Multispecific nanoparticle

Abstract

The present invention addresses the problem of providing a new acellular preparation that need not be prepared for each patient individually and can stably and inexpensively achieve a high killing effect on tumor cells.　Provided is a multispecific nanoparticle capable of binding with two or more target cells, wherein the multispecific nanoparticle contains: (1) a lipid membrane constituting the surface layer of the nanoparticle; and (2) (i) a fusion protein including a membrane-binding region bound to the lipid membrane and two or more target binding regions located outside the lipid membrane, or (ii) two or more fusion proteins including a membrane-binding region bound to the lipid membrane and a target binding region located outside the lipid membrane.

Description

Multispecific Nanoparticles

[0001] The present invention relates to multispecific nanoparticles, methods for producing multispecific nanoparticles, and the like.

[0002] CAR-T cell therapy involves introducing a chimeric antigen receptor (CAR) into T cells collected from peripheral blood to generate CAR-T cells capable of attacking cancer cells, which are then used for treatment (Non-Patent Document 1).

[0003] Chimeric antigen receptors (CARs) are artificial antigen receptors that combine an extracellular domain containing an antibody variable region that recognizes a cancer antigen with the signaling domain of a costimulatory molecule involved in T cell activation. To date, first-generation CARs, whose signaling domain consists solely of CD3ζ, have been followed by second- and third-generation CARs containing different structures and types of signaling domains. CAR-T cell therapy is a groundbreaking cancer treatment that can efficiently kill tumor cells by recognizing tumor cells and activating CAR-expressing T cells (CAR-T cells) themselves.

[0004] Currently, CAR-T cell therapy targeting the CD19 molecule for B-cell leukemia and lymphoma has been approved in Europe, the United States, and Japan. Its response rate is extremely high, with remission rates exceeding 80% reported even in refractory cases. Furthermore, development of CAR-T cell therapy targeting other antigens and cancer types is underway, and it is expected to become a groundbreaking cancer treatment technology following immune checkpoint inhibitor therapy.

[0005] While CAR-T cell therapy has attracted attention as a new treatment for recurrent / refractory tumors, its high cost has become a major problem. The cost per administration is $475,000 for Kymriah (Novartis) and $373,000 for Yescarta (Kite), both exceeding 30 million yen. Despite being a highly promising treatment method, the high cost of treatment is a major obstacle to the widespread adoption of CAR-T cell therapy.

[0006] Another problem with CAR-T cell therapy is the lack of a stable therapeutic effect. CAR-T cells are produced using T cells collected from the patient. Therefore, the quality of CAR-T cells is known to vary significantly depending on the patient's condition. In particular, it has been reported that in patients who have undergone repeated chemotherapy, the quality of the collected T cells declines during the culture stage, and CAR-T cells disappear soon after infusion (Non-Patent Document 2).

[0007] Therefore, there is a need to develop new therapeutic methods that can provide a high killing effect on tumor cells more inexpensively and stably.

[0008] Maude SL, et al., N Engl J Med, 2018, 378(5):439-448.Fraietta JA, et al., Nat Med, 2018, 24(5): 563-571.

[0009] An object of the present invention is to provide a new non-cellular preparation that does not require individual preparation for each patient and that can exhibit a high killing effect against tumor cells inexpensively and stably.

[0010] CAR-T cell therapy requires the collection of a patient's own T cells, transfection with the CAR gene, and then infusing them back into the patient. This series of steps to individually prepare CAR-T cells is necessary to create cells specific to cancer cells, but it also contributes to the high cost of treatment and the fact that the quality of therapeutic cells can vary greatly from patient to patient.

[0011] To solve the above problems, the present inventors developed nanoparticles carrying multiple immune regulatory molecules on their surface. By administering the nanoparticles of the present invention into the body, T cells present in the body can be activated "in situ." Surprisingly, this activation is specifically induced in the presence of tumor cells. Therefore, specificity equivalent to that of CAR-T cells is guaranteed, while simultaneously achieving low cost and stable efficacy.

[0012] Furthermore, the nanoparticles of the present invention can be loaded with additional immune-regulating molecules such as costimulatory molecules and cytokine molecules, making it possible to freely adjust and enhance their effects. Another major advantage is that, unlike protein preparations, they are less susceptible to renal excretion, allowing for long-term efficacy.

[0013] The present invention is based on the above-mentioned research results and provides the following: (1) A multispecific nanoparticle capable of binding to two or more target cells, comprising: [A] a lipid membrane forming the surface layer of the nanoparticle; and [B] (i) a fusion protein comprising a membrane-binding region bound to the lipid membrane and two or more target-binding regions located outside the lipid membrane, or (ii) two or more fusion proteins comprising a membrane-binding region bound to the lipid membrane and a target-binding region located outside the lipid membrane. (2) The multispecific nanoparticle according to (1), wherein the target-binding region comprises: (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. (3) The multispecific nanoparticle according to (2), wherein the T cell receptor complex-binding region is selected from the group consisting of an anti-CD3 antibody or fragment thereof, an anti-T cell receptor (TCR) antibody or fragment thereof, and an HLA / peptide fusion molecule. (4) The anti-CD3 antibody comprises: [1] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively; [2] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively; [3] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively; [4] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 96 to 98, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 99 to 101, respectively;[5] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively; [6] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively; [7] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively; [8] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively; [9] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively;

[10] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively;

[11] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively;

[12] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively;

[13] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 168 to 170, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 171 to 173, respectively;

[14] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 176 to 178, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 179 to 181, respectively;

[15] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 345 to 347, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 348, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 349), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 350,

[16] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 356 to 358, respectively;

[17] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 362 to 364, respectively;

[18] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 370 to 372, respectively;

[19] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380, respectively;

[20] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively;

[21] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively;

[22] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 402 to 404, respectively;

[23] A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434;

[24] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 438 to 440, respectively;

[25] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 446 to 448, respectively;

[26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 451 to 453, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 454, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 455), 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 consisting of the amino acid sequences shown in SEQ ID NOs: 459 to 461, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 462, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 463), 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 consisting of the amino acid sequences set forth in SEQ ID NOs: 519 to 521, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 522, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 523), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 524;

[30] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 530 to 532, respectively;

[31] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 530 to 531 and 533, respectively; or

[32] The multispecific nanoparticle according to (3), wherein the anti-TCR antibody comprises: 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively; or the anti-TCR antibody comprises:

[33] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 187 to 189, respectively. (5) The multispecific nanoparticle according to (2), wherein the NK cell surface antigen-binding region is selected from the group consisting of 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, an MICA protein or a fragment thereof, an MICB protein or a fragment thereof, a B7H6 protein or a fragment thereof, and influenza virus-derived hemagglutinin or a fragment thereof.

[34] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 303 to 305, respectively, wherein the anti-NKG2D antibody comprises:

[35] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 311 to 313, respectively, or the anti-NKp46 antibody comprises:

[36] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 319 to 321, respectively, and the IgG Fc region comprises:

[37] The multispecific nanoparticle according to (5), comprising a human IgG1 Fc region consisting of the amino acid sequence shown in SEQ ID NO: 30, or

[38] a human IgG3 Fc region consisting of the amino acid sequence shown in SEQ ID NO: 61. (7) The multispecific nanoparticle according to (2), wherein the macrophage surface antigen-binding region is selected from the group consisting of an anti-FCγRI (CD64) antibody or a fragment thereof, an anti-FCγRIIA (CD32) antibody or a fragment thereof, an anti-FCγRIIIA (CD16A) antibody or a fragment thereof, an anti-CD40 antibody or a fragment thereof, an IgG Fc region or a fragment thereof, a CD40L protein or a fragment thereof, an anti-CD47 antibody or a fragment thereof, and a TLR4a ligand. (8) The anti-CD40 antibody comprises:

[39] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 327 to 329, respectively; or

[40] 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 335 to 337, respectively; and the IgG Fc region comprises:

[41] a human IgG1 consisting of the amino acid sequence shown in SEQ ID NO: 30.

[42] a human IgG3 Fc region consisting of the amino acid sequence shown in SEQ ID NO: 61, wherein the anti-CD47 antibody or fragment thereof comprises:

[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] a variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 583 to 585, respectively, or the TLR4a ligand comprises:

[45] a TLR4a agonist consisting of the amino acid sequence shown in SEQ ID NO: 582, or

[46] a TLR4a agonist consisting of the amino acid sequence shown in SEQ ID NO: 596. (9) The multispecific nanoparticle of any one of (2) to (8), wherein 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. (10) The multispecific nanoparticle of any one of (2) to (9), further comprising, on the surface and / or inside of the nanoparticle, any 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. (11) The costimulatory molecule is one or more 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, and the cytokine molecule is one or more selected from the group consisting of 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, IFN-γ protein, TNF-α protein, and TGF-β protein,the cytokine receptor is one or more selected from the group consisting of a TGF-β 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 chemotactic molecule is one or more selected from the group consisting of a CXCR4 protein, an ITGA4 protein, an ITGB1 protein, an ICAM-1 protein, a VCAM-1 protein, and an LFA3 protein; the immune checkpoint inhibitor molecule is one or more selected from the group consisting of 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, a VISTA inhibitor, an ICOS inhibitor, an ICOS stimulant, a BTLA inhibitor, a CD47 inhibitor, a KIR inhibitor, an LIR inhibitor, a CD94 inhibitor, and an NKG2A inhibitor; and / or (10) The multispecific nanoparticle according to (10), wherein 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. (12) The multispecific nanoparticle according to any of (1) to (11), wherein the lipid membrane is composed of a cell membrane, or a cell membrane and a liposome. (13) The multispecific nanoparticle according to any of (1) to (12), further comprising a nanoparticle core composed of a polymer nanoparticle, a metal nanoparticle, or a dendrimer, wherein the lipid membrane encapsulates the nanoparticle core. (14) The multispecific nanoparticle according to any of (1) to (13), wherein the particle diameter is 5 nm to 250 nm.

[0014] (15) A pharmaceutical composition comprising the multispecific nanoparticle according to any one of (1) to (14). (16) A fusion protein comprising a membrane-binding domain and, on the N-terminal or C-terminal side thereof, a T cell receptor complex-binding domain and a cancer antigen-binding domain, wherein the T cell receptor complex-binding domain 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. (17) The anti-CD3 antibody comprises: [1] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively; [2] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively; [3] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively; [4] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 99 to 101, respectively; [5] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively; [6] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively;[7] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively; [8] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively; [9] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively;

[10] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively;

[11] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively;

[12] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively;

[13] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 171 to 173, respectively;

[14] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 179 to 181, respectively;

[15] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 345 to 347, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 348, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 349), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 350;

[16] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 356 to 358, respectively;

[17] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 362 to 364, respectively;

[18] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 370 to 372, respectively;

[19] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 375 to 377, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 378 to 380, respectively;

[20] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 383 to 385, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 386 to 388, respectively;

[21] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively;

[22] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 402 to 404, respectively;

[23] 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;

[24] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 438 to 440, respectively;

[25] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 446 to 448, respectively;

[26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 451 to 453, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 454, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 455), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 456;

[27] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 459 to 461, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 462, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 463), and CDR3 consisting of the amino acid sequence set forth 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 consisting of the amino acid sequences set forth in SEQ ID NOs: 519 to 521, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 522, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 523), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 524;

[30] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 530 to 532, respectively;

[31] A fusion protein according to (16), comprising 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively; or

[32] 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively, wherein the anti-TCR antibody comprises:

[33] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 187 to 189, respectively.

[0015] (18) A nucleic acid encoding the fusion protein according to (16) or (17). (19) A gene expression vector comprising, in an expressible state, the nucleic acid according to (18). (20) A host cell comprising the gene expression vector according to (19). (21) A host cell comprising: a gene expression vector comprising, in an expressible state, a nucleic acid encoding a fusion protein comprising a T cell receptor complex-binding region and a membrane-binding region; and a gene expression vector comprising, in an expressible state, a nucleic acid encoding a fusion protein comprising a cancer antigen-binding region and a membrane-binding region, 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. (22) The anti-CD3 antibody comprises: [1] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 75 to 77, respectively; [2] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 83 to 85, respectively; [3] a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 91 to 93, respectively; [4] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 99 to 101, respectively; [5] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively;[6] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 115 to 117, respectively; [7] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 123 to 125, respectively; [8] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 131 to 133, respectively; [9] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 139 to 141, respectively;

[10] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 147 to 149, respectively;

[11] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 155 to 157, respectively;

[12] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 163 to 165, respectively;

[13] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 171 to 173, respectively;

[14] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 176 to 178, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 179 to 181, respectively;

[15] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 345 to 347, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 348, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 349), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 350;

[16] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 356 to 358, respectively;

[17] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 362 to 364, respectively;

[18] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 367 to 369, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 370 to 372, respectively;

[19] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 375 to 377, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 378 to 380, respectively;

[20] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively;

[21] A heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 394 to 396, respectively;

[22] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 399 to 401, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 402 to 404, respectively;

[23] A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434;

[24] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 438 to 440, respectively;

[25] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 446 to 448, respectively;

[26] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 451 to 453, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 454, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 455), and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 456;

[27] A heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 459 to 461, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 462, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 463), and CDR3 consisting of the amino acid sequence set forth 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 sequences shown in SEQ ID NOs: 519 to 521, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 522, 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;

[30] 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 532, respectively;

[31] 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 530 to 531 and 533, respectively; or

[32] 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 a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 569 to 571, respectively; or wherein the anti-TCR antibody comprises:

[33] The host cell according to (21), comprising a heavy chain variable region comprising 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 comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 187 to 189, respectively. (23) The host cell according to any of (20) to (22), further comprising one or more gene expression vectors comprising, in an expressible state, a nucleic acid encoding any protein selected from the group consisting of a costimulatory molecule, a cytokine molecule, a cytokine receptor, a migration molecule, an immune checkpoint inhibitory molecule, and an immune checkpoint molecule.

[0016] (24) A method for producing multispecific nanoparticles capable of binding to two or more target cells, comprising: a cell membrane fragmentation step of disrupting cells expressing (i) two or more fusion proteins comprising a membrane-binding domain and a target-binding domain, or (ii) a fusion protein comprising a membrane-binding domain and two or more target-binding domains on their cell membranes to fragment the cell membrane; a nanoparticle formation step of forming nanoparticles by reconstituting the cell membrane fragments after the fragmentation step; and a recovery step of recovering the nanoparticles. (25) The method according to (24), wherein the target-binding domains comprise: (a) a T-cell receptor complex-binding domain, an NK cell surface antigen-binding domain, or a macrophage surface antigen-binding domain, and (b) a cancer antigen-binding domain. (26) The method according to (24) or (25), wherein fractions other than the cell membrane fragments and / or undisrupted cells are removed between the cell membrane fragmentation step and the nanoparticle formation step. (27) The method according to any one of (24) to (26), wherein the reconstitution in the nanoparticle formation step comprises mixing the cell membrane fragment with a liposome and / or a nanoparticle core to form a nanoparticle having a surface layer constituted by a lipid membrane formed by the fusion of the cell membrane fragment with the liposome, and / or a nanoparticle having a lipid membrane containing the cell membrane fragment encapsulating the nanoparticle core. (28) The method according to any one of (24) to (27), wherein the reconstitution in the nanoparticle formation step comprises ultrasonic treatment of the cell membrane fragment. (29) A fusion protein comprising a membrane-binding region and, on the N-terminal or C-terminal side thereof, a NK cell surface antigen-binding region and a cancer antigen-binding region, wherein the NK cell surface antigen-binding region is selected from the group consisting of 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 influenza virus-derived hemagglutinin or a fragment thereof.(30) A fusion protein comprising a membrane-binding region and, at the N-terminus or C-terminus thereof, a macrophage surface antigen-binding region and a cancer antigen-binding region, wherein the macrophage surface antigen-binding region is selected from the group consisting of an anti-FCγRI (CD64) antibody or a fragment thereof, an anti-FCγRIIA (CD32) antibody or a fragment thereof, an anti-FCγRIIIA (CD16A) antibody or a fragment thereof, an anti-CD40 antibody or a fragment thereof, an IgG Fc region or a fragment thereof, a CD40L protein or a fragment thereof, an anti-CD47 antibody or a fragment thereof, and a TLR4a ligand. This specification includes the disclosure of Japanese Patent Application No. 2022-069831, from which the present application claims priority.

[0017] The multispecific nanoparticles of the present invention provide a new acellular formulation that does not require individual preparation for each patient and can exert a high killing effect on tumor cells inexpensively and stably.

[0018] 1A and 1B show nanoparticles according to one embodiment of the present invention. Figure 1A is a schematic diagram illustrating the effect of nanoparticles according to the present invention, which have fusion proteins containing two or more target-binding domains on their surface, on two target cells (T cells, NK cells, or macrophages, and cancer cells). Figure 1B shows the effect of nanoparticles according to the present invention, which have two or more fusion proteins containing target-binding domains on their surface. Figure 1B is a diagram illustrating an outline of a method for preparing nanoparticles according to one embodiment of the present invention. Figure 3A shows the structure of the CD3 / CD19 binding protein and K562-mOKT3 / FMC63 cells prepared in Example 1. Figure 3B shows the results of detecting the expression of the CD3 / CD19 binding protein on the cell surface by flow cytometry. Figure 3C shows the viability of tumor cells NALM6 after co-culture with T cells and the K562-mOKT3 / FMC63 cell line. Figure 4A shows the appearance of a PLGA nanoparticle core dispersion. Figure 4B shows the spherical shape of PLGA nanoparticle cores observed using a transmission electron microscope. Figure 4C (left) shows the particle size and concentration of PLGA nanoparticle cores immediately after synthesis. Figure 4C (right) shows the particle size and concentration of PLGA nanoparticle cores after cryopreservation and thawing. This figure shows the nanoparticle production process in Example 2. Figure 5A shows the results of detecting CD80 and 41BBL protein expression on the cell surface using flow cytometry. Figure 5B shows each step of nanoparticle preparation. This figure shows the results of analyzing the nanoparticles produced in Example 2. Figure 6A shows the results of observation of nanoparticles using a transmission electron microscope (TEM). This figure shows PLGA nanoparticle cores before (-) and after (+) cell membrane reconstitution. Figure 6B shows the results of Western blot analysis of components contained in protein fractions obtained at each step of nanoparticle preparation. This figure shows 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 nanoparticle (NP) particles to the number of T cells.Figure 8A shows the results of measuring nanoparticle-induced T cell activation in Example 4. Figure 8A shows the results of detecting CD25 protein expression on the cell surface by flow cytometry. Figure 8B shows the results of measuring CD25 expression levels under each condition. Figure 8B shows the results of measuring nanoparticle-induced T cell activation in Example 4. Figure 8C shows the effects of nanoparticles additionally loaded with costimulatory molecules CD80 protein and 41BBL protein in Example 5. Figure 10A shows the cell doubling rate of T cells. "NP / T cell" indicates the ratio of nanoparticle (NP) particle count to T cell cell count. Figure 10B shows the survival rate of tumor cells after co-culture. Figure 10B shows the results of mOKT3 / FMC63 nanoparticles (CD80 / 41BBL (-)) and mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (CD80 / 41BBL (+)). Figure 10C shows the results of measuring the proportion of cytokine-producing cells in CD8-positive T cells in Example 5. The figures show the results for mOKT3 / FMC63 nanoparticles (CD80 / 41BBL (-)) and mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (CD80 / 41BBL (+)). The figures show the results of measuring the percentage of cytokine-producing cells among CD4- or CD8-positive T cells in the presence or absence of target cells (K562-CD19 cells) in Example 5. FIG. 12A shows the results for CD4-positive T cells. FIG. 12B shows the results for CD8-positive T cells. The figures show the results of comparing tandem nanoparticles with 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 separated nanoparticles. FIG. 13C shows the viability of tumor cells after co-culture with tandem nanoparticles, separated nanoparticles, and T cells. 14A shows the effect of nanoparticles into which cytokine molecules, IL7 protein and IL15 protein, have been additionally introduced in Example 7. Fig. 14A shows the results of detecting the expression of each protein by flow cytometry in K562 cells into which IL7 protein and IL15 protein have been additionally introduced.Figure 14B shows the cell doubling rates of T cells treated with 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. Figure 15A shows the results of evaluating the T cell memory phenotype in Example 7. Figure 15A shows the results of evaluating the expression levels of each marker protein, CCR7, CD45RA, CD62L, CD28, FSC, and CD27, by flow cytometry. Figure 15B shows the cell doubling rate of T cells with 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 nanoparticle (NP) particle count to T cell count. Figure 16A shows the results of measuring phosphorylated STAT5 (pSTAT5) levels by flow cytometry. Figure 16B shows the results of quantifying phosphorylated STAT5 (pSTAT5) fluorescence intensity. Figure 17A shows the CD3 / BCMA binding protein and mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles prepared in Example 8. Figure 17A shows the structure of the CD3 / BCMA binding protein. In the figure, SP indicates a signal peptide. FIG. 17B shows the results of detecting CD25 protein expression on the cell surface by flow cytometry. This figure shows the results of evaluating T cell proliferation and induction of effector effects in Example 8. FIG. 18A shows the cell doubling rate of T cells. FIG. 18B shows the survival rate of tumor cells after co-culture. This figure shows the results of measuring the proportion of cytokine-producing cells in Example 8. FIG. 19A shows a representative flow cytometry plot. FIG. 19B shows the IL2 positivity rate (CD4+) in CD4+ T cells. + IL2 + ) and IFNγ positivity rate in CD8-positive T cells (CD8 + IFNγ +20B shows the results of quantifying CD25 protein expression. FIG. 20C shows the viability of K562-CD19 / Mesothelin cells after co-culture. FIG. 21A shows the sustained release effect of molecules encapsulated in PLGA nanoparticles in Example 10. FIG. 21A shows the fluorescence spectrum of FITC-BSA-encapsulated PLGA nanoparticles. Figure 21B shows the time course of FITC-BSA concentration released to the outside of the dialysis membrane, as measured using a fluorometer. This figure shows the in vivo antitumor effect of nanoparticles in Example 11. Figure 22A shows the administration schedule of NALM6-GL cells, T cells, and nanoparticles. Figure 22B shows imaging results of NALM6-GL cells at 2 to 5 weeks after NALM6-GL cell transplantation in the No NP, NPx3, and NPx6 groups. Figure 22C shows logarithmically transformed luminescence intensity. This figure shows the analysis results of mice administered nanoparticles in Example 11. Figure 23A shows the results of measuring the number of CD8+ T cells in peripheral blood. Figure 23B shows the results of survival analysis using the Kaplan-Meier method. This figure shows the results of evaluating T cell memory phenotypes in Example 11. Figure 24A shows a representative flow cytometry image. Figure 24B shows the percentages of memory stem cells (TSCM) and effector memory (TEM) fractions. 25A and 25B show the effects of nanoparticles loaded with bone marrow-homing molecules in Example 12. Figure 25A shows the results of flow cytometry analysis of the expression of CXCR4, ITGA4, and ITGB1 proteins, which were additionally loaded into K562 cells. Figure 25B shows the administration schedules of NALM6 cells, T cells, and nanoparticles.Figure 25C shows the percentage of T cells in the bone marrow or spleen of mice administered mOKT3 / FMC63 / CD80 / 41BBL / CXCR4 / ITGA4 / ITGB1 nanoparticles ("CXCR4+VLA4", (-)) or mOKT3 / FMC63 / CD80 / 41BBL nanoparticles ("CXCR4+VLA4", (+)) containing bone marrow homing molecules. Figure 26A shows the particle size distribution of PLGA nanoparticle cores prepared from 1 mg / mL or 20 mg / mL PLGA. Figure 26B shows the cell doubling rate of T cells. NP size (nm) indicates the particle size of the nanoparticles. Figure 26C shows the results of quantifying the expression level of CD25 protein. Five fusion proteins designed as immunoregulatory molecules are shown. The effect of membrane vesicles (MVs) carrying cytokine molecules on their surface is shown. Figure 28A shows the proliferation potential (proportion of T cells that underwent cell division) of mouse CD8+ T cells cultured without co-culture with A20 cells (A20 cells: - in the figure) or with co-culture with A20 cells (A20 cells: + in the figure) in the absence of membrane vesicles (MV: -), in the presence of MV (2C11 / 1D3 / 41BBL) (MV: WT), or in the presence of MV (2C11 / 1D3 / 41BBL / IL12) with IL12 fusion protein introduced on its surface (MV: IL12). Figure 28B shows the percentage of IFNγ-producing mouse CD8 T cells cultured in the absence of membrane vesicles (MV:-), in the presence of MV(2C11 / 1D3 / 41BBL) (MV:WT), in the presence of MV(2C11 / 1D3 / 41BBL / IL12) (MV:IL12), 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). The effects of TGFβ receptor, anti-PDL1 scFV, and anti-CTLA4 scFV loaded on membrane vesicles (MV) are shown.Figure 29A shows the results of ELISA for TGFβ concentrations in supernatants obtained from TGFβ-containing medium without membrane vesicles (MV:-), with control MV (2C11 / 1D3 / 41BBL) (MV:WT), or with MV (2C11 / 1D3 / 41BBL / TGFBR) (MV:TGFBRI / II). Figure 29B shows the results of flow cytometry analysis of PDL1 expression levels on the PDL1-positive mouse lymphoma cell line A20 after addition of MV (2C11 / 1D3 / 41BBL / anti-PDL1-scFV). In the figure, "Unstained" represents the results of A20 cells analyzed without fluorescent antibody staining. "Knockout" represents the results of A20 cells with PDL1 gene knockout. "Stained" represents the results of A20 cells analyzed without membrane vesicles. "WT 50 μL" represents the results of adding the control MV (2C11 / 1D3 / 41BBL). Figure 29C shows the results of flow cytometry analysis of CTLA4 expression levels on A20 cells after adding MV (2C11 / 1D3 / 41BBL / anti-CTLA4-scFV) to the mouse lymphoma cell line A20, which stably expresses the CTLA4 gene. In the figure, "WT" represents the results of A20 cells without CTLA4 gene transfection. "MV-" represents the results without adding membrane vesicles. "WT-MV" represents the results of adding the control MV (2C11 / 1D3 / 41BBL). Tumor volume (mm) when PBS, WT-MV, or TME-MV was administered to Balb / c mice subcutaneously implanted with CT26-CD19-luc cells 8, 15, and 22 days after implantation. 3) over time. 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. The tumor volume and survival rate in mice administered with PBS, WT-MV, or TME-MV are shown. Figure 31A shows the mean tumor volume 13, 15, and 17 days after CT26-CD19-luc cell transplantation. "Ctrl" indicates the PBS-administered group. Figure 31B shows the results of mouse survival analysis. "Ctrl" indicates the PBS-administered group. Figure 31C shows the results of observing tumor cells in the mouse body over time in a representative example of the TME-MV-administered group. The results of analyzing cells infiltrating into the tumor in Balb / c mice that were subcutaneously transplanted with CT26-CD19-luc cells and then intratumorally administered with PBS, WT-MV, or TME-MV are shown. Figure 32A shows the percentage of CD8+ T cells among CD45+ blood cells in infiltrating cells. Figure 32B shows the percentage of CD8+ T cells (CD45+CD8+ cells) among all cells in the tumor. Representative flow cytometry plots are shown for the PBS-treated, WT-MV-treated, and TME-MV-treated groups. The percentage of M2 macrophages among tumor-infiltrating macrophages is shown. Representative flow cytometry plots are shown for the PBS-treated, WT-MV-treated, and TME-MV-treated groups. The results of analyzing the expression levels of MICA / B and HLA class I molecules on the leukemia cell line K562 are shown. Figure 36A shows that the NK cell-activating ligand MICA / B is expressed on the leukemia cell line K562. Figure 36B shows that HLA class I molecules, which have an NK cell-suppressive effect, are not expressed on the leukemia cell line K562. Figure 39A shows the results of flow cytometry analysis of the expression levels of CD69, an NK cell activation marker, and CD107a, a degranulation marker, in NK cells cultured alone and in NK cells cocultured with MM.1S cells transfected with the CD19 gene and various MVs. Figure 39B shows the results of evaluating the cytotoxic effect of NK cells against the multiple myeloma cell line MM.1S in the presence of various MVs. Figure 39C shows the results of verifying antigen specificity on target cells. Figure 39B shows the results of evaluating the cytotoxic effect against the CD19-negative multiple myeloma cell line MM.1S.Figure 39B shows the results of evaluating the cytotoxic effect on the CD19-positive multiple myeloma cell line MM.1S. Results are shown for three conditions: absence of MV (MV:-), addition of membrane vesicles derived from non-transfected K562 cells (MV:+, FMC63:-), and addition of membrane vesicles expressing scFV derived from the anti-CD19 antibody (clone FMC63) (MV:+, FMC63:+). Flow cytometric analysis of human M2 macrophages that phagocytosed CFSE-labeled K562 cells and became CFSE-positive is shown. Flow cytometric analysis of mouse M2 macrophages that phagocytosed CFSE-labeled A20 cells and became CFSE-positive is shown. Flow cytometric analysis of the ovarian cancer cell line TOV21G is shown. Figure 42A shows the results of analysis of mesothelin expression. Figure 42B shows the results of analysis of EGFR expression. 1 shows the results of flow cytometry analysis of cytotoxic activity against TOV21G cells.

[0019] 1. Multispecific Nanoparticles 1-1. Overview A first aspect of the present invention is a multispecific nanoparticle. The multispecific nanoparticles of the present invention comprise a lipid membrane and a fusion protein that form the surface layer of the nanoparticle, and are capable of binding to two or more target cells. When the target cells include T cells and cancer cells, the multispecific nanoparticles of the present invention can bind to the T cells and the cancer cells, activate the T cells, and induce cytotoxic activity against the cancer cells. Similarly, they can also induce cytotoxic activity against cancer cells in NK cells and macrophages.

[0020] 1-2. Definitions The following terms frequently used in this specification are defined below. As used herein, "nanoparticle" refers to a particle having a particle size on the order of nanometers (nm). Nanoparticles generally refer to particles having a particle size of 1 nm to several hundred nm. The particle size of nanoparticles is preferably large enough to prevent rapid renal excretion. Specifically, the gap between endothelial cells in the glomerulus of the kidney is 43 nm, and it is known that the upper limit of the size that can pass through the multilayer structure formed by endothelial cells and be naturally excreted is approximately 5 to 10 nm (Denn W. M., et al., Am J. Physiol. Renal Physiol., 2001, 281(4):F579-96; Ohlson M., et al., Am J. Physiol. Renal Physiol., 2001, 280(3):F396-405). Therefore, the particle size 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. Furthermore, the particle size of the nanoparticles is preferably small enough to avoid embolization in pulmonary capillaries and / or capture 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 μm, 70 nm to 180 nm, 80 nm to 160 nm, 90 nm to 150 nm, and 100 nm to 140 nm.

[0021] As used herein, the term "multispecific" refers to specificity for two or more biomolecules or cells. Multispecificity may be specific to two or more molecules, for example, specific to two or more proteins or two or more cells. Multispecificity includes, for example, bispecific, trispecific, tetraspecific, pentaspecific, or higher specificity.

[0022] As used herein, the term "target cell" refers to a cell targeted by the multispecific nanoparticles of the present invention. The target cell may be either a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include bacterial cells such as Escherichia coli cells. Examples of 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 are preferred. Specific examples of mammalian cells include immune cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells, skin keratinocytes, epithelial cells, mesenchymal stem cells, mesenchymal cells (bone cells, adipocytes, muscle cells, chondrocytes, neurons, or fibroblasts), hematopoietic stem cells, various cancer cell lines, neural stem cells, neurons, iPS cells, and ES cells.

[0023] As used herein, "immune cells" include cell types that can function as part of the immune system, as well as undifferentiated cells and precursor cells (e.g., immune precursor cells) that can differentiate into such cell types. Specific examples of immune cells include lymphocytes, granulocytes, dendritic cells, macrophages, and monocytes. Examples of lymphocytes include T cells, B cells, and natural killer cells (NK cells). Lymphocytes may be tumor-infiltrating lymphocytes. Examples of T cells include killer T cells (cytotoxic T cells), helper T cells, and regulatory T cells. T cells may be either CD8+ T cells or CD4+ T cells. T cells can also be classified into naive T cells, memory T cells, and effector T cells, but any of these may be used. It is known that naive T cells and memory T cells are abundant in peripheral blood, while effector T cells are rarely found in peripheral blood. Examples of granulocytes include neutrophils, eosinophils, and basophils. Although immune cells may not necessarily include stem cells in the narrow sense, in this specification they are also considered to include stem cells that can differentiate into lymphocytes (for example, hematopoietic stem cells, etc.).

[0024] As used herein, "two or more target cells" is exemplified by any combination of cells including the above-mentioned target cells. The two or more target cells may be a combination of different cell types or a combination of the same cell type. Examples of combinations of different cell types include, but are not limited to, a combination of T cells and cancer cells, a combination of NK cells and cancer cells, and a combination of macrophages and cancer cells. Examples of combinations of the same cell type include a combination of epithelial cells, etc.

[0025] As used herein, the term "lipid membrane" refers to a membrane whose main component is lipid. Examples of lipid membranes include a lipid bilayer, in which lipid molecules are arranged in two layers by associating their hydrophobic portions with each other, and a multilayer structure in which several layers are stacked on top of each other using a lipid bilayer as a basic structure. The lipid membrane preferably 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. Examples of lipid molecules include phospholipids, glycolipids, acylglycerols, sterols, long-chain fatty acids, long-chain aliphatic alcohols, and glycerin fatty acid esters. For example, the lipid membrane may include cell membrane fragments derived from disrupted cells or lipid membranes derived from liposomes.

[0026] As used herein, "T cell receptor (TCR)" refers to a receptor present on the surface of T cells that recognizes antigens bound to major histocompatibility complex (MHC) molecules. Known T cell receptors include a dimer consisting of an α chain and a β chain (called αβTCR) and a dimer consisting of a γ chain and a δ chain (called γδTCR). αβTCR binds to other adaptor proteins such as CD3 protein to form a complex. As used herein, a complex containing a T cell receptor consisting of an α chain and a β chain and an adaptor protein is referred to as a "T cell receptor complex (TCR complex)." Adaptor proteins that form the TCR complex together with αβTCR include CD3δ protein, CD3γ protein, CD3ε protein, and CD3ζ protein.

[0027] As used herein, the term "cancer antigen" refers to an antigen capable of distinguishing cancer cells from normal cells. Cancer antigens are also called tumor antigens or tumor-associated antigens and are typically biomolecules that are specifically and / or overexpressed in cancer cells. The cancer antigens targeted by the multispecific nanoparticles of the present invention are, in principle, biomolecules such as proteins, sugar chains, or lipids that are presented 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. An example of a peptide fragment is a peptide fragment that is degraded by proteasomes in cancer cells and then presented on the surface of cancer cells together with HLA molecules. As used herein, the term "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. Specific examples of cancer antigens include, but are not limited to, A33, BAGE, B-cell maturation antigen (BCMA), Bcl-2, β-catenin, CA19-9, CA125, carboxyanhydrase-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, ephrin B2, estrogen receptor, FAP, folate binding protein, GAGE, G250, disialoganglioside (GD2), GM2, gp75, gp100 (Pmel 17), ERBB2 (HER-2 / neu), HPV E6, HPV Examples include E7, IGF1R, L1-CAM, LRP, MAGE, MART, mesothelin, MUC (MUC1, MUC2, etc.), MUM-1-B, myc, NYESO-1, p53, PRAME, progesterone receptor, PSA, PSCA, PSMA, ras, ROR1, survivin, SV40 T, tenascin, TNF-α, TSTA tyrosinase, VEGF, WT1, hTERT, and PAP.

[0028] As used herein, the term "membrane protein" includes integral membrane proteins, peripheral membrane proteins, and lipid-anchored proteins.

[0029] As used herein, an "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. An integral monotopic protein is a membrane protein that does not completely penetrate the membrane but protrudes from only one side of the membrane. A transmembrane protein is a membrane protein that completely penetrates the membrane. Transmembrane proteins are divided into single-pass transmembrane proteins that have one transmembrane domain and multi-pass transmembrane proteins that have two or more transmembrane domains. A specific example of a single-pass transmembrane protein is an immunoglobulin heavy chain. A specific example of a multi-pass transmembrane protein is a choline transporter, a histamine H1 receptor, and a G protein-coupled receptor.

[0030] As used herein, a "peripheral protein" is a protein that is not itself embedded in the membrane, but is anchored to the membrane by binding to lipids or integral membrane proteins.

[0031] As used herein, the term "lipid-anchored protein" refers to a protein that is anchored to a membrane by a lipid attached thereto through lipid modification. Specific examples include glycosylphosphatidylinositol (GPI)-conjugated proteins, prenylated proteins, cholesterol-conjugated proteins, and fatty acid-acylated (e.g., S-palmitoylated and N-myristoylated) proteins.

[0032] Membrane protein fragments include portions of any of the above membrane proteins that can be anchored to a membrane. For example, fragments containing one or more transmembrane domains of a transmembrane protein include the transmembrane domains of CD3 protein, CD4 protein, CD8 protein, CD28 protein, and IL-2 receptor.

[0033] As used herein, the term "costimulatory molecule" refers to a molecule that mediates an additional signal required for antigen-presenting cells to activate T cells. It is also called a costimulatory factor. Examples of costimulatory molecules include CD28 protein, CD40 protein, CD70 protein, CD80 protein, CD86 protein, ICOS ligand protein, OX40 ligand protein, 4-1BB ligand (also known as 4-1BBL or CD137L) protein, GITR ligand protein, and LIGHT protein.

[0034] As used herein, "cytokine" is a general term for proteins with relatively small molecular weights secreted by cells. Cytokines are mainly secreted by immune cells and are responsible for intercellular signaling. Examples of cytokines include interleukins, interferons, chemokines, hematopoietic factors, cell growth factors, and tumor necrosis factors. 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. Specific examples of hematopoietic factors include GM-CSF. Specific examples of interferons include IFN-γ proteins. Specific examples of cell growth factors include TGF-β proteins. Specific examples of tumor necrosis factors include TNF-α proteins.

[0035] As used herein, the term "cytokine receptor" refers to a protein that functions as a receptor for the cytokine. Examples of cytokine receptors include interleukin receptors, interferon receptors, chemokine receptors, hematopoietic factor receptors, growth factor receptors, and tumor necrosis factor receptors. The cytokine receptor may be a receptor for any of the above cytokines (e.g., TGF-β 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 the IL-4 receptor subunit α (IL4RA) and the IL-2 receptor common γ chain (IL2RG). The IL-10 receptor may be a heterodimer composed of the IL-10 receptor subunit α (IL10RA) and the IL-10 receptor subunit β. The IL-13 receptor may be a heterodimer composed of the IL-13 receptor subunit α1 (IL13RA1) and the IL-4 receptor subunit α (IL4RA). An example of IL10RA is human-derived IL10RA consisting of the amino acid sequence set forth in SEQ ID NO: 597. An example of IL10RB is human-derived IL10RB consisting of the amino acid sequence set forth in SEQ ID NO: 598. An example of IL4RA is human-derived IL4RA consisting of the amino acid sequence set forth in SEQ ID NO: 599. An example of IL2RG is human-derived IL2RG consisting of the amino acid sequence set forth in SEQ ID NO: 600. An example of IL13RA1 is human-derived IL13RA1 consisting of the amino acid sequence set forth in SEQ ID NO: 601. An example of a CSF-1 receptor is human-derived colony-stimulating factor 1 receptor (CSF1R) consisting of the amino acid sequence set forth in SEQ ID NO: 602.

[0036] As used herein, the term "chemotactic molecule" refers to a molecule that induces the migration of migratory cells. Examples of migratory cells include leukocytes such as granulocytes (e.g., neutrophils, eosinophils, or basophils) or mononuclear cells (e.g., monocytes or lymphocytes). Examples of chemotactic molecules include interleukins, cytokines, chemokines, homing molecules, adhesion molecules, and the like.

[0037] As used herein, a "homing molecule" refers to a surface molecule expressed by lymphocytes when homing to a specific organ. Examples of 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, and β2-integrin.

[0038] As used herein, the term "adhesion molecule (cell adhesion molecule)" refers to a molecule that mediates binding between cells or between cells and the extracellular matrix. Examples of adhesion molecules include proteins belonging to the cadherin superfamily, proteins belonging to the immunoglobulin superfamily, and proteins belonging to the integrin family.

[0039] As used herein, the term "immune checkpoint inhibitor molecule" refers to a molecule capable of inhibiting cancer cells from evading the host immune response. Examples of immune checkpoint inhibitor molecules are known in the art, including CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, and BTLA inhibitors. Specific examples include antibodies and fragments thereof, such as pembrolizumab, nivolumab, atezolizumab, durvalumumab, avelumab, tremelimumab, and ipilimumab.

[0040] As used herein, the term "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 avoid attack from the immune system by suppressing the activation of immune cells via immune checkpoint molecules. Examples of immune checkpoint molecules include PD1 protein, Lag-3 protein, Tim-3 protein, TIGIT protein, ICOS protein, and BTLA protein.

[0041] A "signal peptide" is an extracellular localization signal required for the extracellular secretion of a protein biosynthesized by gene expression, and is also called a signal peptide. The signal sequence may contain a region composed of hydrophobic amino acids. After translation, the signal peptide is cleaved and removed by a signal peptidase before being translocated outside the cell. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and are, for example, 15 to 30 amino acids long. The signal peptide may be derived from any biological species, either human or non-human, for example, from insect cells or viruses, but is preferably derived from humans. A specific example of a signal peptide is the signal peptide derived from human Oncostatin M.

[0042] A "linker peptide" is a peptide that can be inserted between the fused moieties in a fusion protein such as the chimeric cytokine receptor of the present invention so that each fused moiety can perform its intended function. There are no limitations on the length of the linker peptide, but examples of such a peptide are 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.

[0043] A "tag peptide" is a short peptide consisting of a dozen to several tens of amino acids that can be used to label proteins, and is used for protein detection and purification. Typically, a base sequence encoding the tag peptide is linked to the 5'-end or 3'-end of a gene encoding the protein to be labeled, and the protein is expressed as a fusion protein with the tag peptide, thereby achieving labeling. Various types of tag peptides have been developed in the field, and any tag peptide may be used. Specific examples of tag peptides include FLAG, HA, His, PA, and myc.

[0044] As used herein, the term "antibody" refers to a protein that exhibits immune response to an antigen. Unless otherwise specified, the term refers to a monoclonal antibody. The 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, and human.

[0045] Furthermore, as used herein, the term "monoclonal antibody" refers to a single type of immunoglobulin that contains a framework region (hereinafter referred to as "FR") and a complementarity determining region (hereinafter referred to as "CDR") and is capable of specifically binding to and recognizing an antigen, or at least one set of light chain variable regions (V) contained in an immunoglobulin. L region) and heavy chain variable region (V H A recombinant or synthetic antibody refers to an antibody that contains a specific region.

[0046] When an antibody is composed of immunoglobulin molecules, the immunoglobulin can be of any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0047] "Recombinant antibody" refers to a chimeric antibody or a humanized antibody. A "chimeric antibody" is an antibody created by combining the amino acid sequences of antibodies from different animals, in which the constant region (C region) of one antibody is replaced with the C region of another antibody. For example, an antibody in which the C region of a rat monoclonal antibody is replaced with the C region of a human antibody falls into this category. Specific examples include antibodies in which the heavy chain variable region of a human antibody against a given antigen is replaced with the heavy chain variable region of an antibody against a target antigen, and the light chain variable region of a human antibody is replaced with the light chain variable region of an antibody against a target antigen. This can reduce the immune response to the antibody in the human body. A "humanized antibody" is a mosaic antibody in which the CDRs of a human antibody are replaced with the CDRs of an antibody derived from a mammal other than human. The variable region (V region) of an immunoglobulin molecule is composed of four FRs (FR1, FR2, FR3, and FR4) and three CDRs (CDR1, CDR2, and CDR3) linked in the following order from the N-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The FRs are relatively conserved regions that form the framework of the variable region, while the CDRs directly contribute to the antigen-binding specificity of the antibody. Humanized antibodies can be constructed by, for example, replacing a set of CDR1, CDR2, and CDR3 in the light or heavy chain of an antibody against an antigen of interest with a set of CDR1, CDR2, and CDR3 in the light or heavy chain of a human antibody against any antigen, thereby inheriting the antigen-binding specificity of the antibody against the antigen of interest. Because such humanized antibodies are derived from human antibodies except for the CDRs, they can reduce the immune response to the antibody in the human body more than chimeric antibodies.

[0048] "Synthetic antibody" refers to an antibody synthesized chemically or by using recombinant DNA technology. For example, it includes antibodies newly synthesized using recombinant DNA technology. Specific examples include scFv (single chain fragment of variable region). In an immunoglobulin molecule, a set of variable regions (light chain variable region V) that form a functional antigen-binding site is called a "synthetic antibody." L and heavy chain variable region V H) are located on separate polypeptide chains, known as light and heavy chains. scFvs are immunoglobulin molecules with V L and V H scFv is a synthetic antibody with a molecular weight of approximately 35 kDa or less, in which a set of variable regions are linked by a sufficiently long flexible linker and contained within a single polypeptide chain. Within an scFv, a pair of variable regions can self-assemble to form a functional antigen-binding site. scFv can be obtained by inserting recombinant DNA encoding it into a vector using known techniques and expressing it.

[0049] The antibody can also be modified. The term "modification" as used herein includes functional modifications necessary for antigen-specific binding activity, such as glycosylation, and labeling modifications necessary for antibody detection.

[0050] Glycosylation modifications on antibodies are performed to adjust the affinity of the antibody for its target antigen. Specific examples include substitutions of glycosylated amino acid residues in the antibody FR to remove the glycosylation site, thereby eliminating glycosylation at that site.

[0051] The dissociation constant of an antibody with an antigen is 10 -7 It is preferable that the value is equal to or less than M, for example, 10 -8 It is preferable that the affinity is as high as 10 M or less, and more preferably 10 -9 M or less, particularly preferably 10 -10 The dissociation constant is equal to or less than M. The dissociation constant can be measured using techniques known in the art. For example, it may be measured using a Biacore system (GE Healthcare) with rate evaluation kit software.

[0052] As used herein, the term "fragment" of an antibody refers to an antibody fragment that consists of a part of an antibody and exhibits immunoreactivity to an antigen, similar to an antibody, and is an antigen-binding fragment. Examples of such fragments include Fab, Fab', F(ab')2, Fv fragment, Fv fragment stabilized by a disulfide bond (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), diabody stabilized by a disulfide bond (dsdiabody), single-chain antibody molecule (scFv), dimeric scFv (bivalent diabody), multispecific antibody, heavy chain antibody such as camelized single domain antibody (camelized antibody; VHH antibody), nanobody, domain antibody, and bivalent domain antibody. Fab is an antibody fragment generated by cleavage of an IgG molecule with papain at the N-terminal side of the hinge disulfide bond, and is composed of an H-chain constant region (heavy chain constant region: hereinafter referred to as C H The three domains that make up the H 1. C H 2. C H 3) V H C adjacent to H 1 and V H , and a full-length L chain. Fab' has a slightly longer H chain than Fab, including the hinge region, but has essentially the same structure as Fab. Fab' can be obtained by reducing the Fab' dimer (F(ab')2) produced when IgG molecules are cleaved with pepsin at the C-terminal side of the disulfide bond in the hinge region under mild conditions to cleave the disulfide bond in the hinge region. All of these antibody fragments contain the antigen-binding site and therefore have the ability to specifically bind to antigen epitopes.

[0053] As used herein, the term "nanoparticle core" refers to a core particle that constitutes the interior of a nanoparticle. In the multispecific nanoparticles of the present invention, this refers to a core particle encapsulated in a lipid membrane that constitutes the surface layer of the nanoparticle. The material of the core particle is not particularly limited, and it may be a polymer nanoparticle, a metal nanoparticle, or a dendrimer. Note that, as used herein, multispecific nanoparticles may or may not encapsulate a nanoparticle core.

[0054] The polymer that constitutes the polymer nanoparticles is preferably a biocompatible and biodegradable polymer, and examples thereof include natural polymers such as gelatin, collagen, fibrin, polyaspartic acid, polyglutamic acid, polyleucine, polysaccharides, cellulose, alginate, dextran, amylose, pectin, chitosan, chitin, heparin, and hyaluronic acid, as well as synthetic polymers such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), PLGA-ethylene oxide fumarate, PLGA-TGPS, pCPH, PHB-PVA, PEG-PLA, PCL, PAC, PEC, polyisobutyl cyanoacrylate, poly(HPMA), PHB, PHA, poly-β-R-malic acid, ethyl cellulose, and polybutylene succinate (PBS). Polylactic acid-polyglycolic acid copolymer (PLGA) is a polyester containing glycolic acid and lactic acid monomers, and is a well-known biocompatible and biodegradable polymer approved by the U.S. Food and Drug Administration (FDA) for use in tissue engineering and pharmaceutical delivery systems.

[0055] The material of the metal nanoparticles is not particularly limited, but may be, for example, gold, platinum, silver, or iron.

[0056] The dendrimer is not particularly limited as long as it is a pharmaceutically acceptable dendrimer, and examples thereof include PAMAM dendrimers, G5 dendrimers, acylated dendrimers, PEGylated dendrimers, and acetylated dendrimers.

[0057] As used herein, the term "liposome" refers to a vesicle comprising 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 or the like.

[0058] As used herein, the term "sustained release" refers to the gradual release of a substance into space. In this specification, the term "sustained release" refers to the gradual diffusion of a component within the lipid membrane from the multispecific nanoparticle of the present invention to its exterior. The period during which the component continues to be diffused includes, for example, one hour or more, two hours or more, three hours or more, six hours or more, half a day or more, one day or more, two days or more, three days or more, one week or more, two weeks or more, or one month or more.

[0059] As used herein, when a protein is identified by a specific amino acid sequence, it is intended to also refer to the corresponding wild-type and mutant proteins derived from any biological species. For example, it is intended to include proteins consisting of amino acid sequences in which one or more amino acids are deleted, substituted, or added, or amino acid sequences that are 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequences described herein. The same applies to nucleotide sequences.

[0060] As used herein, "amino acid identity (amino acid sequence identity)" refers to the percentage (%) of matching amino acid residues in the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when the sequences are aligned by inserting appropriate gaps into one or both sequences as needed to maximize the number of identical amino acid residues. "Nucleotide identity (nucleotide sequence identity)" can be determined in a similar manner.

[0061] As used herein, "amino acid substitution" refers to substitutions among the 20 types of amino acids that constitute natural proteins. Amino acid substitutions are preferably within conservative amino acid groups that have similar properties, such as charge, side chain, polarity, and aromaticity. Examples of such substitutions include substitutions within the group of uncharged polar amino acids with low polarity 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), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp).

[0062] 1-3. Configuration The multispecific nanoparticles of the present invention comprise a lipid membrane that forms the surface layer of the nanoparticle, and (i) a fusion protein comprising a membrane-binding region bound to the lipid membrane and two or more target-binding regions located outside the lipid membrane, or (ii) two or more fusion proteins comprising a membrane-binding region bound to the lipid membrane and target-binding regions located outside the lipid membrane.

[0063] In one embodiment, the multispecific nanoparticles of the present invention comprise the fusion protein (i) above. In the following description of this specification, a fusion protein having the configuration (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, is referred to as a "type I fusion protein." Type I fusion proteins can comprise two or more target-binding regions on the N-terminus and a membrane-binding region on the C-terminus, or a membrane-binding region on the N-terminus and two or more target-binding regions on the C-terminus.

[0064] As used herein, multispecific nanoparticles comprising type I fusion proteins 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.

[0065] In one embodiment, the multispecific nanoparticle of the present invention comprises the fusion protein (ii) above. In (ii) above, each of the two or more fusion proteins comprises a membrane-binding region bound to a lipid membrane and one or more target-binding regions located outside the lipid membrane. In the following description of this specification, a fusion protein having the configuration (ii) above, specifically a fusion protein comprising a membrane-binding region bound to a lipid membrane and one or more target-binding regions located outside the lipid membrane, is referred to as a "type II fusion protein." Specifically, a type II fusion protein may comprise a target-binding region on the N-terminus and a membrane-binding region on the C-terminus, or a membrane-binding region on the N-terminus and a target-binding region on the C-terminus.

[0066] Herein, multispecific nanoparticles containing type II fusion proteins are referred to as "type II multispecific nanoparticles." Type II multispecific nanoparticles are characterized by containing two or more fusion proteins, each containing at least one target-binding region. Therefore, it is understood that type II multispecific nanoparticles contain a total of two or more target-binding regions.

[0067] In a further embodiment, the multispecific nanoparticles of the present invention can comprise both the Type I and Type II fusion proteins described above, with it being understood that such embodiments are encompassed by and not excluded from either the Type I or Type II multispecific nanoparticles described above.

[0068] As used herein, unless otherwise specified, the term "fusion protein" refers to either the type I fusion protein or the type II fusion protein described above. In addition to the membrane-binding domain and target-binding domain described in detail below, the type I fusion protein and the type II fusion protein may optionally contain a cytoplasmic domain, a signal peptide, a linker peptide, and / or a tag peptide.

[0069] As used herein, a "membrane-binding region" refers to a region that stably incorporates or attaches a fusion protein to a lipid membrane. A "membrane-binding region bound to a lipid membrane" means that the membrane-binding region is in contact with, attached to, or bound to a lipid membrane through hydrophobic interactions. Examples of membrane-binding regions include the above-mentioned membrane proteins or fragments thereof, and artificial transmembrane domains that primarily contain hydrophobic amino acid residues. The membrane-binding region may be derived from the same species as the signal sequence and / or from the same protein.

[0070] As used herein, the term "target-binding region" refers to a region in the type I fusion protein or type II fusion protein that can bind to a target molecule or cell. More specifically, it refers to a region that can bind to a surface antigen expressed on the surface of a target cell. In the above configurations (i) and (ii), 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 molecule. However, if the molecule is the same, it is preferable that they bind to different epitopes. Examples of two or more target molecules include a combination of (a) a T cell receptor complex, an NK cell surface antigen, or a macrophage surface antigen, and (b) a cancer antigen.

[0071] In one embodiment, the multispecific nanoparticles of the present invention target T cells and cancer cells, and the target-binding region comprises a T cell receptor complex-binding region and a cancer antigen-binding region. Here, "the target-binding region comprises a T cell receptor complex-binding region and a cancer antigen-binding region" means that, in the above-described type I multispecific nanoparticles, two or more target-binding regions contained in the type I fusion protein comprise a T cell receptor complex-binding region and a cancer antigen-binding region, or that, in the above-described type II multispecific nanoparticles, separate type II fusion proteins comprise a T cell receptor complex-binding region and a cancer antigen-binding region. In the case of type I multispecific nanoparticles, the positional relationship between the T cell receptor complex-binding region and the cancer antigen-binding region does not matter; the cancer antigen-binding region may be located either N-terminally or C-terminally of the T cell receptor complex-binding region.

[0072] As used herein, the term "T cell receptor complex-binding region" refers to a region on the cell surface of a T cell that can bind to the T cell receptor complex. The T cell receptor complex-binding region is preferably a region that activates T cells by binding to the T cell receptor complex and can induce effector activity such as cytotoxicity against cancer cells. The protein to which the T cell receptor complex-binding region binds is not particularly limited, as long as it is a component of the T cell receptor complex. Examples of proteins to which the T cell receptor complex-binding region binds include any component of the T cell receptor (e.g., the T cell receptor α chain, the T cell receptor β chain, the T cell receptor γ chain, or the T cell receptor δ chain), as well as any adaptor protein contained in the T cell receptor complex, such as CD3δ protein, CD3γ protein, CD3ε protein, and CD3ζ protein. Specific examples of T cell receptor complex binding regions include anti-T cell receptor (TCR) antibodies (e.g., anti-T cell receptor α chain antibodies, anti-T cell receptor β chain antibodies, anti-T cell receptor γ chain antibodies, and anti-T cell receptor δ chain antibodies) and anti-CD3 antibodies (e.g., anti-CD3δ antibodies, anti-CD3γ antibodies, anti-CD3ε antibodies, and anti-CD3ζ antibodies). Further examples of T cell receptor complex binding regions that bind to T cell receptors include HLA / peptide fusion molecules.

[0073] As used herein, the term "HLA / peptide fusion molecule" refers to a fusion molecule comprising an HLA class I molecule or an HLA class II molecule and a peptide. Here, an HLA class I molecule has a structure in which an α chain and a β2-microglobulin are associated. Furthermore, an HLA class II molecule has a structure in which an α chain and a β chain are associated. As used herein, the HLA antigens of both HLA class I molecules and HLA class II molecules are not particularly limited. The peptide contained in the HLA / peptide fusion molecule is a peptide consisting of several to several dozen amino acid residues, e.g., 5 to 20 amino acid residues, that binds to the antigen-presenting groove in an HLA class I molecule or an HLA class II molecule. In addition to the HLA class I molecule or an HLA class II molecule and the peptide, the HLA / peptide fusion molecule may also contain a linker peptide, such as a flexible linker, connecting 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 these are not particularly limited. An example of an α chain of an HLA class I molecule is the human α chain consisting of the amino acid sequence shown in SEQ ID NO: 26 and belonging to HLA-A:0201. An example of β2 microglobulin is the human β2 microglobulin consisting of the amino acid sequence shown in SEQ ID NO: 27. An example of an α chain of an HLA class II molecule is the human α chain consisting of the amino acid sequence shown in SEQ ID NO: 28 and belonging to HLA-DRA*01:01. An example of a β chain of an HLA class II molecule is the human β chain consisting of the amino acid sequence shown in SEQ ID NO: 29 and belonging to HLA-DRB*01:01.

[0074] In one embodiment, the HLA / peptide fusion molecule comprises an α chain of an HLA class I molecule, β2 microglobulin, and an antigenic peptide, and optionally a linker peptide. Honda, T., et al., Protein Eng Des Sel., 2015, 28(2):53-8, discloses a fusion molecule comprising, from the N-terminus, an antigenic peptide, a peptide linker, β2 microglobulin, and an α chain of an HLA class I molecule, as well as a fusion molecule comprising, from the N-terminus, β2 microglobulin, the α chain of an HLA class I molecule, a peptide linker, and an antigenic peptide.

[0075] In another embodiment, the HLA / peptide fusion molecule comprises β2 microglobulin and an antigenic peptide, and optionally a linker peptide. A literature article (Tafuro, S., et al., Eur J Immunol., 2001, 31(2):440-9) discloses a fusion molecule comprising, in order from the N-terminus, an antigenic peptide, a peptide linker, and β2 microglobulin, and that the fusion molecule functions as an HLA molecule together with the α chain of an HLA class I molecule.

[0076] In one embodiment, the HLA / peptide fusion molecule comprises the α chain, β chain, and antigenic peptide of an HLA class II molecule, and optionally a linker peptide.

[0077] HLA / peptide fusion molecules can activate T cells that have a T cell receptor that recognizes a peptide as an antigen, such as CD8-positive T cells and CD4-positive T cells. Unlike anti-CD3 antibodies and the like, which can nonspecifically activate T cells regardless of the antigen, HLA / peptide fusion molecules have the advantage of being able to specifically activate only T cells that have a T cell receptor for a specific antigen.

[0078] In one embodiment, the T cell receptor complex binding region is an anti-CD3 antibody or a fragment thereof. Examples of anti-CD3 antibodies include anti-CD3δ antibodies, anti-CD3γ antibodies, anti-CD3ε antibodies, and anti-CD3ζ 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 Table 1 below illustrates an anti-CD3ε antibody. CDRs are numbered according to the Kabat antibody numbering system.

[0079]

[0080] Further specific examples of anti-CD3 antibodies include anti-CD3ε antibodies disclosed in WO 2021 / 224499 and comprising the same set of CDRs as the above-mentioned hXR32 antibody, i.e., anti-CD3ε antibodies comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 106, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 107 to 109, respectively (for example, an anti-CD3ε antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 343 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 344); and anti-CD3ε antibodies disclosed in WO 2017 / 112762 and comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 345 to 347, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 348, CDR2 consisting of the amino acid sequence GAS (SEQ ID NO: 349), and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 350. an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 351 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 352; an anti-CD3ε antibody disclosed in WO 2015 / 095392, comprising a heavy chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 353 to 355, respectively, and a light chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 356 to 358, respectively; an anti-CD3ε antibody disclosed in WO 2017 / 210485, comprising a heavy chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 359 to 361, respectively, and a light chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 362 to 364, respectively (e.g., an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 365 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 366);an anti-CD3ε antibody disclosed in WO 2016 / 020309, which comprises 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 a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 370 to 372, respectively (e.g., an anti-CD3ε antibody comprising 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); an anti-CD3ε antibody disclosed in WO 2016 / 141303, which comprises the amino acid sequences shown in SEQ ID NOs: 375 to 377, respectively; an anti-CD3ε antibody comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 378 to 380 (for example, an anti-CD3ε antibody comprising 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); an anti-CD3ε antibody disclosed in WO 2017 / 134140, comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 383 to 385, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 386 to 388, respectively; an anti-CD3ε antibody (for example, an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 389 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 390); an anti-CD3ε antibody (for example, an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 391 to 393, respectively, and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 394 to 396, respectively, as disclosed in U.S. Patent Application Publication No. 2019 / 0002568; for example, an anti-CD3ε antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 397 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 398), and an anti-CD3ε antibody comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 399 to 401, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 402 to 404, respectively (for example, an anti-CD3ε antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 405 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 406);an anti-CD3ε antibody disclosed in WO 2013 / 072415, which comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 407 to 426, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 427 to 434; an anti-CD3ε antibody (e.g., an anti-CD3ε antibody) disclosed in WO 2017 / 134134, which comprises a heavy chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 435 to 437, respectively, and a light chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 438 to 440, respectively (e.g., an anti-CD3ε antibody) an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 443 to 445, respectively, and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 446 to 448, respectively, as disclosed in WO 2021 / 228783 and WO 2019 / 220368 (e.g., an anti-CD3ε antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 449 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 450);an anti-CD3ε antibody disclosed in WO 2021 / 113701, comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 451 to 453, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 454, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 455), and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 456 (e.g., an anti-CD3ε 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 an anti-CD3ε antibody comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 459 to 461, respectively, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 462, CDR2 consisting of the amino acid sequence AAS (SEQ ID NO: 463), and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 464 (for example, an anti-CD3ε 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); an anti-CD3ε antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 492 and 492, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 494 to 518; an anti-CD3ε antibody disclosed in U.S. Patent Application Publication No. 2022 / 0025047, comprising a heavy chain variable region comprising CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 519 to 521, respectively, and a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 522, CDR2 comprising the amino acid sequence GAS (SEQ ID NO: 523), and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 524; an anti-CD3ε antibody comprising a variable region (e.g., an anti-CD3ε antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 525 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 526); an anti-CD3ε antibody disclosed in WO 2016 / 205520 comprising a heavy chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 527 to 529, respectively, and a light chain variable region comprising CDR1 to CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 530 to 532 or the amino acid sequences set forth in SEQ ID NOs: 530 to 531 and 533, respectively;and anti-CD3ε antibodies comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 566 to 568, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 569 to 571, respectively (for example, an anti-CD3ε antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 572 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 573; or anti-CD3ε antibody clone 2C11).

[0081] In one embodiment, 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 α chain, the T cell receptor β chain, or both. Anti-TCR monoclonal antibodies such as WT31 and BMA031 are known to be able to 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 the heavy and light chain variable regions and CDRs of exemplary anti-TCR antibodies are shown in Table 2 below. CDRs are numbered according to the Kabat antibody numbering system. In addition to the examples in Table 2 below, the heavy chain variable region and light chain variable region, as well as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, chain CDR2, and light chain CDR3 of WT31 are also exemplified.

[0082]

[0083] As used herein, the term "cancer antigen-binding region" refers to a region on the cell surface of a cancer cell that can bind to a cancer antigen. The type of cancer antigen is not particularly limited and can be arbitrarily selected from, for example, the cancer antigens described above.

[0084] In one embodiment, the cancer antigen is CD19, BCMA, mesothelin, GD2, CD20, CD22, EGFR, CD33, CD123, ERBB2, CD133, CEA, CEACAM5, MUC1, or PSMA, all of which have been developed as CARs and are undergoing clinical trials.

[0085] In further embodiments, 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 fragment thereof, an anti-EGFR antibody or fragment thereof, an anti-CD33 antibody or fragment thereof, an anti-CD123 antibody or fragment thereof, an anti-ERBB2 antibody or fragment thereof, an anti-CD133 antibody or fragment thereof, an anti-CEA antibody or fragment thereof, an anti-CEACAM5 antibody or fragment thereof, an anti-MUC1 antibody or fragment thereof, or an anti-PSM antibody or fragment thereof.

[0086] In one embodiment, the cancer antigen-binding region is an anti-CD19 antibody or fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-CD19 antibodies are shown in Table 3 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0087]

[0088] Further examples of 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 light chain variable region comprising CDR1, CDR2, and CDR3 contained in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 581 (for example, an anti-CD19 antibody comprising 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).

[0089] In one embodiment, the cancer antigen-binding region is an anti-mesothelin antibody or fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-mesothelin antibodies are shown in Table 4 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0090]

[0091] In one embodiment, the cancer antigen-binding region is an anti-BCMA antibody or fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-BCMA antibodies are shown in Table 5 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0092]

[0093] In one embodiment, the cancer antigen-binding region is an anti-EGFR antibody or a fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-EGFR antibodies are shown in Table 6 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0094]

[0095] In one embodiment, the cancer antigen-binding region is an anti-GD2 antibody or a fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-GD2 antibodies are shown in Table 7 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0096]

[0097] In one embodiment, the multispecific nanoparticles of the present invention comprise 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 comprises an anti-CD3 antibody or a fragment thereof, and the cancer antigen-binding region comprises an anti-CD19 antibody or a fragment thereof. An example of such a fusion protein is a type I fusion protein comprising the amino acid sequence set forth in SEQ ID NO:5.

[0098] In one embodiment, the multispecific nanoparticles of the present invention comprise 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, wherein the T cell receptor complex-binding region comprises an anti-CD3 antibody or a fragment thereof, and the cancer antigen-binding region comprises an anti-CD19 antibody or a fragment thereof. Examples of such fusion proteins include a type II fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:9 and a type II fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:11.

[0099] In one embodiment, the multispecific nanoparticles of the present invention comprise 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 comprises an anti-CD3 antibody or a fragment thereof, and the cancer antigen-binding region comprises an anti-mesothelin antibody or a fragment thereof. Examples of such a fusion protein include a type II fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:9 and a type II fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:21.

[0100] In one embodiment, the multispecific nanoparticles of the present invention comprise 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 comprises an anti-CD3 antibody or fragment thereof, and the cancer antigen binding region comprises an anti-BCMA antibody or fragment thereof. An example of such a type I fusion protein is the amino acid sequence set forth in SEQ ID NO: 17.

[0101] In one embodiment, the multispecific nanoparticles of the present invention target NK cells and cancer cells, and the target-binding region comprises an NK cell surface antigen-binding region and a cancer antigen-binding region, as defined above.

[0102] As used herein, the term "NK cell surface antigen-binding region" refers to a region on the cell surface of an NK cell that can bind to any surface antigen of that cell. Examples of NK cell surface antigens include, but are not limited to, CD16, NKG2D, Nkp30, Nkp44, Nkp46, and 2B4. Examples of these antigens are disclosed in the literature (Gauthier, L., et al., Cell, 2019, 177(7):1701-1713.e16.). Other examples include KIR family molecules, Ly49 family molecules, CD94-NKG2C / E / H heterodimeric receptor, 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.

[0103] In one embodiment, 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 anti-2B4 antibody or fragment thereof.

[0104] Furthermore, ligand molecules or binding fragments thereof that bind to NK cell surface antigens can also be used as NK cell surface antigen-binding regions. Specific examples include the IgG Fc region or fragment thereof that binds to CD16, the MICA protein or fragment thereof and the MICB protein or fragment thereof that bind to NKG2D, the B7H6 protein or fragment thereof that binds to Nkp30, and influenza virus hemagglutinin or fragment thereof that binds to NKp44 or NKp46. The IgG Fc region may be the Fc region of any subclass (IgG1, IgG2, IgG3, and IgG4). Although not limited thereto, IgG1 and IgG3 Fc regions are preferred, as they can transmit potent activation signals to NK cells and macrophages. Specific examples of the above include the 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), human B7H6 protein (SEQ ID NO: 33), and influenza virus hemagglutinin (SEQ ID NO: 34).

[0105] In one embodiment, the NK cell surface antigen-binding region is an anti-CD16 antibody or fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-CD16 antibodies are shown in Table 8 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0106]

[0107] In one embodiment, the NK cell surface antigen-binding region is an anti-NKG2D antibody or fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-NKG2D antibodies are shown in Table 9 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0108]

[0109] In one embodiment, the NK cell surface antigen-binding region is an anti-NKp46 antibody or a fragment thereof. The sequences of the heavy and light chain variable regions and CDRs of exemplary anti-NKp46 antibodies are shown in Table 10 below. The CDRs are numbered according to the Kabat antibody numbering system.

[0110]

[0111] In one embodiment, the multispecific nanoparticles of the present invention target macrophage cells and cancer cells, and the target-binding regions comprise a macrophage cell surface antigen-binding region and a cancer antigen-binding region, as defined above.

[0112] As used herein, the term "macrophage surface antigen-binding region" refers to a region on the cell surface of a macrophage that can bind to any surface antigen thereof. Examples of macrophage surface antigens include, but are not limited to, FCγRI (CD64), FCγRIIA (CD32), FCγRIIIA (CD16), CD40, CD47, and TLR4a.

[0113] In one embodiment, the macrophage surface antigen-binding region is an anti-FCγRI (CD64) antibody or fragment thereof, an anti-FCγRIIA (CD32A) antibody or fragment thereof, an anti-FCγRIIIA (CD16) antibody or fragment thereof, an anti-CD40 antibody or fragment thereof, an anti-CD47 antibody or fragment thereof, or an anti-TLR4a antibody or fragment thereof. Examples of anti-CD47 antibodies include anti-CD47 antibodies comprising 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 (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); and anti-CD47 VHH comprising a variable region comprising CDR1 to CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 583 to 585, respectively (e.g., an anti-CD47 VHH comprising a variable region shown in SEQ ID NO: 586).

[0114] Furthermore, a ligand molecule or a binding fragment thereof that binds to a macrophage surface antigen can also be used as a macrophage surface antigen-binding region. Specific examples include an IgG Fc region that binds to FcγRI (CD64) (e.g., the Fc regions of IgG1, IgG2, IgG3, and IgG4, preferably the Fc regions of IgG1 and IgG3; for example, a human IgG1 Fc region consisting of the amino acid sequence set forth in SEQ ID NO: 30) or a fragment thereof, a CD40L protein that binds to CD40 or a fragment thereof, and a TLR4a ligand that binds to TLR4a. While an IgG Fc region is not normally expressed on the cell membrane, it can be expressed on the cell membrane as a fusion polypeptide linked to a type II membrane protein or a portion thereof whose C-terminus is extracellularly located. The use of an IgG Fc region as a macrophage surface antigen-binding region is preferred because it enables the use of a signal that potently activates macrophages via FcγRI (CD64). Specific examples of these include the aforementioned human IgG Fc region and human CD40L protein (SEQ ID NO: 35). Specific examples of TLR4a ligands include TLR4a agonists consisting of the amino acid sequence shown in SEQ ID NO: 582 or SEQ ID NO: 596.

[0115] In one embodiment, the macrophage surface antigen-binding region is an anti-CD40 antibody or 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. The CDRs are numbered according to the Kabat antibody numbering system.

[0116]

[0117] In one embodiment, the multispecific nanoparticles of the present invention can further comprise, on the surface and / or interior of the nanoparticle, one or more molecules selected from the group consisting of costimulatory molecules, cytokine molecules, chemotactic molecules, immune checkpoint inhibitory molecules, and immune checkpoint molecules. The chemotactic molecules may be homing molecules, chemokine molecules, or adhesion molecules. Preferably, the multispecific nanoparticles of the present invention comprise 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. Therefore, the multispecific nanoparticles of the present invention preferably comprise costimulatory molecules and cytokine molecules. When the nanoparticles contain the above molecules on their surface, the cytokine molecules, chemotactic molecules, or homing molecules can be introduced onto the surface of the nanoparticles as fusion proteins in which any of these molecules is linked to a membrane-binding domain. Similarly, immune checkpoint inhibitory molecules can be introduced onto the surface of the nanoparticles as fusion proteins in which a corresponding antibody, such as an scFV, or an antibody fragment is linked to a membrane-binding domain. When the nanoparticles contain the above molecules internally, they can be encapsulated within the lipid membrane without being linked to a membrane-binding domain. Immune checkpoint inhibitor molecules can also be encapsulated within lipid membranes.

[0118] In one embodiment, the costimulatory molecule is a CD80 protein, a CD86 protein, a 4-1BB ligand protein, an ICOS ligand protein, a CD40 protein, a CD70 protein, an OX40 ligand protein, a GITR ligand protein, and / or a LIGHT protein, or a fusion molecule comprising any of these. Specific examples 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), 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).

[0119] In one embodiment, the cytokine molecule is an IL-7 protein, an IL-15 protein, an IL-21 protein, an IL-2 protein, an IL-12 protein, an IL-4 protein, an IL-10 protein, an IL-18 protein, a GM-CSF protein, an IFN-γ protein, a TNF-α protein, and / or a TGF-β protein, or a fusion molecule containing any of these. Specific examples 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-γ protein (SEQ ID NO: 52), human TNF-α protein (SEQ ID NO: 53), and human TGF-β protein (SEQ ID NO: 54). IFNγ and GM-CSF are preferred cytokine molecules, particularly in embodiments involving a macrophage surface antigen binding region.

[0120] In one embodiment, 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 containing 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: 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).

[0121] In one embodiment, the immune checkpoint inhibitor molecule is a CTLA-4 inhibitor (e.g., tremelimumab, ipilimumab; for example, an anti-CTLA-4 antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 558 to 560, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 561 to 563, respectively; or an anti-CTLA-4 antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 564 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 565), a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, durvalumumab), a PD-L1 inhibitor (e.g., atezolizumab, durvalumumab, avelumab; for example, an anti-CTLA-4 antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 558 to 560, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 561 to 563, respectively), a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, durvalumumab), a PD-L1 inhibitor (e.g., atezolizumab, durvalumumab, avelumab; for example, an anti-CTLA-4 antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 561 to 563, respectively), a PD-L1 inhibitor (e.g., atezolizumab, durvalu an anti-PD-L1 antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 550-552, and a light chain variable region comprising CDR1, CDR2, and CDR3, respectively, consisting of the amino acid sequences set forth in SEQ ID NOs: 553-555; or an anti-PD-L1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 556 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 557), a Lag-3 inhibitor (e.g., Relatlimab), a Tim-3 inhibitor, a TIGIT inhibitor (e.g., Ociperlimab), a B7-H3 inhibitor, a VISTA inhibitor, an ICOS inhibitor, an ICOS stimulant, a BTLA inhibitor, a CD47 inhibitor, a KIR inhibitor, an LIR inhibitor, a CD94 inhibitor, or an NKG2A inhibitor.

[0122] In one embodiment, the immune checkpoint molecule may be PD1 protein, Lag-3 protein, Tim-3 protein, TIGIT protein, ICOS protein, or BTLA protein. These immune checkpoint molecules are normally expressed in immune cells, but when contained in the surface layer of the multispecific nanoparticles of the present invention, they can bind to inhibitory ligands presented on cancer cells and inhibit their action on immune cells, thereby functioning similarly to the above-mentioned immune checkpoint inhibitor molecules. 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), human TIGIT protein (SEQ ID NO: 67), human ICOS protein (SEQ ID NO: 68), and human BTLA protein (SEQ ID NO: 69).

[0123] Further examples of immune checkpoint molecules include CD47 protein or fusion molecules containing the same. CD47 protein is known to function as an immune checkpoint molecule for macrophages. Therefore, when the multispecific nanoparticles of the present invention contain CD47 protein or a fusion molecule containing the same on the surface layer of the nanoparticles, they can avoid phagocytosis by macrophages. Avoidance of phagocytosis of nanoparticles by macrophages is preferred when the target-binding region contains a T cell receptor complex-binding region or an NK cell surface antigen-binding region. An example of a CD47 protein is human CD47 protein (SEQ ID NO: 63).

[0124] In one embodiment, the lipid membrane that constitutes the surface layer of the multispecific nanoparticle of the present invention is composed of a cell membrane, or a cell membrane and a liposome.

[0125] The multispecific nanoparticles of the present invention can contain a nanoparticle core and / or a medium such as water or physiological saline within the lipid membrane that forms the surface layer of the nanoparticle. The nanoparticle core or medium can contain cytokines, chemotactic molecules, etc. The molecules contained within the lipid membrane can be slowly released to the outside of the lipid membrane.

[0126] In one embodiment, the lipid membrane that constitutes the surface layer of the multispecific nanoparticles of the present invention can encapsulate a nanoparticle core, such as a polymer nanoparticle, a metal nanoparticle, or a dendrimer.

[0127] In another embodiment, the lipid membrane that constitutes the surface layer of the multispecific nanoparticles of the present invention comprises an aqueous solution or buffer solution such as water or physiological saline, and does not encapsulate the nanoparticle core.

[0128] In a further embodiment, the lipid membrane forming the surface layer of the multispecific nanoparticles of the present invention forms a liposome, and the liposome can encapsulate a nanoparticle core. Nanoparticles in which the liposome encapsulates a nanoparticle core such as a PLGA polymer are disclosed in the literature (Mandal, B., Nanomedicine, 2013, 9(4):474-91.).

[0129] The multispecific nanoparticles of the present invention can induce effector activity against cancer cells in T cells present in the body. Therefore, unlike CAR-T cell therapy, there is no need to prepare cells individually for each patient, and they can provide an inexpensive, off-the-shelf treatment method.

[0130] The multispecific nanoparticles of the present invention surprisingly can activate T cells only in the presence of tumor cells, thus reducing non-specific toxicity in normal tissues and providing high safety.

[0131] Another major advantage of the multispecific nanoparticles of the present invention is that various regulatory molecules can be freely added in addition to the fusion protein containing the target-binding domain. For example, the addition of immunoregulatory molecules such as costimulatory molecules and cytokine molecules can improve T cell proliferation and long-term survival. Furthermore, the addition of immune checkpoint inhibitory molecules can suppress cancer cell evasion of host immunity. Furthermore, the addition of chemotactic molecules such as homing molecules and adhesion molecules can enhance the ability of cancer cells to migrate to tumor sites. By simultaneously displaying these regulatory molecules on the nanoparticle surface, the multispecific nanoparticles of the present invention can provide a "field" for highly efficient immune response induction.

[0132] The multispecific nanoparticles of the present invention are too large for the intercellular spaces present in the glomeruli of the kidney and are therefore less susceptible to renal excretion, which is advantageous in that they can maintain high efficacy for a long period of time compared to protein preparations and the like whose active ingredients are excreted by the kidney.

[0133] 2. Pharmaceutical Compositions 2-1. Overview A second aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of this aspect comprises the multispecific nanoparticles of the first aspect as an active ingredient and can be used for cancer treatment, etc. In one embodiment, the pharmaceutical composition of this aspect is a pharmaceutical composition for cancer treatment. In another embodiment, the pharmaceutical composition of this aspect is a non-cellular preparation.

[0134] 2-2. Definitions As used herein, the term "subject" refers to a target to which the pharmaceutical composition of this embodiment is applied. For example, it refers to a tissue, an organ, or an individual. In the case of an individual, it is, for example, a mammal, preferably a human individual. The human individual may be a patient such as a cancer patient.

[0135] As used herein, "subject information" refers to various information relating to the characteristics and condition of a subject. For example, when the subject is a human individual, information may include age, weight, sex, general health condition, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant medication, and resistance to treatment.

[0136] As used herein, the term "treatment" refers to alleviating or eliminating symptoms associated with disease, and / or preventing or suppressing the progression of disease, as well as curing the disease.

[0137] As used herein, the term "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.

[0138] The type of "cancer" used herein is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver 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, pediatric tumors such as neuroblastoma, blood cancer, lymphoma, and myeloma. Examples of blood cancers include leukemias (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphomas (e.g., non-Hodgkin's lymphoma), and myelomas (e.g., multiple myeloma).

[0139] 2-3. Composition 2-3-1. Constituent Components The constituent components of the pharmaceutical composition of this embodiment will be explained. The pharmaceutical composition of this embodiment contains, as essential components, one or more active ingredients, and a solvent and / or carrier. Each constituent component will be explained in detail below.

[0140] (Active ingredient) The pharmaceutical composition of this embodiment includes, as an essential active ingredient, the multispecific nanoparticles described in embodiment 1. The pharmaceutical composition of this embodiment can contain one or more types of multispecific nanoparticles.

[0141] The content of the active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited. Generally, the content varies depending on the type of active ingredient, the dosage form, and the type of solvent and carrier, which are other components described below. Therefore, it may be determined appropriately taking into account each condition. It is sufficient that an effective amount of the active ingredient is contained in a single dose of the pharmaceutical composition. However, if a large amount of the pharmaceutical composition needs to be administered to a subject to obtain the pharmacological effect of the active ingredient, it may be administered in several divided doses to reduce the burden on the subject. In this case, the total amount of the active ingredient is sufficient as long as it contains an effective amount.

[0142] The term "effective amount" refers to the amount necessary for the active ingredient to function and which causes little or no harmful side effects in the subject to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of application, and the 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, etc.

[0143] The amount of multispecific nanoparticles contained in the pharmaceutical composition of this embodiment is, for example, 10 4 Particle ~10 16 particles, 10 6 Particle ~10 15 particles, or 10 9 Particle ~10 14 particles, preferably 10 11 Particle ~10 13 particles, e.g. 1 x 10 12 Particle ~5×10 12 It is a particle.

[0144] (Solvent) The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable solvent, if necessary. "Pharmaceutically acceptable solvent" refers to a solvent commonly used in the pharmaceutical formulation field. Examples include water, an aqueous solution, or an organic solvent. Aqueous solutions include, for example, saline, an isotonic solution containing glucose or other adjuvants, phosphate buffer, phosphate-buffered saline, sodium acetate buffer, glycol, or ethanol solutions. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Organic solvents include ethanol.

[0145] (Carrier) The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as needed. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include excipients, human serum albumin, etc.

[0146] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides; inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, or sulfates; salts of organic acids such as acetates, propionates, malonates, or benzoates; metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, kaolin, silicic acid, or combinations thereof.

[0147] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, absorption enhancers, bulking agents, preservatives, antiseptics, antioxidants, buffers, isotonicity agents, and the like that are commonly used in pharmaceutical compositions.

[0148] The carrier is used to avoid or suppress the decomposition of the active ingredient by enzymes and the like in the body of the subject, as well as to facilitate formulation and administration methods and maintain the dosage form and pharmacological efficacy, and may be used appropriately as needed.

[0149] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, as long as it is in a form that allows the active ingredient to be delivered to the target site in the body of the subject without being inactivated.

[0150] The specific dosage form varies depending on the application method, which will be described later. The application method can be roughly divided into parenteral administration and oral administration, with parenteral administration being preferred.

[0151] If the administration method is parenteral administration, a preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. A good example of a liquid is an injection. An injection can be formulated by appropriately combining a solvent, excipient, suspending agent, surfactant, stabilizer, pH adjuster, etc., and mixing them in a unit dose form required for generally accepted pharmaceutical practice.

[0152] 2-3-3. Application Method The application method of the pharmaceutical composition of the present invention is not particularly limited, and the administration route is not limited, but may be, for example, parenteral administration. Parenteral administration 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, while systemic administration in parenteral administration includes intracirculatory administration, for example, intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration. The preferred administration route is intravenous administration, which may also be administered by infusion (e.g., single intravenous infusion).

[0153] 2-4. Effect The pharmaceutical composition of this embodiment provides a method for treating and / or preventing a disease, a method for inducing effector activity in T cells, and a method for inducing cytotoxic activity against cancer cells in T cells, NK cells, and / or macrophages, all of which comprise a step of administering the pharmaceutical composition of the present invention to a subject. The disease may be, for example, cancer, and the subject may be a cancer patient.

[0154] There is also provided the use of a multispecific nanoparticle or pharmaceutical composition of the invention in the manufacture of a medicament for treating and / or preventing a disease, such as cancer.

[0155] 3. Method for Producing Multispecific Nanoparticles 3-1. Overview A third aspect of the present invention is a method for producing multispecific nanoparticles capable of binding to two or more target cells. The production method of this aspect includes, as essential steps, a cell membrane fragmentation step, a nanoparticle formation step, and a recovery step, and, as selection steps, a nanoparticle core preparation step and a vector introduction step.

[0156] The gene expression vectors (type I fusion protein expression vector and type II fusion protein expression vector described below) and host cells described below in the production method of this embodiment are also provided as further embodiments of the present invention.

[0157] 3-2. Method Each step of the method for producing the multispecific nanoparticles of this embodiment will be specifically explained.

[0158] (Nanoparticle Core Preparation Step) The "nanoparticle preparation step" refers to a step of preparing nanoparticle cores to be mixed with cell membrane fragments in the nanoparticle formation step described below. In this step, nanoparticle cores may be prepared by methods known in the art depending on the material, such as polymer nanoparticles, metal nanoparticles, or dendrimers. For example, in the case of polymer nanoparticles, nanoparticle cores can be obtained by contacting or mixing an organic solvent, such as acetone, in which a polymer, such as PLGA, has been dissolved with an aqueous solution by dropwise addition, followed by removal of the organic solvent. Ultrasonication or stirring can also be performed as needed to promote the formation of nanoparticle cores.

[0159] In one embodiment, cytokine molecules, chemotactic molecules, and / or immune checkpoint inhibitory molecules can be added to the solvent used to prepare the nanoparticle cores, thereby encapsulating each molecule in the nanoparticle cores. For example, in the case of polymer nanoparticles such as PLGA, cytokine molecules can be added to an organic solvent such as acetone or an aqueous solution in which the polymer has been dissolved, and then the mixture is contacted or mixed by dropwise addition to obtain nanoparticle cores encapsulating cytokine molecules.

[0160] (Vector introduction process) The "vector introduction process" refers to a process 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 a process of introducing two or more gene expression vectors containing a nucleic acid encoding a type II fusion protein (hereinafter referred to as "type II fusion protein expression vectors") into a host cell.

[0161] The nucleic acid encoding the type I or type II fusion protein may be any nucleic acid encoding any of the type I or type II fusion proteins described in the first aspect. The base sequence of such a nucleic acid is not limited. For example, the nucleic acid may comprise, 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. Other examples include codon-optimized base sequences and base sequences with an initiation codon (ATG) added to the 5'-end.

[0162] The type I fusion protein expression vector or type II fusion protein expression vector is a gene expression vector that contains a nucleic acid encoding the above-mentioned 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. In addition to the nucleic acid and promoter, which are the components, the gene expression vector may also contain components such as a marker gene (selection marker), an enhancer, a terminator, a replication origin, and a polyA signal, as necessary.

[0163] As used herein, the term "gene expression vector" refers to a vector that contains a gene or a 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. The gene expression vector may be a plasmid vector or a viral vector.

[0164] As used herein, "in an expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter. Known vectors include plasmid vectors and viral vectors, and either vector can be used. Generally, a plasmid vector that is easy to manipulate for genetic recombination or a viral vector that can easily introduce a gene into immune cells will suffice.

[0165] The plasmid vector may be, for example, 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 E. coli.

[0166] Viral vectors that can be used include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Replication-deficient viral vectors that do not autonomously replicate in infected cells may also be used.

[0167] When a retroviral vector is used, appropriate packaging cells and packaging signal sequences can be selected based on the LTR sequence to produce retroviral particles. Examples of packaging cells include PG13 (ATCC® CRL-10686™), PA317 (ATCC® CRL-9078™), GP+E-86 and GP+envAm-12 (U.S. Patent 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, which have high transfection efficiency. Many types of viral vectors produced based on packaging cells that can be used to package retroviruses and retroviral vectors are commercially available from many companies.

[0168] As used herein, a "promoter" refers to a gene expression regulatory region capable of controlling the expression of a gene or the like located downstream (3'-end side) in a cell 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 at which the gene or the like under their expression control is expressed. A ubiquitous promoter is a promoter that controls the expression of a target gene or the like (a target gene or the like) in all cells, i.e., the entire host organism. A site-specific promoter is a promoter that controls the expression of a target gene or the like only in specific cells or tissues. The promoter contained in the gene expression vector of the present invention may be either a ubiquitous promoter or a site-specific promoter, but it is preferable that it can induce expression in immune cells.

[0169] Furthermore, promoters are classified into constitutively active promoters, expression-inducible promoters, and stage-specifically active promoters based on the timing of expression. Constitutively active promoters can constitutively express a target gene, etc. in cells. Expression-inducible promoters can induce the expression of a target gene, etc. in cells at any stage. Stage-specifically active promoters can induce the expression of a target gene, etc. in cells only at a specific stage of development. Any of these promoters can be considered as overexpression promoters, as they can cause excessive expression of a target gene in host cells. The promoter contained in the gene expression vector of the present invention is preferably a constitutively active promoter, which enables long-term persistence of therapeutic effects.

[0170] The promoter in the gene expression vector used in this step is a promoter capable of inducing the expression of a nucleic acid encoding a type I fusion protein or a type II fusion protein in host cells. Since the host cells into which the gene expression vector in this step is introduced are, in principle, mammalian cells, particularly human-derived cells, such as human immune cells, any promoter capable of expressing a downstream gene in these cells may be used. Examples include the CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, EF1α promoter, Ub promoter, and 5' LTR promoter. In the case of a retroviral vector, a nucleic acid encoding a chimeric cytokine receptor can be placed downstream of the 5' LTR promoter to induce its gene expression.

[0171] As used herein, a "marker gene" refers to a gene encoding a marker protein, also known as a selection marker or reporter protein. A "marker protein" refers to a peptide whose activity can be used to determine the presence or absence of expression of a marker gene. Detection of activity may involve direct detection of the activity of the marker protein itself, or indirect detection via a metabolite, such as a dye, generated by the activity of the marker protein. Detection may be biological (including detection via binding of peptides or nucleic acids, such as antibodies or aptamers), chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), or sensory detection by the detector (including detection via vision, touch, smell, hearing, and taste).

[0172] The type of labeled protein encoded by the marker gene is not particularly limited, as long as its activity can be detected by methods known in the art. Labeled proteins that are less invasive to transformants during detection are preferred. Examples include tag peptides, drug-resistance proteins, chromoproteins, fluorescent proteins, and luminescent proteins.

[0173] As used herein, the term "enhancer" is not particularly limited as long as it can enhance the expression efficiency of a gene or a fragment thereof in a vector.

[0174] As used herein, a "terminator" refers to a sequence that can terminate transcription of a gene or the like expressed by the activity of the promoter. The type of terminator is not particularly limited. Preferably, the terminator is derived from the same organism as the promoter. Particularly preferred is a terminator that is paired with the promoter on the genome in a single gene expression control system.

[0175] In one embodiment, in this step, in addition to the type I fusion protein expression vector or type II fusion protein expression vector, a gene expression vector encoding one or more selected from the group consisting of costimulatory molecules, cytokine molecules, migration molecules, homing molecules, adhesion molecules, and immune checkpoint inhibitory molecules can be additionally introduced. The composition of each of these molecules is as described in the first aspect, but cytokine molecules, migration molecules, homing molecules, and immune checkpoint inhibitory molecules are preferably introduced as fusion molecules containing a membrane-binding domain. The composition of the gene expression vector is also as described above. There are no particular limitations on the method for introducing each of the above vectors into host cells.

[0176] The type of host cell used in this step is not limited. The host cell may be any cell capable of expressing a type I fusion protein or a type II fusion protein on its membrane, and therefore is not limited to mammalian cells. The host cell may be either a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include bacterial cells such as Escherichia coli cells. Examples of 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 are preferred. Specific examples of mammalian cells include, but are not limited to, CHO cells, COS cells, Vero cells, HEK293 cells and their derivatives (e.g., 293F cells, 293T cells, Freestyle 293 cells, etc.), HeLa cells, NIH3T3 cells, K562 cells, etc. The host cell may be either an adherent cell or a suspension cell. Alternatively, the host cells may be cells (autologous cells) previously isolated from the subject to whom the multispecific nanoparticles are to be administered.

[0177] When each vector is a viral vector, methods for infecting cells with the virus are known in the art. A functional substance that improves viral infection efficiency, such as fibronectin or a fibronectin fragment (e.g., RetroNectin (registered trademark) or Vecofusin-1 (registered trademark), which are fibronectin fragments having a heparin-binding site), may be used for the introduction of the viral vector.

[0178] The viral infection method using RetroNectin is exemplified as follows: After treating a cell culture plate with RetroNectin, the bottom of the plate is blocked with a 2% BSA / PBS solution for 30 minutes. After washing with PBS, a retrovirus solution derived from PG13 packaging cells is added, and the culture plate is centrifuged at 32°C and 2000 g for 2 hours. After centrifugation, the viral solution is removed, and cells are seeded onto the plate.

[0179] When each vector is a non-viral vector such as a plasmid, a gene transfer method (transformation method) known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used. Examples of such methods include lipofection, electroporation, microinjection, calcium phosphate method, DEAE-Dextran method, and particle bombardment.

[0180] 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 isolated.

[0181] The host cell into which the vector is introduced in this process expresses on its cell membrane either (i) a fusion protein comprising a membrane-binding domain and two or more target-binding domains, or (ii) two or more fusion proteins comprising a membrane-binding domain and a target-binding domain. Because both the extracellular and intracellular portions of the cell membrane fragment can be located on the surface of the nanoparticles formed in the nanoparticle formation process described below, the target-binding domain of the fusion protein in the host cell may, in principle, be located on either the extracellular or cytoplasmic side. However, it is preferred that the target-binding domain be located on the extracellular side. This is because glycosylation of proteins on the outer side of the cell membrane facilitates the extracellular portion of the cell membrane fragment to be located on the surface of the nanoparticles (Luk et al., Nanoscale, 2014, 6(5):2730-7).

[0182] (Cell membrane fragmentation step) The "cell membrane fragmentation step" refers to a step of disrupting cells expressing, on their cell membranes, two or more fusion proteins each containing a membrane-binding domain and a target-binding domain, or a fusion protein each containing a membrane-binding domain and two or more target-binding domains, to fragment the cell membrane. The purpose of this step is to obtain fragmented cell membranes containing type I fusion proteins or type II fusion proteins.

[0183] The method for fragmenting the cell membrane in this step is not limited. For example, cells can be disrupted and the cell membrane fragmented by physical methods or methods using a lysis solution. Physical fragmentation methods include disrupting cells by osmotic pressure using a hypotonic solution or the like, mechanically disrupting cells using glass beads or the like, sonication, and freeze-thawing. Fragmentation methods using a lysis solution include disrupting cells using a surfactant. However, in this step, it is preferable to disrupt cells under conditions that do not degrade or denature the fusion protein expressed on the cell membrane, or that make it difficult to degrade or denature it. Therefore, disruption is preferably performed in the presence of a protease inhibitor. Furthermore, in mechanical disruption methods, it is preferable to disrupt cells while cooling to prevent thermal denaturation of the protein.

[0184] In one embodiment, after this step and before the following nanoparticle formation step, fractions other than cell membrane fragments and / or undisrupted cells can be removed. Removal of fractions other than cell membrane fragments and / or undisrupted cells can be performed by centrifugation, filtration, sedimentation, decantation, magnetic separation, or a combination thereof. Magnetic separation may be performed, if necessary, by using antibody-bound magnetic beads or the like to collect cell membrane fragments or remove undisrupted cells.

[0185] (Nanoparticle formation process) The "nanoparticle formation process" is a process of forming nanoparticles by reconstituting the cell membrane fragments after the fragmentation process. The specific method for reconstituting the cell membrane fragments in this process is not limited, and for example, the cell membrane fragments can be reconstituted by applying physical stimulation (e.g., stirring, shaking, and / or ultrasonic treatment) to the cell membrane fragments. Alternatively, by mixing the cell membrane fragments with liposomes and / or nanoparticle cores and applying physical stimulation as necessary, nanoparticles whose surface is constituted by a lipid membrane formed by the fusion of the cell membrane fragments and liposomes, or nanoparticles whose nanoparticle core is encapsulated in a lipid membrane containing the cell membrane fragments, can be formed.

[0186] Such nanoparticles are formed by mixing cell membrane fragments with liposomes, either by fusing the liposomes with the cell membrane fragments or by mixing the cell membrane fragments with nanoparticle cores, resulting in the cell membrane fragments assembling together to encapsulate the nanoparticle cores. This results in the formation of nanoparticles with a specific particle size range. For information on the fusion of cell membrane fragments with liposomes, see, for example, Liu, Z., et al., Proc Natl Acad Sci US A., 2021, 118(30):e2022769118.

[0187] Particle formation can be promoted by physical and / or chemical methods. A convenient method is to use a physical stimulus to break the bonds between cell membrane fragments and / or between liposome or nanoparticle cores, thereby increasing the contact between them. The physical stimulus can be, for example, stirring, shaking, or ultrasonic treatment.

[0188] In one embodiment, this step involves sonication of the cell membrane fragments and liposome and / or nanoparticle cores.

[0189] In a further embodiment, this step can also involve mixing cell membrane fragments with both liposomes and nanoparticle cores to form nanoparticles in which the lipid membrane formed by the fusion of the cell membrane fragments and liposomes constitutes the surface layer and the lipid membrane encapsulates the nanoparticle core. Nanoparticles in which liposomes encapsulate nanoparticle cores such as PLGA polymers can be prepared with reference to literature (Mandal, B., Nanomedicine, 2013, 9(4):474-91.).

[0190] In one embodiment, the solvent used to fuse the liposome with the cell membrane fragment contains cytokine molecules, chemotactic molecules, and / or immune checkpoint inhibitory molecules, so that the nanoparticles after fusion can encapsulate these molecules.

[0191] (Recovery step) The "recovery step" is a step of recovering the nanoparticles formed in the nanoparticle formation step. The recovery method in this step is not limited as long as it is a method for separating cell membrane fragments or cell membrane vesicles that did not form nanoparticles from the nanoparticles formed in the nanoparticle formation step. For example, the target nanoparticles can be obtained by centrifugation, filtration, sedimentation, decantation, or a combination thereof. In either case, the method can basically be performed according to a conventional method in the field. For example, when recovering by centrifugation, centrifugation can be performed under conditions where the target nanoparticles settle but the cell membrane vesicles that did not form nanoparticles do not settle. Furthermore, when recovering by filtration, a filter with a pore size that allows the target nanoparticles to pass through but does not allow the cell membrane vesicles that did not form nanoparticles to pass through can be used.

[0192] In this step, if necessary, nanoparticle cores that are not encapsulated in the lipid membrane composed of cell membrane fragments and liposomes that are not induced with cell membrane fragments can also be removed. The collected target nanoparticles can be stored refrigerated or frozen until use.

[0193] 3-3. Effects The multispecific nanoparticles produced by the production method of this embodiment contain a type I fusion protein or type II fusion protein derived from the cell membrane of the host cell used to produce the nanoparticles in their surface layer. As a result, nanoparticles can be produced that display two or more target binding domains on their surface. Furthermore, nanoparticles can also be obtained that have additional co-stimulatory molecules, cytokine molecules, etc., introduced into their surface layer and / or interior.

[0194] Example 1: Design and cell surface expression of a CD3 / CD19 binding protein (Objective) A fusion protein containing two scFVs derived from an anti-CD3ε antibody and an anti-CD19 antibody (hereinafter referred to as the "CD3 / CD19 binding protein") was prepared and transfected into the leukemia cell line K562. Furthermore, the expression of the CD3 / CD19 binding protein on the cell surface was detected. Furthermore, the cytotoxic activity against the CD19-positive leukemia cell line NALM6 was verified.

[0195] (Methods and Results) (1) Preparation of CD3 / CD19 binding protein The CD3 / CD19 binding protein was prepared by linking, from the N-terminus, a signal peptide (hereinafter referred to as "SP"), a single-chain variable fragment (scFV) derived from the mouse anti-CD3ε antibody clone OKT3 (hereinafter referred to as "mOKT3 scFV"), a linker peptide, an scFV derived from the anti-CD19 antibody clone FMC63 (hereinafter referred to as "FMC63 scFV"), and a fragment derived from the CD8α protein (Fig. 3A).

[0196] SP is a peptide for transporting the CD3 / CD19 binding protein to the cell surface, and a signal peptide (SEQ ID NO: 1) derived from silkworm fibroin L protein was used.

[0197] The mOKT3 scFV is derived from the mouse anti-CD3ε antibody clone OKT3 and is an scFV containing a sequence with two amino acid mutations introduced, based on the literature (Kipriyanov SM, et al., Protein Eng., 1997, 10(4):445-53). To indicate that the mutations have been introduced into OKT3, it is referred to as "mOKT3" herein. Specifically, the mOKT3 scFV contains a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 80 to 82, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 83 to 85, and its full length consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0198] FMC63 scFV is an scFV derived from the 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 comprises a heavy chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 192 to 194, and a light chain variable region including CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOs: 195 to 197, and its full length consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0199] The CD8α protein-derived fragment is contained in the CD3 / CD19 binding protein for the purpose of anchoring it to the cell membrane, and comprises the region from the hinge, including the transmembrane domain, to the cytoplasmic domain of the human CD8α protein. Specifically, it consists of the amino acid sequence shown in SEQ ID NO:4.

[0200] The full-length amino acid sequence of the CD3 / CD19 binding protein is shown in SEQ ID NO: 5. The nucleotide sequence of the gene encoding the CD3 / CD19 binding protein is shown in SEQ ID NO: 6.

[0201] (2) Introduction into the leukemia cell line K562. The gene encoding the CD3 / CD19 binding protein was introduced into the leukemia cell line K562 to express the CD3 / CD19 binding protein in the K562 cell line. Expression of the CD3 / CD19 binding protein on the cell surface was detected using Protein L, which binds to scFV. Specifically, cells were labeled with biotin-labeled Protein L and streptavidin-PE and then analyzed by flow cytometry.

[0202] Figure 3B shows flow cytometry plots of cells transfected with the CD3 / CD19 binding protein (Fig. 3B, "K562-mOKT3 / FMC63") and control cells (Fig. 3B, "K562") that were not transfected with the CD3 / CD19 binding protein. Most of the cells transfected with the CD3 / CD19 binding protein were positive, indicating that the CD3 / CD19 binding protein was expressed on the cell surface.

[0203] (3) Cytotoxicity against the CD19-positive leukemia cell line NALM6: We verified that the K562 cell line transfected with a CD3 / CD19 binding protein (hereafter referred to as the "K562-mOKT3 / FMC63 cell line") can induce cytotoxic activity in T cells against tumor cells. The tumor cells used were the CD19-positive leukemia cell line NALM6 (Tohoku University Institute of Development, Aging and Cancer, Medical Cell Resource Center, TKG 0413) stably transfected with EGFP-P2A-Luc2 (GL) (hereafter referred to as the "NALM6-GFP cell line"), and nonspecific cultured human T cells were used.

[0204] Specifically, human cultured T cells, the NALM6 cell line, and the K562-mOKT3 / FMC63 cell line were cultured at a ratio of 3:3:1 (K562-OKT3 / FMC63 cell line: 3.3 × 10 4 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 the wells cultured with NALM6 cells alone as 100%.

[0205] The results are shown in Figure 3C. Compared with the negative control (not including the K562 cell line, Figure 3C, "-") and K562 cells transfected with the CD3-binding protein used in Example 6 (described below) instead of the K562-mOKT3 / FMC63 cell line (hereinafter referred to as "K562-mOKT3 cells"; Figure 3C, "K562-mOKT3"), the viability of tumor cells was significantly reduced when cocultured with the K562-mOKT3 / FMC63 cell line (Figure 3C, "K562-mOKT3 / FMC63") (n = 4, mean ± SD, one-way ANOVA with multiple comparison test).

[0206] Therefore, it was demonstrated that the cell line transfected with the CD3 / CD19 binding protein can induce cytotoxic activity in T cells against tumor cells.

[0207] Example 2: Preparation of nanoparticles (Objective) Nanoparticles were prepared from the cell membrane of a cell line transfected with CD3 / CD19 binding protein. Specifically, the cell line was disrupted, and the resulting cell membrane fragments were reconstituted on PLGA nanoparticle cores to prepare nanoparticles containing the CD3 / CD19 binding protein on their surface (Figure 2).

[0208] (Methods and Results) (1) Preparation of PLGA Nanoparticle Cores. PLGA nanoparticle cores were prepared using the nanoprecipitation method. This method was performed with some modifications to the method described in the literature (Barichello et al., Drug Dev Ind Pharm, 1999; Hu et al., Nature, 2015). Specifically, PLGA (Resomer® RG 502H, poly(D,L-lactide-co-glycolide), Sigma-Aldrich) was dissolved in acetone at a concentration of 10 mg / mL. The resulting PLGA solution was added dropwise to a 2% (w / v) aqueous polyvinyl alcohol (PVA) solution with stirring. The solution was then sonicated using a Bioruptor ultrasonicator, and the acetone was evaporated overnight in a fume hood. The resulting solution was centrifuged at 20,000 rpm for 1 hour, the supernatant was removed, and the nanoparticles were washed with purified water. Sterile spherical nanoparticle cores (hereinafter referred to as "PLGA nanoparticle cores") were obtained by passing through a 0.22 μm filter.

[0209] The nanoparticle cores prepared are shown in Figure 4. Figure 4A shows the appearance of the PLGA nanoparticle core dispersion. Observation using a transmission electron microscope confirmed the spherical shape of the PLGA nanoparticle cores (Figure 4B). Measurement of the particle size and concentration of the PLGA nanoparticle cores using a NanoSight (Malvern Instruments) revealed that the particle size immediately after synthesis was approximately 117 nm (Figure 4C, left). No change in particle size was observed even after freezing and thawing, and the particles remained stable (Figure 4C, right).

[0210] (2) Preparation of Nanoparticles A gene encoding the CD3 / CD19 binding protein described in Example 1, a gene encoding the CD80 protein consisting of the amino acid sequence shown in SEQ ID NO: 7, and a gene encoding the 41BBL protein consisting of the amino acid sequence shown in SEQ ID NO: 8 were introduced into the leukemia cell line K562. The CD80 protein and the 41BBL protein are costimulatory molecules. Expression of the CD80 protein and the 41BBL protein on the cell surface was confirmed by flow cytometry using the same method as in Example 1 (Figure 5A).

[0211] The process for preparing nanoparticles using cell lines is shown in Figure 5B. Specifically, the cell lines (1 × 10 cells) were suspended in a hypotonic buffer solution (20 mM Tris-HCl pH 7.5, 10 mM KCl, 2 mM MgCl, EDTA-free protease inhibitors (Fang et al. Nano Lett. 2014)). 8 Cells were homogenized using a homogenizer, and microscopic observations confirmed that almost all cells were lysed. The cell lysate was centrifuged at 3,200 g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was then ultracentrifuged at 100,000 g for 60 minutes at 4°C. The supernatant containing cytoplasmic proteins was removed, and the precipitate containing cell membrane fragments was suspended in washing buffer (10 mM Tris-HCl pH 7.5, 2 mM EDTA). This procedure was repeated, and the precipitate containing cell membrane fragments was then suspended in pure water.

[0212] The PLGA nanoparticle cores were coated with cell membrane fragments using 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 while sonicating for approximately 2 minutes. The mixture was then subjected to ultracentrifugation at 20,000 rpm for 30 minutes at 4°C, and the resulting pellet was suspended in PBS under sonication. Further sonication yielded nanoparticles. The resulting nanoparticles were stored at -80°C.

[0213] The above nanoparticle preparation methods were based on literature (Fang et al. Nano Lett. 2014; Fusciello et al. Nat Commun 2019; Fontana et al. Adv Mater 2017; Abas et al. Anal Biochem. 2010; Hu et al. Nature. 2015).

[0214] The components contained in the protein fractions obtained at each step of nanoparticle preparation were analyzed by Western blot, and the results are shown in Figure 6B. The results shown in Figure 6B indicate that the protein fractions used for reconstitution contained plasma membrane proteins CD80 and 41BBL, while SDHA contained in mitochondria and β-actin contained mainly in the cytoplasm were not, indicating that plasma membranes containing plasma membrane proteins were selectively used for nanoparticle preparation.

[0215] Figure 6A shows the results of observation of the resulting nanoparticles by transmission electron microscopy (TEM). The nanoparticle dispersion was dropped onto a TEM grid, dried, and fixed, then stained with uranyl acetate and observed under TEM. Compared to the PLGA nanoparticle core before cell membrane reconstitution (Figure 6A, (-)), the TEM image of the resulting nanoparticles (Figure 6A, (+)) confirmed the formation of a new layer on the particle surface, indicating the reconstitution of cell membranes on the particle surface.

[0216] Nanoparticles prepared by the above method from the K562 cell line transfected with the CD3 / CD19 binding protein, CD80 protein, and 41BBL protein are referred to as "mOKT3 / FMC63 / CD80 / 41BBL nanoparticles" in the following examples. Nanoparticles prepared by a similar method from the K562 cell line transfected with the CD3 / CD19 binding protein are referred to as "mOKT3 / FMC63 nanoparticles" in the following examples.

[0217] Example 3: Induction of effector effect by nanoparticles (Objective) To verify that mOKT3 / FMC63 nanoparticles can induce cytotoxic activity in T cells against tumor cells.

[0218] (Methods and Results) (1) Preparation of T Cells. T cells were prepared by culturing cryopreserved peripheral blood mononuclear cells (PBMCs) for approximately one week after priming. Cultures were performed in RPMI 1640 medium containing 10% FBS, penicillin (100 units / mL), streptomycin (100 μg / mL), and recombinant IL-2 (100 IU / mL). The same medium was used in the following examples unless otherwise noted. On the first day of culture, PBMCs were stimulated with anti-CD3 antibody (priming). For priming, K562 cells were used, expressing a single-chain variable fragment (scFV) derived from an anti-CD3 antibody (mOKT3, an antibody derived from clone OKT3 with amino acid mutations) and the costimulatory molecule CD80 on their cell surface. After treatment with mitomycin to arrest cell division, the K562 cells were co-cultured with PBMCs at a ratio of 1:7.

[0219] (2) Preparation of CAR-T Cells In the following examples, CAR-T cells were used as a control. When introducing the CAR gene into T cells, the CAR gene was introduced on day 2 of T cell culture. The retroviral plasmid, pMX plasmid (Kitamura T. et al., Exp Hematol., 2003, 31:1007-1014), was transiently transfected into Plat-E packaging cells using TranslT293 (Mirus Bio). The resulting ecotropic retroviral vector was stably transfected into PG13 packaging cells. The resulting PG13 cell-derived viral vector was then transfected into T cells using RetroNectin (Takara Bio).

[0220] (3) Induction of effector effects by nanoparticles. Human T cells and the CD19-positive leukemia cell line NALM6 were co-cultured at 1x10 5 After mixing, nanoparticles (NPs) were added to each culture plate at a ratio of 10 to the number of T cells. 3 times to 10 7The tumor cells were co-cultured with NALM6 cells at twice the number of particles administered. 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 the wells cultured with NALM6 cells alone as 100%.

[0221] The results are shown in Figure 7. mOKT3 / FMC63 nanoparticles (NPs) were administered at 10 4 times to 10 7 Tumor cell viability was significantly reduced when mOKT3 / FMC63 nanoparticles were administered at twice the number of particles (n = 3, mean ± standard deviation; * P < 0.01 (one-way ANOVA with multiple comparison test for results when the NP / T cell ratio was 0)). In Figure 7, "CART" indicates tumor cell viability when CAR-T cells and NALM6 cells were mixed at a 1:1 ratio as a control. The CAR-T cells used here were second-generation CARs already approved for B-cell tumors. Specifically, they were T cells transfected with a chimeric antigen receptor (CAR) containing FMC63 scFV, a CD28 protein-derived fragment (the region from the hinge to the cytoplasmic domain containing the transmembrane domain of human CD28 protein), and the cytoplasmic domain of CD3ζ. These results demonstrate that mOKT3 / FMC63 nanoparticles can induce effector effects comparable to or greater than those of CAR-T cells.

[0222] Example 4: Mechanism of T cell activation by nanoparticles (Objective) 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. Because its expression level can change sensitively depending on the strength of T cell receptor stimulation, it is commonly used as a marker for measuring the strength of T cell receptor stimulation (Schuh et al. J Exp Med. 1998;188(7):1369-73). In this example, we specifically examined whether T cell activation by mOKT3 / FMC63 nanoparticles depends on the presence of tumor cells, which are the target cells.

[0223] (Methods and Results) The target cells used were the CD19-positive leukemia cell line NALM6 or the leukemia cell line K562 transfected with CD19 protein (hereinafter referred to as "K562-CD19 cells"). T cells were cultured with mOKT3 / FMC63 nanoparticles in the presence or absence of either of these target cells, and the expression of CD25 protein in the CD8-positive T cell fraction after 24 hours was analyzed by flow cytometry. The number of mOKT3 / FMC63 nanoparticle particles was 1 x 10 relative to the number of T cells. 6 Double or 5 x 10 6 NALM6 cells were co-cultured at the same cell number as the T cells, and K562-CD19 cells were co-cultured at one-third the number of T cells.

[0224] Figure 8A shows the 5 × 10 6 Representative flow cytometry results are shown for T cells cultured with mOKT3 / FMC63 nanoparticles at twice the particle number. In the presence of K562-CD19 cells or NALM6 cells, mOKT3 / FMC63 nanoparticles activated T cells. In contrast, mOKT3 / FMC63 nanoparticles did not activate T cells in the absence of target cells. These results indicate that mOKT3 / FMC63 nanoparticles can activate T cells only in the presence of target cells.

[0225] Figure 8B shows the results of measuring the CD25 expression level under each condition (n = 3, mean ± standard deviation; * P < 0.01 (one-way ANOVA with multiple comparison test for the results when the NP / T cell ratio was 0)). As controls, the expression levels of CD25 protein when CAR-T cells targeting CD19 protein were co-cultured with NALM6 cells at a 1:1 ratio and when CAR-T cells targeting CD19 protein were co-cultured with K562-CD19 cells at a 3:1 ratio are also shown. The CAR-T cells used were the same as those described in Example 3. As shown in Figure 8B, T cells were activated by mOKT3 / FMC63 nanoparticles in the presence of NALM6 cells or K562-CD19 cells. In contrast, T cells were not activated by mOKT3 / FMC63 nanoparticles in the absence of target cells.

[0226] Figure 9 shows that T cells were treated with mOKT3 / FMC63 nanoparticles (5 × 10 6 The mOKT3 / FMC63 nanoparticles were cultured in the presence of the CD19-negative K562 cell line or K562-CD19 cells, and co-cultured with the CD19-negative K562 cell line or K562-CD19 cells. After 24 hours of culture, the expression level of CD25 protein in the CD8-positive T cell fraction was measured by flow cytometry. As a result, T cells were not activated by the mOKT3 / FMC63 nanoparticles in the presence of the CD19-negative K562 cell line. In contrast, T cells were activated by the mOKT3 / FMC63 nanoparticles in the presence of K562-CD19 cells (n = 4, mean ± standard deviation; one-way ANOVA with multiple comparison test; ns, not significant).

[0227] These results demonstrate that 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 can activate T cells only in the presence of tumor cells, while T cells are not activated in normal tissues. Without being bound by theory, it is thought that tumor cells provide a scaffold for presenting mOKT3 / FMC63 nanoparticles to T cells, resulting in concentrated binding of mOKT3 / FMC63 nanoparticles between tumor cells and T cells, thereby allowing activation signals to be transmitted to T cells above a threshold.

[0228] Example 5: Introduction of costimulatory molecules into nanoparticles (Objective) CD80 protein and 41BBL protein were additionally introduced into mOKT3 / FMC63 nanoparticles, which are costimulatory molecules. The effects on T cell proliferation, effector effects, and cytokine production were examined.

[0229] (Methods and Results) (1) Effect on T Cell Proliferation Using a method similar to that used in Example 2, mOKT3 / FMC63 nanoparticles, which are nanoparticles into which a CD3 / CD19 binding protein has been introduced, and mOKT3 / FMC63 / CD80 / 41BBL nanoparticles, which are nanoparticles into which a CD3 / CD19 binding protein, a CD80 protein, and a 41BBL protein have been introduced, were prepared.

[0230] 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 relative to the number of T cells. 6 Double or 5 x 10 6 The doubling rate was calculated by adding NALM6 cells to the T cells at the same cell number as the T cells. Three days after the start of culture, the cytokine IL2 was added at a concentration of 100 IU / mL. One week after the start of culture, the T cells were counted and the cell doubling rate was calculated.

[0231] The results are shown in Figure 10A. Compared with mOKT3 / FMC63 nanoparticles, mOKT3 / FMC63 / CD80 / 41BBL nanoparticles enhanced T cell proliferation (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure). Therefore, it was demonstrated that the addition of costimulatory molecules to nanoparticles enhanced T cell proliferation.

[0232] (2) Effect on effector activity T cells, NALM6 cells, and nanoparticles were mixed at a ratio of 1:1:1 × 10 6 After overnight co-culture at a ratio of 0.1 to 0.1, the number of surviving NALM6 cells was measured by flow cytometry. The ratio of the number of surviving cells to the number of NALM6 cells at the start of culture was calculated.

[0233] The results are shown in Figure 10B. Compared with mOKT3 / FMC63 nanoparticles, mOKT3 / FMC63 / CD80 / 41BBL nanoparticles significantly reduced the viability of NALM6 cells and enhanced their effector effects (n = 4, mean ± standard deviation; p values ​​from Student's t-test are shown in the figure). Therefore, the addition of costimulatory molecules to nanoparticles enhanced their effector effects.

[0234] (3) Effect on cytokine production in CD8+ T cells. T cells, K562-CD19 cells, and nanoparticles were mixed at a ratio of 1:0.5:5×10 6 After culturing for 6 hours at a ratio of 1:1, the ratio of cytokine (IFNγ, IL2, and TNFα)-producing cells among CD8-positive T cells was analyzed by flow cytometry.

[0235] The results are shown in Figure 11. Compared with mOKT3 / FMC63 nanoparticles (Figure 11, CD80 / 41BBL (-)), the number of cells producing IFNγ, IL2, and TNFα was significantly increased with mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (Figure 11, CD80 / 41BBL (+)) (n = 4, mean ± standard deviation; p values ​​from Student's t-test are shown in the figure). Therefore, it was demonstrated that the addition of costimulatory molecules to nanoparticles enhances cytokine production by T cells.

[0236] (4) Tumor cell dependency study: T cells and nanoparticles were mixed at a ratio of 1:0.5:5×10 in the presence or absence of K562-CD19 cells. 6 After culturing for 6 hours at a ratio of 1000 to 1000, the ratio of cells producing cytokines (IFNγ, IL2, and TNFα) in CD4-positive T cells and CD8-positive T cells was analyzed by flow cytometry.

[0237] The results are shown in Figure 12. For both CD4-positive T cells (Figure 12A) and CD8-positive T cells (Figure 12B), the cytokine secretion of T cells induced by nanoparticles containing costimulatory molecules required the presence of tumor cells (n = 3, mean ± standard deviation; p values ​​for each cytokine obtained by one-way ANOVA with multiple comparison test are shown in the figure).

[0238] Example 6 Comparison of Tandem Nanoparticles and Separate Nanoparticles (Objective) The mOKT3 / FMC63 nanoparticles developed in the above examples contain a CD3 / CD19 binding protein containing two scFVs in tandem on their surface (Figure 13A, "tandem expression"; hereinafter referred to as "tandem nanoparticles"). In this example, two fusion proteins containing each of the two scFVs are simultaneously introduced onto the nanoparticle surface (Figure 13A, "separate expression"; hereinafter referred to as "separate nanoparticles"), and the effects of the tandem nanoparticles and the separate nanoparticles are compared.

[0239] (Methods and Results) (1) Preparation of Tandem Nanoparticles The tandem nanoparticles in this example were mOKT3 / FMC63 / CD80 / 41BBL nanoparticles prepared by the same method as in Examples 1 and 2. Therefore, the tandem nanoparticles contain CD3 / CD19 binding protein, CD80 protein, and 41BBL protein on their surface (Figure 13A, "tandem expression"). The CD3 / CD19 binding protein has a structure in which, from the N-terminus, SP, mOKT3 scFV, linker peptide, FMC63 scFV, and a fragment derived from CD8α protein are linked.

[0240] (2) Preparation of Separate Nanoparticles The separate nanoparticles contain, on their surface, a fusion protein (hereinafter referred to as "CD3-binding protein") consisting of SP, mOKT3 scFV, and a fragment derived from CD8α protein linked in order from the N-terminus, a fusion protein (hereinafter referred to as "CD19-binding protein") consisting of SP, FMC63 scFV, and a fragment derived from CD8α protein linked in order from the N-terminus, as well as CD80 protein and 41BBL protein (Figure 13A, "separate expression"). The specific composition of each component is as described in Examples 1 and 2.

[0241] The full-length amino acid sequence of the CD3 binding protein is shown in SEQ ID NO: 9. The nucleotide 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 in SEQ ID NO: 11. The nucleotide sequence of the gene encoding the CD19 binding protein is shown in SEQ ID NO: 12. Separable nanoparticles were produced from the leukemia cell line K562 by the same method as in Examples 1 and 2, after introducing the genes encoding the CD3 binding protein and the CD19 binding protein, as well as the genes encoding the CD80 protein and the 41BBL protein.

[0242] (3) T cell activation by tandem nanoparticles and separated nanoparticles. CD19-positive leukemia cell line NALM6 was used as target cells, and T cells, NALM6 cells, and nanoparticles were mixed at a ratio of 1:1:5×10 6 After overnight co-culture at a ratio of 1 / 10, CD8+ T cells were analyzed by flow cytometry for CD25 protein expression.

[0243] The results are shown in Figure 13B. Tandem nanoparticles (Figure 13B, mOKT3 / FMC63 / CD80 / 41BBL (tandem expression)) and separate nanoparticles (Figure 13B, mOKT3 / FMC63 / CD80 / 41BBL (separate expression)) induced comparable T cell activation.

[0244] (4) Induction of cytotoxicity by tandem nanoparticles and isolated nanoparticles. After overnight co-culture, the percentage of surviving NALM6 cells was measured by flow cytometry based on GFP fluorescence. The survival rate was calculated by setting the number of tumor cells in the wells containing only NALM6 cells as 100%.

[0245] The results are shown in Figure 13C. It was shown that the tandem nanoparticles (Figure 13C, "T cell + NP (tandem scFV)") and the separate nanoparticles (Figure 13C, "T cell + NP (separate scFV)") were able to induce equivalent cytotoxic activity (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure).

[0246] Example 7: Introduction of cytokine molecules into nanoparticles and their effects (Objective) The cytokine molecules IL7 protein and IL15 protein are additionally introduced into nanoparticles. The effects on T cell proliferation, T cell memory phenotype, and phosphorylated STAT5 are examined.

[0247] (Methods and Results) (1) Introduction of cytokine molecules into nanoparticles Nanoparticles containing CD3 / CD19 binding protein, CD80 protein, 41BBL protein, IL7 protein, and IL15 protein on the surface layer were prepared.

[0248] To deliver IL7 protein to the cell membrane, a fusion protein (hereinafter referred to as "IL7 fusion protein") was used, which had a structure in which, from the N-terminus, SP, IL7 protein, and a fragment derived from CD8α protein were linked. The full-length amino acid sequence of the IL7 fusion protein is shown in SEQ ID NO: 13. The nucleotide sequence of the gene encoding the IL7 fusion protein is shown in SEQ ID NO: 14.

[0249] To deliver IL15 protein to the cell membrane, a fusion protein (hereinafter referred to as "IL15 fusion protein") was used, which had a structure in which, from the N-terminus, SP, IL15 protein, and a fragment derived from CD8α protein were linked. The full-length amino acid sequence of the IL15 fusion protein is shown in SEQ ID NO: 15. The nucleotide sequence of the gene encoding the IL15 fusion protein is shown in SEQ ID NO: 16.

[0250] Genes encoding CD3 / CD19 binding protein, CD80 protein, 41BBL protein, IL7 fusion protein, and IL15 fusion protein were introduced into the leukemia cell line K562 using methods similar to those described in Examples 1 and 2. Expression of the IL7 fusion protein and IL15 fusion protein on the cell surface was confirmed by flow cytometry using the same method as in Example 1 ( Figure 14A ). Nanoparticles containing these proteins on their surface layer were prepared from these cells using the same method as in Example 2 (hereinafter referred to as "mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles").

[0251] In this example, the mOKT3 / FMC63 / CD80 / 41BBL nanoparticles prepared in Example 2 were used as a control (referred to as "mOKT3 / FMC63 / CD80 / 41BBL nanoparticles" in this example).

[0252] (2) Effect on T cell proliferation T cells and the CD19-positive leukemia cell line NALM6 were co-cultured in the presence of nanoparticles. The number of nanoparticles was 2 × 10 relative to the number of T cells. 6 times or 1×10 7 NALM6 cells were co-cultured with T cells at the same cell number. After one week of culture in cytokine-free medium, the T cells were counted and the cell doubling rate was calculated.

[0253] The results are shown in Figure 14B. Compared with mOKT3 / FMC63 / CD80 / 41BBL nanoparticles, mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles enhanced T cell proliferation (n = 4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure). Therefore, it was demonstrated that the addition of cytokine molecules to nanoparticles enhanced T cell proliferation.

[0254] (3) Evaluation of T cell memory phenotypes Normally, T cells acquire undifferentiated memory phenotypes (CD45RA) as they proliferate. + / - CCR7 + CD28 + CD62L + CD27+ ) is known to be lost during the course of T cell proliferation. Therefore, the memory phenotype of T cells expanded in the above (1) after the culture in (1) was analyzed by flow cytometry. Specifically, the expression levels of the marker proteins CCR7, CD45RA, CD62L, CD28, FSC, and CD27 were evaluated by flow cytometry.

[0255] 2×10 6 Representative FACS plots of nanoparticle administration under double conditions are shown in Figure 15A. Despite the enhanced cell proliferation, T cells expanded in the presence of mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles maintained these markers at approximately the same levels compared to T cells expanded in the presence of mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (Figure 15A).

[0256] The cell doubling rate of T cells with undifferentiated memory phenotypes is shown in Figure 15B. The results showed that proliferation of T cells with undifferentiated memory phenotypes was significantly enhanced in the presence of mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles (n=4, mean±SD; *P<0.01 (one-way ANOVA with multiple comparison test)).

[0257] (4) Evaluation of phosphorylated STAT5 To evaluate cytokine stimulation of T cells by mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles, the level of phosphorylated STAT5 (pSTAT5) in T cells was measured. Specifically, T cells were incubated overnight in cytokine-free medium, and then 2 × 10 6 After administration of 200 μg of nanoparticles, phosphorylated STAT5 was quantified by flow cytometry 30 minutes later.

[0258] Representative flow cytometry plots are shown in Figure 16A. Quantitative fluorescence intensity derived from phosphorylated STAT5 (pSTAT5) is shown in Figure 16B (n = 4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison tests are shown in the figure; ns, not significant). These results demonstrate that mOKT3 / FMC63 / CD80 / 41BBL / IL7 / IL15 nanoparticles can activate cytokine signals in T cells.

[0259] Example 8: Development of nanoparticles targeting BCMA protein (Objective) A fusion protein containing two scFVs derived from an anti-CD3ε antibody and an anti-BCMA (B cell maturation antigen) antibody (hereinafter referred to as a "CD3 / BCMA binding protein") will be produced. Furthermore, nanoparticles containing the CD3 / BCMA binding protein on their surface will be produced, and their effects on RPMI8226 cells, a BCMA-positive multiple myeloma cell line, will be examined.

[0260] (Methods and Results) (1) Preparation of CD3 / BCMA-binding protein The CD3 / BCMA-binding protein has a structure consisting of, from the N-terminus, SP, mOKT3 scFV, a linker peptide, an scFV derived from the anti-BCMA antibody clone BCMA98 (hereinafter referred to as "BCMA98 scFV"), and a fragment derived from the CD8α protein (Figure 17A). The components other than BCMA98 scFV were as described in Example 1.

[0261] BCMA98 scFV is an scFV derived from the anti-BCMA antibody clone BCMA98. Specifically, BCMA98 scFV comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 240 to 242, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 243 to 245, and its full length consists of the amino acid sequence shown in SEQ ID NO: 17.

[0262] The full-length amino acid sequence of the CD3 / BCMA binding protein is shown in SEQ ID NO: 18. The nucleotide sequence of the gene encoding the CD3 / BCMA binding protein is shown in SEQ ID NO: 19.

[0263] (2) Preparation of mOKT3 / BCMA98 / CD80 / 41BBL Nanoparticles Nanoparticles containing CD3 / BCMA binding protein, CD80 protein, and 41BBL protein on their surface were prepared. Specifically, genes encoding CD3 / BCMA binding protein, CD80 protein, and 41BBL protein were introduced into the leukemia cell line K562 using the same method as in Examples 1 and 2. Nanoparticles containing these proteins on their surface were prepared from these cells using the same method as in Example 2 (hereinafter referred to as "mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles").

[0264] (3) Effect on T cell activation RPMI8226 cells (BCMA-positive multiple myeloma cell line; JCRB, JCRB0034) were used as target cells. T cells and RPMI8226 cells were co-cultured in the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles. The number of nanoparticles was 5 × 10 relative to the number of T cells. 6 The RPMI8226 cells were co-cultured with the same number of T cells as the T cells. After overnight co-culture, the expression of CD25 protein in the CD8+ T cell fraction was analyzed by flow cytometry.

[0265] 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.

[0266] (4) Effects on T cell proliferation. T cells and RPMI8226 cells were co-cultured in the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles using the same method as in (3) above. One week after the start of culture, T cell counts were measured and cell doubling rates were calculated. As a control, cell proliferation was also analyzed when BCMA-specific CAR-T cells were co-cultured with RPMI8226 cells. The CAR-T cells used here were transfected with chimeric antigen receptors (CARs) containing mOKT3 scFV, BCMA98 scFV, the same CD28 protein-derived fragment as above, and the cytoplasmic domain of CD3ζ.

[0267] The results are shown in Figure 18A. In the presence of RPMI8226 cells, mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles enhanced T cell proliferation, whereas in the absence of RPMI8226 cells, mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles did not significantly enhance T cell proliferation (n = 4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure; ns, not significant). These results demonstrate that mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles induce T cell proliferation in a target cell-dependent manner.

[0268] (5) Induction of Effector Effects: T cells and RPMI8226 cells were co-cultured in the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles using the same method as in (3) above. After overnight co-culture, the number of viable RPMI8226 cells was analyzed by flow cytometry. As a control, the viability of RPMI8226 cells was also measured when RPMI8226 cells were co-cultured with BCMA-specific CAR-T cells. The viability was calculated by setting the number of tumor cells in wells containing only RPMI8226 cells as 100%.

[0269] The results are shown in Figure 18B. In the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles, the viability of RPMI8226 cells was significantly reduced and the effector effect was enhanced (n = 3, p values ​​from one-way ANOVA with multiple comparison test are shown in the figure).

[0270] (6) Effect on cytokine production: T cells and RPMI8226 cells were co-cultured in the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles using the same method as described in (3) above. After 6 hours of co-culture, the amounts of cytokines (IL2 and IFNγ) produced by CD4+ T cells and CD8+ T cells were analyzed by flow cytometry.

[0271] Representative flow cytometry plots are shown in Figure 19A. Figure 19B shows the IL2 positivity rate in CD4-positive T cells (Figure 19B, "CD4 + IL2 + " ) and the IFNγ positivity rate in CD8-positive T cells (Figure 19B, "CD8 + IFNγ + In the presence of mOKT3 / BCMA98 / CD80 / 41BBL nanoparticles, the IL2 and IFNγ positive rates were shown to increase in an RPMI8226 cell-dependent manner (n=3, mean ± standard deviation; *P<0.01 (one-way ANOVA with multiple comparison test compared with the RPMI8226 cell (-) and nanoparticle (+) groups)).

[0272] Example 9: Development of nanoparticles targeting mesothelin (Objective) To prepare nanoparticles in which two fusion proteins, each containing two scFVs derived from an anti-CD3ε antibody and an anti-mesothelin antibody, are simultaneously introduced onto the nanoparticle surface, and to examine their effect on K562-CD19 / Mesothelin cells. The nanoparticles in this example have the structure shown in Figure 13A under "separate expression."

[0273] (Methods and Results) (1) Preparation of CD3-binding protein and mesothelin-binding protein A fusion protein (hereinafter referred to as "CD3-binding protein") having a structure in which, from the N-terminus, SP, mOKT3 scFV, and a fragment derived from CD8α protein are linked together, and a fusion protein (hereinafter referred to as "mesothelin-binding protein") having a structure in which, from the N-terminus, SP, scFV derived from anti-mesothelin antibody clone ss1 (hereinafter referred to as "ss1 scFV"), and a fragment derived from CD8α protein were linked together (hereinafter referred to as "mesothelin-binding protein").

[0274] The ss1 scFV is an scFV derived from the anti-mesothelin antibody clone ss1. Specifically, the ss1 scFV comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 224 to 226, and a light chain variable region comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 227 to 229, and its full length consists of the amino acid sequence shown in SEQ ID NO: 20.

[0275] The full-length amino acid sequence of the mesothelin-binding protein is shown in SEQ ID NO: 21. The nucleotide sequence of the gene encoding the mesothelin-binding protein is shown in SEQ ID NO: 22. The structure of the CD3-binding protein and the gene encoding it is as described in Example 6.

[0276] (2) Preparation of mOKT3 / ss1 / CD80 / 41BBL / IL7 / IL15 Nanoparticles Nanoparticles containing CD3-binding protein, mesothelin-binding protein, CD80 protein, 41BBL protein, IL7 protein, and IL15 protein on their surface layers were prepared. Specifically, genes encoding CD3-binding protein, mesothelin-binding protein, CD80 protein, 41BBL protein, IL7 protein, and IL15 protein were introduced into the leukemia cell line K562 using the same method as in Examples 1 and 2. Nanoparticles containing these proteins on their surface layers were prepared from these cells using the same method as in Example 2 (hereinafter referred to as "mOKT3 / ss1 / CD80 / 41BBL / IL7 / IL15 nanoparticles").

[0277] (3) Effect on T cell activation. K562 cells stably transfected with the CD19 gene and mesothelin gene were used as target cells (hereafter 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 x 10 relative to the number of T cells. 6 K562-CD19 / Mesothelin cells were co-cultured with T cells at a cell number of 0.33 times the number of T cells. After overnight co-culture, CD25 protein expression in the CD8+ T cell fraction was analyzed by flow cytometry.

[0278] Figure 20A shows representative flow cytometry results from four experiments. Figure 20B shows the results of quantifying CD25 protein expression (n=4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison tests are shown in the figure). It was shown that mOKT3 / ss1 / CD80 / 41BBL / IL7 / IL15 nanoparticles (Figure 20, "ss1") induced T cell activation in the presence of target cells to a similar extent as mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (Figure 20, "FMC63").

[0279] (4) Induction of effector effects. T cells and K562-CD19 / Mesothelin cells were co-cultured in the presence of mOKT3 / ss1 / CD80 / 41BBL / IL7 / IL15 nanoparticles using the same method as in (3) above. After overnight co-culture, the number of surviving K562-CD19 / Mesothelin cells was analyzed by flow cytometry. The viability was calculated by setting the number of K562-CD19 / Mesothelin cells in wells cultured with only K562-CD19 / Mesothelin cells as 100%.

[0280] The results are shown in Figure 20C. In the presence of mOKT3 / ss1 / CD80 / 41BBL / IL7 / IL15 nanoparticles, the viability of K562-CD19 / Mesothelin cells was significantly reduced, and the effector effect was enhanced (n = 3, p values ​​from Student's t-test are shown in the figure).

[0281] Example 10: Sustained release effect of molecules encapsulated in PLGA nanoparticles (Objective) To verify the sustained release of molecules encapsulated in PLGA nanoparticles from the nanoparticles, and to examine the effect of the molecular weight of PLGA on the sustained release rate.

[0282] (Methods and Results) (1) Encapsulation of FITC-BSA into PLGA nanoparticles. Fluorescently modified bovine serum albumin (FITC-BSA) was encapsulated inside PLGA nanoparticles. An FITC-BSA aqueous solution was added dropwise to PLGA dissolved in dichloromethane (DCM) under ultrasonic irradiation to obtain a w / o emulsion (oil-in-water emulsion). The resulting w / o emulsion was then added dropwise to a PVA aqueous solution under ultrasonic irradiation. The DCM was then evaporated, and the nanoparticles were separated and washed using an ultracentrifuge to obtain FITC-BSA-encapsulated PLGA nanoparticles.

[0283] The fluorescence spectrum of the FITC-BSA-encapsulated PLGA nanoparticles is shown in Figure 21 A. Fluorescence from FITC-BSA was observed around 530 nm, confirming that FITC-BSA was encapsulated within the PLGA nanoparticles.

[0284] (2) Evaluation of the sustained release rate of FITC-BSA from PLGA nanoparticles The dispersion of FITC-BSA-encapsulated PLGA nanoparticles obtained in (1) above was encapsulated in a dialysis membrane and immersed in phosphate buffer, and the change over time in the concentration of FITC-BSA released outside the dialysis membrane was measured using a fluorometer.

[0285] The results are shown in Figure 21B. Approximately 50% of the encapsulated FITC-BSA was released over 7 days, demonstrating sustained release over a long period. Furthermore, increasing the molecular weight of PLGA from 10 kDa to 40 kDa reduced the sustained release rate from approximately 50% to 35% after 7 days.

[0286] Example 11: In vivo antitumor effect of nanoparticles (Objective) The tandem mOKT3 / FMC63 / CD80 / 41BBL nanoparticles prepared in Example 2 were administered to mice to examine the antitumor effect against the CD19-positive leukemia cell line NALM6 in the mice.

[0287] (Methods and Results) (1) Administration of nanoparticles. 1 x 10 cells of the CD19-positive leukemia cell line NALM6 (referred to as "NALM6-GL cells") transfected with the EGFP-P2A-Luc2 gene were administered. 6 Culture human T cells with 4 x 10 6 1 × 10 nanoparticles were administered into the tail vein of NSG mice (Charles River Japan, JAX Mice Stock No: 005557). Since NSG mice do not have T cells, human T cells were introduced from the outside by infusion to prepare T cells that could be activated by nanoparticles in the mouse body. From the next day, 1 × 10 nanoparticles were administered at intervals of 3 to 4 days. 12 mOKT3 / FMC63 / CD80 / 41BBL nanoparticles were administered intravenously three times. Starting 15 days after NALM6-GL cell administration, mOKT3 / FMC63 / CD80 / 41BBL nanoparticles were administered three additional times weekly (Figure 22A). Hereinafter, the group receiving the first three doses is referred to as "NPx3," and the group receiving six doses, including the first three and the last three, is referred to as "NPx6." A group that received no nanoparticles at all (hereinafter referred to as "No NP") served as a negative control.

[0288] (2) Time-lapse observation of NALM6-GL cells The proliferation of NALM6-GL cells in vivo was observed over time by luciferase luminescence using an IVIS (registered trademark) Spectrum in vivo imaging system (PerkinElmer).

[0289] Figure 22B shows imaging results from 2 to 5 weeks after NALM6-GL cell implantation in the No NP, NPx3, and NPx6 groups. Figure 22C shows the results of measuring NALM6 tumor burden as luciferase luminescence using IVIS Imaging. Figure 22C shows logarithmically transformed luminescence intensities. Significance was tested for each time point for each group of mice (n = 6) by one-way ANOVA with multiple comparison test (* P < 0.05, ** P < 0.01). At week 2, the NPx3 and NPx6 groups showed significantly reduced NALM6-GL cell mass compared with the No NP group (indicated as "No treatment" in the figure). At week 3, the NPx6 group showed significantly reduced NALM6-GL cell mass compared with the NPx3 group.

[0290] (3) Measurement of CD8+ T cell counts in peripheral blood. Peripheral blood was collected from mice in each group 2 and 4 weeks after NALM6-GL cell transplantation, and the percentage of CD8+ T cells was analyzed. The results are shown in Figure 23A. Repeated administration of nanoparticles significantly increased the number of human T cells administered to the mice over time (Student t-test, P<0.01).

[0291] (4) Kaplan-Meier Survival Analysis. Survival analysis of mice in each group was performed using the Kaplan-Meier method. The results are shown in Figure 23B (P values ​​are based on the log-rank test). The survival rate was significantly increased in the NPx3 group compared with the No NP group (indicated as "No treatment" in the figure). Furthermore, the survival rate was significantly increased in the NPx6 group compared with the NPx3 group.

[0292] (5) Evaluation of T cell memory phenotypes In the NPx6 group, the memory differentiation state of peripheral blood CD8+ T cells was analyzed at weeks 2 and 4. Specifically, the expression levels of marker proteins CCR7, CD45RA, CD62L, and FSC were assessed by flow cytometry.

[0293] The results are shown in Figure 24. Figure 24A shows a representative flow cytometry image. Figure 24B shows the percentage of memory stem cells (TSCM) and effector memory (TEM) fractions (n ​​= 6, mean ± SD; p values ​​for each fraction are shown in the figure by paired t-test). At week 2, the undifferentiated CD45RA+CD62L+CCR7+ fraction (memory stem cells, TSCM) accounted for approximately 40% of the total. However, by week 4, this percentage decreased, and the effector memory (TEM) fraction (CD45RA-CD62L-CCR7-) expanded. These results demonstrate that T cells were stimulated by nanoparticles in vivo and proliferated and differentiated into effector cells.

[0294] Example 12: Introduction of chemotactic molecules into nanoparticles and their effects (Objective) Nanoparticles containing bone marrow homing molecules were administered to mice to examine the effect on T cell proliferation in the bone marrow. CXCR4 protein and VLA4 (a heterodimer of ITGA4 protein and ITGB1 protein) were introduced into the nanoparticles as bone marrow homing molecules.

[0295] (Methods and Results) (1) Preparation of Nanoparticles Incorporating Bone Marrow-Homing Molecules Genes encoding the CD3 / CD19 binding protein, CD80 protein, and 41BBL protein corresponding to the tandem structures described in Examples 1 and 2, as well as genes encoding the mouse CXCR4 protein consisting of the amino acid sequence shown in SEQ ID NO: 23, the mouse ITGA4 protein consisting of the amino acid sequence shown in SEQ ID NO: 24, and the mouse ITGB1 protein consisting of the amino acid sequence shown in SEQ ID NO: 25, were introduced into the leukemia cell line K562. Expression of the CXCR4 protein, ITGA4 protein, and ITGB1 protein on the cell surface was confirmed by flow cytometry using the same method as in Example 1 ( Figure 25A ). Nanoparticles containing these proteins on their surface were prepared from these cells using the same method as in Example 2 (hereinafter referred to as "mOKT3 / FMC63 / CD80 / 41BBL / CXCR4 / ITGA4 / ITGB1 nanoparticles"). In this example, as a control, nanoparticles were used that did not contain the above-mentioned proteins CXCR4 protein, ITGA4 protein, and ITGB1 protein, but contained CD3 / CD19 binding protein, CD80 protein, and 41BBL protein (referred to as "mOKT3 / FMC63 / CD80 / 41BBL nanoparticles" in this example).

[0296] (2) Homing molecule-based effects in bone marrow. Cultured T cells and CD19-positive leukemia cell line NALM6 were incubated at 3 × 10 6 3 × 10 mOKT3 / FMC63 / CD80 / 41BBL / CXCR4 / ITGA4 / ITGB1 nanoparticles or mOKT3 / FMC63 / CD80 / 41BBL nanoparticles were administered intravenously to NSG mice, and 7 days later, 3 × 10 mOKT3 / FMC63 / CD80 / 41BBL nanoparticles were administered intravenously. 12 After a further 5 days, the bone marrow and spleen of the NSG mice were removed, and the proportion of human T cells relative to total bone marrow and spleen cells was calculated as CD45-positive cells by flow cytometry.

[0297] The results are shown in Figure 25C (n = 5, mean ± standard deviation). The mOKT3 / FMC63 / CD80 / 41BBL / CXCR4 / ITGA4 / ITGB1 nanoparticles containing bone marrow homing molecules significantly increased T cells in the bone marrow compared with the mOKT3 / FMC63 / CD80 / 41BBL nanoparticles (Figure 25C, Bone marrow, CXCR4+VLA4 (-) vs. (+); p values ​​from Student's t-test are shown in the figure). However, no significant difference was observed in the spleen (ns, not significant). These results demonstrate that nanoparticles can be delivered to specific organs by incorporating homing molecules into them as chemotactic molecules.

[0298] Example 13: Effect of nanoparticles with different particle sizes (Objective) To prepare nanoparticles with different particle sizes and to examine their influence on efficacy.

[0299] (Method and Results) In the method for preparing PLGA nanoparticle cores described in Example 2, the concentration of PLGA dissolved in acetone was set to 1 mg / mL or 20 mg / mL. The particle size of the obtained nanoparticle cores was measured, and the results are shown in Figure 26A. It was demonstrated that the particle size could be controlled based on different PLGA concentrations.

[0300] Next, nanoparticles were prepared by the same method as in Example 2 from PLGA nanoparticle cores with particle diameters of 105 nm, 153 nm, or 237 nm and cell membrane fragments derived from the K562 cell line into which the CD3 / CD19 binding protein, CD80 protein, and 41BBL protein described in Example 2 had been introduced. T cells and the NALM6 cell line were co-cultured in the presence of nanoparticles with different particle sizes. The T cells and NALM6 cells were co-cultured at the same cell numbers, with a ratio of 5 × 10 to the number of T cells. 6 After one week of co-culture, the number of T cells was measured by flow cytometry, and T cell proliferation was analyzed.

[0301] The results are shown in Figure 26B. A significant proliferation of T cells was observed when the particle diameter was 105 nm, 153 nm, or 237 nm (n = 4, mean ± standard deviation; * P < 0.01 (one-way ANOVA with multiple comparison test compared to the culture of T cells alone)).

[0302] Figure 26C shows the results of a similar co-culture performed for one day, followed by flow cytometry analysis of CD25 expression in the CD8+ T cell fraction to assess T cell activation. Regardless of particle size, T cell activation was induced only in the presence of target tumor cells, NALM6 cells (n = 4, mean ± standard deviation; * P < 0.01, NS not significant (one-way ANOVA with multiple comparison test compared with T cell-only culture)).

[0303] Example 14: Modification of tumor microenvironment based on immune regulators (Objective) Membrane vesicles are prepared with surface-mounted cytokine molecules or immune checkpoint inhibitor molecules as immune regulators. In this example, the cytokine molecules IL12 and IL18, TGF-β receptor, and immune checkpoint inhibitor molecules anti-PDL1 antibody and anti-CTLA4 antibody are introduced into the membrane vesicles, and the effects of these immune regulators on tumor microenvironment modification are evaluated.

[0304] (Methods and Results) (1) Preparation of Membrane Vesicles (MVs) The following fusion proteins (a) to (e) were designed as immunoregulatory molecules for modifying the tumor microenvironment ( Figure 27 ): (a) IL12 Fusion Protein: A fusion protein (hereinafter referred to as the "IL12 fusion protein") was designed by linking, from the N-terminus, SP, mouse-derived IL12-p40 protein, IL12-p35 protein, and the transmembrane and cytoplasmic domains derived from mouse-derived CD8α protein (hereinafter referred to as the "CD8α protein-derived fragment"). The full-length amino acid sequence of the IL12 fusion protein is shown in SEQ ID NO: 534. (b) IL18 Fusion Protein: A fusion protein (hereinafter referred to as the "IL18 fusion protein") was designed by linking, from the N-terminus, SP, mouse-derived IL18 protein, and a fragment derived from human-derived CD8α protein. The full-length amino acid sequence of the IL18 fusion protein is shown in SEQ ID NO: 535. (c) TGFBRI / II fusion protein: A fusion protein (hereinafter referred to as "TGFBRI / II fusion protein") was designed by linking, from the N-terminus, SP, the extracellular domains of mouse TGFBRI and mouse TGFBRII proteins, and a fragment derived from human CD8α protein. The full-length amino acid sequence of the TGFBRI / II fusion protein is shown in SEQ ID NO: 536. (d) Anti-PDL1-scFV fusion protein: A fusion protein (hereinafter referred to as "anti-PDL1-scFV fusion protein") was designed by linking, from the N-terminus, SP, anti-PDL1-scFV derived from anti-PDL1 antibody clone A09-246-2, and a fragment derived from human CD8α protein. The full-length amino acid sequence of the anti-PDL1-scFV fusion protein is shown in SEQ ID NO: 537. (e) Anti-CTLA4-scFV fusion protein. A fusion protein (hereinafter referred to as "anti-CTLA4-scFV fusion protein") was designed by linking, from the N-terminus, SP, anti-CTLA4-scFV derived from the anti-CTLA4 antibody clone 9D9, and a fragment derived from mouse CD8α protein. The full-length amino acid sequence of the anti-CTLA4-scFV fusion protein is shown in SEQ ID NO: 538.

[0305] In addition to any of the fusion proteins (a) to (e) above, genes encoding the following (i) to (iii): (i) a fusion protein linking, in order from the N-terminus, SP, 2C11 scFV (a single-chain variable region fragment derived from mouse anti-CD3ε antibody clone 2C11), and a fragment derived from human-derived CD8α protein (hereinafter referred to as "CD3 binding protein (2C11)"; the amino acid sequence of which is shown in SEQ ID NO: 539); (ii) a fusion protein linking, in order from the N-terminus, SP, 1D3 scFV (a single-chain variable region fragment derived from mouse anti-CD19 antibody clone 1D3), and a fragment derived from human-derived CD8α protein (hereinafter referred to as "CD19 binding protein (1D3)"; the amino acid sequence of which is shown in SEQ ID NO: 540); and (iii) a mouse-derived 41BBL protein consisting of the amino acid sequence shown in SEQ ID NO: 587 were transfected into a mouse lymphoma cell line A20 in which the CD19 gene and PDL1 gene had been knocked out.

[0306] The cells were suspended (1 × 10) in a hypotonic buffer solution (composition: 20 mM Tris-HCl pH 7.5, 10 mM KCl, 2 mM MgCl, EDTA-free protease inhibitors (Fang et al. Nano Lett. 2014)). 8 Cells were disrupted using a homogenizer, and it was confirmed under a microscope that almost all cells had been disrupted. The cell lysate was centrifuged at 3,200 g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was then ultracentrifuged at 100,000 g for 60 minutes at 4°C. The supernatant containing cytoplasmic proteins was removed, and the precipitate containing cell membrane fragments was suspended in washing buffer (10 mM Tris-HCl pH 7.5, 2 mM EDTA). This procedure was repeated, and the precipitate containing cell membrane fragments was thoroughly suspended in PBS under ultrasonic irradiation to obtain a concentration of 5 x 10 cells per μL. 4 ~3x10 5An MV solution containing cell-equivalent membrane vesicles (vesicles with a diameter of approximately 50 nm to approximately 200 nm and a sac-like structure, whose surface is composed of a lipid bilayer membrane in which proteins derived from cell membrane fragments are embedded) was prepared and used in the following examples. In the following examples, membrane vesicles that do not contain PLGA nanoparticle cores are referred to as "MVs (membrane vesicles)" to distinguish them from the nanoparticles containing PLGA nanoparticle cores in the above examples. Hereinafter, the MV containing the above (a) and (i) to (iii) on its surface layer will be referred to as "MV(2C11 / 1D3 / 41BBL / IL12)", the MV containing the above (b) and (i) to (iii) on its surface layer will be referred to as "MV(2C11 / 1D3 / 41BBL / IL18)", the MV containing the above (a), (b), and (i) to (iii) on its surface layer will be referred to as "MV(2C11 / 1D3 / 41BBL / IL12 / IL18)", and the MV containing the above (c) An MV containing (i) and (iii) on its surface layer is referred to as "MV(2C11 / 1D3 / 41BBL / TGFBR)," an MV containing (d) and (i) to (iii) on its surface layer is referred to as "MV(2C11 / 1D3 / 41BBL / anti-PDL1-scFV)," and an MV containing (e) and (i) to (iii) on its surface layer is referred to as "MV(2C11 / 1D3 / 41BBL / anti-CTLA4-scFV)." Furthermore, an MV containing only (i) to (iii) on its surface layer, without (a), was prepared by the same method as above and was referred to as "MV(2C11 / 1D3 / 41BBL)" as a control.

[0307] (2) Effect of IL-12 fusion protein / proliferation of CD8-positive T cells 5 × 10 per μL prepared in (1) above 4 Mouse CD8+ T cells (5 × 10) were cultured in a culture medium containing 50 μL of MV solution containing MVs equivalent to 10 cells. 4 cells) and the PDL1 gene knockout mouse lymphoma cell line A20 (5 × 10 4 The T cells were co-cultured for four days to assess their proliferation. CFSE dye was incorporated into the T cells before co-culture began, and the amount of CFSE dye diluted with cell division was analyzed using a flow cytometer after co-culture to calculate the percentage of T cells that underwent cell division.

[0308] The results are shown in Figure 28A. When T cells were cocultured with A20 cells (Figure 28, A20 cells:+), the proliferation of T cells was significantly enhanced in the presence of MVs (2C11 / 1D3 / 41BBL / IL12) carrying IL12 fusion protein on their surface (Figure 28, MV:IL12) compared with the presence of control MVs (2C11 / 1D3 / 41BBL) (Figure 28, MV:WT) (n = 4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure as *** P < 0.001, **** P < 0.0001).

[0309] (3) Effect of IL-12 fusion protein and IL-18 fusion protein / IFNγ production by CD8 positive T cells Mouse spleen-derived T cells were expressed in the mouse lymphoma cell line A20 expressing the CD3 binding protein (2C11) described in (i) above and the 41BBL protein described in (iii) above, and were treated with mitomycin, then co-cultured at a T cell:A20 cell ratio of 3:1 for 1 week and expanded. After expansion, 2 × 10 T cells were 5 5 x 10 cells per μL 4 After 24 hours of culture in a culture medium containing 50 μL of MV solution containing MVs equivalent to the number of cells, IFNγ production by CD8-positive T cells was evaluated using a flow cytometer.

[0310] The results are shown in Figure 28B. IFNγ production by T cells was induced in the presence of MVs carrying IL12 fusion protein (2C11 / 1D3 / 41BBL / IL12) or IL18 fusion protein (2C11 / 1D3 / 41BBL / IL18), significantly enhancing effector function (Figure 28B, MV:IL12 or MV:IL18). Furthermore, IFNγ production was further enhanced in the presence of MVs (2C11 / 1D3 / 41BBL / IL12 / IL18) (Figure 28B, MV:IL12+IL18) compared with either MV alone (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown as *P<0.05, **P<0.01, ***P<0.001).

[0311] (4) Effect of TGFβ receptor / adsorption of TGFβ. 200 μL of medium containing 10 ng / mL mouse TGFβ (Biolegend, #763102) was added with 5 × 10 cells per μL of the medium prepared in (1) above. 4 20 μL of MV (2C11 / 1D3 / 41BBL / TGFBR) solution containing MVs equivalent to the number of cells was added. After 1 hour, the TGF-β concentration in the supernatant was measured by ELISA (mouse TGF-beta 1 DuoSet, R&D systems #DY1679).

[0312] The results are shown in Figure 29A. When MV (2C11 / 1D3 / 41BBL / TGFBR) was added (Figure 29A, MV:TGFBR1 / II), the TGF-β content in the culture supernatant was reduced compared to the absence of MV (Figure 28, MV:-) and the presence of the control MV (2C11 / 1D3 / 41BBL) (Figure 28, MV:WT), demonstrating a significant TGFβ adsorption capacity (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure as **P<0.01, ***P<0.001).

[0313] (5) Effect of Anti-PDL1-scFV fusion protein 5 × 10 4 For each PDL1-positive mouse lymphoma cell line A20, 5 × 10 4 MV (2C11 / 1D3 / 41BBL / anti-PDL1-scFV) solution containing MVs equivalent to the number of cells was added at 5 μL, 10 μL, 20 μL, or 50 μL. After 4 hours, the PDL1 expression level in A20 cells was analyzed by flow cytometry.

[0314] The results are shown in Figure 29B. It was shown that the greater the dose of MV added, the more the PDL1 expression level on A20 cells decreased (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are shown in the figure as *** P < 0.001, **** P < 0.0001, and ns indicates that no significant difference was detected).

[0315] (6) Effect of Anti-CTLA4-scFV fusion protein. 5 × 10 4 For each mouse lymphoma cell line A20, 5 × 10 per μL of the 4 MV (2C11 / 1D3 / 41BBL / anti-CTLA4-scFV) solution containing the equivalent number of MVs to cells was added at 5 μL, 10 μL, 20 μL, or 50 μL. After 4 hours, the expression level of CTLA4 in A20 cells was analyzed by flow cytometry.

[0316] The results are shown in Figure 29C. It was shown that the expression level of CTLA4 on A20 cells decreased with increasing dose of MV (n = 3, mean ± standard deviation; p values ​​obtained by one-way ANOVA with multiple comparison test are shown in the figure as * P < 0.05, *** P < 0.001, **** P < 0.0001).

[0317] Example 15: In vivo antitumor effect of MV (Objective) The MV prepared in Example 14 was administered to mice to examine the antitumor effect on the colon cancer cell line CT26 in the mouse body.

[0318] (Methods and Results) In this example, MVs (hereinafter referred to as "TME-MVs") containing all of (a) to (e) and (i) to (iii) described in Example 14 on their surface were prepared in the same manner as described in Example 14 and administered to mice. That is, TME-MVs were MVs that had been introduced with genes encoding an IL12 fusion protein, an IL18 fusion protein, a TGFBRI / II fusion protein, an anti-PDL1-scFV fusion protein, an anti-CTLA4-scFV fusion protein, and a CD3-binding protein (2C11), a CD19-binding protein (1D3), and a 41BBL protein, and were prepared in the same manner as described in Example 14 from the mouse lymphoma cell line A20 in which the CD19 gene and PDL1 gene had been knocked out.

[0319] In this example, the MV (2C11 / 1D3 / 41BBL) prepared in Example 14 was used as a control MV (hereinafter referred to as "WT-MV" in this example).

[0320] The colon cancer cell line CT26, into which the mouse CD19 gene and luciferase gene had been introduced (hereinafter referred to as "CT26-CD19-luc cells"), was subcutaneously transplanted into Balb / c mice. PBS or the above-mentioned WT-MV or TME-MV was administered intratumorally 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. At each administration, 1.5 × 10 8 Mice were administered 50 μL of a 500 μL MV solution containing MVs equivalent to the cells. Time course observation of tumor size was performed based on luciferase luminescence detection and direct measurement of subcutaneous tumor diameter, as in Example 11. Survival analysis of mice was performed based on the tumor volume reaching 400 mm in succession. 3 The Kaplan-Meier method was used to determine the endpoint when the time point exceeded 100 μg / kg. The number of mice used in the experiment was n = 6 for the PBS-administered group, and n = 13 for each of the WT-MV-administered group and the TME-MV-administered group.

[0321] The results of measuring tumor diameter over time in the groups administered PBS, WT-MV, or TME-MV are shown in Figure 30. The mean tumor volumes in each group on days 13, 15, and 17 after CT26 cell inoculation are shown in Figure 31 (mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison tests are shown in the figure as *P<0.05, **P<0.01, and ns indicates no significant difference). The WT-MV-administered group had a lower tumor volume than the PBS-administered group, and the TME-MV-administered group showed even more effective suppression of tumor volume increase than the WT-MV-administered group.

[0322] The results of survival analysis of mice in each group are shown in Figure 31B. The survival rate was increased in the WT-MV-treated group compared with the PBS-treated group, and the survival rate was even higher in the TME-MV-treated group compared with the WT-MV-treated group (p values ​​by log-rank test are indicated in the figure as *P<0.05, **P<0.01). Figure 31C shows a representative example of tumor disappearance after 29 days in the TME-MV-treated group, analyzed over time by an in vivo imaging system to determine the tumor cell burden and localization in the mice.

[0323] Next, CT26-CD19-luc cells were subcutaneously implanted into Balb / c mice, and 8 days after implantation, PBS, WT-MV, or TME-MV was administered intratumorally. Tumor infiltrating cells were analyzed 13 days after implantation. The number of mice used in this experiment was n = 5 for the PBS-treated group, and n = 8 for the WT-MV-treated group and TME-MV-treated group. Figures 32 to 35 show the percentage of CD8+ T cells among CD45+ blood cells in the infiltrating cells (Figure 32A), the percentage of CD8+ T cells (CD45+CD8+ cells) among all tumor cells (Figure 32B), and a representative flow cytometry plot (Figure 33). The percentage of M2 macrophages (F4 / 80+CD206+ cells) among tumor-infiltrating macrophages (F4 / 80+ cells) (Figure 34) and a representative flow cytometry plot (Figure 35) are also shown (mean ± standard deviation; p-values ​​from one-way ANOVA with multiple comparison tests are indicated as *P<0.05, **P<0.01). Compared with the PBS- and WT-MV-treated groups, the TME-MV-treated group showed an increased percentage of CD8+ T cells among tumor-infiltrating cells and a decreased percentage of M2 macrophages among tumor-infiltrating macrophages. Comparison of the TME-MV and WT-MV groups suggests that the decrease in the proportion of M2 macrophages is due to the function of the immune regulatory molecules additionally loaded onto TME-MV. Example 16: Induction of NK cell effector function (Objective) Membrane vesicles loaded with NK cell activating ligands were prepared and the effector function of NK cells was evaluated. In this example, anti-NKp46 antibody, anti-NKG2D antibody, and IgG1 Fc region were used as NK cell activating ligands.

[0324] (Methods and Results) (1) Preparation of membrane vesicles (MVs) We confirmed by flow cytometry that the NK cell-activating ligand MICA / B was physiologically expressed on the leukemia cell line K562 (Fig. 36A). Furthermore, we confirmed by flow cytometry that HLA class I molecules, which have an NK cell-suppressive effect, were not expressed on the leukemia cell line K562 (Fig. 36B).

[0325] A gene encoding the following (a) and a gene encoding any one of the following (b) to (d) were introduced into the leukemia cell line K562: (a) a fusion protein linking, in order from the N-terminus, SP, FMC63 scFV (a single-chain variable region fragment derived from mouse anti-CD19 antibody clone FMC63), and a fragment derived from human-derived CD8α protein (this is the same as the "CD19 binding protein" described in Example 6 and consists of the amino acid sequence shown in SEQ ID NO: 11); (b) a fusion protein linking, in order from the N-terminus, SP, Nkp46-1 scFV (a single-chain variable region fragment derived from anti-NKp46 antibody clone Nkp46-1), and a fragment derived from human-derived CD8α protein (hereinafter referred to as "NKp46 binding protein", the amino acid sequence of which is shown in SEQ ID NO: 541); (c) a fusion protein linking, in order from the N-terminus, SP, NKG2D (d) a fusion protein linking, from the N-terminus, the cytoplasmic and transmembrane domains of the human transferrin receptor (human TFRC) and the IgG1 Fc region (CH2+CH3), which is an activating ligand for CD16 (hereinafter referred to as the "IgG1 Fc fusion protein"; the amino acid sequence of which is shown in SEQ ID NO: 543).

[0326] MVs were produced from the leukemia cell line K562 transfected with a gene encoding the above (a) and a gene encoding any one of the following (b) to (d) by the same method as in Example 14. Of the resulting MVs, MVs containing the above (a) and (b) on their surface are referred to as "Nkp46Ab-MVs," MVs containing the above (a) and (c) on their surface are referred to as "NKG2DAb-MVs," and MVs containing the above (a) and (d) on their surface are referred to as "IgG1Fc-MVs." In addition, MVs were produced from the leukemia cell line K562 transfected with only the above (a) and used as a comparative control in the following examples (hereinafter referred to as "WT-MVs" in these examples).

[0327] Furthermore, MV was produced from the leukemia cell line K562, into which all of the genes encoding (a) to (d) above had been introduced, by the same method as in Example 14. This MV is referred to as "IgG1Fc / NKG2DAb / Nkp46Ab-MV."

[0328] (2) Induction of effector function on NK cells. 3 × 10 per μL of the medium prepared in (1) above. 5 NK cells (1 × 10) were cultured in a culture medium containing 10 μL of MV solution containing MVs equivalent to 10 cells. 5 cells) and CD19-transfected multiple myeloma cell line MM.1S (1 × 10 5 The cells were co-cultured, and after 3 hours, the expression levels of activation markers CD69 and CD107a were analyzed by flow cytometry.

[0329] The results of flow cytometry are shown in Figure 37. NK cells cocultured with MM.1S-CD19 cells (MM.1S cells transfected with the CD19 gene) in the presence of Nkp46Ab-MV, NKG2DAb-MV, or IgG1Fc-MV showed elevated levels of activation markers compared with NK cells cultured alone, indicating activation. NK cells were also activated in the presence of WT-MV, demonstrating that IgG1Fc / NKG2DAb / Nkp46Ab-MV, which is loaded with multiple antibodies against activating ligands, can induce stronger effector functions than MV loaded with each antibody.

[0330] Next, 3 × 10 per μL of the solution prepared in (1) above 5 NK cells (1 × 10) were cultured in a culture medium containing 10 μL of MV solution containing MVs equivalent to 10 cells. 5 cells) and CD19-transfected multiple myeloma cell line MM.1S (1 × 10 5MM.1S cells were co-cultured with NKp46Ab-MV, NKG2DAb-MV, or IgG1Fc-MV, and the cytotoxic effect on MM.1S cells was analyzed by flow cytometry 24 hours later. MM.1S cell viability was significantly reduced after co-culture in the presence of Nkp46Ab-MV, NKG2DAb-MV, or IgG1Fc-MV, demonstrating that MV enhances the antitumor effect of NK cells (Fig. 38; n = 4, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison test are indicated as ***P < 0.001 in the figure).

[0331] (3) Verification of antigen specificity To verify that MV-mediated NK cell activation is specific to the antigen present on target cells, the cytotoxic effect was evaluated using the CD19-negative multiple myeloma cell line MM.1S (hereafter referred to as "MM.1S (CD19-negative)") and MM.1S stably transfected with the CD19 gene (hereafter referred to as "MM.1S (CD19-positive)"). MVs used were the FMC63-CD8α-transfected MVs prepared in (1) above and MVs isolated from non-transfected K562 cells.

[0332] 10 μL (1 × 10) of FMC63-CD8α-transfected MV or non-transfected MV 8 NK cells (1 x 10) were cultured in a medium supplemented with 10 µL of MVs (500 µL containing 1 x 10 cells). 5 1 × 10 cells) and MM.1S (CD19 negative) or MM.1S (CD19 positive) 5 After 24 hours of co-culture, the cytotoxic effect on MM.1S cells was analyzed by flow cytometry.

[0333] The results are shown in Figure 39 (n = 3, mean ± standard deviation; p values ​​from one-way ANOVA with multiple comparison tests are shown as ****P<0.0001). None of the MVs induced cytotoxic activity against MM.1S (CD19-negative) cells, regardless of whether they were transfected with FMC63-CD8α (Figure 39A). However, only MVs expressing FMC63-CD8α induced cytotoxic activity against MM.1S (CD19-positive) cells (Figure 39B). These results demonstrate that NK cell activation is induced specifically in an antigen-specific manner on target cells.

[0334] Example 17: Induction of phagocytic activity in macrophages (Objective) Nanoparticles loaded with an activating ligand for macrophages are prepared, and the induction of phagocytic activity in human and mouse macrophages is examined.

[0335] (Methods and Results) (1) Preparation of Nanoparticles A gene encoding the following (a), or a gene encoding the following (a) and a gene encoding the following (b) were introduced into the leukemia cell line K562: (a) a fusion protein linking, in order from the N-terminus, SP, FMC63 scFV (a single-chain variable region fragment derived from the mouse anti-CD19 antibody clone FMC63), and a fragment derived from human CD8α protein (this is the same as the “CD19 binding protein” described in Example 6 and consists of the amino acid sequence shown in SEQ ID NO: 11); (b) a fusion protein linking, in order from the N-terminus, the cytoplasmic and transmembrane domains of the human transferrin receptor (human TFRC) and the IgG1 Fc region (CH2 + CH3), which is an activating ligand for CD16 (hereinafter referred to as “IgG1 Fc fusion protein”, the amino acid sequence of which is shown in SEQ ID NO: 543).

[0336] Furthermore, a gene encoding the following (c), or a gene encoding the following (c) and a gene encoding the following (d) or (e) were introduced into the mouse lymphoma cell line A20: (c) a fusion protein linking, in order from the N-terminus, SP, 1D3 scFV (a single-chain variable region fragment derived from mouse anti-CD19 antibody clone 1D3), and a fragment derived from human-derived CD8α protein (this protein is identical to the "CD19 binding protein (1D3)" described in Example 14 and consists of the amino acid sequence shown in SEQ ID NO: 540); (d) a fusion protein linking, in order from the N-terminus, SP, anti-CD47 VHH (a camelized antibody derived from anti-CD47 antibody clone A4, which contains 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. 2016;113:E2646-54), and a fragment derived from human-derived CD8α protein (hereinafter referred to as "anti-CD47 (e) mouse CD40L consisting of the amino acid sequence set forth in SEQ ID NO: 546);

[0337] Nanoparticles (NPs) containing a PLGA nanoparticle core with a reconstituted cell membrane on its surface were prepared from transfected leukemia cell line K562 and mouse lymphoma cell line A20 by the same method as in Example 2. Of the resulting nanoparticles, NPs containing the above (a) on their surface layer are referred to as "NP(FMC63)," NPs containing the above (a) and (b) on their surface layer are referred to as "NP(FMC63 / IgG1 Fc)," NPs containing the above (c) on their surface layer are referred to as "NP(1D3)," NPs containing the above (c) and (d) on their surface layer are referred to as "NP(1D3 / CD40L)," and NPs containing the above (c) and (e) on their surface layer are referred to as "NP(1D3 / anti-CD47 VHH)."

[0338] (2) Induction of phagocytic activity in human macrophages. Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) using anti-CD14 microbeads (Miltenyi Biotec). The isolated monocytes were then differentiated into macrophages by culturing them for 6 days in medium supplemented with M-CSF (Peprotech, #300-25). Subsequently, the macrophages were differentiated into M2-type macrophages by culturing them for 2 days in medium supplemented with 20 ng / mL IL-4 (Peprotech, #200-04).

[0339] The nanoparticles (5 × 10 11 Human M2 macrophages (3 × 10 4 cells) and CD19-transfected, CFSE-labeled K562 cells (1 × 10 5 After 4 hours of co-culture, the percentage of macrophages (CD14+CFSE+) that had phagocytosed K562 cells and turned CFSE-positive was analyzed by flow cytometry.

[0340] The results of flow cytometry are shown in Figure 40. In the presence of NPs (FMC63 / IgG1 Fc) containing the IgG1 Fc fusion protein on their surface, the percentage of macrophages that phagocytosed K562 cells and became CFSE-positive was significantly increased compared to the control NPs (FMC63), demonstrating that the phagocytic ability of macrophages was significantly enhanced.

[0341] (3) Induction of phagocytic activity in mouse macrophages. Monocytes isolated from mouse bone marrow were differentiated into macrophages by culturing them for 7 days in a medium supplemented with 10 ng / mL M-CSF (Peprotech, #300-25). The macrophages were then differentiated into M2-type macrophages by culturing them for 2 days in a medium supplemented with 20 ng / mL IL-4 (Peprotech, #200-04).

[0342] The nanoparticles (5 × 10 11 M2-type macrophages (3 × 10 4cells) and CFSE-labeled CD19-positive lymphoma cell line A20 cells (1 × 10 5 After 4 hours of co-culture, the percentage of macrophage cells (F4 / 80+CFSE+) that had phagocytosed the CFSE-labeled A20 cells and turned CFSE-positive was analyzed by flow cytometry.

[0343] The results of flow cytometry are shown in Figure 41. In the presence of NP (1D3 / CD40L) containing a CD40L fusion protein on its surface layer or NP (1D3 / Anti-CD47 VHH) containing an anti-CD47 VHH fusion protein on its surface layer, the percentage of macrophages that phagocytosed A20 cells and became CFSE-positive was significantly increased compared to the control NP (1D3), indicating that the phagocytic ability of macrophages was significantly enhanced.

[0344] Example 18: Evaluation of sequences derived from various anti-CD3 antibody clones (Objective) Membrane vesicles carrying scFV derived from the anti-CD3 antibody clones OKT3, L2K, or UCHT1 on their surface were prepared, and their effect on inducing effector function in T cells was evaluated.

[0345] (Methods and Results) (1) Preparation of Membrane Vesicles (MVs) A gene encoding any one of the following (a) to (c), a gene encoding either one of the following (d) or (e), and further genes encoding the following (f) to (i) were introduced into the leukemia cell line K562: (a) a fusion protein linking, in order from the N-terminus, SP, OKT3 scFV (a single-chain variable region fragment derived from the mouse anti-CD3ε antibody clone OKT3), and a fragment derived from human-derived CD8α protein (this is the same as the “CD3 binding protein” described in Example 6, and consists of the amino acid sequence shown in SEQ ID NO: 9; this example will be referred to as “CD3 binding protein (OKT3)”); (b) a fusion protein linking, in order from the N-terminus, SP, L2K scFV (a single-chain variable region fragment derived from the mouse anti-CD3ε antibody clone L2K), and a fragment derived from human-derived CD8α protein (this will be referred to as “CD3 binding protein (L2K)” hereinafter, and the amino acid sequence of this protein will be shown in SEQ ID NO: 547); (c) (d) a fusion protein linking, in order from the N-terminus, SP, UCHT1 scFV (a single-chain variable region fragment derived from mouse anti-CD3ε antibody clone UCHT1), and a fragment derived from human-derived CD8α protein (hereinafter referred to as "CD3 binding protein (UCHT1)", the amino acid sequence of which is shown by SEQ ID NO: 548); (e) a fusion protein linking, in order from the N-terminus, SP, ss1 scFV (a single-chain variable region fragment derived from anti-mesothelin antibody clone ss1), and a fragment derived from human-derived CD8α protein (this is the same as the "mesothelin binding protein" described in Example 9, and consists of the amino acid sequence shown by SEQ ID NO: 21); (f) a CD80 protein consisting of the amino acid sequence shown by SEQ ID NO: 7; (g) a 41BBL protein consisting of the amino acid sequence shown in SEQ ID NO: 8; (h) an IL-7 protein consisting of the amino acid sequence shown in SEQ ID NO: 43;(i) an IL-15 protein consisting of the amino acid sequence shown in SEQ ID NO: 44;

[0346] MVs were prepared from the leukemia cell line K562 into which the above genes had been introduced, in the same manner as in Example 14. Of the obtained MVs, MVs containing the above (a), (d), and (f) to (i) on their surface layer are referred to as "ss1-OKT3-MVs"; MVs containing the above (a), (e), and (f) to (i) on their surface layer are referred to as "AbA-OKT3-MVs"; MVs containing the above (b), (d), and (f) to (i) on their surface layer are referred to as "ss1-L2K-MVs"; MVs containing the above (b), (e), and (f) to (i) on their surface layer are referred to as "AbA-L2K-MVs"; MVs containing the above (c), (d), and (f) to (i) on their surface layer are referred to as "ss1-UCHT1-MVs"; and MVs containing the above (c), (e), and (f) to (i) on their surface layer are referred to as "AbA-UCHT1-MVs". In addition, MV was prepared from the leukemia cell line K562, which had not been transfected with any of the above (a) to (i), and used as a comparative control in the following examples (hereinafter referred to as "MV (No Ab)").

[0347] (2) Induction of effector function in T cells Flow cytometry analysis of the ovarian cancer cell line TOV21G (ATCC #CRL-11730) confirmed the expression of mesothelin (MSLN) and EGFR on the cell surface ( Figure 42 ).

[0348] 5 × 10 per 1 μL prepared in (1) above 5 TOV21G cells (1 x 10) were cultured in a medium containing 10 μL of MV solution containing MVs equivalent to 10 cells. 5 ) and T cells (1 × 10 5 After 24 hours, the cytotoxic activity against TOV21G cells was analyzed by flow cytometry.

[0349] The results are shown in Figure 43. It was shown that all of the MVs, ss1-OKT3-MV, AbA-OKT3-MV, ss1-L2K-MV, AbA-L2K-MV, ss1-UCHT1-MV, and AbA-UCHT1-MV, were able to induce significant cytotoxic effects compared to MV (No Ab), and that the three anti-CD3 antibody clones (OKT3, L2K, and UCHT1) were able to induce comparable effects (n = 3, mean ± standard deviation; one-way ANOVA with multiple comparison test; ns indicates no significant difference). All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A multispecific nanoparticle capable of binding to two or more target cells, (1) The lipid film constituting the surface layer of the nanoparticles, and (2) (i) A fusion protein comprising a membrane-binding region derived from CD8α protein bound to the lipid membrane, and two or more target-binding regions located outside the lipid membrane, or (ii) Two or more fusion proteins comprising a membrane-binding region derived from the CD8α protein bound to the lipid membrane, and a target-binding region located outside the lipid membrane. The multispecific nanoparticles comprising the above.

2. The aforementioned target binding region is (a) T cell receptor complex binding region, NK cell surface antigen binding region, or macrophage surface antigen binding region, and (b) Cancer antigen binding region The multispecific nanoparticles according to claim 1, comprising:

3. The multispecific nanoparticle according to claim 2, 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.

4. The aforementioned anti-CD3 antibody, [1] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 72 to 74, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 75 to 77, respectively. [2] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 80 to 82, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 83 to 85, respectively. [3] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 88 to 90, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 91 to 93, respectively. [4] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 96 to 98, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 99 to 101, respectively. [5] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 104 to 106, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 107 to 109, respectively. [6] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 112 to 114, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 115 to 117, respectively. [7] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 120 to 122, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 123 to 125, respectively. [8] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 128-130, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 131-133, respectively. [9] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 136 to 138, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 139 to 141, respectively. [10] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 144 to 146, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 147 to 149, respectively. [11] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 152 to 154, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 155 to 157, respectively. [12] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 160 to 162, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 163 to 165, [13] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 168 to 170, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 171 to 173, [14] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 176 to 178, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 179 to 181, respectively. [15] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 345 to 347, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 348, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs.

350. [16] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 353 to 355, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 356 to 358, respectively. [17] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 359 to 361, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 362 to 364, respectively. [18] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 367 to 369, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 370 to 372, respectively. [19] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 375 to 377, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 378 to 380, respectively. [20] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 383 to 385, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 386 to 388, respectively. [21] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 391 to 393, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 394 to 396, respectively. [22] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 399 to 401, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 402 to 404, respectively. [23] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407-426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427-434, [24] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 435 to 437, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 438 to 440, respectively. [25] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 443 to 445, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 446 to 448, respectively. [26] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 451 to 453, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 454, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 456 [27] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 459 to 461, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 462, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 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] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 519 to 521, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 522, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 524, [30] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 530 to 532, respectively. [31] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 530 to 531 and 533, respectively, or [32] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 566 to 568, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 569 to 571, respectively. including, or The aforementioned anti-TCR antibody [33] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 184 to 186, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 187 to 189, respectively. The multispecific nanoparticles according to claim 3, comprising:

5. The multispecific nanoparticle according to claim 2, wherein the NK cell surface antigen-binding region is selected from the group consisting of anti-CD16 antibody or fragment thereof, anti-NKG2D antibody or fragment thereof, anti-Nkp30 antibody or fragment thereof, anti-Nkp44 antibody or fragment thereof, anti-Nkp46 antibody or fragment thereof, anti-2B4 antibody or fragment thereof, IgG Fc region or fragment thereof, MICA protein or fragment thereof, MICB protein or fragment thereof, B7H6 protein or fragment thereof, and influenza virus-derived hemagglutinin or fragment thereof.

6. The aforementioned anti-CD16 antibody [34] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 300 to 302, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 303 to 305, respectively. Includes, The aforementioned anti-NKG2D antibody [35] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 308 to 310, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 311 to 313, respectively. Includes, The aforementioned anti-NKp46 antibody [36] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 316 to 318, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 319 to 321, respectively. Includes, The IgG Fc region is [37] Human IgG1 Fc region consisting of the amino acid sequence shown in Sequence ID No. 30, or [38] Human IgG3 Fc region consisting of the amino acid sequence shown in Sequence ID No. 61 The multispecific nanoparticles according to claim 5, comprising:

7. The multispecific nanoparticle according to claim 2, wherein the macrophage surface antigen-binding region is selected from the group consisting of an anti-FcγRI (CD64) antibody or fragment thereof, an anti-FcγRIIA (CD32) antibody or fragment thereof, an anti-FcγRIIIA (CD16A) antibody or fragment thereof, an anti-CD40 antibody or fragment thereof, an IgG Fc region or fragment thereof, a CD40L protein or fragment thereof, an anti-CD47 antibody or fragment thereof, and a TLR4a ligand.

8. The aforementioned anti-CD40 antibody [39] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 324 to 326, respectively, and light chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 327 to 329, respectively, or [40] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 332 to 334, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 335 to 337, respectively. Includes, The IgG Fc region is [41] Human IgG1 Fc region consisting of the amino acid sequence shown in Sequence ID No. 30, or [42] Human IgG3 Fc region consisting of the amino acid sequence shown in Sequence ID No. 61 Includes, The anti-CD47 antibody or fragment thereof [43] Heavy chain variable regions including CDR1 to CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 590 to 592, and light chain variable regions including CDR1 to CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 593 to 595, or [44] Variable regions including CDR1 to CDR3, which consist of amino acid sequences shown in SEQ ID NOs. 583 to 585, respectively. including, or The TLR4a ligand mentioned above [45] A TLR4a ligand consisting of the amino acid sequence shown in Sequence ID No. 582, or [46] TLR4a ligand consisting of the amino acid sequence shown in SEQ ID NO: 596 The multispecific nanoparticles according to claim 7, comprising:

9. The multispecific nanoparticle according to any one of claims 2 to 8, wherein 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.

10. The multispecific nanoparticle according to any one of claims 2 to 8, further comprising one or more selected from the group consisting of costimulatory molecules, cytokine molecules, cytokine receptors, migratory molecules, immune checkpoint inhibitor molecules, and immune checkpoint molecules in the surface and / or interior of the nanoparticle.

11. The aforementioned co-stimulatory molecule is one or more 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 one or more selected from the group consisting of 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, IFN-γ protein, TNF-α protein, and TGF-β protein. The migratory 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 cytokine receptor is one or more selected from the group consisting of TGF-β receptor, IL-6 receptor, IL-1 receptor, TNF receptor, IL-4 receptor, IL-10 receptor, IL-13 receptor, and CSF-1 receptor. The immune checkpoint inhibitor molecule is one or more selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, B7-H3 inhibitors, VISTA inhibitors, ICOS inhibitors, ICOS stimulants, BTLA inhibitors, CD47 inhibitors, KIR inhibitors, LIR inhibitors, CD94 inhibitors, and NKG2A inhibitors, and / or The multispecific nanoparticles according to claim 10, wherein 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.

12. The multispecific nanoparticle according to any one of claims 1 to 8, wherein the lipid membrane is composed of a cell membrane, or a cell membrane and liposomes.

13. A multispecific nanoparticle according to any one of claims 1 to 8, further comprising a nanoparticle core made of polymer nanoparticles, metal nanoparticles, or a dendrimer, wherein the lipid membrane encapsulates the nanoparticle core.

14. Multiple specificity nanoparticles according to any one of claims 1 to 8, wherein the particle size is 5 nm to 250 nm.

15. A pharmaceutical composition comprising multispecific nanoparticles according to any one of claims 1 to 8.

16. A fusion protein comprising a membrane-binding region derived from CD8α protein, and a T cell receptor complex-binding region and a cancer antigen-binding region on its N-terminal 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.

17. The aforementioned anti-CD3 antibody, [1] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 72 to 74, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 75 to 77, respectively. [2] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 80 to 82, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 83 to 85, respectively. [3] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 88 to 90, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 91 to 93, respectively. [4] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 96 to 98, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 99 to 101, respectively. [5] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 104 to 106, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 107 to 109, respectively. [6] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 112 to 114, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 115 to 117, respectively. [7] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 120 to 122, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 123 to 125, respectively. [8] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 128-130, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 131-133, respectively. [9] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 136 to 138, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 139 to 141, respectively. [10] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 144 to 146, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 147 to 149, respectively. [11] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 152 to 154, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 155 to 157, respectively. [12] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 160 to 162, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 163 to 165, [13] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 168 to 170, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 171 to 173, [14] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 176 to 178, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 179 to 181, respectively. [15] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 345 to 347, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 348, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs.

350. [16] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 353 to 355, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 356 to 358, respectively. [17] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 359 to 361, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 362 to 364, respectively. [18] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 367 to 369, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 370 to 372, respectively. [19] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 375 to 377, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 378 to 380, respectively. [20] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 383 to 385, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 386 to 388, respectively. [21] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 391 to 393, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 394 to 396, respectively. [22] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 399 to 401, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 402 to 404, respectively. [23] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407-426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427-434, [24] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 435 to 437, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 438 to 440, respectively. [25] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 443 to 445, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 446 to 448, respectively. [26] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 451 to 453, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 454, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 456 [27] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 459 to 461, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 462, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 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] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 519 to 521, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 522, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 524, [30] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 530 to 532, respectively. [31] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 530 to 531 and 533, respectively, or [32] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 566 to 568, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 569 to 571, respectively. Includes, The aforementioned anti-TCR antibody [33] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 184 to 186, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 187 to 189, respectively. The fusion protein according to claim 16, comprising:

18. A nucleic acid encoding the fusion protein according to claim 16 or 17.

19. A gene expression vector comprising the nucleic acid described in claim 18 in an expressible state.

20. A host cell comprising the gene expression vector according to claim 19.

21. A gene expression vector containing nucleic acids in an expressionable state that encode a fusion protein including a T cell receptor complex binding domain and a membrane-binding domain derived from CD8α protein, and A gene expression vector containing nucleic acids encoding a fusion protein including a cancer antigen-binding region and a membrane-binding region, in a state capable of expression. Includes, A host cell in which 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.

22. The aforementioned anti-CD3 antibody, [1] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 72 to 74, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 75 to 77, respectively. [2] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 80 to 82, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 83 to 85, respectively. [3] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 88 to 90, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 91 to 93, respectively. [4] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 96 to 98, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 99 to 101, respectively. [5] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 104 to 106, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 107 to 109, respectively. [6] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 112 to 114, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 115 to 117, respectively. [7] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 120 to 122, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 123 to 125, respectively. [8] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 128-130, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 131-133, respectively. [9] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 136 to 138, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 139 to 141, respectively. [10] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 144 to 146, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 147 to 149, respectively. [11] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 152 to 154, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 155 to 157, respectively. [12] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 160 to 162, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 163 to 165, [13] Heavy chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 168 to 170, and light chain variable regions including CDR1, CDR2, and CDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 171 to 173, [14] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 176 to 178, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 179 to 181, respectively. [15] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 345 to 347, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 348, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs.

350. [16] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 353 to 355, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 356 to 358, respectively. [17] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 359 to 361, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 362 to 364, respectively. [18] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 367 to 369, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 370 to 372, respectively. [19] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 375 to 377, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 378 to 380, respectively. [20] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 383 to 385, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 386 to 388, respectively. [21] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 391 to 393, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 394 to 396, respectively. [22] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 399 to 401, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 402 to 404, respectively. [23] A heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 407-426, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 427-434, [24] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 435 to 437, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 438 to 440, respectively. [25] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 443 to 445, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 446 to 448, respectively. [26] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 451 to 453, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 454, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 456 [27] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 459 to 461, respectively, and light chain variable regions comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 462, CDR2 consisting of the amino acid sequence AAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 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] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 519 to 521, respectively, and light chain variable regions including CDR1 consisting of the amino acid sequence shown in SEQ ID NOs. 522, CDR2 consisting of the amino acid sequence GAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NOs. 524, [30] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 530 to 532, respectively. [31] Heavy chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 527 to 529, respectively, and light chain variable regions including CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 530 to 531 and 533, respectively, or [32] Heavy chain variable regions comprising CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 566 to 568, respectively, and light chain variable regions comprising CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 569 to 571, respectively, or the anti-TCR antibody, [33] Heavy chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 184 to 186, respectively, and light chain variable regions including CDR1, CDR2, and CDR3, which consist of the amino acid sequences shown in SEQ ID NOs. 187 to 189, respectively. A host cell according to claim 21, comprising:

23. The host cell according to claim 20, further comprising one or more gene expression vectors capable of expressing nucleic acids encoding any protein selected from the group consisting of costimulatory molecules, cytokine molecules, cytokine receptors, migratory molecules, immune checkpoint inhibitor molecules, and immune checkpoint molecules.

24. A method for producing multispecific nanoparticles that can bind to two or more target cells, (i) Two or more fusion proteins containing a membrane-binding domain and a target-binding domain derived from the CD8α protein, or (ii) A fusion protein containing a membrane-binding domain derived from the CD8α protein and two or more target-binding domains. A cell membrane fragmentation step involves lysing cells that express on their cell membranes to fragment the cell membranes. A nanoparticle formation step, which involves reconstructing the cell membrane fragments after the fragmentation step to form nanoparticles, and Recovery process for recovering the aforementioned nanoparticles. The method, including the method described above.

25. The aforementioned target binding region is (a) T cell receptor complex binding region, NK cell surface antigen binding region, or macrophage surface antigen binding region, and (b) Cancer antigen binding region The method according to claim 24, including the method described in claim 24.

26. The method according to claim 24, wherein fractions other than cell membrane fragments and / or undisrupted cells are removed between the cell membrane fragmentation step and the nanoparticle formation step.

27. The method according to any one of claims 24 to 26, wherein the reconstruction in the nanoparticle formation step includes mixing the cell membrane fragment with liposomes and / or nanoparticle cores to form nanoparticles in which a lipid membrane formed by the fusion of the cell membrane fragment and the liposomes constitutes the surface layer thereof, and / or nanoparticles in which the lipid membrane containing the cell membrane fragment encapsulates the nanoparticle core.

28. The method according to claim 24, wherein the reconstruction in the nanoparticle formation step includes sonication of the cell membrane fragment.