (2C11/1D3/41BBL/IL12/IL18)
  • MV:IL12+IL18 MV 2C11/1D3/41BBL/IL12/IL18
  • TGF ⁇ receptor anti-PDL1 scFV
  • anti-CTLA4 scFV mounted on membrane vesicles
  • Figure 29A shows conditions in which membrane vesicles are not added to the medium containing TGF ⁇ (MV:-), conditions in which MV (2C11/1D3/41BBL) as a comparison control is added (MV:WT), and MV (2C11/1D3/ 41BBL/TGFBR) was added (MV:TGFBRI/II), and the TGF ⁇ concentration in the supernatant was measured by ELISA.
  • Figure 29B shows the results of measuring the PDL1 expression level on A20 cells by flow cytometry after adding MV (2C11/1D3/41BBL/anti-PDL1-scFV) to the PDL1-positive mouse lymphoma cell line A20. .
  • WT is the result of A20 cells in which the CTLA4 gene has not been introduced
  • MV- is the result when membrane vesicles are not added
  • WT-MV is the result of the comparison control MV (2C11/1D3 /41BBL) is shown.
  • Tumor volume mm 3
  • Figure 30A shows the results of PBS administration.
  • Figure 30B shows the results of WT-MV administration.
  • Figure 30C shows the results of TME-MV administration.
  • FIG. 31A shows the average tumor volume at 13, 15, and 17 days after transplantation of CT26-CD19-luc cells.
  • Ctrl indicates the PBS administration group.
  • Figure 31B shows the results of mouse survival analysis.
  • Ctrl indicates the PBS administration group.
  • FIG. 31C shows the results of time-lapse observation of tumor cells in mouse bodies in a representative example of the TME-MV administration group. The results of analyzing cells infiltrating into tumors in Balb/c mice in which PBS, WT-MV, or TME-MV was intratumorally administered after subcutaneously transplanting CT26-CD19-luc cells are shown.
  • Figure 32A shows the ratio of CD8-positive T cells to CD45-positive blood cells in infiltrating cells.
  • Figure 32B shows the percentage of CD8 positive T cells (CD45+CD8+ cells) among the total cells within the tumor.
  • Representative flow cytometry plots for the PBS administration group, WT-MV administration group, and TME-MV administration group are shown.
  • the percentage of M2 macrophages among macrophages infiltrating into the tumor is shown.
  • Representative flow cytometry plots for the PBS administration group, WT-MV administration group, and TME-MV administration group are shown.
  • the results of analyzing the expression levels of MICA/B and HLA class I molecules on leukemia cell line K562 are shown.
  • Figure 36A shows that the NK cell activation ligand MICA/B is expressed on leukemia cell line K562.
  • Figure 36B shows that HLA class I molecules that act as NK cell suppressors are not expressed on leukemia cell line K562.
  • Expression levels of CD69, an activation marker of NK cells, and CD107a, a degranulation marker, were determined in NK cells cultured alone and in NK cells co-cultured with MM.1S cells to which various MVs were added and the CD19 gene was introduced. The results of analysis by flow cytometry are shown. The results of evaluating the cytotoxic effect of NK cells on multiple myeloma cell line MM.1S in the presence of various MVs are shown.
  • Figure 39A shows the results of evaluating the cytotoxic effect on the CD19-negative multiple myeloma cell line MM.1S.
  • Figure 39B shows the results of evaluating the cytotoxic effect on CD19-positive multiple myeloma cell line MM.1S.
  • MV-absent conditions MV:-
  • conditions in which membrane vesicles derived from K562 without gene introduction were added MV:+, FMC63:-
  • scFV derived from anti-CD19 antibody clone FMC63
  • the first aspect of the invention is a multispecific nanoparticle.
  • the multispecific nanoparticles of the present invention include a lipid membrane and a fusion protein that constitute the surface layer of the nanoparticles, and are capable of binding to two or more target cells.
  • the target cells include T cells and cancer cells
  • the multispecific nanoparticles of the present invention can bind to the T cells and cancer cells, activate the T cells, and induce cytotoxic activity against the cancer cells. can.
  • cytotoxic activity against cancer cells can also be induced against NK cells and macrophages.
  • Nanoparticles refer to particles having a particle diameter on the order of nanometers (nm). Nanoparticles generally mean particles having a particle size of 1 nm to several hundred nm. The particle size of the nanoparticles is preferably large enough to prevent rapid renal excretion. Specifically, the gap between endothelial cells in the kidney glomerulus is 43 nm, and the upper limit of the size that can pass through the multilayered structure formed by endothelial cells and be naturally excreted is approximately 5 to 10 nm.
  • the particle diameter of the nanoparticles is preferably 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more.
  • the particle size of the nanoparticles is preferably small enough not to form emboli in pulmonary capillaries and/or not to be trapped in the liver, for example, 400 nm or less, 300 nm or less, or 250 nm or less.
  • Preferred particle size ranges include 50 nm to 250 nm, 60 nm to 200 ⁇ m, 70 nm to 180 nm, 80 to 160 nm, 90 nm to 150 nm, or 100 nm to 140 nm.
  • multispecificity refers to specificity for two or more biomolecules or cells. Multispecificity need only be specific to two or more, for example, specific to two or more proteins or two or more cells. Multispecificity includes, for example, bispecificity, trispecificity, quadruplespecificity, quintuspecificity, or more specificity.
  • target cells refer to cells targeted by the multispecific nanoparticles of the present invention.
  • Target cells may be either prokaryotic or eukaryotic.
  • prokaryotic cells include bacterial cells such as E. coli cells.
  • eukaryotic cells include fungal cells (e.g. yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g. chicken cells), and mammalian cells (e.g. , mouse cells, chimpanzee cells, and human cells). Preferably they are mammalian cells.
  • mammalian cells include immune cells, peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells, skin keratinocytes, epithelial cells, mesenchymal stem cells, and mesenchymal cells (bone cells, fat cells, muscle cells). , chondrocytes, nerve cells, or fibroblasts), hematopoietic stem cells, various cancer cell lines, neural stem cells, nerve cells, iPS cells, and ES cells.
  • PBMCs peripheral blood mononuclear cells
  • keratinocytes epithelial cells
  • mesenchymal stem cells mesenchymal stem cells
  • mesenchymal cells mesenchymal cells (bone cells, fat cells, muscle cells).
  • chondrocytes, nerve cells, or fibroblasts) hematopoietic stem cells
  • various cancer cell lines various cancer cell lines, neural stem cells, nerve cells, iPS cells, and ES cells.
  • immune cells include cell types that can function as part of the immune system, as well as undifferentiated cells and progenitor cells (e.g., immune progenitor cells) that can differentiate into such cell types.
  • specific examples of immune cells include lymphocytes, granulocytes, dendritic cells, macrophages, and monocytes.
  • lymphocytes include T cells, B cells, and natural killer cells (NK cells).
  • the lymphocytes may be tumor-infiltrating lymphocytes.
  • T cells include killer T cells (cytotoxic T cells), helper T cells, and regulatory T cells.
  • the T cells may be either CD8-positive T cells or CD4-positive T cells.
  • T cells can be classified into na ⁇ ve T cells, memory T cells, and effector T cells, but they may be any of them. It is known that while na ⁇ ve T cells and memory T cells are abundantly contained in peripheral blood, effector T cells are hardly contained in peripheral blood. Examples of granulocytes include neutrophils, eosinophils, and basophils. Although immune cells may not include stem cells in a narrow sense, in this specification they also include stem cells that can differentiate into lymphocytes (eg, hematopoietic stem cells, etc.).
  • two or more target cells is exemplified by any combination of cells including the above target cells.
  • the two or more target cells may be a combination of different cell types or a combination of the same cell types.
  • combinations of different cell types include, but are not limited to, combinations of T cells and cancer cells, NK cells and cancer cells, and macrophages and cancer cells.
  • An example of a combination of the same cell type is a combination of epithelial cells.
  • lipid membrane means a membrane whose main constituent is lipid.
  • lipid membranes include a lipid bilayer in which lipid molecules are arranged in two layers by associating their hydrophobic parts with each other, and a multilayer structure in which lipid bilayers are the basic structure and are stacked several times.
  • the lipid membrane includes a lipid bilayer.
  • the type of lipid molecules constituting the lipid membrane is not particularly limited, and may be, for example, a simple lipid, a complex lipid, or a derived lipid.
  • lipid molecules include phospholipids, glycolipids, acylglycerols, sterols, long chain fatty acids, long chain fatty alcohols, glycerin fatty acid esters, and the like.
  • the lipid membrane can include cell membrane fragments derived from disrupted cells and lipid membranes derived from liposomes.
  • T cell receptor is a receptor present on the surface of T cells that recognizes antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • T cell receptors a dimer consisting of an ⁇ chain and a ⁇ chain (referred to as ⁇ TCR) and a dimer consisting of a ⁇ chain and a ⁇ chain (referred to as ⁇ TCR) are known.
  • ⁇ TCR binds to other adapter proteins such as CD3 protein to form a complex.
  • TCR complex a complex containing a T cell receptor composed of an ⁇ chain and a ⁇ chain, and an adapter protein is referred to as a “T cell receptor complex (TCR complex)”.
  • Adapter proteins that constitute the TCR complex together with ⁇ TCR include CD3 ⁇ protein, CD3 ⁇ protein, CD3 ⁇ protein, and CD3 ⁇ protein.
  • cancer antigen refers to an antigen that can distinguish cancer cells from normal cells. Cancer antigens are also called tumor antigens or tumor-associated antigens, and usually refer to biomolecules that are specific and/or overexpressed in cancer cells. Cancer antigens targeted by the multispecific nanoparticles of the present invention are, in principle, biomolecules such as proteins, sugar chains, or lipids displayed on the surface of cancer cells. Proteins that can be presented on the surface of cancer cells are not limited to full-length proteins consisting of wild-type amino acid sequences, but also include peptide fragments and neoantigens.
  • peptide fragments include peptide fragments that are degraded by proteasomes within cancer cells and then presented on the surface of cancer cells together with HLA molecules.
  • "neoantigen” refers to a cancer antigen containing a mutant amino acid sequence expressed from a mutated gene in cancer cells. Neoantigens are antigens that are not present in normal cells and are therefore highly specific to cancer cells.
  • cancer antigens include, but are not limited to, A33, BAGE, B cell maturation antigen (BCMA), Bcl-2, ⁇ -catenin, CA19-9, CA125, carboxy-anhydrase-IX ( CAIX), CCR4, CD5, CD19, CD20, CD21, CD22, CD24, CD33, CD37, CD45, CD123, CD133, CEA, CEACAM5, c-Met, CS-1, Cyclin B1, DAGE, EBNA, EGFR, EpCAM, EphrinB2, estrogen receptor, FAP, folate binding protein, GAGE, G250, disialoganglioside (GD2), GM2, gp75, gp100 (Pmel 17), ERBB2 (HER-2/neu), HPV E6, HPV E7, IGF1R , L1-CAM, LRP, MAGE, MART, mesothelin, MUC (MUC1, MUC2, etc.), MUM-1-B, myc, NY
  • membrane proteins include integral membrane proteins, superficial membrane proteins, and lipid-anchored proteins.
  • integral membrane protein refers to a protein, at least a portion of which can be embedded in a membrane, and includes integral monotopic proteins and transmembrane proteins. Integral monotopic proteins are membrane proteins that do not completely penetrate the membrane, but only project on one side of the membrane. Transmembrane proteins are membrane proteins that completely span the membrane. Transmembrane proteins are divided into single transmembrane proteins having one transmembrane domain and multitransmembrane proteins having two or more transmembrane domains. Specific examples of single-transmembrane proteins include immunoglobulin heavy chains. Specific examples of multiple transmembrane proteins include choline transporters, histamine H1 receptors, and G protein-coupled receptors.
  • a “superficial protein” is a protein that is not embedded in the membrane itself, but is fixed to the membrane by binding to lipids or integral membrane proteins.
  • lipid-anchored protein is a protein that is fixed to a membrane by a lipid added by lipid modification.
  • GPI glycosylphosphatidylinositol
  • prenylated proteins prenylated proteins
  • cholesterolylated proteins cholesterolylated proteins
  • fatty acid acylated eg, S-palmitoylated and N-myristoylated
  • membrane protein fragments include fragments of any of the above membrane proteins that can be fixed to the membrane. Examples include fragments containing one or more transmembrane domains in transmembrane proteins. Examples include CD3 protein, CD4 protein, CD8 protein, CD28 protein, and the transmembrane domain of the IL-2 receptor.
  • co-stimulatory molecule refers to a molecule that mediates auxiliary signals necessary for antigen-presenting cells to activate T cells. Also called co-stimulatory factor. Co-stimulatory molecules include CD28 protein, CD40 protein, CD70 protein, CD80 protein, CD86 protein, ICOS ligand protein, OX40 ligand protein, 4-1BB ligand (also called 4-1BBL or CD137L) protein, GITR ligand protein, and LIGHT protein. etc.
  • cytokine is a general term for proteins with a relatively small molecular weight that are secreted from cells. Cytokines are mainly secreted by immune cells and are responsible for information transmission between cells. Examples of cytokines include interleukins, interferons, chemokines, hematopoietic factors, cell growth factors, tumor necrosis factors, and the like. Specific examples of interleukins include IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, and IL- 21 are listed. A specific example of a hematopoietic factor is GM-CSF. A specific example of interferon is IFN- ⁇ protein. A specific example of a cell growth factor is TGF- ⁇ protein. A specific example of tumor necrosis factor is TNF- ⁇ protein.
  • cytokine receptor refers to a protein that functions as a receptor for the cytokine.
  • cytokine receptors include interleukin receptors, interferon receptors, chemokine receptors, hematopoietic factor receptors, cell growth factor receptors, and tumor necrosis factor receptors.
  • Cytokine receptors include any of the cytokine receptors listed above (e.g., TGF- ⁇ receptor, IL-6 receptor, IL-1 receptor, TNF receptor, IL-4 receptor, IL-10 receptor, IL-13 receptor, and CSF-1 receptor).
  • the IL-4 receptor may be a heterodimer composed of IL-4 receptor subunit ⁇ (IL4RA) and IL-2 receptor common ⁇ chain (IL2RG).
  • the IL-10 receptor may be a heterodimer composed of IL-10 receptor subunit ⁇ (IL10RA) and IL-10 receptor subunit ⁇ .
  • the IL-13 receptor may be a heterodimer composed of IL-13 receptor subunit ⁇ 1 (IL13RA1) and IL-4 receptor subunit ⁇ (IL4RA).
  • An example of IL10RA is human-derived IL10RA consisting of the amino acid sequence shown in SEQ ID NO: 597.
  • An example of IL10RB includes human-derived IL10RB consisting of the amino acid sequence shown in SEQ ID NO: 598.
  • An example of IL4RA includes human-derived IL4RA consisting of the amino acid sequence shown in SEQ ID NO: 599.
  • IL2RG human-derived IL2RG consisting of the amino acid sequence shown in SEQ ID NO: 600.
  • IL13RA1 human-derived IL13RA1 consisting of the amino acid sequence shown in SEQ ID NO: 601.
  • CSF-1 receptor human-derived colony stimulating factor 1 receptor (CSF1R), which has the amino acid sequence shown in SEQ ID NO: 602.
  • migrating molecule refers to a molecule that induces migration of migratory cells.
  • migrating cells include leukocytes such as granulocytes (eg, neutrophils, eosinophils, or basophils) or mononuclear cells (eg, monocytes or lymphocytes).
  • leukocytes such as granulocytes (eg, neutrophils, eosinophils, or basophils) or mononuclear cells (eg, monocytes or lymphocytes).
  • chemotactic molecules include interleukins, cytokines, chemokines, homing molecules, adhesion molecules, and the like.
  • a "homing molecule” is a surface molecule that is expressed when lymphocytes home to a specific organ.
  • homing molecules include chemokine receptors, integrin receptors, interleukin receptors, growth factor receptors, and hormone receptors.
  • Specific examples of homing molecules include CX3CR1 protein, CXCR2 protein, CXCR4 protein, ITGA4 protein, ITGB1 protein, ICAM-1 protein, VCAM-1 protein, CCR5 protein, IGFR2 protein, ⁇ 2-integrin, and the like.
  • adhesion molecule means a molecule that mediates the bond between cells or the bond between cells and extracellular matrix.
  • adhesion molecules include proteins belonging to the cadherin superfamily, proteins belonging to the immunoglobulin superfamily, and proteins belonging to the integrin family.
  • immune checkpoint inhibitory molecule refers to a molecule that can inhibit evasion of host immune responses by cancer cells.
  • immune checkpoint inhibitor molecules are known in the art, such as CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors. agents, and BTLA inhibitors.
  • Specific examples include antibodies such as pembrolizumab, nivolumab, atezolizumab, durvalmumab, avelumab, tremelimumab, and ipilimumab, and fragments thereof.
  • immune checkpoint molecule refers to a molecule that can regulate the activation of immune cells such as T cells, NK cells, and macrophages. Cancer cells can evade attack from the immune system by suppressing immune cell activation via immune checkpoint molecules.
  • immune checkpoint molecules include PD1 protein, Lag-3 protein, Tim-3 protein, TIGIT protein, ICOS protein, or BTLA protein.
  • a “signal peptide” is an extracellular transfer signal that is necessary when a protein biosynthesized by gene expression is secreted out of the cell, and is also called a signal peptide.
  • the signal sequence may include a region composed of hydrophobic amino acids. After translation, the signal peptide is cleaved and removed by a signal peptidase before being exported to the outside of the cell. Signal peptide sequences are present at the N-terminus of many secreted and membrane proteins and are, for example, 15-30 amino acids long.
  • the signal peptide may be derived from any species, human or non-human, such as an insect cell or a virus, but is preferably human. A specific example of the signal peptide is the signal peptide derived from human Oncostatin M.
  • linker peptide is a peptide that can be inserted between each part of the fusion protein, such as the chimeric cytokine receptor of the present invention, in order for each part to be fused to perform a desired function.
  • the length of the linker peptide is not limited, but is typically 3 to 100 amino acids long, preferably 5 to 50 amino acids long. Peptides containing many amino acids with relatively small side chains, such as serine and glycine, are often used.
  • a "tag peptide” is a short peptide consisting of a dozen to several dozen amino acids that can label a protein, and is used for protein detection and purification. Usually, a base sequence encoding a tag peptide is linked to the 5' or 3' end of a gene encoding the protein to be labeled, and the protein is labeled by expressing it as a fusion protein with the tag peptide.
  • tag peptides have been developed in the art, and any tag peptide may be used. Specific examples of tag peptides include FLAG, HA, His, PA, myc, and the like.
  • antibody refers to a protein that exhibits immunoreactivity to an antigen.
  • monoclonal antibodies are referred to.
  • the biological species from which the antibody is derived is not particularly limited. Antibodies derived from birds and mammals are preferred. Examples include chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, or human.
  • a “monoclonal antibody” includes a framework region (hereinafter referred to as "FR”) and a complementarity determining region (hereinafter referred to as "CDR"). , a single type of immunoglobulin that can specifically bind to and recognize an antigen, or at least one set of a light chain variable region (V L region) and a heavy chain variable region (V H A recombinant or synthetic antibody that encompasses a region).
  • FR framework region
  • CDR complementarity determining region
  • immunoglobulins can be of any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY) or of any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1). , and IgA2).
  • Recombinant antibody refers to a chimeric antibody or a humanized antibody.
  • a “chimeric antibody” is an antibody produced by combining the amino acid sequences of antibodies derived from different animals, and is an antibody in which the constant region (C region) of one antibody is replaced with the C region of another antibody.
  • C region constant region
  • an antibody in which the C region of a rat monoclonal antibody is replaced with the C region of a human antibody is applicable.
  • the heavy chain variable region of a human antibody against any antigen is replaced with the heavy chain variable region of an antibody against the antigen of interest, and the light chain variable region of a human antibody is replaced with the light chain variable region of an antibody against the antigen of interest.
  • a "humanized antibody” is a mosaic antibody in which the CDRs in a human antibody are replaced with the CDRs in an antibody derived from a non-human mammal.
  • the variable region (V region) of an immunoglobulin molecule consists of four FRs (FR1, FR2, FR3, and FR4) and three CDRs (CDR1, CDR2, and CDR3) starting from the N-terminus: FR1-CDR1-FR2-CDR2-FR3- It is composed of CDR3-FR4 concatenated in the order.
  • a humanized antibody is, for example, a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of an antibody against an antigen of interest, and a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a human antibody against any antigen. By substituting each of these with , it is possible to construct a human antibody that inherits the antigen-binding specificity of the antibody for the target antigen. Since such humanized antibodies are derived from human antibodies except for CDRs, they can reduce the immune reaction against the antibodies in the human body more than chimeric antibodies.
  • “Synthetic antibody” refers to an antibody that is synthesized chemically or by using recombinant DNA methods. Examples include antibodies newly synthesized using recombinant DNA methods. Specific examples include scFv (single chain fragment of variable region: single chain antibody). In immunoglobulin molecules, a pair of variable regions (light chain variable region V L and heavy chain variable region V H ) that form a functional antigen-binding site are located on separate polypeptide chains, the light chain and the heavy chain. do. scFv is a synthetic antibody with a molecular weight of about 35 kDa or less that has a structure in which V L and V H are linked by a sufficiently long flexible linker and included in one polypeptide chain in an immunoglobulin molecule. Within a scFv, a set of variable regions can self-assemble together to form a functional antigen binding site. scFv can be obtained by incorporating recombinant DNA encoding it into a vector using known techniques and expressing it
  • Antibodies can also be modified.
  • modification includes functional modifications necessary for antigen-specific binding activity such as glycosylation, and modifications on labels necessary for antibody detection.
  • Glycosylation modifications on antibodies are performed to adjust the affinity of the antibody for its target antigen. Specifically, for example, in the FR of an antibody, a modification may be made in which glycosylation at that site is lost by introducing substitutions into amino acid residues constituting glycosylation and removing the glycosylation site.
  • the antibody preferably has a dissociation constant with the antigen of 10 -7 M or less, for example, preferably has a high affinity of 10 -8 M or less, more preferably 10 -9 M or less, particularly preferably 10 -10 M or less.
  • the above dissociation constant can be measured using techniques known in the art. For example, it may be measured using the Biacore system (GE Healthcare) using speed evaluation kit software.
  • fragment of an antibody refers to an antibody fragment that consists of a part of an antibody and exhibits immunoreactivity to an antigen like an antibody, and is an antigen-binding fragment.
  • Fab fragment fragment
  • Fv fragment fragment stabilized by disulfide bonds
  • dsFv disulfide bonds
  • dsFv-dsFv' bispecific dsFv
  • heavy-duty diabodies ds diabodies
  • scFv single chain antibody molecules
  • dimeric scFv bivalent diabodies
  • multispecific antibodies camelized single domain antibodies (camelized antibodies; VHH antibodies), etc.
  • Fab is an antibody fragment produced when an IgG molecule is cleaved by papain on the N-terminal side of the disulfide bond in the hinge region, and constitutes the H chain constant region (heavy chain constant region: hereinafter referred to as C H ). It consists of three domains (C H 1, C H 2, and C H 3), which are adjacent to V H , and the full-length L chain.
  • C H H chain constant region
  • Fab' has a slightly longer H chain than Fab, including the hinge region, but has substantially the same structure as Fab.
  • Fab' is produced by reducing the Fab' dimer (F(ab') 2 ), which is produced by pepsin cleaving the IgG molecule at the C-terminal side of the disulfide bond in the hinge region, under mild conditions. It can be obtained by cleaving disulfide linkages. Since all of these antibody fragments contain an antigen-binding site, they have the ability to specifically bind to an antigen epitope.
  • nanoparticle core means a core particle that constitutes the inside of a nanoparticle.
  • the multispecific nanoparticle of the present invention corresponds to a core particle encapsulated in a lipid membrane that constitutes the surface layer of the nanoparticle.
  • the material of the core particles is not particularly limited, and may be polymer nanoparticles, metal nanoparticles, or dendrimers. Note that in this specification, the multispecific nanoparticles may or may not include a nanoparticle core.
  • the polymer constituting the polymer nanoparticles is preferably a biocompatible and biodegradable polymer, such as gelatin, collagen, fibrin, polyaspartic acid, polyglutamic acid, polyleucine, polysaccharide, cellulose, alginate, dextran, amylose, pectin, Natural polymers such as chitosan, chitin, heparin, and hyaluronic acid, as well as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-polyglycolic acid copolymer (PLGA), PLGA-ethylene oxide fumarate, PLGA-TGPS, pCPH , PHB-PVA, PEG-PLA, PCL, PAC, PEC, polyisobutylcyanoacrylate, poly(HPMA), PHB, PHA, poly- ⁇ -R-malic acid, ethylcellulose, and polybutylene succinate (PBS), etc.
  • Synthetic polymers include: Polylactic acid-polyglycolic acid copolymer (PLGA) is a polyester containing glycolic acid monomer and lactic acid monomer, and is used in tissue engineering and pharmaceutical delivery systems approved by the US Food and Drug Administration (FDA). Well known biocompatible and biodegradable polymers are used.
  • PLGA Polylactic acid-polyglycolic acid copolymer
  • FDA US Food and Drug Administration
  • the material of the metal nanoparticles is not particularly limited, and may be, for example, gold, platinum, silver, or iron.
  • the dendrimer is not particularly limited as long as it is a pharmaceutically acceptable dendrimer.
  • examples include PAMAM dendrimers, G5 dendrimers, acylated dendrimers, PEGylated dendrimers, and acetylated dendrimers.
  • liposome refers to a vesicle containing a lipid membrane and an aqueous medium encapsulated in the lipid membrane.
  • the lipid membrane of a liposome is composed of one or more lipid layers. For example, it is composed of a lipid bilayer containing phospholipids.
  • sustained release refers to a substance being gradually released into space. In this specification, this refers to the gradual release of components inside the lipid membrane from the multispecific nanoparticles of the present invention to the outside.
  • the period during which the component continues to be diffused is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, half a day or more, 1 day or more, 2 days or more, 3 days or more, 1 week or more, 2 weeks or more, or This applies to periods of one month or more.
  • a protein when specified by a specific amino acid sequence, the corresponding wild-type and mutant proteins derived from any biological species are also referred to.
  • amino acid sequence described herein one or more amino acids are deleted, substituted, or added, or 60% or more, 70% or more, 80% or more, 85% or more of the amino acid sequence. It is intended to include proteins consisting of amino acid sequences having % or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The same applies to base sequences.
  • amino acid identity refers to the amino acid sequences of two polypeptides to be compared, so that the number of amino acid residues that match is maximized, as necessary for one or both polypeptides. It refers to the ratio (%) of the number of identical amino acid residues to the total number of amino acid residues when aligned with appropriate gaps inserted.
  • Base identity base sequence identity
  • amino acid substitution refers to substitution between the 20 types of amino acids that make up natural proteins. Amino acid substitutions are preferably within a group of conservative amino acids with similar properties such as charge, side chain, polarity, aromaticity, etc.
  • uncharged polar amino acids with low polar side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile), , Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), and basic amino acids (Asp, Glu).
  • Examples include substitutions within aromatic amino acid groups (Phe, Tyr, Trp).
  • the multispecific nanoparticle of the present invention comprises a lipid membrane constituting the surface layer of the nanoparticle, (i) a membrane binding region bound to the lipid membrane, and two or more target binding regions located outside the lipid membrane. (ii) a membrane binding region associated with a lipid membrane, and a target binding region located external to the lipid membrane.
  • the multispecific nanoparticle of the present invention comprises the fusion protein of (i) above.
  • a fusion protein having the structure (i) above specifically a fusion protein comprising a membrane binding region bound to a lipid membrane and two or more target binding regions located outside the lipid membrane.
  • the protein is called a "type I fusion protein.”
  • Type I fusion proteins contain two or more target-binding regions at the N-terminus and a membrane-binding region at the C-terminus, or a membrane-binding region at the N-terminus and two or more target-binding regions at the C-terminus. can be included.
  • multispecific nanoparticles containing a type I fusion protein are referred to as "type I multispecific nanoparticles.”
  • Type I multispecific nanoparticles are characterized in that at least one fusion protein contains two or more target binding regions.
  • the multispecific nanoparticle of the present invention comprises the fusion protein (ii) above.
  • each of the two or more fusion proteins includes a membrane binding region bound to a lipid membrane and one or more target binding regions located outside the lipid membrane.
  • the protein is called a "type II fusion protein.”
  • the type II fusion protein contains a target binding region at the N-terminus and a membrane-binding region at the C-terminus, or a membrane-binding region at the N-terminus and a target-binding region at the C-terminus. be able to.
  • type II multispecific nanoparticles containing a type II fusion protein are referred to as "type II multispecific nanoparticles.”
  • Type II multispecific nanoparticles are characterized by comprising two or more fusion proteins containing at least one target binding region. It is therefore understood that a type II multispecific nanoparticle includes a total of two or more target binding regions.
  • the multispecific nanoparticles of the invention can include both the type I and type II fusion proteins described above. It is understood that such embodiments are encompassed by, and not excluded from, either the Type I and Type II multispecific nanoparticles described above.
  • fusion protein shall mean either the type I fusion protein or the type II fusion protein described above, unless otherwise specified.
  • Type I fusion proteins and type II fusion proteins may contain a cytoplasmic domain, a signal peptide, a linker peptide, and/or a tag peptide, etc., as necessary, in addition to the membrane binding region and target binding region specifically explained below. I can do it.
  • a “membrane binding region” is a region that stably incorporates or attaches a fusion protein to a lipid membrane.
  • the term "membrane-binding region bound to a lipid membrane” means that the membrane-binding region contacts, adheres to, or binds to a lipid membrane through hydrophobic interaction.
  • membrane-binding regions include the above-mentioned membrane proteins or fragments thereof, and artificial transmembrane domains containing mainly hydrophobic amino acid residues.
  • the membrane binding region may be derived from the same biological species and/or the same protein as the signal sequence.
  • target binding region refers to a region capable of binding to a target molecule or cell in the above type I fusion protein or type II fusion protein. More specifically, it is a region that can target and bind to a surface antigen expressed on the surface of a target cell.
  • the two or more target molecules to which the two or more target binding regions bind are not limited.
  • the two or more target molecules may be a combination of different molecules or a combination of the same molecules. However, it is preferable that the same molecules bind to different epitopes.
  • Examples of two or more target molecules include a combination of (a) a T cell receptor complex, a NK cell surface antigen, or a macrophage surface antigen, and (b) a cancer antigen.
  • the multispecific nanoparticles of the invention target T cells and cancer cells, and the target binding region includes a T cell receptor complex binding region and a cancer antigen binding region.
  • the target binding region includes a T cell receptor complex binding region and a cancer antigen binding region means that in the type I multispecific nanoparticle described above, two or more targets contained in the type I fusion protein the binding region comprises a T cell receptor complex binding region and a cancer antigen binding region, or in two or more type II fusion proteins contained in the type II multispecific nanoparticles described above, separate type II fusion proteins; includes a T cell receptor complex binding region and a cancer antigen binding region.
  • the cancer antigen binding region is located on the N-terminal side of the T cell receptor complex binding region, regardless of the positional relationship between the T cell receptor complex binding region and the cancer antigen binding region. or the cancer antigen binding region may be located on the C-terminal side of the T cell receptor complex binding region.
  • T cell receptor complex binding region refers to a region that can bind to the T cell receptor complex on the cell surface of a T cell.
  • the T cell receptor complex binding region is preferably a region that can activate T cells by binding to the T cell receptor complex and induce effector activity such as cytotoxic activity against cancer cells.
  • the protein bound by the T cell receptor complex binding region is not particularly limited as long as it is a component of the T cell receptor complex.
  • T cell receptor complex binding region examples include any component of the T cell receptor (e.g., T cell receptor alpha chain, T cell receptor beta chain, T cell receptor gamma chain, or T cell receptor ⁇ chain), and any adapter protein included in the T cell receptor complex, such as CD3 ⁇ protein, CD3 ⁇ protein, CD3 ⁇ protein, and CD3 ⁇ protein.
  • T cell receptor complex binding regions include anti-T cell receptor (TCR) antibodies (e.g., anti-T cell receptor ⁇ chain antibody, anti-T cell receptor ⁇ chain antibody, anti-T cell receptor gamma and anti-CD3 antibodies (eg, anti-CD3 ⁇ antibodies, anti-CD3 ⁇ antibodies, anti-CD3 ⁇ antibodies, and anti-CD3 ⁇ antibodies).
  • T cell receptor complex binding regions that bind to T cell receptors include HLA/peptide fusion molecules.
  • HLA/peptide fusion molecule refers to a fusion molecule containing an HLA class I molecule or HLA class II molecule and a peptide.
  • the HLA class I molecule has a structure in which ⁇ chain and ⁇ 2 microglobulin are associated.
  • HLA class II molecules have a structure in which ⁇ chains and ⁇ chains are associated.
  • HLA class I molecules and HLA class II molecules are not particularly limited to HLA antigens.
  • the peptide contained in the HLA/peptide fusion molecule is a peptide consisting of several to several tens of amino acid residues, for example, 5 to 20 amino acid residues, that binds to the antigen-presenting groove of HLA class I molecules or HLA class II molecules. It is.
  • the HLA/peptide fusion molecule can include a linker peptide, such as a flexible linker, that connects the two. Note that HLA class I molecules and HLA class II molecules are highly polymorphic. Three subclasses of HLA class I molecules, HLA-A, HLA-B, and HLA-C, are known, but they are not particularly limited.
  • An example of the ⁇ chain of an HLA class I molecule is the human ⁇ chain, which consists of the amino acid sequence shown in SEQ ID NO: 26 and belongs to HLA-A:0201.
  • An example of ⁇ 2 microglobulin is human ⁇ 2 microglobulin consisting of the amino acid sequence shown in SEQ ID NO: 27.
  • An example of the ⁇ chain of an HLA class II molecule is the human ⁇ chain, which consists of the amino acid sequence shown in SEQ ID NO: 28 and belongs to HLA-DRA*01:01.
  • An example of the ⁇ chain of an HLA class II molecule is the human ⁇ chain consisting of the amino acid sequence shown in SEQ ID NO: 29 and belonging to HLA-DRB*01:01.
  • the HLA/peptide fusion molecule comprises the ⁇ chain of an HLA class I molecule, ⁇ 2 microglobulin, and an antigenic peptide, and optionally a linker peptide.
  • a linker peptide in the literature (Honda, T., et al., Protein Eng Des Sel., 2015, 28(2):53-8.), in order from the N-terminus, antigen peptide, peptide linker, ⁇ 2 microglobulin, and HLA
  • a fusion molecule containing an ⁇ chain of a class I molecule, and a fusion molecule containing, in order from the N-terminus, ⁇ 2 microglobulin, an ⁇ chain of an HLA class I molecule, a peptide linker, and an antigen peptide are disclosed.
  • the HLA/peptide fusion molecule comprises ⁇ 2 microglobulin and an antigenic peptide, and optionally a linker peptide.
  • a linker peptide In the literature (Tafuro, S., et al., Eur J Immunol., 2001, 31(2):440-9.), fusions containing an antigen peptide, a peptide linker, and ⁇ 2 microglobulin in order from the N-terminus are described.
  • a molecule is disclosed that functions as an HLA molecule together with the alpha chain of an HLA class I molecule.
  • the HLA/peptide fusion molecule comprises the alpha chain, beta chain of an HLA class II molecule, and an antigenic peptide, and optionally a linker peptide.
  • HLA/peptide fusion molecules can activate T cells containing T cell receptors that recognize peptides as antigens, such as CD8-positive T cells and CD4-positive T cells.
  • HLA/peptide fusion molecules have the advantage of being able to specifically activate only T cells that contain T cell receptors for specific antigens, unlike anti-CD3 antibodies that can activate T cells non-specifically regardless of the antigen. has.
  • the T cell receptor complex binding region is an anti-CD3 antibody or fragment thereof.
  • anti-CD3 antibodies include anti-CD3 ⁇ antibodies, anti-CD3 ⁇ antibodies, anti-CD3 ⁇ antibodies, and anti-CD3 ⁇ antibodies.
  • the sequences of the heavy and light chain variable regions and CDRs of exemplary anti-CD3 antibodies are shown in Table 1 below. Note that in Table 1 below, anti-CD3 ⁇ antibodies are exemplified. CDRs shall follow the Kabat antibody numbering system.
  • anti-CD3 antibodies include anti-CD3 ⁇ antibodies disclosed in WO 2021/224499 and containing the same set of CDRs as the hXR32 antibody described above, i.e., consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 106, respectively.
  • An anti-CD3 ⁇ antibody containing a heavy chain variable region comprising CDR1 to CDR3 and a light chain variable region comprising CDR1 to CDR3 comprising the amino acid sequence shown in SEQ ID NO: 107 to 109, respectively for example, a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 343
  • Anti-CD3 ⁇ antibody comprising a chain variable region and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 344; disclosed in International Publication No.
  • Anti-CD3 ⁇ antibody comprising a region; disclosed in WO 2017/210485, and a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 359 to 361, respectively, and SEQ ID NOs: 362 to 364, respectively.
  • An anti-CD3 ⁇ antibody comprising a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown in (for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 365, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 366) anti-CD3 ⁇ antibody); disclosed in International Publication No.
  • 2016/020309 includes a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 367 to 369, respectively, and SEQ ID NOs: 370 to 372, respectively.
  • An anti-CD3 ⁇ antibody comprising a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown in (for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 373, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 374) anti-CD3 ⁇ antibody); disclosed in International Publication No.
  • An anti-CD3 ⁇ antibody comprising a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown in (for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 381, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 382) anti-CD3 ⁇ antibody); disclosed in International Publication No.
  • An anti-CD3 ⁇ antibody comprising a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown in (for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 389, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 390) anti-CD3 ⁇ antibody); disclosed in US Patent Application Publication No.
  • Anti-CD3 ⁇ antibody comprising a light chain variable region comprising a light chain variable region (for example, an anti-CD3 ⁇ antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 405 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 406); International publication No. 2013/072415, the heavy chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and the light chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434.
  • Anti-CD3 ⁇ antibody comprising chain variable region disclosed in International Publication No.
  • An anti-CD3 ⁇ antibody comprising a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 440 (for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 441, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 442)
  • Anti-CD3 ⁇ antibody containing a variable region disclosed in International Publication No. 2021/228783 and International Publication No.
  • an anti-CD3 ⁇ antibody for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 449, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 449, and SEQ ID NO: 450
  • anti-CD3 ⁇ antibody comprising a light chain variable region consisting of the amino acid sequence shown in WO 2021/113701
  • an anti-CD3 ⁇ antibody for example, an anti-CD3 ⁇ antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 457 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 458), and a CDR1 consisting of the amino acid sequences shown in SEQ ID NOs: 459 to 461, respectively.
  • an anti-CD3 ⁇ antibody e.g., an anti-CD3 ⁇ antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 465 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 466; disclosed in US Patent No.
  • an anti-CD3 ⁇ comprising a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 467 to 492, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 494 to 518.
  • Antibody disclosed in US Patent Application Publication No. 2022/0025047, and includes a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NO: 519 to 521, respectively, and CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 522.
  • an anti-CD3 ⁇ antibody for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 525) comprising a light chain variable region including CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 523), and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 524.
  • anti-CD3 ⁇ antibody comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 526); disclosed in WO 2016/205520;
  • An anti-CD3 ⁇ antibody comprising a heavy chain variable region comprising CDR3 and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 532 or the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively; and
  • a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 566 to 568, respectively, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively.
  • an anti-CD3 ⁇ antibody for example, an anti-CD3 ⁇ antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 572 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 573; or anti-CD3 ⁇ antibody clone 2C11
  • an anti-CD3 ⁇ antibody for example, an anti-CD3 ⁇ antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 572 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 573; or anti-CD3 ⁇ antibody clone 2C11
  • the T cell receptor complex binding region is an anti-T cell receptor antibody (anti-TCR antibody) or a fragment thereof.
  • the anti-TCR antibody may be an antibody against the T cell receptor ⁇ chain, the T cell receptor ⁇ chain, or both. It is known that anti-TCR monoclonal antibodies such as WT31 and BMA031 can activate T cells similarly to anti-CD3 antibodies (Gupta, S., et al., Cell Immunol., 1991, 132(1): 26-44.; Knobloch, C., et al., Cell Immunol., 1991, 138(1):150-64.). The sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-TCR antibody are shown in Table 2 below.
  • CDRs shall follow the Kabat antibody numbering system.
  • Table 2 the heavy chain variable region and light chain variable region of WT31, as well as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, chain CDR2, and light chain CDR3 are also illustrated.
  • cancer antigen-binding region refers to a region that can bind to a cancer antigen on the cell surface of a cancer cell.
  • the type of cancer antigen is not particularly limited, and can be arbitrarily selected from the cancer antigens mentioned above, for example.
  • the cancer antigen is CD19, BCMA, mesothelin, GD2, CD20, CD22, EGFR, CD33, CD123, ERBB2, CD133, CEA, CEACAM5, MUC1, or PSMA. All of these have a track record of development as CARs and are undergoing clinical trials.
  • the cancer antigen binding region is an anti-CD19 antibody or fragment thereof, an anti-mesothelin antibody or fragment thereof, an anti-BCMA antibody or fragment thereof, an anti-GD2 antibody or fragment thereof, an anti-CD20 antibody or fragment thereof, an anti-CD22 antibody or its fragment, anti-EGFR antibody or its fragment, anti-CD33 antibody or its fragment, anti-CD123 antibody or its fragment, anti-ERBB2 antibody or its fragment, anti-CD133 antibody or its fragment, anti-CEA antibody or its fragment, anti-CEACAM5 antibody or its fragment, anti-MUC1 antibody or fragment thereof, or anti-PSM antibody or fragment thereof.
  • the cancer antigen binding region is an anti-CD19 antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-CD19 antibody are shown in Table 3 below. CDRs shall follow the Kabat antibody numbering system.
  • anti-CD19 antibodies or fragments thereof include a heavy chain variable region comprising CDR1, CDR2, and CDR3 contained in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 580, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 581.
  • a light chain variable region containing CDR1, CDR2, and CDR3 contained in the light chain variable region for example, a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 580, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 581) or anti-CD19 antibody clone 1D3).
  • the cancer antigen binding region is an anti-mesothelin antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-mesothelin antibody are shown in Table 4 below. CDRs shall follow the Kabat antibody numbering system.
  • the cancer antigen binding region is an anti-BCMA antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-BCMA antibody are shown in Table 5 below. CDRs shall follow the Kabat antibody numbering system.
  • the cancer antigen binding region is an anti-EGFR antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-EGFR antibody are shown in Table 6 below. CDRs shall follow the Kabat antibody numbering system.
  • the cancer antigen binding region is an anti-GD2 antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-GD2 antibody are shown in Table 7 below. CDRs shall follow the Kabat antibody numbering system.
  • a multispecific nanoparticle of the invention comprises a type I fusion protein comprising a T cell receptor complex binding region and a cancer antigen binding region, wherein the T cell receptor complex binding region is an anti-CD3 antibody. or a fragment thereof, and the cancer antigen-binding region consists of an anti-CD19 antibody or a fragment thereof.
  • a type I fusion protein is the amino acid sequence shown in SEQ ID NO:5.
  • a multispecific nanoparticle of the invention comprises a type II fusion protein comprising a T cell receptor complex binding region and a type II fusion protein comprising a cancer antigen binding region;
  • the body-binding region consists of an anti-CD3 antibody or a fragment thereof, and the cancer antigen-binding region consists of an anti-CD19 antibody or a fragment thereof.
  • Examples of such a type II fusion protein include a type II fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 9, and a type II fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 11.
  • a multispecific nanoparticle of the invention comprises a type II fusion protein comprising a T cell receptor complex binding region and a cancer antigen binding region, wherein the T cell receptor complex binding region is an anti-CD3 antibody binding region. or a fragment thereof, and the cancer antigen-binding region consists of an anti-mesothelin antibody or a fragment thereof.
  • a type II fusion protein include a type II fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 9, and a type II fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 21.
  • a multispecific nanoparticle of the invention comprises a type I fusion protein comprising a T cell receptor complex binding region and a cancer antigen binding region, wherein the T cell receptor complex binding region is an anti-CD3 antibody. or a fragment thereof, and the cancer antigen-binding region consists of an anti-BCMA antibody or a fragment thereof.
  • a type I fusion protein is the amino acid sequence shown in SEQ ID NO: 17.
  • the multispecific nanoparticles of the present invention target NK cells and cancer cells, and the target binding region includes a NK cell surface antigen binding region and a cancer antigen binding region.
  • the cancer antigen binding region shall be as described above.
  • NK cell surface antigen-binding region refers to a region on the cell surface of a NK cell that can bind to any surface antigen thereof.
  • NK cell surface antigens include, but are not limited to, CD16, NKG2D, Nkp30, Nkp44, Nkp46, and 2B4. Examples of these are disclosed in the literature (Gauthier, L., et al., Cell, 2019, 177(7):1701-1713.e16.).
  • KIR family molecules Ly49 family molecules, CD94-NKG2C/E/H heterodimeric receptors, DNAM-1/CD226, CRTAM, CD94-NKG2A, TIGIT, CD96, SLAM family receptors, 2B4/ CD244, CRACC/SLAMF7, NTB-A/SLAMF6, CD27, CD100/Semaphorin 4D, CD160, Siglec, Siglec-3, Siglec-7, Siglec-9, ILT2/LILRB1, KLRG1, LAIR-1, CD161/NKR-P1A , and CEACAM-1.
  • the NK cell surface antigen binding region is an anti-CD16 antibody or fragment thereof, an anti-NKG2D antibody or fragment thereof, an anti-Nkp30 antibody or fragment thereof, an anti-Nkp44 antibody or fragment thereof, an anti-Nkp46 antibody or fragment thereof, or an 2B4 antibody or its fragment.
  • a ligand molecule that binds to an NK cell surface antigen or a binding fragment thereof can also be used as the NK cell surface antigen-binding region.
  • Specific examples include IgG Fc regions or fragments thereof that bind to CD16, MICA proteins or fragments thereof and MICB proteins or fragments thereof that bind to NKG2D, B7H6 proteins or fragments thereof that bind to Nkp30, and influenza virus that binds to NKp44 or NKp46. derived hemagglutinin or a fragment thereof.
  • the IgG Fc region may be of any subclass (IgG1, IgG2, IgG3, and IgG4), including but not limited to IgG1 and IgG1, which can send strong activation signals to NK cells and macrophages.
  • the Fc region of IgG3 is preferred. Specific examples of the above examples include human IgG1 Fc region (SEQ ID NO: 30), human IgG3 Fc region (SEQ ID NO: 61), human MICA protein (SEQ ID NO: 31), human MICB protein (SEQ ID NO: 32), and human B7H6 protein (SEQ ID NO: 32). No. 33), and influenza virus-derived hemagglutinin (SEQ ID No. 34).
  • the NK cell surface antigen binding region is an anti-CD16 antibody or a fragment thereof.
  • the sequences of each heavy and light chain variable region and each CDR of an exemplary anti-CD16 antibody are shown in Table 8 below. CDRs shall follow the Kabat antibody numbering system.
  • the NK cell surface antigen binding region is an anti-NKG2D antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-NKG2D antibody are shown in Table 9 below. CDRs shall follow the Kabat antibody numbering system.
  • the NK cell surface antigen binding region is an anti-NKp46 antibody or a fragment thereof.
  • the sequences of each heavy chain and light chain variable region and each CDR of an exemplary anti-NKp46 antibody are shown in Table 10 below. CDRs shall follow the Kabat antibody numbering system.
  • the multispecific nanoparticles of the present invention target macrophage cells and cancer cells, and the target binding region includes a macrophage cell surface antigen binding region and a cancer antigen binding region.
  • the cancer antigen binding region shall be as described above.
  • macrophage surface antigen-binding region refers to a region on the cell surface of a macrophage that can bind to any surface antigen thereof.
  • macrophage surface antigens include, but are not limited to, FC ⁇ RI (CD64), FC ⁇ RIIA (CD32), FC ⁇ RIIIA (CD16), CD40, CD47, and TLR4a.
  • the macrophage surface antigen binding region comprises an anti-FC ⁇ RI (CD64) antibody or fragment thereof, an anti-FC ⁇ RIIA (CD32A) antibody or fragment thereof, an anti-FC ⁇ RIIIA (CD16) antibody or fragment thereof, an anti-CD40 antibody or fragment thereof, an anti-FC ⁇ RIIA (CD32A) antibody or fragment thereof, CD47 antibody or fragment thereof, or anti-TLR4a antibody or fragment thereof.
  • anti-CD47 antibodies include a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 590 to 592, respectively, and a light chain comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 593 to 595, respectively.
  • an anti-CD47 antibody comprising a variable region e.g., an anti-CD47 antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 589 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 588
  • each SEQ ID NO: Examples include anti-CD47 VHH containing a variable region comprising CDR1 to CDR3 consisting of the amino acid sequence shown by 583 to 585 (for example, anti-CD47 VHH including a variable region shown by SEQ ID NO: 586).
  • a ligand molecule that binds to a macrophage surface antigen or a binding fragment thereof can also be used as the macrophage surface antigen-binding region.
  • an IgG Fc region that binds to FC ⁇ RI (CD64) (e.g., an IgG1, IgG2, IgG3, and IgG4 Fc region, preferably an IgG1 and IgG3 Fc region; for example, a human IgG1 having the amino acid sequence shown in SEQ ID NO: 30) Fc region) or a fragment thereof, CD40L protein or a fragment thereof that binds to CD40, and TLR4a ligand that binds to TLR4a.
  • FC ⁇ RI CD64
  • IgG1, IgG2, IgG3, and IgG4 Fc region e.g., an IgG1, IgG2, IgG3, and IgG4 Fc region, preferably an IgG1 and IgG3 Fc region; for example,
  • the IgG Fc region is not normally expressed on the cell membrane, it can be expressed on the cell membrane as a fusion polypeptide in which the C-terminal side is linked to a type II membrane protein or a portion thereof located outside the cell.
  • a signal that strongly activates macrophages via FC ⁇ RI CD64
  • Specific examples of these include the human IgG Fc region described above and the human CD40L protein (SEQ ID NO: 35).
  • Specific examples of TLR4a ligands include TLR4a agonists having the amino acid sequence shown by SEQ ID NO: 582 or SEQ ID NO: 596.
  • the macrophage surface antigen binding region is an anti-CD40 antibody or a fragment thereof.
  • the sequences of the heavy and light chain variable regions and CDRs of exemplary anti-CD40 antibodies are shown in Table 11 below. CDRs shall follow the Kabat antibody numbering system.
  • the multispecific nanoparticle of the present invention comprises a co-stimulatory molecule, a cytokine molecule, a chemotactic molecule, an immune checkpoint inhibitory molecule, and an immune checkpoint molecule on the surface and/or interior of the nanoparticle. It may further include one or more of the selected ones.
  • a chemotactic molecule may be a homing molecule, a chemokine molecule, or an adhesion molecule.
  • the multispecific nanoparticles of the invention include costimulatory molecules. T cell receptor signals, costimulatory molecule signals, and cytokine signals are referred to as signals 1, 2, and 3, and their combination results in strong T cell activation.
  • the multispecific nanoparticles of the invention preferably include co-stimulatory molecules and cytokine molecules.
  • a cytokine molecule, migration molecule, or homing molecule can be introduced into the surface layer of the nanoparticle as a fusion protein in which any of these molecules is linked to a membrane binding region.
  • immune checkpoint inhibitor molecules can be introduced onto the surface layer of nanoparticles as a fusion protein in which a corresponding antibody or antibody fragment such as scFV is linked to a membrane binding region.
  • the nanoparticle contains the above molecule, it can be encapsulated inside the lipid membrane without being linked to the membrane binding region.
  • Immune checkpoint inhibitor molecules can similarly be encapsulated inside lipid membranes.
  • the costimulatory molecule is CD80 protein, CD86 protein, 4-1BB ligand protein, ICOS ligand protein, CD40 protein, CD70 protein, OX40 ligand protein, GITR ligand protein, and/or LIGHT protein, or any of these.
  • costimulatory molecules include human CD80 protein (SEQ ID NO: 7), human CD86 protein (SEQ ID NO: 36), human 4-1BB ligand protein (SEQ ID NO: 8), human ICOS ligand protein (SEQ ID NO: 37), Mentioning human CD40 protein (SEQ ID NO: 38), human CD70 protein (SEQ ID NO: 39), human OX40 ligand protein (SEQ ID NO: 40), human GITR ligand protein (SEQ ID NO: 41), and human LIGHT protein (SEQ ID NO: 42) I can do it.
  • human CD80 protein SEQ ID NO: 7
  • human CD86 protein SEQ ID NO: 36
  • human 4-1BB ligand protein SEQ ID NO: 8
  • human ICOS ligand protein SEQ ID NO: 37
  • Mentioning human CD40 protein SEQ ID NO: 38
  • human CD70 protein SEQ ID NO: 39
  • human OX40 ligand protein SEQ ID NO: 40
  • human GITR ligand protein SEQ ID NO: 41
  • the cytokine molecule is IL-7 protein, IL-15 protein, IL-21 protein, IL-2 protein, IL-12 protein, IL-4 protein, IL-10 protein, IL-18 protein, GM - A fusion molecule comprising CSF protein, IFN- ⁇ protein, TNF- ⁇ protein, and/or TGF- ⁇ protein, or any of these.
  • cytokine molecules include human IL-7 protein (SEQ ID NO: 43), human IL-15 protein (SEQ ID NO: 44), human IL-21 protein (SEQ ID NO: 45), human IL-2 protein (SEQ ID NO: 46), human IL-12A protein (SEQ ID NO: 47), human IL-12B protein (SEQ ID NO: 62), human IL-4 protein (SEQ ID NO: 48), human IL-10 protein (SEQ ID NO: 49), human IL- 18 protein (SEQ ID NO: 50), human GM-CSF protein (SEQ ID NO: 51), human IFN- ⁇ protein (SEQ ID NO: 52), human TNF- ⁇ protein (SEQ ID NO: 53), and human TGF- ⁇ protein (SEQ ID NO: 53). 54). Particularly in embodiments involving macrophage surface antigen binding regions, IFN ⁇ and GM-CSF are preferred as cytokine molecules.
  • the chemotactic molecule is a CXCR4 protein, an ITGA4 protein, an ITGB1 protein, an ICAM-1 protein, a VCAM-1 protein, and/or an LFA3 protein, or a fusion molecule comprising any of these.
  • Specific examples of these chemotactic molecules include mouse CXCR4 protein (SEQ ID NO: 23), human CXCR4 protein (SEQ ID NO: 58), mouse ITGA4 protein (SEQ ID NO: 24), human ITGA4 protein (SEQ ID NO: 59), mouse ITGB1 protein (SEQ ID NO: 59), and mouse CXCR4 protein (SEQ ID NO: 58). No. 25), human ITGB1 protein (SEQ ID No. 60), human ICAM-1 protein (SEQ ID No. 55), human VCAM-1 protein (SEQ ID No. 56), and human LFA3 protein (SEQ ID No. 57).
  • the immune checkpoint inhibitory molecule is a CTLA-4 inhibitor (e.g., tremelimumab, ipilimumab; for example, a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 558-560, respectively; and an anti-CTLA-4 antibody comprising a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NO: 561 to 563, respectively; or a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 564, and anti-CTLA-4 antibody containing a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 565), PD-1 inhibitors (e.g., pembrolizumab, nivolumab, durvalumumab), PD-L1 inhibitors (e.g., atezolizumab, durvalmumab, Avelum), PD
  • Lag-3 inhibitors e.g. Relatlimab
  • Tim-3 inhibitors e.g. TIGIT inhibitors
  • B7-H3 inhibitors VISTA inhibitors
  • ICOS inhibitors ICOS stimulators
  • BTLA inhibitors CD47 It may be an inhibitor, a KIR inhibitor, a LIR inhibitor, a CD94 inhibitor, or an NKG2A inhibitor.
  • the immune checkpoint molecule may be a PD1 protein, Lag-3 protein, Tim-3 protein, TIGIT protein, ICOS protein, or BTLA protein.
  • These immune checkpoint molecules are normally expressed on immune cells, but when included in the surface layer of the multispecific nanoparticles of the present invention, they can It may function similarly to the immune checkpoint inhibitor molecules described above, as its effects on immune cells can be suppressed.
  • Specific examples of these immune checkpoint molecules include human PD1 protein (SEQ ID NO: 64), human Lag-3 protein (SEQ ID NO: 65), human Tim-3 protein (SEQ ID NO: 66), and human TIGIT protein (SEQ ID NO: 67).
  • human ICOS protein SEQ ID NO: 68
  • human BTLA protein SEQ ID NO: 69.
  • immune checkpoint molecules include CD47 protein or fusion molecules containing it.
  • the CD47 protein is known to function as an immune checkpoint molecule for macrophages. Therefore, when the multispecific nanoparticle of the present invention contains CD47 protein or a fusion molecule containing it on the surface layer of the nanoparticle, phagocytosis of the nanoparticle by macrophages can be avoided. Avoidance of phagocytosis of nanoparticles by macrophages is preferred when the target binding region comprises a T cell receptor complex binding region or a NK cell surface antigen binding region.
  • An example of CD47 protein is human CD47 protein (SEQ ID NO: 63).
  • the lipid membrane constituting the surface layer of the multispecific nanoparticle of the present invention is composed of a cell membrane, or a cell membrane and a liposome.
  • the multispecific nanoparticle of the present invention can contain a nanoparticle core and/or a medium such as water or physiological saline inside the lipid membrane that constitutes its surface layer.
  • Nanoparticle cores and vehicles can include cytokines, chemotactic molecules, and the like. Molecules contained within the lipid membrane can be slowly released to the exterior of the lipid membrane.
  • the lipid membrane that constitutes the surface layer of the multispecific nanoparticle of the present invention can encapsulate the nanoparticle core.
  • nanoparticle cores include polymeric nanoparticles, metal nanoparticles, and dendrimers.
  • the lipid membrane constituting the surface layer of the multispecific nanoparticle of the present invention contains an aqueous solution or a buffer solution such as water or physiological saline, and does not encapsulate the nanoparticle core.
  • the lipid membrane constituting the surface layer of the multispecific nanoparticle of the invention constitutes a liposome, and the liposome can encapsulate the nanoparticle core.
  • Nanoparticles in which a liposome encapsulates a nanoparticle core such as a PLGA polymer are disclosed in the literature (Mandal, B., Nanomedicine, 2013, 9(4):474-91.).
  • the multispecific nanoparticles of the present invention can induce effector activity against cancer cells in T cells existing in vivo. Therefore, unlike CAR-T cell therapy, there is no need to individually prepare cells for each patient, making it possible to provide an inexpensive and off-the-shelf treatment method.
  • the multispecific nanoparticles of the present invention are surprisingly able to activate T cells only in the presence of tumor cells. Therefore, non-specific toxicity in normal tissues is reduced and safety is high.
  • multispecific nanoparticles of the present invention can be added at will.
  • T cell proliferation and long-term survival can be improved by adding immune regulatory molecules such as costimulatory molecules and cytokine molecules.
  • immune checkpoint inhibitor molecules it is also possible to suppress evasion of host immunity by cancer cells.
  • migration molecules such as homing molecules or adhesion molecules, it is also possible to enhance the migration ability to the tumor site.
  • the multispecific nanoparticles of the present invention can provide a "field" for highly efficiently inducing an immune response.
  • the multispecific nanoparticles of the present invention are large in size relative to the spaces between endothelial cells present in the glomerulus of the kidney, and are difficult to be excreted by the kidneys. Therefore, it is possible to maintain high drug efficacy over a long period of time, which is advantageous compared to protein preparations whose active ingredients are excreted by the kidneys.
  • composition 2-1. Summary The second aspect of the invention is a pharmaceutical composition.
  • the pharmaceutical composition of this embodiment contains the multispecific nanoparticles of the first embodiment as an active ingredient, and can be used for cancer treatment and the like.
  • the pharmaceutical composition of this aspect is a pharmaceutical composition for treating cancer. Additionally, in one embodiment, the pharmaceutical composition of this aspect is a non-cellular preparation.
  • the term "subject” refers to a subject to which the pharmaceutical composition of this embodiment is applied.
  • a tissue for example, a tissue, an organ, or an individual.
  • an individual it is, for example, a mammal, preferably a human individual.
  • the human individual may be a patient, such as a cancer patient.
  • subject information refers to various information regarding the characteristics and condition of the subject. For example, if the subject is a human individual, age, weight, gender, general health condition, presence or absence of disease, degree of progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, resistance to treatment, etc. .
  • treatment refers to the alleviation or elimination of symptoms associated with a disease, the prevention or suppression of the progression of a disease, and the cure of a disease.
  • disease is not limited. Examples of diseases include cancer, hepatitis, and infectious diseases. Infectious diseases include viral infections such as influenza and HIV, bacterial infections, and fungal infections. The disease is preferably cancer.
  • cancer is not limited, but includes, for example, adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma.
  • Specific cancer types include, for example, malignant melanoma, oral cavity cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer (colon cancer and (including rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver cancer Cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, soft tissue sarcoma, glioma, neuroblastoma, etc.
  • Examples include pediatric tumors, blood cancers, lymphoma, myeloma, etc.
  • blood cancers include leukemia (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma), and myeloma (e.g., multiple myeloma). Can be mentioned.
  • leukemia e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia
  • lymphoma e.g., non-Hodgkin's lymphoma
  • myeloma e.g., multiple myeloma
  • composition 2-3 Configuration 2-3-1.
  • Components The components of the pharmaceutical composition of this embodiment will be explained.
  • the pharmaceutical composition of this embodiment includes one or more active ingredients and a solvent and/or carrier as essential components. Each component will be specifically explained below.
  • the pharmaceutical composition of this embodiment includes the multispecific nanoparticles described in the first embodiment as an essential active ingredient.
  • Pharmaceutical compositions of this embodiment can include one or more multispecific nanoparticles.
  • the content of the active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited. In general, the content varies depending on the type of active ingredient, the dosage form, and the type of solvent and carrier that are other constituents described below. Therefore, it may be determined as appropriate in consideration of each condition. It is sufficient that a single dose of the pharmaceutical composition contains an effective amount of the active ingredient. However, if it is necessary to administer a large amount of the pharmaceutical composition to the subject in order to obtain the pharmacological effects of the active ingredient, the administration may be divided into several doses to reduce the burden on the subject. In this case, the amount of the active ingredient may be such that the total amount includes an effective amount.
  • Effective amount refers to the amount necessary to exert its function as an active ingredient, and the amount that causes little or no harmful side effects to the subject to whom it is applied. This effective amount may vary depending on various conditions such as subject information, route of application, and number of applications. Therefore, when the pharmaceutical composition of this embodiment is used as a medicine, the content of the active ingredient is ultimately determined by the judgment of a doctor, pharmacist, or the like.
  • the amount of multispecific nanoparticles included in the pharmaceutical composition of this embodiment may be, for example, from 10 4 particles to 10 16 particles, from 10 6 particles to 10 15 particles, or from 10 9 particles to 10 14 particles, preferably from 10 11 particles to 10 13 particles, for example 1 ⁇ 10 12 particles to 5 ⁇ 10 12 particles.
  • the pharmaceutical composition of the present invention can contain a pharmaceutically acceptable solvent as necessary.
  • “Pharmaceutically acceptable solvent” refers to a solvent commonly used in the pharmaceutical art. Examples include water or an aqueous solution, or an organic solvent. Aqueous solutions include, for example, saline, isotonic solutions containing dextrose or other adjuvants, phosphate buffers, phosphate buffered saline, sodium acetate buffers, glycol or ethanol solutions. Examples of the adjuvant include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. Organic solvents include ethanol.
  • composition of the present invention can optionally contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to excipients commonly used in the pharmaceutical art. Examples include excipients, human serum albumin, and the like.
  • Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, acetates, propions, etc. Salts of organic acids such as acid salts, malonate or benzoate salts, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, kaolin, silicic acid, or combinations thereof.
  • sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides
  • inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, acetates, propions, etc.
  • Salts of organic acids such as acid salts, malonate or benzoate salts, metal salts, citric acid, tartaric acid, glycine, polyethylene
  • solubilizers suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption enhancers, bulking agents, etc. commonly used in pharmaceutical compositions, etc.
  • Preservatives, preservatives, antioxidants, buffers, tonicity agents, etc. may also be included as appropriate.
  • the carrier is used to avoid or suppress the decomposition of the active ingredient by enzymes, etc. in the subject's body, to facilitate formulation and administration methods, and to maintain the dosage form and drug efficacy. It may be used as appropriate.
  • Dosage Form The dosage form of the pharmaceutical composition of the present invention is not particularly limited. It may be in any form as long as it can be delivered to the target site without deactivating the active ingredient within the body of the subject.
  • the specific dosage form varies depending on the application method described below. Application methods can be broadly classified into parenteral administration and oral administration, with parenteral administration being preferred.
  • the preferred dosage form is a liquid preparation that can be administered directly to the target site or systemically via the circulatory system.
  • a liquid preparation is an injection.
  • injectables are prepared by combining appropriate solvents, excipients, suspending agents, surfactants, stabilizers, pH regulators, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. It can be formulated into a formulation.
  • the method of applying the pharmaceutical composition of the present invention is not particularly limited, and the route of administration is not limited, but parenteral administration may be used, for example.
  • Parenteral administration methods can be further subdivided into systemic administration and local administration.
  • Local administration includes, for example, intradermal administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intranasal administration, intratumoral administration, tissue administration, and organ administration
  • systemic administration of parenteral administration includes:
  • Intracirculatory administration includes, for example, intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration.
  • the preferred route of administration is intravenous administration, and may also be an infusion administered by drip (eg, a single intravenous infusion).
  • a method for treating and/or preventing a disease, a method for inducing effector activity in T cells, and a method for inducing effector activity in T cells which includes the step of administering the pharmaceutical composition of the present invention to a subject.
  • a method of inducing NK cells and/or macrophages to have cytotoxic activity against cancer cells is provided.
  • the disease may be cancer, for example, and the subject may be a cancer patient.
  • multispecific nanoparticles or pharmaceutical compositions of the invention in the manufacture of medicaments for treating and/or preventing diseases such as cancer.
  • the third aspect of the invention is a method of making multispecific nanoparticles that are capable of binding to two or more target cells.
  • the production method of this embodiment includes a cell membrane fragmentation step, a nanoparticle formation step, and a recovery step as essential steps, and a nanoparticle core preparation step and a vector introduction step as selection steps.
  • gene expression vectors type I fusion protein expression vector and type II fusion protein expression vector described below
  • host cells which will be explained below in the production method of this embodiment, are also provided as a further embodiment of the present invention.
  • nanoparticle core preparation process is a step of preparing nanoparticle cores to be mixed with cell membrane fragments in the nanoparticle formation step described below.
  • nanoparticle cores may be prepared by a method known in the art depending on the material of the polymer nanoparticle, metal nanoparticle, dendrimer, or the like.
  • nanoparticle cores can be obtained by contacting or mixing an aqueous solution with an organic solvent such as acetone in which a polymer such as PLGA is dissolved by dropping or the like, and then removing the organic solvent. Formation of nanoparticle cores can also be promoted by performing ultrasonic treatment or stirring as necessary.
  • each molecule can be encapsulated in the nanoparticle core by adding cytokine molecules, chemotactic molecules, and/or immune checkpoint inhibitor molecules to the solvent used for nanoparticle core preparation.
  • cytokine molecules such as PLGA
  • cytokine molecules, etc. are added to an organic solvent such as acetone or an aqueous solution in which the polymer is dissolved, and then the cytokine molecules, etc. are encapsulated by contacting or mixing such as dropping.
  • a nanoparticle core can be obtained.
  • Vector introduction step refers to the step of introducing a gene expression vector containing a nucleic acid encoding a type I fusion protein (hereinafter referred to as "type I fusion protein expression vector") into a host cell, or the step of introducing a gene expression vector containing a nucleic acid encoding a type I fusion protein into a host cell, or This is a step of introducing two or more gene expression vectors (hereinafter referred to as "type II fusion protein expression vectors”) containing the encoding nucleic acids into host cells.
  • the nucleic acid encoding the type I fusion protein or type II fusion protein may be any nucleic acid encoding the type I fusion protein or type II fusion protein described in the first aspect.
  • the base sequence of such a nucleic acid is not limited.
  • a nucleic acid containing in frame a nucleic acid encoding a target binding region at the 5' end and a nucleic acid encoding a membrane binding region at the 3' end can be mentioned.
  • Further examples include a codon-optimized base sequence and a base sequence with an initiation codon (ATG) added to the 5' end.
  • the type I fusion protein expression vector or the type II fusion protein expression vector contains a nucleic acid encoding the above type I fusion protein or type II fusion protein and a promoter, and is capable of expressing the type I fusion protein or type II fusion protein in cells. It is a gene expression vector.
  • the gene expression vector may contain constituent elements such as a marker gene (selection marker), an enhancer, a terminator, an origin of replication, and a polyA signal, as necessary. .
  • gene expression vector refers to a vector that contains a gene or gene fragment (hereinafter referred to as “gene, etc.”) in an expressible state and includes an expression unit that can control the expression of the gene, etc. .
  • Gene expression vectors may be plasmid vectors or viral vectors.
  • the term "expressible state” refers to a gene to be expressed, etc. placed in the downstream region of the promoter under the control of the promoter.
  • Known vectors include plasmid vectors and virus vectors, and any of these vectors can be used. Generally, any plasmid vector that can be easily genetically recombined or a viral vector that can easily introduce genes into immune cells may be used.
  • the plasmid vector may be a commercially available expression vector for mammalian cells, such as Promega's pCI vector or pSI vector, or a shuttle vector that can replicate between mammalian cells and bacteria such as Escherichia coli.
  • Viral vectors include, for example, retrovirus vectors (including oncoretrovirus vectors, lentivirus vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Viral vectors such as Epstein-Barr virus (EBV) vectors and HSV vectors can be used. Replication-deficient viral vectors may also be used so that they do not replicate autonomously within infected cells.
  • retrovirus vectors including oncoretrovirus vectors, lentivirus vectors, and pseudotyped vectors
  • AAV adeno-associated virus
  • simian virus vectors simian virus vectors
  • vaccinia virus vectors Sendai virus vectors
  • Sendai virus vectors Sendai virus vectors
  • Viral vectors such as Epstein-Barr virus (EBV) vectors and HSV vectors can be used.
  • a suitable packaging cell and packaging signal sequence can be selected based on the LTR sequence to produce retrovirus particles.
  • packaging cells include PG13 (ATCC® CRL-10686(TM)), PA317 (ATCC® CRL-9078(TM)), GP+E-86 and GP+envAm-12( No. 5,278,056), and Psi-Crip (Proceedings of the National Academy of Sciences of the United States of America, vol. 85, pp. 6460-6464 (1988)).
  • Retroviral particles can also be produced using 293 cells or 293T cells with high transfection efficiency. Many types of viral vectors produced based on retroviruses and packaging cells that can be used to package retroviral vectors are commercially available from many companies.
  • promoter refers to a gene expression regulatory region that can control the expression of genes located downstream (3' end side) in cells into which a gene expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location where genes under expression control are expressed.
  • a ubiquitous promoter is a promoter that controls the expression of a target gene (target gene, etc.) in all cells, that is, in the entire host individual.
  • a site-specific promoter is a promoter that controls the expression of a target gene, etc. only in a specific cell or tissue.
  • the promoter contained in the gene expression vector of the present invention may be either a ubiquitous promoter or a site-specific promoter, but is preferably capable of inducing expression in immune cells.
  • promoters are classified into constitutively active promoters, expression-induced promoters, and time-specific active promoters based on the timing of expression.
  • a constitutively active promoter can constantly express a target gene, etc. within cells.
  • An expression-inducible promoter can induce expression of a target gene or the like at any time within a cell.
  • a stage-specific active promoter can induce expression of a target gene, etc. within cells only at a specific stage of development. Any promoter can be considered an overexpression type promoter since it can lead to excessive expression of the target gene within the host cell.
  • the promoter contained in the gene expression vector of the present invention is preferably a constitutively active promoter that enables long-term sustainability of therapeutic effects.
  • the promoter is a promoter that can induce expression of a nucleic acid encoding a type I fusion protein or a type II fusion protein in host cells.
  • the host cells into which the gene expression vector in this step is introduced are, in principle, mammalian cells, especially human-derived cells, such as human-derived immune cells, so any promoter that can express the downstream gene in these cells may be used.
  • Examples include CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, EF1 ⁇ promoter, Ub promoter, 5' LTR promoter, and the like.
  • a nucleic acid encoding a chimeric cytokine receptor can be placed downstream of the 5' LTR promoter to drive its gene expression.
  • a "marker gene” is a gene that encodes a mark protein, also called a selectable marker or reporter protein.
  • a "labeled protein” refers to a peptide that can determine whether a labeled gene is expressed or not based on its activity. Detection of activity may be performed by directly detecting the activity of the labeled protein itself, or indirectly through metabolites generated by the activity of the labeled protein, such as dyes. good. Detection may include biological detection (including detection by binding of peptides and nucleic acids such as antibodies and aptamers), chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), or detection. It may be any sensory detection (including detection by sight, touch, smell, hearing, and taste) of the person.
  • labeled protein encoded by the labeled gene is not particularly limited as long as its activity can be detected by methods known in the art.
  • a labeled protein that is less invasive to transformants upon detection is preferred. Examples include tag peptides, drug-resistant proteins, chromoproteins, fluorescent proteins, luminescent proteins, and the like.
  • the term “enhancer” is not particularly limited as long as it can enhance the expression efficiency of a gene or a fragment thereof within a vector.
  • a "terminator” is a sequence capable of terminating the transcription of a gene, etc. expressed by the activity of the promoter.
  • the type of terminator is not particularly limited.
  • the terminator is derived from the same species as the promoter.
  • a terminator paired with the promoter on the genome in a single gene expression control system is particularly preferred.
  • this step comprises, in addition to the type I fusion protein expression vector or type II fusion protein expression vector, costimulatory molecules, cytokine molecules, migration molecules, homing molecules, adhesion molecules, and immune checkpoint inhibitor molecules.
  • a gene expression vector encoding one or more selected from the group can be additionally introduced.
  • the structure of each of these molecules shall be as described in the first embodiment, but cytokine molecules, migration molecules, homing molecules, immune checkpoint inhibitor molecules, etc. are preferably introduced as fusion molecules containing a membrane binding region. .
  • the structure of the gene expression vector shall be similar to the above-mentioned structure. There are no particular limitations on the method of introducing each of the above-mentioned vectors into host cells.
  • the type of host cell in this step is not limited.
  • the host cell may be any cell in which the type I fusion protein or type II fusion protein can be expressed on its membrane, and is therefore not limited to mammalian cells.
  • the host cell may be either prokaryotic or eukaryotic.
  • prokaryotic cells include bacterial cells such as E. coli cells.
  • eukaryotic cells include fungal cells (e.g. yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g. chicken cells), and mammalian cells (e.g. , mouse cells, chimpanzee cells, and human cells).
  • mammalian cells e.g. , mouse cells, chimpanzee cells, and human cells.
  • mammalian cells e.g. , mouse cells, chimpanzee cells, and human cells.
  • mammalian cells e.g. , mouse cells, chimpanzee
  • mammalian cells include, but are not limited to, CHO cells, COS cells, Vero cells, HEK293 cells and their derivative cells (e.g., 293F cells, 293T cells, Freestyle 293 cells, etc.), HeLa cells, NIH3T3 cells, and K562 cells. etc.
  • Host cells may be adherent cells or floating cells. Further, the host cell may be a cell (autologous cell) that has been isolated in advance from a subject to whom the multispecific nanoparticles are administered.
  • each vector is a viral vector
  • methods of viral infection of cells are known in the art.
  • a functional substance that improves virus infection efficiency such as fibronectin or a fibronectin fragment (for example, Retronectin (registered trademark) or Vecofusin-1 (registered trademark), which is a fibronectin fragment having a heparin binding site), etc.
  • fibronectin or a fibronectin fragment for example, Retronectin (registered trademark) or Vecofusin-1 (registered trademark), which is a fibronectin fragment having a heparin binding site
  • Functional substances may be used.
  • virus infection methods using retronectin include the following methods. After treating the cell culture plate with RetroNectin, block the bottom of the plate with 2% BSA/PBS solution for 30 minutes. Thereafter, after washing with PBS, a retrovirus solution derived from PG13 packaging cells is loaded, and the culture plate is centrifuged at 32°C and 2000g for 2 hours. After centrifugation, the virus solution is removed and the cells are plated.
  • each vector is a non-viral vector such as a plasmid
  • plasmid a non-viral vector
  • examples include lipofection method, electroporation method, microinjection method, calcium phosphate method, DEAE-Dextran method, particle bombardment, and the like.
  • the host cells into which the vector has been introduced in this step can usually be used as is, but if necessary, only the cells into which the vector has been introduced may be separated.
  • the host cell into which the vector has been introduced in this step is capable of transducing (i) a fusion protein containing a membrane-binding region and two or more target-binding regions, or (ii) a fusion protein containing two or more membrane-binding regions and a target-binding region into a cell membrane. It is expressed above. Note that both extracellular and intracellular cell membrane fragments can be placed on the surface of nanoparticles formed in the nanoparticle formation step described below, so in host cells, the target binding region of the fusion protein is, in principle, located outside the cell. The target binding region may be located either on the cytoplasmic side or on the cytoplasmic side, but it is preferable that the target binding region be located on the outside of the cell. This is because proteins on the outside of the cell membrane are modified with sugar chains, making it easier for the outside of the cell membrane fragment to be placed on the surface side of the nanoparticle (Luk et al., Nanoscale, 2014, 6(5): 2730-7.).
  • Cell membrane fragmentation step refers to disrupting cells expressing two or more fusion proteins containing a membrane-binding region and a target-binding region, or a fusion protein containing a membrane-binding region and two or more target-binding regions on their cell membranes. , a step of fragmenting the cell membrane. This step aims to obtain fragmented cell membranes containing type I or type II fusion proteins.
  • the method of fragmenting the cell membrane in this step is not limited.
  • cells can be disrupted by a physical method or a method using a lysis solution to fragment the cell membrane.
  • physical fragmentation methods include a method of disrupting cells by osmotic pressure using a hypotonic solution, a method of mechanically disrupting cells using glass beads, etc., ultrasonication, and freezing and thawing. It will be done.
  • the fragmentation method using a lysate include a method in which cells are disrupted using a surfactant.
  • the mechanical crushing method it is preferable to crush the protein while cooling it to prevent thermal denaturation of the protein.
  • fractions other than cell membrane fragments and/or unbroken cells can be removed. Removal of fractions other than cell membrane fragments and/or unbroken cells can be performed by centrifugation, filtration, sedimentation, decantation, magnetic separation, or a combination thereof. Magnetic separation may be performed by collecting cell membrane fragments using antibody-conjugated magnetic beads or the like, or by removing unbroken cells, if necessary.
  • the “nanoparticle forming step” is a step of forming nanoparticles by reconstituting the cell membrane fragments after the fragmentation step.
  • the specific method for reconstituting the cell membrane fragments in this step is not limited, and for example, by applying physical stimulation (e.g., stirring treatment, shaking treatment, and/or ultrasonication treatment, etc.) to the cell membrane fragments, the cell membrane fragments can be reconstituted. Fragments can be reassembled.
  • Nanoparticles whose surface layer is composed of a lipid membrane in which cell membrane fragments and liposomes are fused, or Nanoparticles can also be formed in which a lipid membrane containing cell membrane fragments encapsulates a nanoparticle core.
  • Such nanoparticles can be produced by mixing cell membrane fragments with liposomes such that the liposomes fuse with the cell membrane fragments, or by mixing cell membrane fragments and nanoparticle cores so that the cell membrane fragments aggregate with each other so as to encapsulate the nanoparticle cores. It is formed by this. This forms nanoparticles with a specific range of particle sizes.
  • Particle formation can be promoted by physical and/or chemical methods.
  • a simple method is to release the bonds between cell membrane fragments and/or the bonds between liposomes or nanoparticle cores by physical stimulation, thereby increasing the efficiency of contact between the two.
  • Physical stimulation can be provided, for example, by stirring, shaking, or ultrasonication.
  • this step includes sonication of cell membrane fragments and liposomes and/or nanoparticle cores.
  • the step includes mixing the cell membrane fragment with both the liposome and the nanoparticle core, such that the lipid membrane in which the cell membrane fragment and the liposome are fused constitutes its surface layer, and the lipid membrane encapsulates the nanoparticle core.
  • Nanoparticles can also be formed. Nanoparticles in which a liposome encapsulates a nanoparticle core such as a PLGA polymer can be produced with reference to literature (Mandal, B., Nanomedicine, 2013, 9(4):474-91.).
  • the solvent used to fuse the liposome and the cell membrane fragment contains a cytokine molecule, a migration molecule, and/or an immune checkpoint inhibitor molecule.
  • the nanoparticles after fusion can encapsulate these molecules.
  • the “recovery process” is a process of recovering the nanoparticles formed in the nanoparticle formation process.
  • the recovery method in this step is not limited as long as it is a method of separating cell membrane fragments and cell membrane vesicles that did not form nanoparticles from the nanoparticles formed in the nanoparticle formation step.
  • the desired nanoparticles can be obtained by centrifugation, filtration, sedimentation, decantation, or a combination thereof. All of these steps can basically be carried out in accordance with conventional methods in the field.
  • centrifugation may be performed under conditions in which the target nanoparticles precipitate and cell membrane vesicles that have not formed nanoparticles do not precipitate.
  • a filter may be used that has a pore size through which the target nanoparticles can pass, but through which cell membrane vesicles that have not formed nanoparticles cannot pass.
  • nanoparticle cores that were not encapsulated in the lipid membrane composed of cell membrane fragments and liposomes that were not induced with cell membrane fragments can also be removed, if necessary.
  • the collected nanoparticles of interest can be stored refrigerated or frozen until their use.
  • the surface layer of the nanoparticles contains type I fusion protein or type II fusion protein derived from the cell membrane of the host cell used for nanoparticle production.
  • nanoparticles can be produced in which two or more target binding regions are displayed on the surface.
  • costimulatory molecules, cytokine molecules, etc. are additionally introduced into the surface layer and/or inside.
  • Example 1 Design and cell surface expression of CD3/CD19 binding protein> (the purpose) A fusion protein containing two scFVs derived from an anti-CD3 ⁇ antibody and an anti-CD19 antibody (hereinafter referred to as "CD3/CD19 binding protein") is produced, and the gene is introduced into leukemia cell line K562. Furthermore, the expression of CD3/CD19 binding protein on the cell surface is detected. In addition, cytotoxic activity against the CD19-positive leukemia cell line NALM6 will be verified.
  • the CD3/CD19 binding protein consists of a signal peptide (hereinafter referred to as "SP"), a single chain variable region fragment derived from mouse anti-CD3 ⁇ antibody clone OKT3 (from the N-terminus), single chain variable fragment; scFV) (hereinafter referred to as "mOKT3 scFV”), linker peptide, scFV derived from anti-CD19 antibody clone FMC63 (hereinafter referred to as "FMC63 scFV”), and CD8 ⁇ protein-derived fragment. ( Figure 3A).
  • SP signal peptide
  • mOKT3 scFV single chain variable fragment derived from mouse anti-CD3 ⁇ antibody clone OKT3 (from the N-terminus)
  • mOKT3 scFV single chain variable fragment
  • linker peptide linker peptide
  • FMC63 scFV anti-CD19 antibody clone FMC63
  • SP is a peptide for transporting CD3/CD19 binding protein to the cell surface, and a signal peptide derived from silkworm Fibroin L protein (SEQ ID NO: 1) was used.
  • mOKT3 scFV is an scFV derived from the mouse anti-CD3 ⁇ antibody clone OKT3 and contains a sequence in which two amino acid mutations have been introduced. :445-53.). In order to indicate that a mutation has been introduced in OKT3, it is referred to herein as "mOKT3".
  • mOKT3 scFV has a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 80 to 82, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85. It contains a light chain variable region including CDR3, and its entire length consists of the amino acid sequence shown in SEQ ID NO:2.
  • FMC63 scFV is an scFV derived from anti-CD19 antibody clone FMC63, and is based on the literature (Nicholson et al., Mol Immunol. 1997, 34(16-17):1157-65.). Specifically, FMC63 scFV has a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 192 to 194, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 195 to 197. It contains a light chain variable region including CDR3, and its entire length consists of the amino acid sequence shown in SEQ ID NO:3.
  • the CD8 ⁇ protein-derived fragment is included in the CD3/CD19 binding protein for the purpose of retaining it on the cell membrane, and includes the region from the hinge containing the cell membrane-spanning domain to the cytoplasmic domain in the human CD8 ⁇ protein. Specifically, it consists of the amino acid sequence shown in SEQ ID NO:4.
  • the full-length amino acid sequence of the CD3/CD19 binding protein is shown in SEQ ID NO: 5. Furthermore, the base sequence of the gene encoding the CD3/CD19 binding protein is shown as SEQ ID NO: 6.
  • Figure 3B shows flow cytometry plots of cells transfected with CD3/CD19 binding protein (Figure 3B, "K562-mOKT3/FMC63") and control cells without it ( Figure 3B, "K562"). . Most of the cells into which the CD3/CD19 binding protein was introduced were positive, indicating that the CD3/CD19 binding protein was expressed on the cell surface.
  • K562 cell line (hereinafter referred to as "K562-mOKT3/FMC63 cell line") into which CD3/CD19 binding protein has been introduced has cytotoxic activity against tumor cells. We verified that it could be induced into cells.
  • human cultured T cells, NALM6 cell line, and K562-mOKT3/FMC63 cell line were co-cultured at a ratio of 3:3:1 (K562-OKT3/FMC63 cell line is 3.3 ⁇ 10 4 cells/well). did. After overnight culture, the percentage of surviving NALM6 cells was measured by flow cytometry. The survival rate was calculated by setting the number of tumor cells in wells in which only NALM6 cells were cultured as 100%.
  • the cell line introduced with the CD3/CD19 binding protein was able to induce T cells to have cytotoxic activity against tumor cells.
  • Nanoparticles are produced from the cell membrane of a cell line into which the CD3/CD19 binding protein has been introduced. Specifically, by reconstituting cell membrane fragments obtained by disrupting cell lines onto PLGA nanoparticle cores, nanoparticles containing CD3/CD19 binding proteins on the surface layer are prepared ( Figure 2).
  • PLGA nanoparticle core was prepared based on the nanoprecipitation method.
  • the nanoprecipitation method was performed by partially modifying the method described in the literature (Barichello et al., Drug Dev Ind Pharm, 1999; Hu et al., Nature, 2015).
  • PLGA Resomer® RG 502H, poly(D,L-lactide-co-glycolide), Sigma-Aldrich
  • PVA polyvinyl alcohol
  • PLGA nanoparticle cores sterile spherical nanoparticle cores
  • FIG. 4A shows the appearance of the PLGA nanoparticle core dispersion. Observation using a transmission electron microscope confirmed the spherical shape of the PLGA nanoparticle core ( Figure 4B). As a result of measuring the particle size and concentration of the PLGA nanoparticle core using NanoSight (Malvern), the particle size immediately after synthesis was approximately 117 nm ( Figure 4C, left). Even after freezing/thawing, no change in particle size was observed and the particles remained stable ( Figure 4C, right).
  • the process of preparing nanoparticles using cell lines is shown in Figure 5B.
  • the cell line was suspended in a low osmotic buffer (composition: 20mM Tris-HCl pH 7.5, 10mM KCl, 2mM MgCl 2 , EDTA-free protease inhibitor (Fang et al. Nano Lett. 2014)). (1 ⁇ 10 8 cells/2 mL).
  • the cells were crushed using a homogenizer, and it was confirmed using a microscope that almost all the cells were crushed.
  • the cell disruption solution was centrifuged at 3,200g for 5 minutes at 4°C, and the supernatant was collected.
  • the supernatant was centrifuged at 10,000g and 4°C for 10 minutes, and then the supernatant was ultracentrifuged at 100,000g and 4°C for 60 minutes. After removing the supernatant containing cytoplasmic proteins, suspend the precipitate containing cell membrane fragments in washing buffer (10mM Tris-HCl pH 7.5, 2mM EDTA), repeat this operation, and then add the precipitate containing cell membrane fragments to pure water. Suspended.
  • Coating of PLGA nanoparticle cores with cell membrane fragments was performed by the following method.
  • the PLGA nanoparticle core dispersion obtained in (1) above and the cell membrane fragments obtained above were mixed at a mass ratio of 2:1, and suspended using a micropipette for about 2 minutes while irradiating with ultrasound.
  • the mixture was then subjected to ultracentrifugation at 20,000 rpm for 30 minutes at 4°C, and the resulting pellets were suspended in PBS under ultrasonic irradiation.
  • Nanoparticles were obtained by further performing ultrasonic irradiation.
  • the obtained nanoparticles were stored at -80°C.
  • Figure 6B shows the results of western blotting analysis of the components contained in the protein fractions obtained in each step of nanoparticle preparation. From the results shown in Figure 6B, CD80 protein and 41BBL protein, which are cell membrane proteins, are contained in the protein fraction used for reconstitution, while SDHA protein contained in mitochondria and ⁇ - actin protein was not included, indicating that cell membranes containing cell membrane proteins were selectively used for nanoparticle preparation.
  • Figure 6A shows the results of observing the obtained nanoparticles with a transmission electron microscope (TEM). After the nanoparticle dispersion was dropped onto a TEM grid, dried and fixed, it was stained with uranium acetate and TEM observation was performed. The TEM image of the obtained nanoparticles ( Figure 6A, (+)) confirmed the formation of a new layer on the particle surface compared to the PLGA nanoparticle core before cell membrane reconstitution ( Figure 6A, (-)). , it was shown that the cell membrane was reconstituted on the particle surface layer.
  • TEM transmission electron microscope
  • Nanoparticles produced from the K562 cell line into which CD3/CD19 binding protein, CD80 protein, and 41BBL protein were introduced by the above method are referred to as “mOKT3/FMC63/CD80/41BBL nanoparticles” in the following examples.
  • nanoparticles produced from the K562 cell line into which CD3/CD19 binding protein has been introduced by the same method are referred to as “mOKT3/FMC63 nanoparticles” in the following examples.
  • Example 3 Induction of effector effect by nanoparticles> (the purpose) We will verify that mOKT3/FMC63 nanoparticles can induce T cells to have cytotoxic activity against tumor cells.
  • T cells were cryopreserved peripheral blood mononuclear cells cultured for about one week after initial stimulation. Cultivation was performed in RPMI 1640 medium containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 ⁇ g/mL), and recombinant IL2 (100 IU/mL). Similar media were used in the following Examples unless otherwise specified. On the first day of culture, peripheral blood mononuclear cells were stimulated with anti-CD3 antibody (priming).
  • K562 cell line that expressed a single chain variable region fragment (scFV) derived from an anti-CD3 antibody (mOKT3, which is an antibody obtained by introducing amino acid mutations into clone OKT3) and the costimulatory molecule CD80 on the cell surface.
  • scFV single chain variable region fragment
  • mOKT3 anti-CD3 antibody
  • CD80 costimulatory molecule
  • CAR-T cells were used as a control.
  • the CAR gene was introduced on the second day of T cell culture.
  • the pMX plasmid (Kitamura T. et al., Exp Hematol., 2003, 31:1007-1014.) as a retroviral plasmid was introduced into Plat-E packaging cells by transient transfection using TranslT293 (Mirus Bio).
  • the resulting ecotropic retroviral vector was stably introduced into PG13 packaging cells.
  • the obtained PG13 cell-derived viral vector was introduced into T cells using RetroNectin (Takara Bio).
  • nanoparticles were added to each culture plate at 10 3 for the number of T cells.
  • the particles were administered at ⁇ 10 7 times the number of particles and co-cultured.
  • the percentage of surviving NALM6 cells was measured by flow cytometry. The survival rate was calculated by setting the number of tumor cells in wells in which only NALM6 cells were cultured as 100%.
  • Figure 7 shows the results.
  • mOKT3/FMC63 nanoparticles NPs
  • NPs mOKT3/FMC63 nanoparticles
  • FIG. 7 “CART” indicates the survival rate of tumor cells when CAR-T cells and NALM6 cells were mixed at a ratio of 1:1 as a control.
  • the CAR-T cells used here were second-generation CARs, which were already approved for B-cell tumors.
  • FMC63 scFV a CD28 protein-derived fragment (including the region from the hinge to the cytoplasmic domain containing the transmembrane domain in the human CD28 protein), and a chimeric antigen receptor (CAR) containing the cytoplasmic domain of CD3 ⁇ were introduced.
  • CD28 protein-derived fragment including the region from the hinge to the cytoplasmic domain containing the transmembrane domain in the human CD28 protein
  • CAR chimeric antigen receptor
  • Example 4 Mechanism of T cell activation by nanoparticles> (the purpose) The mechanism of T cell activation by mOKT3/FMC63 nanoparticles was investigated using the expression of CD25 protein, a marker of T cell activation, as an indicator.
  • CD25 is regulated at the gene expression level by NFAT, a transcription factor activated by T cell receptor stimulation. It is commonly used as a marker to measure the intensity of T cell receptor stimulation because its expression level can change sensitively depending on the intensity of T cell receptor stimulation (Schuh et al. J Exp Med. 1998;188(7):1369-73.)
  • NFAT a transcription factor activated by T cell receptor stimulation
  • the CD19-positive leukemia cell line NALM6 or the leukemia cell line K562 into which CD19 protein was introduced (hereinafter referred to as "K562-CD19 cells") was used.
  • T cells were cultured with mOKT3/FMC63 nanoparticles in the presence or absence of any of these target cells, and 24 hours later, the expression of CD25 protein in the CD8-positive T cell fraction was analyzed by flow cytometry. Note that the number of mOKT3/FMC63 nanoparticles was 1 ⁇ 10 6 times or 5 ⁇ 10 6 times the number of T cells.
  • NALM6 cells were co-cultured at the same number of T cells, and K562-CD19 cells were co-cultured at one-third the number of T cells.
  • Figure 8A shows representative flow cytometry results when T cells were cultured with mOKT3/FMC63 nanoparticles at 5x10 6 times the number of particles.
  • mOKT3/FMC63 nanoparticles activated T cells.
  • mOKT3/FMC63 nanoparticles did not activate T cells in the absence of target cells. This result indicated that T cells could be activated by mOKT3/FMC63 nanoparticles only in the presence of target cells.
  • expression of CD25 protein when CAR-T cells and NALM6 cells were co-cultured at 1:1 for CD19 protein and when CAR-T cells and K562-CD19 cells were co-cultured at 3:1 for CD19 protein. It also indicates the amount.
  • the same CAR-T cells as described in Example 3 were used as CAR-T cells.
  • T cells were activated by mOKT3/FMC63 nanoparticles in the presence of NALM6 cells or K562-CD19 cells. In contrast, in the absence of target cells, mOKT3/FMC63 nanoparticles did not activate T cells.
  • mOKT3/FMC63 nanoparticles can activate T cells only in the presence of tumor cells. Conversely, T cells were not activated in the absence of tumor cells. Therefore, mOKT3/FMC63 nanoparticles are an extremely safe technology that does not activate T cells in normal tissues and can activate T cells only in the presence of tumor cells. It was revealed. Without being bound by theory, it is possible that mOKT3/FMC63 nanoparticles intensively bind between tumor cells and T cells by providing a scaffold for tumor cells to present mOKT3/FMC63 nanoparticles to T cells. This is thought to cause activation signals to be input to T cells above the threshold.
  • CD80 protein and 41BBL protein are additionally introduced into the nanoparticles, which are costimulatory molecules, mOKT3/FMC63 nanoparticles. Examine the effects on T cell proliferation, effector effects, and cytokine production.
  • T cells were cultured with nanoparticles in the presence or absence of the CD19-positive leukemia cell line NALM6.
  • the number of nanoparticles was 1 ⁇ 10 6 times or 5 ⁇ 10 6 times the number of T cells.
  • NALM6 cells were co-cultured at the same number of T cells.
  • Three days after the start of culture cytokine IL2 was added at a concentration of 100 IU/mL.
  • the number of T cells was measured and the cell doubling rate was calculated.
  • Example 6 Comparison of tandem nanoparticles and separated nanoparticles> (the purpose)
  • the mOKT3/FMC63 nanoparticle developed in the above example contains a CD3/CD19 binding protein containing two scFVs in tandem on its surface layer ( Figure 13A, "tandem expression”; hereinafter referred to as “tandem nanoparticle”).
  • Figure 13A "tandem expression”; hereinafter referred to as “tandem nanoparticle”
  • two fusion proteins containing each of the two scFVs were simultaneously introduced into the nanoparticle surface layer (Fig. 13A, "separate expression”; hereinafter referred to as “separate nanoparticles”), and tandem nanoparticles and Compare the effects of separated nanoparticles.
  • tandem nanoparticles are mOKT3/FMC63/CD80/41BBL nanoparticles prepared by the same method as in Examples 1 and 2. Therefore, the tandem nanoparticle contains CD3/CD19 binding protein, CD80 protein, and 41BBL protein on its surface layer (FIG. 13A, "tandem expression").
  • the CD3/CD19 binding protein has a structure in which SP, mOKT3 scFV, linker peptide, FMC63 scFV, and CD8 ⁇ protein-derived fragment are linked in order from the N-terminus.
  • Isolated nanoparticles consist of a fusion protein (hereinafter referred to as "CD3 binding protein") having a structure in which SP, mOKT3 scFV, and CD8 ⁇ protein-derived fragment are linked in order from the N-terminus. and a fusion protein having a structure in which SP, FMC63 scFV, and a CD8 ⁇ protein-derived fragment are linked in order from the N-terminal side (hereinafter referred to as "CD19 binding protein”), and the surface layer contains CD80 protein and 41BBL protein ( Figure 13A , "separate expression").
  • CD3 binding protein a fusion protein having a structure in which SP, mOKT3 scFV, and CD8 ⁇ protein-derived fragment are linked in order from the N-terminus.
  • CD19 binding protein a fusion protein having a structure in which SP, FMC63 scFV, and a CD8 ⁇ protein-derived fragment are linked in order from the N-terminal side
  • the full-length amino acid sequence of the CD3 binding protein is shown in SEQ ID NO: 9.
  • the base sequence of the gene encoding the CD3 binding protein is shown in SEQ ID NO: 10.
  • the full-length amino acid sequence of the CD19 binding protein is shown as SEQ ID NO: 11.
  • the base sequence of the gene encoding the CD19 binding protein is shown as SEQ ID NO: 12.
  • Isolated nanoparticles were prepared using a leukemia cell line in which a gene encoding a CD3 binding protein, a gene encoding a CD19 binding protein, a gene encoding a CD80 protein, and a gene encoding a 41BBL protein were obtained by the same method as in Examples 1 and 2. It was introduced into K562 cells and produced from the cells in the same manner as in Example 2.
  • T cell activation using tandem nanoparticles and separated nanoparticles using the CD19-positive leukemia cell line NALM6 as the target cell, T cells, NALM6 cells, and nanoparticles were added at a ratio of 1:1:5 ⁇ 10 6 cells. After overnight co-culture, the expression of CD25 protein in CD8-positive T cells was analyzed by flow cytometry.
  • Example 7 Introduction of cytokine molecules into nanoparticles and its effects> (the purpose) Cytokine molecules IL7 and IL15 proteins are additionally introduced into the nanoparticles. Examine the effects on T cell proliferation, T cell memory traits, and phosphorylated STAT5.
  • IL7 fusion protein having a structure in which SP, IL7 protein, and CD8 ⁇ protein-derived fragments were linked in order from the N-terminus was used.
  • the full-length amino acid sequence of the IL7 fusion protein is shown in SEQ ID NO: 13.
  • the base sequence of the gene encoding the IL7 fusion protein is shown as SEQ ID NO: 14.
  • IL15 fusion protein For the introduction of IL15 protein into the cell membrane, a fusion protein (hereinafter referred to as "IL15 fusion protein") having a structure in which SP, IL15 protein, and CD8 ⁇ protein-derived fragments were linked in order from the N-terminus was used.
  • the full-length amino acid sequence of the IL15 fusion protein is shown in SEQ ID NO: 15.
  • the base sequence of the gene encoding the IL15 fusion protein is shown in SEQ ID NO: 16.
  • mOKT3/FMC63/CD80/41BBL nanoparticles produced in Example 2 were used as a control (referred to as "mOKT3/FMC63/CD80/41BBL nanoparticles" in this example).
  • T cells and CD19-positive leukemia cell line NALM6 were co-cultured in the presence of nanoparticles.
  • the number of nanoparticles was 2 ⁇ 10 6 times or 1 ⁇ 10 7 times the number of T cells.
  • NALM6 cells were co-cultured at the same number of T cells. After culturing for one week in a medium without adding cytokines, the number of T cells was measured and the cell doubling rate was calculated.
  • T cell memory traits It is known that T cells normally lose their undifferentiated memory traits (CD45RA +/- CCR7 + CD28 + CD62L + CD27 + ) as they proliferate. Therefore, the memory traits of the T cells proliferated in (1) above after culturing in (1) above were analyzed by flow cytometry. Specifically, the expression levels of each marker protein of CCR7, CD45RA, CD62L, CD28, FSC, and CD27 were evaluated by flow cytometry.
  • FIG. 15A A representative FACS plot when nanoparticles were administered at 2 ⁇ 10 6 times is shown in FIG. 15A.
  • mOKT3/FMC63/CD80/41BBL/IL7/IL15 nanoparticles compared to T cells proliferated in the presence of mOKT3/FMC63/CD80/41BBL nanoparticles.
  • Figure 15B shows the measurement results of the cell doubling rate of T cells with undifferentiated memory traits.
  • FIG. 16A A representative flow cytometry plot is shown in Figure 16A.
  • Example 8 Development of nanoparticles targeting BCMA protein> (the purpose) A fusion protein (hereinafter referred to as "CD3/BCMA binding protein") containing two scFVs derived from an anti-CD3 ⁇ antibody and an anti-BCMA (B Cell Maturation Antigen) antibody is produced. Furthermore, we will create nanoparticles containing CD3/BCMA binding protein on their surface layer and verify their effect on RPMI8226 cells, a BCMA-positive multiple myeloma cell line.
  • CD3/BCMA binding protein A fusion protein (hereinafter referred to as "CD3/BCMA binding protein") containing two scFVs derived from an anti-CD3 ⁇ antibody and an anti-BCMA (B Cell Maturation Antigen) antibody is produced. Furthermore, we will create nanoparticles containing CD3/BCMA binding protein on their surface layer and verify their effect on RPMI8226 cells, a BCMA-positive multiple myeloma cell line.
  • CD3/BCMA binding protein consists of SP, mOKT3 scFV, linker peptide, and scFV derived from anti-BCMA antibody clone BCMA98 (hereinafter referred to as "BCMA98 scFV") in order from the N-terminal side. , and a structure in which CD8 ⁇ protein-derived fragments are linked (FIG. 17A). Components other than BCMA98 scFV are as described in Example 1.
  • BCMA98 scFV is an scFV derived from anti-BCMA antibody clone BCMA98.
  • BCMA98 scFV has a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 240 to 242, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 243 to 245. It contains a light chain variable region including CDR3, and its entire length consists of the amino acid sequence shown in SEQ ID NO: 17.
  • the full-length amino acid sequence of the CD3/BCMA binding protein is shown in SEQ ID NO: 18. Furthermore, the base sequence of the gene encoding the CD3/BCMA binding protein is shown as SEQ ID NO: 19.
  • Nanoparticles containing CD3/BCMA binding protein, CD80 protein, and 41BBL protein on the surface layer were prepared. Specifically, each gene encoding the CD3/BCMA binding protein, CD80 protein, and 41BBL protein was introduced into the leukemia cell line K562 by the same method as in Examples 1 and 2. Nanoparticles containing these proteins on their surface layer were produced from these cells in the same manner as in Example 2 (hereinafter referred to as "mOKT3/BCMA98/CD80/41BBL nanoparticles").
  • RPMI8226 cells BCMA-positive multiple myeloma cell line; JCRB, JCRB0034
  • T cells and RPMI8226 cells were co-cultured in the presence of mOKT3/BCMA98/CD80/41BBL nanoparticles. The number of nanoparticles was 5 ⁇ 10 6 times the number of T cells.
  • RPMI8226 cells were co-cultured at the same number of T cells. Co-culture was performed overnight, and the expression of CD25 protein in the CD8-positive T cell fraction was analyzed by flow cytometry.
  • Figure 17B shows representative flow cytometry results from three experiments. mOKT3/BCMA98/CD80/41BBL nanoparticles were shown to induce T cell activation in the presence of target cells.
  • T cells and RPMI8226 cells were co-cultured in the presence of mOKT3/BCMA98/CD80/41BBL nanoparticles by the same method as in (3) above. One week after the start of culture, the number of T cells was measured and the cell doubling rate was calculated. As a control, we also analyzed cell proliferation when CAR-T cells and RPMI8226 cells were co-cultured against BCMA.
  • the CAR-T cells used here were T cells transfected with mOKT3 scFV, BCMA98 scFV, the same CD28 protein-derived fragment as above, and a chimeric antigen receptor (CAR) containing the cytoplasmic domain of CD3 ⁇ .
  • FIG. 19A shows the IL2 positive rate in CD4-positive T cells (FIG. 19B, "CD4 + IL2 + ”) and the IFN ⁇ -positive rate in CD8-positive T cells (FIG. 19B, "CD8 + IFN ⁇ + ").
  • Nanoparticles targeting mesothelin> (the purpose) Nanoparticles will be created in which two fusion proteins containing each of two scFVs derived from an anti-CD3 ⁇ antibody and an anti-mesothelin antibody are simultaneously introduced into the nanoparticle surface layer, and the effect on K562-CD19/Mesothelin cells will be verified. Note that the nanoparticles of this example have the configuration shown in "separate expression" in FIG. 13A.
  • a fusion protein having a structure in which SP, mOKT3 scFV, and CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (hereinafter referred to as "CD3-binding protein"), and the N-terminus.
  • ss1 scFV is an scFV derived from anti-mesothelin antibody clone ss1.
  • ss1 scFV has a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 224 to 226, and CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 227 to 229. It contains a light chain variable region including CDR3, and its entire length consists of the amino acid sequence shown in SEQ ID NO: 20.
  • the full-length amino acid sequence of mesothelin binding protein is shown in SEQ ID NO: 21. Furthermore, the base sequence of the gene encoding mesothelin binding protein is shown as SEQ ID NO: 22. Note that the structure of the CD3 binding protein and the gene encoding it is as described in Example 6.
  • Nanoparticles containing CD3 binding protein, mesothelin binding protein, CD80 protein, 41BBL protein, IL7 protein, and IL15 protein on the surface layer were prepared. Specifically, each gene encoding a CD3 binding protein, mesothelin binding protein, CD80 protein, 41BBL protein, IL7 protein, and IL15 protein was introduced into the leukemia cell line K562 by the same method as in Examples 1 and 2. Nanoparticles containing these proteins on their surface layers were produced from these cells in the same manner as in Example 2 (hereinafter referred to as "mOKT3/ss1/CD80/41BBL/IL7/IL15 nanoparticles").
  • K562-CD19/Mesothelin cells Effect on T cell activation K562 cells into which CD19 gene and mesothelin gene were each stably introduced were used as target cells (hereinafter referred to as "K562-CD19/Mesothelin cells"). T cells and K562-CD19/Mesothelin cells were co-cultured in the presence of mOKT3/ss1/CD80/41BBL/IL7/IL15 nanoparticles. The number of nanoparticles was 5 ⁇ 10 6 times the number of T cells. In addition, K562-CD19/Mesothelin cells were co-cultured at a number 0.33 times the number of T cells. Co-culture was performed overnight, and the expression of CD25 protein in the CD8-positive T cell fraction was analyzed by flow cytometry.
  • FIG. 20A shows representative flow cytometry results from four experiments.
  • mOKT3/ss1/CD80/41BBL/IL7/IL15 nanoparticles ( Figure 20, "ss1") showed similar T It has been shown to induce cell activation.
  • Example 10 Sustained release effect of molecules encapsulated in PLGA nanoparticles> (the purpose) We will verify that molecules encapsulated within PLGA nanoparticles are slowly released from the PLGA nanoparticles to the outside. We also examine the effect of the molecular weight of PLGA on the sustained release rate.
  • the obtained w/o emulsion was dropped into a PVA aqueous solution under ultrasound irradiation, and then DCM was evaporated and separated and washed using an ultracentrifuge to obtain FITC-BSA-encapsulated PLGA nanoparticles. .
  • the fluorescence spectrum of FITC-BSA encapsulated PLGA nanoparticles is shown in Figure 21A.
  • FITC-BSA-derived fluorescence was observed around 530 nm, confirming that FITC-BSA was encapsulated within the PLGA nanoparticles.
  • Example 11 In vivo antitumor effect of nanoparticles> (the purpose) The tandem mOKT3/FMC63/CD80/41BBL nanoparticles produced in Example 2 are administered to mice, and the antitumor effect on the CD19-positive leukemia cell line NALM6 in the mouse body is examined.
  • Nanoparticles 1 ⁇ 10 6 CD19-positive leukemia cell line NALM6 (denoted as “NALM6-GL cells”) into which the EGFP-P2A-Luc2 gene has been introduced and 4 ⁇ 10 6 cultured human T cells were NSGed. It was administered to mice (Charles River Japan, JAX Mice Stock No: 005557) through the tail vein. Since T cells do not exist in NSG mice, human T cells were introduced externally by infusion to create T cells that can be activated by nanoparticles within the mouse body.
  • NP x 3 the group administered the first three times
  • NP x 6 the group administered six times in total, including the first three times and the latter three times
  • No NP a group to which no nanoparticles were administered
  • FIG. 22B Imaging results at 2 to 5 weeks after NALM6-GL cell transplantation in the No NP group, NP ⁇ 3 group, and NP ⁇ 6 group are shown in FIG. 22B. Furthermore, the results of measuring the amount of NALM6 tumor as the amount of luciferase luminescence using IVIS Imaging are shown in FIG. 22C.
  • the amount of NALM6-GL cells was significantly decreased in the NP ⁇ 3 group and the NP ⁇ 6 group compared to the No NP group (indicated as “No treatment” in the figure).
  • the amount of NALM6-GL cells was significantly decreased in the NP ⁇ 6 group compared to the NP ⁇ 3 group.
  • Figure 24A shows a representative flow cytometry diagram.
  • TSCM memory stem cell
  • TEM effector memory
  • Nanoparticles containing a bone marrow homing molecule additionally introduced as a migration molecule will be administered to mice, and the effect on the proliferation of T cells in the bone marrow will be examined.
  • bone marrow homing molecules CXCR4 protein and VLA4 (a heterodimer of ITGA4 protein and ITGB1 protein) are introduced into nanoparticles.
  • Nanoparticles containing these proteins on their surface layer were produced from these cells in the same manner as in Example 2 (hereinafter referred to as "mOKT3/FMC63/CD80/41BBL/CXCR4/ITGA4/ITGB1 nanoparticles").
  • mOKT3/FMC63/CD80/41BBL/CXCR4/ITGA4/ITGB1 nanoparticles as a control, we used nanoparticles that did not contain CXCR4 protein, ITGA4 protein, and ITGB1 protein among the above proteins, but contained CD3/CD19 binding protein, CD80 protein, and 41BBL protein (in this example, (referred to as “mOKT3/FMC63/CD80/41BBL nanoparticles”).
  • Nanoparticles were produced from the same method as in Example 2.
  • T cells and the NALM6 cell line were co-cultured in the presence of nanoparticles with different particle sizes. Note that T cells and NALM6 cells were co-cultured with the same number of cells, and nanoparticles were used in a number 5 ⁇ 10 6 times as large as the number of T cells. After co-culturing for one week, the number of T cells was measured by flow cytometry, and the proliferation of T cells was analyzed.
  • Example 14 Modification of tumor microenvironment based on immune regulatory factors> (the purpose) Membrane vesicles carrying cytokine molecules or immune checkpoint inhibitor molecules on their surface as immune regulators are prepared.
  • cytokine molecules IL12 and IL18, TGF- ⁇ receptor, immune checkpoint inhibitor molecules anti-PDL1 antibody and antibody CTLA4 antibody are introduced into membrane vesicles, and created a tumor microenvironment based on these immune regulatory factors. Evaluate the modification effect.
  • MVs membrane vesicles
  • fusion proteins (a) to (e) as immune regulatory molecules to modify the tumor microenvironment ( Figure 27):
  • (a) IL12 fusion protein In order from the N-terminus, SP, mouse-derived IL12-p40 protein and IL12-p35 protein, and the transmembrane domain and cytoplasmic domain derived from mouse-derived CD8 ⁇ protein (hereinafter referred to as “CD8 ⁇ protein-derived fragment”)
  • CD8 ⁇ protein-derived fragment mouse-derived CD8 ⁇ protein-derived fragment
  • a fusion protein (hereinafter referred to as "IL12 fusion protein") was designed.
  • the full-length amino acid sequence of the IL12 fusion protein is shown as SEQ ID NO: 534.
  • IL18 fusion protein A fusion protein (hereinafter referred to as "IL18 fusion protein") was designed in which SP, mouse-derived IL18 protein, and human-derived CD8 ⁇ protein-derived fragment were linked in order from the N-terminal side.
  • the full-length amino acid sequence of the IL18 fusion protein is shown as SEQ ID NO: 535.
  • TGFBRI/II fusion protein A fusion protein (hereinafter referred to as “TGFBRI/II fusion protein) was designed.
  • the full-length amino acid sequence of the TGFBRI/II fusion protein is shown as SEQ ID NO: 536.
  • Anti-PDL1-scFV fusion protein A fusion protein (hereinafter referred to as , referred to as “anti-PDL1-scFV fusion protein”).
  • the full-length amino acid sequence of the anti-PDL1-scFV fusion protein is shown as SEQ ID NO: 537.
  • Anti-CTLA4-scFV fusion protein A fusion protein (hereinafter referred to as ⁇ anti- CTLA4-scFV fusion protein was designed.
  • the full-length amino acid sequence of the anti-CTLA4-scFV fusion protein is shown as SEQ ID NO: 538.
  • fusion protein in which SP, 2C11 scFV (single chain variable region fragment derived from mouse anti-CD3 ⁇ antibody clone 2C11), and human CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (hereinafter referred to as "CD3 binding protein").
  • CD19 binding protein 1D3 A fusion protein (hereinafter referred to as “CD19 binding protein 1D3), whose amino acid sequence is shown as SEQ ID NO: 540); and (iii) a mouse in which the CD19 gene and PDL1 gene are knocked out in each gene encoding the mouse-derived 41BBL protein consisting of the amino acid sequence shown in SEQ ID NO: 587. It was introduced into lymphoma cell line A20.
  • the cells were suspended (1 ⁇ 10 8 cells/2mL). The cells were crushed using a homogenizer, and it was confirmed using a microscope that almost all the cells were crushed.
  • the cell disruption solution was centrifuged at 3,200g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was centrifuged at 10,000g and 4°C for 10 minutes, and then the supernatant was ultracentrifuged at 100,000g and 4°C for 60 minutes.
  • the precipitate containing cell membrane fragments excluding the supernatant containing cytoplasmic proteins, is suspended in washing buffer (10mM Tris-HCl pH 7.5, 2mM EDTA), and after repeating this operation, the precipitate containing cell membrane fragments is supernatant in PBS.
  • membrane vesicles By thoroughly suspending the cells under sonic irradiation, membrane vesicles (the surface layer of which is composed of a lipid bilayer membrane embedded with proteins derived from cell membrane fragments) are produced, which are equivalent to 5 ⁇ 10 4 to 3 ⁇ 10 5 cells per 1 ⁇ L.
  • a MV solution containing vesicles with a diameter of about 50 nm to about 200 nm and a bag-like structure was prepared and used in the following examples.
  • MV membrane vesicle that does not contain a PLGA nanoparticle core
  • the MV containing the above (a) and (i) to (iii) in its surface layer is referred to as "MV(2C11/1D3/41BBL/IL12)", and the above (b) and (i) to (iii) are referred to as "MV(2C11/1D3/41BBL/IL12)".
  • the MV that contains the above is called “MV(2C11/1D3/41BBL/IL18)”
  • the MV that contains the above (a), (b), and (i) to (iii) on its surface layer is called “MV(2C11/1D3/41BBL/IL18)".
  • MV(2C11/1D3/41BBL/TGFBR) MV containing (c) and (i) to (iii) above in its surface layer, "MV(2C11/1D3/41BBL/TGFBR)", and (d) and (i) to (iii) above
  • the MV containing in its surface layer is called "MV(2C11/1D3/41BBL/anti-PDL1-scFV)”
  • the MV containing the above (e) and (i) to (iii) in its surface layer is called "MV(2C11/1D3/anti-PDL1-scFV)". 41BBL/anti-CTLA4-scFV).
  • an MV containing only (i) to (iii) in its surface layer without the above (a) was produced by the same method as above and referred to as "MV(2C11/1D3/41BBL)".
  • mice spleen-derived T cells were treated with the CD3 binding protein (2C11) in (i) above and the 41BBL protein in (iii) above.
  • IFN ⁇ production of CD8-positive T cells was measured using a flow cytometer. Evaluation was made using a meter.
  • TGF ⁇ receptor/adsorption of TGF ⁇ MV containing MV equivalent to 5 ⁇ 10 4 cells per 1 ⁇ L prepared in (1) above in 200 ⁇ L of medium containing 10 ng/mL mouse TGF ⁇ (Biolegend, #763102) 2C11/1D3/41BBL/TGFBR) solution was added. After 1 hour, the TGF ⁇ concentration in the supernatant was measured by ELISA (mouse TGF-beta 1 DuoSet, R&D systems #DY1679).
  • Example 15 In vivo antitumor effect of MV> (the purpose) The MV produced in Example 14 will be administered to mice, and the antitumor effect on the colon cancer cell line CT26 in the mice will be examined.
  • TBE-MV MV containing all of (a) to (e) and (i) to (iii) described in Example 14 on its surface layer was used as the MV described in Example 14. It was prepared in the same manner as described above and administered to mice.
  • TME-MV contains IL12 fusion protein, IL18 fusion protein, TGFBRI/II fusion protein, Anti-PDL1-scFV fusion protein, and Anti-CTLA4-scFV fusion protein, as well as CD3 binding protein (2C11), CD19 binding protein ( 1D3) and 41BBL protein were introduced, and the MV was prepared in the same manner as in Example 14 from the mouse lymphoma cell line A20 in which the CD19 gene and PDL1 gene were knocked out.
  • CD3 binding protein 2C11
  • CD19 binding protein 1D3
  • 41BBL protein 41BBL protein
  • the MV (2C11/1D3/41BBL) produced in Example 14 was used as a control MV (hereinafter referred to as "WT-MV" in this example).
  • Colon cancer cell line CT26 (hereinafter referred to as "CT26-CD19-luc cells") into which mouse CD19 gene and luciferase gene were introduced was subcutaneously transplanted into Balb/c mice.
  • PBS or the above WT-MV or TME-MV was intratumorally administered 8, 15, and 22 days after transplantation, and the therapeutic effect was evaluated by measuring the tumor diameter (tumor volume) and survival rate over time. evaluated.
  • mice received 50 ⁇ L of a 500 ⁇ L dose of MV solution containing MV equivalent to 1.5 ⁇ 10 8 cells.
  • the tumor diameter was observed over time based on the detection of luciferase luminescence and direct measurement of the subcutaneous tumor diameter.
  • mice survival analysis was performed by the Kaplan-Meier method, with the end point being when the tumor volume continuously exceeded 400 mm 3 .
  • Figure 30 shows the results of measuring the tumor diameter over time in the groups administered with PBS, WT-MV, or TME-MV.
  • Figure 31 shows the mean values of tumor volume in each group 13, 15, and 17 days after CT26 cell transplantation (mean ⁇ standard deviation; p value by one way ANOVA with multiple comparison test * P ⁇ 0.05, ** Showed in the figure as P ⁇ 0.01, ns indicates that no significant difference was detected).
  • the WT-MV administration group the tumor volume was suppressed lower than in the PBS administration group, and in the TME-MV administration group, the increase in tumor volume was further effectively suppressed compared to the WT-MV administration group.
  • FIG 31B The results of survival analysis in each group of mice are shown in Figure 31B.
  • the survival rate was increased in the WT-MV-treated group compared to the PBS-treated group, and the survival rate was further increased in the TME-MV-treated group compared to the WT-MV-treated group (p-value by log-rank test * P ⁇ 0.05, ** Shown as P ⁇ 0.01 in the figure).
  • Figure 31C shows a typical example in which the amount/localization of tumor cells in the mouse body in the TME-MV administration group was analyzed over time using an in vivo imaging system, and the tumor disappeared after 29 days.
  • CT26-CD19-luc cells were subcutaneously transplanted into Balb/c mice, and 8 days after transplantation, PBS, WT-MV, or TME-MV was intratumorally administered, and 13 days after transplantation, the cells infiltrated into the tumor.
  • Figures 32 to 35 show the ratio of CD8-positive T cells to CD45-positive blood cells in infiltrating cells (Figure 32A) and the ratio of CD8-positive T cells (CD45+CD8+ cells) to all cells in the tumor ( Figure 32B).
  • Example 16 Induction of effector function on NK cells> (the purpose)
  • anti-NKp46 antibody, anti-NKG2D antibody, and IgG1 Fc region are used as NK cell activation ligands.
  • a gene encoding the following (a) and a gene encoding any of the following (b) to (d) were introduced into the leukemia cell line K562: (a) A fusion protein in which SP, FMC63 scFV (single chain variable region fragment derived from mouse anti-CD19 antibody clone FMC63), and human-derived CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (as described in Example 6).
  • CD19 binding protein consisting of the amino acid sequence shown in SEQ ID NO: 11);
  • a fusion protein hereinafter referred to as “NKp46 binding protein, whose amino acid sequence is shown as SEQ ID NO: 541);
  • a fusion protein hereinafter referred to as “NKG2D binding protein ” and its amino acid sequence is shown as SEQ ID NO: 542);
  • a fusion protein hereinafter referred to as "IgG1 Fc fusion protein, whose amino acid sequence is shown as SEQ ID NO: 543).
  • MV was produced by the same method as in Example 14 from the leukemia cell line K562 into which the gene encoding the above (a) and the gene encoding any of the following (b) to (d) were introduced.
  • the MV containing the above (a) and (b) on its surface layer is called “Nkp46Ab-MV”
  • the MV containing the above (a) and (c) on its surface layer is called “NKG2DAb-MV”
  • IgG1Fc-MV the above ( MV containing a) and (d) on its surface layer
  • MV was produced from leukemia cell line K562 into which only (a) above was introduced, and used as a control in the following examples (hereinafter referred to as "WT-MV" in this example).
  • MV was produced by the same method as in Example 14 from the leukemia cell line K562 into which all of the genes encoding (a) to (d) above were introduced. This MV is called "IgG1Fc/NKG2DAb/Nkp46Ab-MV.”
  • NK cells (1 x 10 5 cells ) and CD19 in a culture medium added with 10 ⁇ L of the MV solution containing MV equivalent to 3 x 10 5 cells per 1 ⁇ L prepared in (1) above.
  • the multiple myeloma cell line MM.1S (1 ⁇ 10 5 cells) into which the gene had been introduced was co-cultured, and 3 hours later, the expression levels of activation markers CD69 and CD107a were analyzed by flow cytometry.
  • NK cells co-cultured with MM.1S-CD19 cells (MM.1S cells transfected with the CD19 gene) in the presence of Nkp46Ab-MV, NKG2DAb-MV, or IgG1Fc-MV compared to NK cells cultured alone.
  • the levels of activation markers increased, indicating activation.
  • NK cells showed activation even in the presence of WT-MV, and IgG1Fc/NKG2DAb/Nkp46Ab-MV loaded with multiple antibodies against activating ligands induced stronger effector functions compared to MV loaded with each antibody. It became clear that it could be done.
  • NK cells (1 ⁇ 10 5 cells) and CD19 gene-transfected multiple myeloma were cultured in a culture medium to which 10 ⁇ L of the MV solution containing MV equivalent to 3 ⁇ 10 5 cells per 1 ⁇ L prepared in (1) above was added.
  • Cell line MM.1S (1 ⁇ 10 5 cells) was co-cultured, and 24 hours later, the cytotoxic effect on MM.1S cells was analyzed by flow cytometry. The survival rate was significantly decreased in MM.1S cells after co-culture in the presence of Nkp46Ab-MV, NKG2DAb-MV, or IgG1Fc-MV, indicating that MV enhances the antitumor effect of NK cells.
  • MM.1S CD19-negative multiple myeloma cell line MM.1S
  • the cytotoxic effect was evaluated using MM.1S (hereinafter referred to as "MM.1S (CD19 negative)") and MM.1S into which the CD19 gene was stably introduced (hereinafter referred to as "MM.1S (CD19 positive)").
  • the MV used was the FMC63-CD8 ⁇ -introduced MV prepared in (1) above, and the MV isolated from K562 cells to which no gene had been introduced.
  • NK cells (1 ⁇ 10 5 cells) were added to a culture solution containing 10 ⁇ L of FMC63-CD8 ⁇ -transfected MVs or non-transfected MVs (10 ⁇ L of 500 ⁇ L containing MVs equivalent to 1 ⁇ 10 8 cells). ) and 1 ⁇ 10 5 MM.1S (CD19 negative) or MM.1S (CD19 positive) were co-cultured. After 24 hours of co-culture, the cytotoxic effect on MM.1S cells was analyzed by flow cytometry.
  • Example 17 Induction of phagocytosis to macrophages> (the purpose) We will create nanoparticles loaded with activating ligands for macrophages and verify their ability to induce phagocytosis in human and mouse macrophages.
  • CD19 binding protein consisting of the amino acid sequence shown in SEQ ID NO: 11);
  • a fusion protein hereinafter referred to as "IgG1 Fc fusion protein, whose amino acid sequence is shown as SEQ ID NO: 543).
  • a gene encoding the following (c), or a gene encoding the following (c) and a gene encoding the following (d) or (e) was introduced into the mouse lymphoma cell line A20: (c) A fusion protein in which SP, 1D3 scFV (single chain variable region fragment derived from mouse anti-CD19 antibody clone 1D3), and human-derived CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (as described in Example 14).
  • SP single chain variable region fragment derived from mouse anti-CD19 antibody clone 1D3
  • human-derived CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (as described in Example 14).
  • CD19 binding protein (1D3) consisting of the amino acid sequence shown in SEQ ID NO: 540;
  • SP anti-CD47 VHH (camelized antibody derived from anti-CD47 antibody clone A4, containing a variable region consisting of the amino acid sequence shown in SEQ ID NO: 586; Sockolosky et al. Proc Natl Acad) Sci U S A.
  • anti-CD47 VHH fusion protein a fusion protein in which human-derived CD8 ⁇ protein-derived fragments are linked
  • anti-CD47 VHH fusion protein the amino acid sequence of which is shown as SEQ ID NO: 545
  • Mouse CD40L consisting of the amino acid sequence shown in SEQ ID NO: 546.
  • nanoparticles (NP) containing a PLGA nanoparticle core and having a reconstituted cell membrane on the surface layer were produced using the same method as in Example 2.
  • NPs containing the above (a) on their surface layer are called “NP(FMC63)”
  • NPs containing the above (a) and (b) on their surface layer are called “NP(FMC63/IgG1 Fc)”
  • NP containing the above (c) in its surface layer is "NP(1D3)”
  • NP containing the above (c) and (d) in its surface layer is "NP(1D3/CD40L)”
  • the above (c) and (e) NP containing in its surface layer is called “NP(1D3/anti-CD47 VHH)”.
  • Monocytes were isolated from human peripheral blood mononuclear cells using anti-CD14 microbeads (Miltenyi Biotec). The isolated monocytes were then cultured for 6 days in a medium supplemented with M-CSF (Peprotech, #300-25) to differentiate into macrophages. Thereafter, the macrophages were differentiated into M2 type macrophages by culturing for 2 days in a medium supplemented with 20 ng/mL IL-4 (Peprotech, #200-04).
  • Human-derived M2 type macrophages (3 x 10 4 cells) and the CD19 gene were introduced into the culture solution containing the nanoparticles (5 x 10 11 particles) prepared in (1) above, and labeled with CFSE.
  • K562 cells (1 ⁇ 10 5 cells) were co-cultured. After 4 hours of co-culture, the percentage of macrophages (CD14+CFSE+) that phagocytosed K562 cells and became CFSE positive was analyzed by flow cytometry.
  • mouse-derived M2 type macrophages (3 x 10 4 ) and CFSE-labeled CD19-positive lymphoma cell line A20 were incubated. cells (1 ⁇ 10 5 cells) were co-cultured. After 4 hours of co-culture, the percentage of macrophage cells (F4/80+CFSE+) that phagocytosed the CFSE-labeled A20 cells and became CFSE-positive was analyzed by flow cytometry.
  • Example 18 Evaluation of sequences derived from various anti-CD3 antibody clones> (the purpose) Membrane vesicles carrying scFV derived from anti-CD3 antibody clones OKT3, L2K, or UCHT1 on the surface layer will be prepared, and the effector function induction effect on T cells will be evaluated.
  • MV membrane vesicles
  • CD3 binding protein is identical and consists of the amino acid sequence shown in SEQ ID NO: 9, and is referred to as "CD3 binding protein (OKT3)" in this example);
  • a fusion protein hereinafter referred to as “CD3 binding protein L2K), whose amino acid sequence is shown as SEQ ID NO: 547);
  • a fusion protein hereinafter referred to as “CD3 binding protein UCHT1, whose amino acid sequence is shown as SEQ ID NO: 548);
  • a fusion protein (“mesothelin binding protein, consisting of the amino acid sequence shown in SEQ ID NO: 21),
  • e A fusion protein in which SP, AbA scFV (single chain variable region fragment derived from anti-EGFR antibody clone AbA), and human-derived CD8 ⁇ protein-derived fragment are linked in order from the N-terminus (hereinafter referred to as "EGFR binding protein”).
  • SEQ ID NO: 549 CD80 protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
  • CD80 protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
  • 41BBL protein consisting of the amino acid sequence shown in SEQ ID NO: 8;
  • IL-7 protein consisting of the amino acid sequence shown in SEQ ID NO: 43;
  • IL-15 protein consisting of the amino acid sequence shown in SEQ ID NO: 44.
  • MV was produced by the same method as in Example 14 from the leukemia cell line K562 into which the above gene had been introduced.
  • the MV containing the above (a), (d), and (f) to (i) on its surface layer is called “ss1-OKT3-MV”; the above (a), (e), and (f ) to (i) in its surface layer is "AbA-OKT3-MV”; MV containing the above (b), (d), and (f) to (i) in its surface layer is "ss1-L2K-MV".
  • MV containing the above (b), (e), and (f) to (i) on its surface layer is "AbA-L2K-MV”; the above (c), (d), and (f) to (i) ) on its surface layer is called “ss1-UCHT1-MV”; MV containing the above (c), (e), and (f) to (i) on its surface layer is called “AbA-UCHT1-MV”.
  • MV was produced from the leukemia cell line K562 into which none of the above (a) to (i) had been introduced, and was used as a control in the following examples (hereinafter referred to as "MV (No Ab)").
  • TOV21G cells (1 x 10 5 cells) and T cells (1 x 10 5 cells) were co-cultured in a culture medium to which 10 ⁇ L of the MV solution containing 5 x 10 5 cells per 1 ⁇ L of MV prepared in (1) above was added. Cultured. After 24 hours, cytotoxic activity against TOV21G cells was analyzed by flow cytometry.

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Abstract

La présente invention a pour but de procurer une nouvelle préparation acellulaire pouvant être préparée individuellement pour chaque patient et permettant d'obtenir, de manière stable et à peu de frais, un effet de destruction élevé sur les cellules tumorales.　L'invention concerne une nanoparticule multispécifique capable de se lier à au moins deux cellules cibles, la nanoparticule multispécifique contenant : (1) une membrane lipidique constituant la couche de surface de la nanoparticule; et (2) (i) une protéine de fusion comprenant une région de liaison à la membrane liée à la membrane lipidique et au moins deux régions de liaison cibles situées à l'extérieur de la membrane lipidique, ou (ii) au moins deux protéines de fusion comprenant une région de liaison à la membrane liée à la membrane lipidique et une région de liaison cible située à l'extérieur de la membrane lipidique.
PCT/JP2023/015857 2022-04-21 2023-04-21 Nanoparticule multispécifique WO2023204290A1 (fr)

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