CN116635519A

CN116635519A - Modified immune cell and application thereof

Info

Publication number: CN116635519A
Application number: CN202180079469.7A
Authority: CN
Inventors: 李本尚; 王天怡; 万欣宇; 杨晓敏
Original assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd
Current assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd
Priority date: 2020-11-26
Filing date: 2021-11-25
Publication date: 2023-08-22
Also published as: WO2022111562A1

Abstract

The present application relates to a fusion protein, and in particular to a modified immune cell comprising the fusion protein and uses thereof. The fusion proteins comprise an antigen or functionally active fragment thereof, a co-stimulatory domain and an intracellular signaling domain. The modified cells comprising the fusion protein bind to multispecific antibodies, have good killing effect on tumor cells, and are named antigen-coupled antibody-directed T cell immunotherapy (Antigen Coupling Antibody Redirect T cell Immunotherapy, agCAR-T).

Description

Modified immune cell and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a fusion protein, a modified immune cell and application thereof.

Background

Chimeric antigen receptor T cell immunotherapy (Chimeric antigen receptor T-cell immunotherapy, CART) is an effective adoptive cell immunotherapy developed in recent years, and the method achieves the aim of eliminating tumor cells in vivo by carrying out genetic engineering on autologous-derived T cells to make effector T lymphocytes produce targeted cytotoxicity against tumor cells.

The targeting of CART depends on engineering T cells to express specific single chain antibodies (scFv) recognizing self tumor cell associated antigens (Tumor associated antigen, TAA), consisting of the light chain variable region (VL) and the heavy chain variable region (VH) of TAA specific monoclonal antibodies, with a soft hinge region between VL and VH. In addition to expressing specific single-chain antibodies against tumor cells on the surface of T cells, co-stimulatory molecules such as CD28, 4-1BB and OX4, etc. which can synergistically activate T cells are simultaneously expressed in T cells, so that on one hand, tumor cells are recognized by means of the specific single-chain antibodies, and on the other hand, targeted cytotoxicity is generated by the co-stimulatory molecules and CD3 zeta to synergistically activate T cells.

At present, CART technology has achieved great success in liquid tumors, but has not yet been changed significantly in the field of application of solid tumors. Problems faced by CART treatment include: 1) The parts of solid tumors do not spread on the whole body like leukemia, CART cells need to reach the focus of the solid tumors and infiltrate into the inside of the tumors to play a role, which is the space obstruction effect of CART treatment formed by the tissue structure of the solid tumors; 2) Inhibition of the immune microenvironment within the tumor can cause the CART cells to fail to function normally and can produce T cell depletion; 3) The conditions of hypoxia, nutrient deficiency and the like exist in the microenvironment inside the solid tumor, and the large-scale proliferation of CART cells and the targeted cytotoxin effect are not facilitated. There is therefore a need to develop new structures for the treatment of tumors.

Disclosure of Invention

The present application provides a fusion protein, a modified cell comprising and/or expressing the fusion protein and a multispecific antibody having one or more of the following properties: 1) The fusion protein can bind to a first targeting moiety of a multispecific antibody that activates a modified cell comprising the fusion protein, and a second targeting moiety of the multispecific antibody can target a tumor cell, thereby killing the tumor by the modified cell comprising the fusion protein; 2) The range of cellular immunotherapy is widened; 3) The modified cells comprising the fusion protein are effective and uniform for attacking tumor cells; 4) Solving the problem of T cell exhaustion; 5) Can be used for treating solid tumor; 6) The risk of cytokine storm is reduced; 7) Does not cause autoimmune reaction.

In one aspect, the application provides a fusion protein comprising: 1) An antigen or functionally active fragment thereof; 2) A costimulatory domain; and 3) an intracellular signaling domain.

In certain embodiments, the antigen comprises a tumor-associated antigen or a functionally active fragment thereof.

In certain embodiments, the antigen comprises an immune cell activation-related antigen or functionally active fragment thereof.

In certain embodiments, the immune cell activation-related antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of: CD45, CD56 and CD58.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7 and BCMA.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises a CD19 protein or functionally active fragment thereof.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises a CD20 protein or functionally active fragment thereof.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises a CD22 protein or functionally active fragment thereof.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises a CD33 protein or functionally active fragment thereof.

In certain embodiments, the tumor-associated antigen or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 2, 4, 6, 8 and 10.

In certain embodiments, the co-stimulatory domain comprises a co-stimulatory domain derived from a protein selected from the group consisting of: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88.

In certain embodiments, the costimulatory domain comprises a costimulatory domain derived from 4-1 BB.

In certain embodiments, the costimulatory domain comprises the amino acid sequence depicted in SEQ ID NO. 12.

In certain embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fceri γ, fceri β, fcgammaRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.

In certain embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from cd3ζ.

In certain embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO. 19.

In certain embodiments, the C-terminus of the tumor-associated antigen or functionally active fragment thereof is directly or indirectly linked to the N-terminus of the costimulatory domain.

In certain embodiments, the C-terminus of the costimulatory domain is directly or indirectly linked to the N-terminus of the intracellular signaling domain.

In certain embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO. 20.

In another aspect, the application provides one or more isolated nucleic acid molecules encoding the fusion proteins.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO. 21.

In another aspect, the application provides one or more vectors comprising said isolated nucleic acid molecules.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises a lentiviral vector.

In another aspect, the application provides a modified cell comprising said fusion protein, said nucleic acid molecule and/or said vector.

In certain embodiments, the modified cell comprises an immune cell.

In certain embodiments, the modified cell comprises an immune effector cell.

In certain embodiments, the immune effector cell is selected from the group consisting of: t cells, macrophagesNatural killer cells (NK cells) and NKT cells.

In certain embodiments, the modified cell comprises a T cell or a macrophage

In certain embodiments, the modified cell comprises an NK cell or a NKT cell.

In certain embodiments, the modified cells further comprise and/or express one or more multispecific antibodies.

In certain embodiments, the multispecific antibody comprises a first targeting moiety and a second targeting moiety.

In certain embodiments, the first targeting moiety is capable of specifically binding to a tumor-associated antigen.

In certain embodiments, the first targeting moiety recognizes the antigen or functionally active fragment thereof in the fusion protein.

In certain embodiments, the tumor-associated antigen targeted by the first targeting moiety is selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7 and BCMA.

In certain embodiments, the tumor-associated antigen targeted by the second targeting moiety is selected from the group consisting of: GD2, DLL1, DLL3, ROR1, GPC3, HER2, VEGFA, CD1a, CD33, B7H3, CD138, BCMA, EGFRVIII and ERBB2.

In certain embodiments, the first targeting moiety recognizes a CD19 protein or functionally active fragment thereof, and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes CD19 protein or a functionally active fragment thereof, and the second targeting moiety recognizes GD2 protein or a functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes a CD19 protein or functionally active fragment thereof, and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes a CD22 protein or functionally active fragment thereof, and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes CD22 protein or a functionally active fragment thereof, and the second targeting moiety recognizes GD2 protein or a functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes a CD22 protein or functionally active fragment thereof, and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes a CD20 protein or functionally active fragment thereof, and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes CD20 protein or a functionally active fragment thereof, and the second targeting moiety recognizes GD2 protein or a functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes a CD20 protein or functionally active fragment thereof, and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes CD33 protein or a functionally active fragment thereof, and the second targeting moiety recognizes GD2 protein or a functionally active fragment thereof.

In certain embodiments, the first targeting moiety recognizes CD33 protein or a functionally active fragment thereof, and the second targeting moiety recognizes B7H3 protein or a functionally active fragment thereof.

In certain embodiments, the first targeting moiety comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85 and SEQ ID NO: 94.

In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 24, SEQ ID NO. 32, SEQ ID NO. 40, SEQ ID NO. 56, SEQ ID NO. 63, SEQ ID NO. 71, SEQ ID NO. 79, and SEQ ID NO. 95.

In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 52, SEQ ID NO. 57, SEQ ID NO. 64, SEQ ID NO. 72, SEQ ID NO. 80, SEQ ID NO. 86, and SEQ ID NO. 96.

In certain embodiments, the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups:

a) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24 and HCDR3: SEQ ID NO. 25;

b) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

c) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

d) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 41;

e) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 47;

f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 52;

g) HCDR1: SEQ ID NO. 55, HCDR2: SEQ ID NO. 56 and HCDR3: SEQ ID NO. 57;

h) HCDR1: SEQ ID NO. 62, HCDR2: SEQ ID NO. 63 and HCDR3: SEQ ID NO. 64;

i) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

j) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

k) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

l) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

m) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: SEQ ID NO. 96.

In certain embodiments, the VH comprises an amino acid sequence as set forth in any one of SEQ ID NO. 22, SEQ ID NO. 30, SEQ ID NO. 38, SEQ ID NO. 46, SEQ ID NO. 51, SEQ ID NO. 54, SEQ ID NO. 61, SEQ ID NO. 69, SEQ ID NO. 77, SEQ ID NO. 84, SEQ ID NO. 90 and SEQ ID NO. 93.

In certain embodiments, the first targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92 and SEQ ID NO: 98.

In certain embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 44, SEQ ID NO. 67, SEQ ID NO. 75, and SEQ ID NO. 99.

In certain embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 45, SEQ ID NO. 50, SEQ ID NO. 45, SEQ ID NO. 60, SEQ ID NO. 68, SEQ ID NO. 76, SEQ ID NO. 83, SEQ ID NO. 89, and SEQ ID NO. 100.

In certain embodiments, the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any one of the following groups:

a) LCDR1: SEQ ID NO. 27, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 29;

b) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

c) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

d) LCDR1: SEQ ID NO. 43, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

e) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 50;

f) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

g) LCDR1: SEQ ID NO 59, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 60;

h) LCDR1: SEQ ID NO:66, LCDR2: SEQ ID NO 67 and LCDR3: SEQ ID NO. 68;

i) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

j) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

k) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

l) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

m) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100.

In certain embodiments, the VL comprises an amino acid sequence set forth in any one of SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 42, SEQ ID NO. 48, SEQ ID NO. 53, SEQ ID NO. 58, SEQ ID NO. 65, SEQ ID NO. 73, SEQ ID NO. 81, SEQ ID NO. 87, SEQ ID NO. 91, and SEQ ID NO. 97.

In certain embodiments, the second targeting moiety comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:136, SEQ ID NO:143 and SEQ ID NO: 151.

In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:144, and SEQ ID NO: 152.

In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:128, SEQ ID NO:138, SEQ ID NO:145, and SEQ ID NO: 153.

a) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

b) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

c) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

d) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

e) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: 96 of SEQ ID NO;

f) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

g) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

h) HCDR1: SEQ ID NO. 110, HCDR2: SEQ ID NO 111 and HCDR3: 112 of SEQ ID NO;

i) HCDR1: SEQ ID NO:117, HCDR2: SEQ ID NO. 118 and HCDR3: SEQ ID NO. 119;

j) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

k) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

l) HCDR1: SEQ ID NO. 136, HCDR2: SEQ ID NO 137 and HCDR3: 138 of SEQ ID NO;

m) HCDR1: SEQ ID NO:143, HCDR2: SEQ ID NO. 144 and HCDR3: SEQ ID NO. 145;

n) HCDR1: SEQ ID NO:151, HCDR2: SEQ ID NO. 152 and HCDR3: SEQ ID NO. 153. In certain embodiments, the VH comprises an amino acid sequence as set forth in any one of SEQ ID NO 69, SEQ ID NO 77, SEQ ID NO 84, SEQ ID NO 90, SEQ ID NO 93, SEQ ID NO 101, SEQ ID NO 123, SEQ ID NO 109, SEQ ID NO 116, SEQ ID NO 125, SEQ ID NO 133, SEQ ID NO 135, SEQ ID NO 142 and SEQ ID NO 150.

In certain embodiments, the second targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:140, SEQ ID NO:147 and SEQ ID NO: 155.

In certain embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 75, SEQ ID NO. 28, SEQ ID NO. 99, SEQ ID NO. 107, SEQ ID NO. 131, SEQ ID NO. 148, and SEQ ID NO. 156.

In certain embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:76, 83, 89, 100, 108, 115, 122, 132, 141, 149, and 157.

a) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

b) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

c) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

d) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

e) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100;

f) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

g) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

h) LCDR1: SEQ ID NO. 114, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 115;

i) LCDR1: SEQ ID NO:121, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 122;

j) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

k) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

l) LCDR1: SEQ ID NO:140, LCDR2: SEQ ID NO. 28 and LCDR3: 141 of SEQ ID NO;

m) LCDR1: SEQ ID NO:147, LCDR2: SEQ ID NO 148 and LCDR3: 149 of SEQ ID NO;

n) LCDR1: SEQ ID NO:155, LCDR2: SEQ ID NO 156 and LCDR3: SEQ ID NO. 157.

In certain embodiments, the VL comprises an amino acid sequence set forth in any one of SEQ ID NO. 73, 81, 87, 91, 97, 105, 124, 113, 120, 129, 134, 139, 146, and 154.

In certain embodiments, the targeting moiety comprises VHH, scFv, fab, dAb and/or a derivative protein structure.

In certain embodiments, the targeting moiety is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, the first targeting moiety comprises an scFv.

In certain embodiments, the second targeting moiety comprises an scFv.

In certain embodiments, in the multispecific antibody, the first targeting moiety is fused in-frame with the second targeting moiety.

In certain embodiments, the first targeting moiety is linked to the second targeting moiety through a hinge region.

In certain embodiments, the hinge region comprises a hinge region derived from a histone: CD8, CD28 and 4-1BB.

In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO. 17.

In certain embodiments, the modified cell comprises and/or expresses a nucleic acid molecule encoding the fusion protein and/or a nucleic acid molecule encoding the multispecific antibody.

In certain embodiments, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on separate vectors.

In certain embodiments, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on the same vector.

In certain embodiments, the nucleic acid molecule encoding the fusion protein is linked to the nucleic acid molecule encoding the multispecific antibody via a sequence encoding a cleavage peptide.

In certain embodiments, the nucleic acid sequence encoding a cleavage peptide is selected from the group consisting of: P2A sequence, T2A sequence, F2A sequence, E2A sequence, bmCPV2A sequence and BmIFV2A sequence.

In certain embodiments, the cleavage peptide sequence comprises a P2A sequence.

In certain embodiments, the cleavage peptide sequence comprises a T2A sequence.

In certain embodiments, the cleaved peptide sequence in the modified cell comprises the sequence of SEQ ID NO: 13-16.

In certain embodiments, the fusion protein is expressed in a different amount than the multispecific antibody.

In another aspect, the application provides a pharmaceutical composition comprising said modified cells, and/or said one or more multispecific antibodies.

In certain embodiments, the multispecific antibody in the pharmaceutical composition comprises a protein molecule comprising a first targeting moiety and a second targeting moiety that is purified using a bioengineering technique.

In certain embodiments, the multispecific antibody is infused into a blood vessel.

In another aspect, the application provides the use of a modified cell as described and/or one or more multispecific antibodies as described in the manufacture of a medicament.

In another aspect, the application provides a pharmaceutical composition comprising the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions for use in treating tumors.

In certain embodiments, the tumor comprises a hematological tumor.

In certain embodiments, the tumor comprises a solid tumor.

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions for use in treating a disease or disorder associated with expression of CD 33.

In certain embodiments, the disease or disorder associated with expression of CD33 comprises human myeloid leukemia (AML).

In another aspect, the application provides the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition for use in treating a disease or disorder associated with GD2 expression.

In certain embodiments, the disease or disorder associated with GD2 expression comprises neuroblastoma.

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions for use in treating a disease or disorder associated with expression of B7H 3.

In certain embodiments, the disease or disorder associated with expression of B7H3 comprises neuroblastoma.

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition for use in treating a disease or disorder associated with expression of HER 2.

In certain embodiments, the disease or disorder associated with expression of HER2 comprises breast cancer.

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecule, the vector of any one of the claims, the modified cell, and/or the pharmaceutical composition for use in treating a disease or disorder associated with expression of DLL 1.

In certain embodiments, the disease or disorder associated with expression of DLL1 comprises lung cancer.

In a further aspect, the application provides the use of one or more of said fusion proteins, said isolated nucleic acid molecules, said vector, said modified cells, and/or said pharmaceutical composition for the manufacture of a medicament for the treatment of a disease or disorder associated with the expression of CD 33.

In certain embodiments, the disease or disorder associated with expression of CD33 comprises human Acute Myeloid Leukemia (AML).

In another aspect, the application provides the use of one or more of said fusion proteins, said isolated nucleic acid molecules, said vector, said modified cells, and/or said pharmaceutical composition for the preparation of a medicament for the treatment of a disease or disorder associated with GD2 expression.

In a further aspect, the application provides the use of one or more of the fusion proteins, the isolated nucleic acid molecules, the vector, the modified cells, and/or the pharmaceutical composition in the manufacture of a medicament for the treatment of a disease or disorder associated with expression of B7H 3.

In a further aspect, the application provides the use of said fusion protein, said isolated nucleic acid molecule, said vector, said modified cell, and/or said pharmaceutical composition for the manufacture of a medicament for the treatment of a disease or disorder associated with the expression of HER 2.

In a further aspect, the application provides the use of one or more of said fusion proteins, said isolated nucleic acid molecules, said vector, said modified cells, and/or said pharmaceutical composition in the manufacture of a medicament for the treatment of a disease or disorder associated with the expression of DLL 1.

In another aspect, the application provides a method of preventing and/or treating a tumor comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition.

In another aspect, the application provides a method of preventing and/or treating a disease or disorder associated with expression of CD33 comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated nucleic acid molecule, the vector of any of the claims, the modified cell, and/or the pharmaceutical composition.

In another aspect, the present application provides a method for preventing and/or treating a disease or disorder associated with GD2 expression, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition.

In another aspect, the application provides a method of preventing and/or treating a disease or disorder associated with expression of B7H3 comprising administering the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition to a subject in need thereof.

In another aspect, the application provides a method of preventing and/or treating a disease or disorder associated with the expression of HER2 comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition.

In another aspect, the application provides a method for preventing and/or treating a disease or disorder associated with DLL1 expression comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

FIG. 1 shows AgCAR structures of I-1 to I-4, I-1 to I-1-3, I-2-1 to I-2-3, I-3-1 to I-3, I-4-1 to I-4-2 according to the present application.

FIG. 2 shows the AgCAR structure of the application from II-1 to II-11.

FIG. 3 shows the targeted cytotoxicity of cells with engineered vector (AgCAR) structures according to the present application.

FIG. 4 shows a carrier structure of the CD19AgCAR backup of the present application.

FIG. 5 shows a construction of a vector of CD19AgCAR Double antibody according to the present application.

FIG. 6 shows a construction of a vector for CD19AgCAR according to the present application.

FIG. 7 shows the transfection efficiency of lentiviruses with CD19AgCAR, lentiviruses with CD19AgCAR backbone, lentiviruses with CD19AgCAR Double antibody transfected human T lymphocytes according to the application.

FIGS. 8A-J show the effect of the application on killing CD33 positive myeloid leukemia cell line HL60 in vitro with T cells of CD19AgCAR, CD19AgCAR backbone, CD19AgCAR Double antibody, CD19-CART and CD 33-CART.

Figures 9A-L show the effect of the application on killing GD2 positive neuroblastoma cell line CHP134 in vitro with T cells bearing CD19AgCAR, T cells bearing CD19AgCAR backbone, T cells bearing CD19AgCAR Double antibody and GD 2-CART.

FIG. 10 shows T cells with CD22AgCAR, T cells with CD20AgCAR and T cells with CD19AgCAR according to the application together with human acute lymphoblastic leukemia cell line MV4;11 residual MV4 in the co-incubation experiment; 11, and a flow cytometry detection result.

FIG. 11 shows the in vivo efficacy detection of T cells with CD19AgCAR-33 according to the application.

FIGS. 12A-B show in vivo efficacy assays of T cells with CD19AgCAR-B7H3 according to the application.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "antigen" or "Ag" generally refers to a molecule that is responsible for eliciting an immune response. Such an immune response may involve antibody production, or activation of specific immunocompetent cells, or both. In certain instances, the antigen comprises a tumor-associated antigen. For example, a tumor-associated antigen may be expressed on the surface of tumor cells. For example, tumor-associated antigens may also be expressed on the surface of other cells (e.g., immune cells). For example, tumor-associated antigens may include, but are not limited to, CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA. In some cases, the antigen comprises an immune cell activation-related antigen. For example, immune cell activation-related antigens may include, but are not limited to, CD45, CD56, and CD58. For example, the skilled artisan will appreciate that any macromolecule, including almost all proteins or peptides, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. Thus, one of skill in the art will understand that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full length nucleotide sequence of a gene. The present application includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Furthermore, one skilled in the art will appreciate that antigens may not be encoded by a "gene". For example, the antigen may be synthetic or may be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids. In certain instances, the antigen may be from a non-human animal, including but not limited to, mammals, such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates.

In the present application, the term "single chain antibody" or "scFv" generally refers to a molecule of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker. See, e.g., bird et al, science,242:423-426 (1988); and Huston et al proc.Natl. Acad. Sci. USA,85:5879-5883 (1988). In some embodiments, the heavy and light chain variable regions of the scFv may be from CD19, CD20, CD33, GD2, B7H3, or CD22.

In the present application, the term "CD19" generally refers to an important membrane antigen on human B lymphocytes that is involved in proliferation and differentiation. The amino acid sequence of human CD19 can be found in UniProt/Swiss-Prot accession number P15391. In the present application, the fusion protein may comprise part or all of the amino acid sequence of human CD 19.

In the present application, the term "CD22" generally refers to an important membrane antigen on human B lymphocytes that is involved in proliferation and differentiation. The amino acid sequence of human CD22 can be found in UniProt/Swiss-Prot accession number P20273. In the present application, the fusion protein may comprise part or all of the amino acid sequence of human CD22.

In the present application, the term "CD20" generally refers to an important membrane antigen on human B lymphocytes that is involved in proliferation and differentiation. The amino acid sequence of human CD20 can be found in UniProt/Swiss-Prot accession number P11836. In the present application, the fusion protein may comprise part or all of the amino acid sequence of human CD 20.

In the present application, the term "CD33" generally refers to myeloid cell differentiation antigens. The amino acid sequence of human CD33 can be found in UniProt/Swiss-Prot accession number P20138. In the present application, the fusion protein may comprise part or all of the amino acid sequence of human CD 33.

In the present application, the term "GD2" generally refers to a ganglioside. GD2 may be expressed on tumor surfaces generated by neuroectoderm, including but not limited to tumors such as neuroblastoma, melanoma, and osteosarcoma.

In the present application, the term "B7H3" generally refers to a transmembrane protein belonging to the B7 family. B7H3 can be expressed on the surface of a variety of tumor cells. Such as lung cancer, head and neck cancer, esophageal cancer, prostate cancer, endometrial cancer, and breast cancer. The amino acid sequence of human B7H3 can be found in UniProt/Swiss-Prot accession number Q5ZPR3. In the present application, the fusion protein may comprise part or all of the amino acid sequence of human B7H 3.

In the present application, the term "ERBB2" generally refers to one of the members of the epidermal growth factor receptor (epidermal growth factor receptor, EGFR) family. The amino acid sequence of human CD20 can be found in UniProt/Swiss-Prot accession number P04626.

In the present application, the term "functionally active fragment" generally refers to an amino acid sequence having homology to a naturally occurring sequence or a polypeptide encoded by a nucleotide sequence of homology and capable of having one or more activities of the naturally occurring sequence. In the context of the present application, a variant of any given sequence refers to a sequence in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally occurring protein and/or polynucleotide, so long as the original functional activity is maintained.

In the present application, the term "homology" may be equivalent to the sequence "identity". Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Homology may be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions), or homology may be expressed in terms of sequence identity. In the present application, a sequence of any one of the mentioned amino acid sequences or nucleotide sequences of SEQ ID NOs having a percent identity refers to a sequence having said percent identity over the entire length of the mentioned SEQ ID NOs. To determine sequence identity, sequence alignments can be performed in a variety of ways known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. One skilled in the art can determine the appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment in the compared full-length sequences.

In the present application, the term "costimulatory signaling domain" generally refers to an intracellular domain that can provide an immune costimulatory molecule, a cell surface molecule that is required for the effective response of lymphocytes to an antigen. In some cases, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from a protein selected from the group consisting of: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fcεRIgamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40 and MyD88. For example, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from 4-1 BB.

In the present application, the term "intracellular signaling domain" generally refers to a domain located inside a cell that is capable of transducing a signal. In the present application, the intracellular signaling domain may transduce a signal into a cell. In general, an intracellular signaling domain is any one of a number of contiguous amino acid sequences that are used to direct protein targeting. In some cases, the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fceri γ, fceri β, fcgammaRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from cd3ζ.

In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and include any molecule comprising an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., fab', and (Fab) 2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, as well as any antigen-binding antibody fragment described herein and derivatives thereof. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. VH and VL regions can be further distinguished as hypervariable regions called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, called Framework Regions (FR). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

In the present application, the term "complementarity determining region" (CDR) generally refers to complementarity determining regions within the variable region of an antigen binding fragment. In the present application, there are 3 CDRs of the heavy chain variable region, which are designated HCDR1, HCDR2 and HCDR3 for each variable region. The exact boundaries of these CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987) and (1991)) found that, despite the large diversity in amino acid sequence levels, certain subunits within the Kabat CDRs take on nearly identical peptide backbone conformations, these subunits being designated as L1, L2 and L3 or H1, H2 and H3, respectively, where "L" and "H" refer to the light and heavy chain regions, respectively, these regions may be designated as Chothia CDRs (Chothia and Lesk, J.mol. Biol.196:901-917 (1987)) and Chothia et al Nature 342:877-883 (1989)) that do not overlap with the boundaries of the specific Kabat (Kabat et al Nature 342:877-883) or that do not overlap with the boundaries of the specific Kabat (1995) or even with the residues of the other human antibodies (1995) and the amino acid sequence levels.

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing a chimeric antigen receptor as described herein or an antigen binding protein as described herein. The cells may include progeny of a single cell. The daughter cells may not necessarily be identical in morphology or in genome to the original parent cells due to natural, accidental or deliberate mutation, but are capable of expressing the chimeric antigen receptor or antigen binding protein of the application. The cells may be obtained by transfecting cells in vitro using the vectors of the present application. The cells may be prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, the cell may be an immune cell. For example, the immune cells may be selected from the group consisting of: t cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells. For example, the immune cells may be T cells.

In the present application, the term "treatment" generally refers to: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be susceptible to the disease, disorder, and/or condition, but has not been diagnosed with the disease; (ii) Inhibiting the disease, disorder or condition, i.e., inhibiting its development; and (iii) alleviating the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition.

In the present application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation (lipid), acetylation, phosphorylation, or any other manipulation (e.g., in combination with a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as D and L optical isomers, as well as amino acid analogs and peptidomimetics.

In the present application, the terms "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus), exons, introns, messenger RNAs (mRNA), transfer RNAs, ribosomal RNAs, short interfering RNAs (siRNA), short hairpin RNAs (shRNA), micro-RNAs (miRNA), ribozymes, cdnas, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers defined according to ligation analysis. Polynucleotides may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeled component.

In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a hematological tumor. For example, the solid tumor may include solid tumors such as neuroblastoma, wilms' cell tumor, hepatoblastoma, pancreatic blastoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, pulmonary blastoma, germ cell tumor, lung cancer, liver cancer, breast cancer, intestinal cancer, stomach cancer, gallbladder cancer, pancreatic cancer, prostate cancer, thyroid cancer, and central nervous system tumor. For example, the hematological neoplasm may include acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, mast cell leukemia, plasma cell leukemia, myeloma.

In the present application, the term "multispecific antibody" generally refers to an antibody molecule that can recognize two or more antigens or epitopes simultaneously. The multispecific antibody can be obtained in eukaryotic expression systems or prokaryotic expression systems by chemical coupling methods, hybridization-hybridoma methods, genetic engineering antibody preparation methods and the like. For example, the multispecific antibody may be a bispecific antibody.

In the present application, the term "subject" generally refers to any organism, including but not limited to mammals, such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the present application, the subject may be a human, a pet or a livestock.

In the present application, the term "and/or" is understood to mean either one of the selectable items or both of the selectable items.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

In the present application, the term "comprising" generally means containing, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.

Detailed Description

Fusion proteins

In the present application, the fusion protein may include an antigen or a functionally active fragment thereof. In some cases, the antigen or functionally active fragment thereof may comprise an immune cell activation-related antigen or functionally active fragment thereof. For example, the immune cell activation-related antigen or functionally active fragment thereof may comprise an antigen or functionally active fragment thereof selected from the group consisting of: CD45, CD56 and CD58.

In the present application, the antigen or functionally active fragment thereof may comprise a tumor-associated antigen. For example, the tumor-associated antigen or functionally active fragment thereof may comprise an antigen or functionally active fragment thereof selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3. For example, the tumor-associated antigen may be an antigen associated with B lymphocyte development. For example, the tumor-associated antigen or functionally active fragment thereof may comprise a CD19 protein or functionally active fragment thereof. For another example, the tumor-associated antigen or functionally active fragment thereof may comprise a CD20 protein or functionally active fragment thereof. For another example, the tumor-associated antigen or functionally active fragment thereof may comprise a CD22 protein or functionally active fragment thereof. For another example, the tumor-associated antigen or functionally active fragment thereof may comprise a CD33 protein or functionally active fragment thereof. For another example, the tumor-associated antigen or functionally active fragment thereof may comprise SEQ ID NO: 2. 4, 6, 8 and 10, the nucleic acid sequence encoding the tumor-associated antigen or functionally active fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1. 3, 5, 7 and 9.

In the present application, the co-stimulatory domain may comprise a co-stimulatory domain derived from a protein selected from the group consisting of: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88. For example, the costimulatory domain may comprise a costimulatory domain derived from 4-1 BB. For example, the co-stimulatory domain may comprise SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12. The nucleic acid sequence encoding the co-stimulatory domain may comprise SEQ ID NO:11, and a nucleic acid sequence as set forth in seq id no.

In the present application, the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fceri γ, fceri β, fcgammaRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from cd3ζ. For example, the intracellular signaling domain may comprise SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no. The nucleic acid sequence encoding the intracellular signaling domain may comprise SEQ ID NO:18, and a nucleic acid sequence shown in seq id no.

In the present application, the C-terminus of the tumor-associated antigen or functionally active fragment thereof may be directly or indirectly linked to the N-terminus of the costimulatory domain.

In the present application, the C-terminal end of the co-stimulatory domain may be directly or indirectly linked to the N-terminal end of the intracellular signaling domain.

In some cases, the fusion protein may comprise, from the N-terminus to the C-terminus, an antigen or functionally active fragment thereof, a co-stimulatory domain, and an intracellular signaling domain.

In the present application, the antigen or functionally active fragment thereof, the co-stimulatory domain and the intracellular signaling domain may be fused in-frame (as shown in figure 1).

For example, the antigen or functionally active fragment thereof may be a CD19 protein or functionally active fragment thereof, the amino acid sequence of which may comprise the sequence shown in SEQ ID NO. 2. For example, the amino acid sequence of the antigen or functionally active fragment thereof can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 2. The nucleic acid sequence may comprise SEQ ID NO:1, and a sequence shown in 1. For example, the nucleic acid sequence of the antigen or functionally active fragment thereof can comprise a nucleic acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the nucleic acid sequence set forth in SEQ ID NO. 1.

For example, the antigen or functionally active fragment thereof may be a CD20 protein or functionally active fragment thereof, the amino acid sequence of which may comprise the sequence shown in SEQ ID NO. 4. For example, the amino acid sequence of the antigen or functionally active fragment thereof can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 4. The nucleic acid sequence may comprise SEQ ID NO: 3. For example, the nucleic acid sequence of the antigen or functionally active fragment thereof can comprise a nucleic acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the nucleic acid sequence set forth in SEQ ID NO. 3.

For example, the antigen or functionally active fragment thereof may be a CD22 protein or functionally active fragment thereof, the amino acid sequence of which may comprise the sequence shown in SEQ ID NO. 6. For example, the amino acid sequence of the antigen or functionally active fragment thereof can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 6. The nucleic acid sequence may comprise SEQ ID NO: 5. For example, the nucleic acid sequence of the antigen or functionally active fragment thereof can comprise a nucleic acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the nucleic acid sequence set forth in SEQ ID NO. 5.

For example, the antigen or functionally active fragment thereof may be a CD33 protein or functionally active fragment thereof, the amino acid sequence of which may comprise the amino acid sequence of SEQ ID NO: 8. For example, the amino acid sequence of the antigen or functionally active fragment thereof can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 8. The nucleic acid sequence may comprise the sequence shown in SEQ ID NO. 9. For example, the nucleic acid sequence of the antigen or functionally active fragment thereof can comprise a nucleic acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the nucleic acid sequence set forth in SEQ ID NO. 9.

In the present application, the co-stimulatory domain may include a co-stimulatory domain derived from 4-1BB, which may comprise the amino acid sequence of SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12. For example, the costimulatory domain can comprise an amino acid sequence that has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence depicted in SEQ ID NO. 12.

In the present application, the intracellular signaling domain may comprise an intracellular signaling domain derived from cd3ζ, which may comprise the sequence of SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no. For example, the amino acid sequence of the intracellular signaling domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 19.

In some cases, the fusion protein may comprise, from N-terminus to C-terminus, CD19 protein or a functionally active fragment thereof, 4-1BB and CD3 zeta.

In some cases, the fusion protein may comprise, from N-terminus to C-terminus, CD22 protein or a functionally active fragment thereof, 4-1BB and CD3 zeta.

In some cases, the fusion protein may comprise, from N-terminus to C-terminus, CD20 protein or a functionally active fragment thereof, 4-1BB and CD3 zeta.

In some cases, the fusion protein may comprise, from N-terminus to C-terminus, CD33 protein or a functionally active fragment thereof, 4-1BB, and CD3 zeta.

Multispecific antibodies

The present application provides one or more multispecific antibodies that may include a first targeting moiety that recognizes the antigen or functionally active fragment thereof in the fusion protein, and a second targeting moiety.

In the present application, the first targeting moiety may bind to the antigen or a functionally active fragment thereof in the fusion protein.

In some cases, the first targeting moiety can specifically bind to a tumor-associated antigen in the fusion protein.

For example, the tumor-associated antigen targeted by the first targeting moiety may be selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3.

In the present application, the second targeting moiety may target a tumor-associated antigen.

For example, the tumor-associated antigen targeted by the second targeting moiety may be selected from the group consisting of: GD2, DLL1, DLL3, ROR1, GPC3, HER2, VEGFA, CD1a, CD33, B7H3, CD138, BCMA, EGFRVIII.

In the present application, the targeting moiety may include, but is not limited to, VHH, scFv, fv fragments, fab 'fragments, F (ab') 2 fragments, dabs, and/or derived protein structures.

In the present application, the targeting moiety may include, but is not limited to, recombinant antibodies, monoclonal antibodies, fully human antibodies, murine antibodies, humanized antibodies, and chimeric antibodies.

In the present application, the first targeting moiety may comprise an scFv. In the present application, the second targeting moiety may comprise an scFv (as shown in fig. 1).

In the present application, the first targeting moiety may recognize CD19 protein or a functionally active fragment thereof, and the second targeting moiety may recognize CD33 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD19 protein or a functionally active fragment thereof, and the second targeting moiety may recognize GD2 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD19 protein or a functionally active fragment thereof, and the second targeting moiety may recognize B7H3 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize a CD22 protein or functionally active fragment thereof, and the second targeting moiety may recognize a CD33 protein or functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD22 protein or a functionally active fragment thereof, and the second targeting moiety may recognize GD2 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD22 protein or a functionally active fragment thereof, and the second targeting moiety may recognize B7H3 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize a CD20 protein or functionally active fragment thereof, and the second targeting moiety may recognize a CD33 protein or functionally active fragment thereof.

In the present application, the first targeting moiety may recognize a CD20 protein or a functionally active fragment thereof, and the second targeting moiety may recognize a GD2 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize a CD20 protein or functionally active fragment thereof, and the second targeting moiety may recognize a B7H3 protein or functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD33 protein or a functionally active fragment thereof, and the second targeting moiety may recognize GD2 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize CD33 protein or a functionally active fragment thereof, and the second targeting moiety may recognize B7H3 protein or a functionally active fragment thereof.

In the present application, the first targeting moiety may recognize a CD19 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as set forth in any one of SEQ ID nos. 22, 30. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 22, SEQ ID NO. 30.

In the present application, the first targeting moiety may recognize a CD22 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 54, 61. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO: 61.

In the present application, the first targeting moiety may recognize a CD20 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as shown in any one of SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO: 51. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO: 51.

In the present application, the first targeting moiety may recognize a CD33 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as shown in any one of SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO: 93. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequences set forth in SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90, and SEQ ID NO: 93.

In the present application, the second targeting moiety may recognize a CD33 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as shown in any one of SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO: 93. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequences set forth in SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90, and SEQ ID NO: 93.

In the present application, the second targeting moiety may recognize a GD2 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as shown in any one of SEQ ID NOs 101, 123, 109, 116. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO:101, 123, 109, 116.

In the present application, the second targeting moiety may recognize a B7H3 protein or functionally active fragment thereof, comprising a heavy chain variable region VH, which VH may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 125, 133. For example, the first targeting moiety can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO:125, SEQ ID NO: 133.

In the present application, the first targeting moiety may comprise a heavy chain variable region VH, which heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 23, SEQ ID NO. 31, SEQ ID NO. 39, SEQ ID NO. 55, SEQ ID NO. 62, SEQ ID NO. 70, SEQ ID NO. 78, SEQ ID NO. 85 and SEQ ID NO. 94. For example, the HCDR1 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any of SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85, and SEQ ID NO: 94.

In the present application, the HCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, and SEQ ID NO: 95. For example, the HCDR2 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any of SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, and SEQ ID NO: 95.

In the present application, the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 52, SEQ ID NO. 57, SEQ ID NO. 64, SEQ ID NO. 72, SEQ ID NO. 80, SEQ ID NO. 86 and SEQ ID NO. 96. For example, the HCDR3 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, and SEQ ID NO: 96.

In the present application, the first targeting moiety may comprise a heavy chain variable region VH, which may include HCDR1, HCDR2 and HCDR3. For example, the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence selected from any one of the following groups, or the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence selected from any one of the following groups:

a) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24 and HCDR3: SEQ ID NO. 25;

b) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

c) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

d) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 41;

e) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 47;

f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 52;

g) HCDR1: SEQ ID NO. 55, HCDR2: SEQ ID NO. 56 and HCDR3: SEQ ID NO. 57;

h) HCDR1: SEQ ID NO. 62, HCDR2: SEQ ID NO. 63 and HCDR3: SEQ ID NO. 64;

i) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

j) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

k) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

l) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

m) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: SEQ ID NO. 96.

In the present application, the first targeting moiety may comprise a light chain variable region VL, and the heavy chain variable region may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 43, SEQ ID NO 49, SEQ ID NO 59, SEQ ID NO 66, SEQ ID NO 74, SEQ ID NO 82, SEQ ID NO 88, SEQ ID NO 92 and SEQ ID NO 98. For example, the LCDR1 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any one of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, and SEQ ID NO: 98.

In the present application, the LCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 44, SEQ ID NO. 28, SEQ ID NO. 67, SEQ ID NO. 75, and SEQ ID NO. 99. For example, the LCDR2 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any one of SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:28, SEQ ID NO:67, SEQ ID NO:75, and SEQ ID NO: 99.

In the present application, the LCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 45, SEQ ID NO. 50, SEQ ID NO. 45, SEQ ID NO. 60, SEQ ID NO. 68, SEQ ID NO. 76, SEQ ID NO. 83, SEQ ID NO. 89 and SEQ ID NO. 100. For example, the LCDR3 may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology with the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:45, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:89, and SEQ ID NO: 100.

In the present application, the light chain variable region VL may be included, and the heavy chain variable region may include LCDR1, LCDR2 and LCDR3. For example, the LCDR1, LCDR2, and LCDR3 can comprise an amino acid sequence selected from any one of the following groups, or the LCDR1, LCDR2, and LCDR3 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence selected from any one of the following groups:

a) LCDR1: SEQ ID NO. 27, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 29;

b) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

c) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

d) LCDR1: SEQ ID NO. 43, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

e) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 50;

f) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

g) LCDR1: SEQ ID NO 59, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 60;

h) LCDR1: SEQ ID NO:66, LCDR2: SEQ ID NO 67 and LCDR3: SEQ ID NO. 68;

i) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

j) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

k) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

l) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

m) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100.

In the present application, the second targeting moiety may comprise a heavy chain variable region VH, which heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:136, SEQ ID NO:143 and SEQ ID NO: 151. For example, the HCDR1 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any of SEQ ID NO:70, 78, 85, 94, 102, 110, 117, 126, 136, 143, and 151.

In the present application, the HCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:144, and SEQ ID NO: 152. For example, the HCDR2 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any of SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:144, and SEQ ID NO: 152.

In the present application, the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:128, SEQ ID NO:138, SEQ ID NO:145, and SEQ ID NO: 153. For example, the HCDR3 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequences set forth in SEQ ID NO:72, 80, 86, 96, 104, 112, 119, 128, 138, 145, and 153.

In the present application, the heavy chain variable region VH may be comprised, and the heavy chain variable region may include HCDR1, HCDR2 and HCDR3. For example, the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence selected from any one of the following groups, or the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence having at least 80% (e.g. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence selected from any one of the following groups:

a) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

b) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

c) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

d) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

e) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: 96 of SEQ ID NO;

f) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

g) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

h) HCDR1: SEQ ID NO. 110, HCDR2: SEQ ID NO 111 and HCDR3: 112 of SEQ ID NO;

i) HCDR1: SEQ ID NO:117, HCDR2: SEQ ID NO. 118 and HCDR3: SEQ ID NO. 119;

j) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

k) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

l) HCDR1: SEQ ID NO. 136, HCDR2: SEQ ID NO 137 and HCDR3: 138 of SEQ ID NO;

m) HCDR1: SEQ ID NO:143, HCDR2: SEQ ID NO. 144 and HCDR3: SEQ ID NO. 145;

n) HCDR1: SEQ ID NO:151, HCDR2: SEQ ID NO. 152 and HCDR3: SEQ ID NO. 153.

In the present application, the second targeting moiety may comprise a light chain variable region VL, and the heavy chain variable region may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:140, SEQ ID NO:147 and SEQ ID NO: 155. For example, the LCDR1 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any one of SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:140, SEQ ID NO:147, and SEQ ID NO: 155.

In the present application, the LCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 75, SEQ ID NO 28, SEQ ID NO 99, SEQ ID NO 107, SEQ ID NO 99, SEQ ID NO 28, SEQ ID NO 131, SEQ ID NO 28, SEQ ID NO 148, and SEQ ID NO 156. For example, the LCDR2 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to an amino acid sequence set forth in any one of SEQ ID NO:75, SEQ ID NO:28, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:99, SEQ ID NO:28, SEQ ID NO:131, SEQ ID NO:28, SEQ ID NO:148, and SEQ ID NO: 156.

In the present application, the LCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 76, SEQ ID NO 83, SEQ ID NO 89, SEQ ID NO 100, SEQ ID NO 108, SEQ ID NO 115, SEQ ID NO 122, SEQ ID NO 132, SEQ ID NO 141, SEQ ID NO 149 and SEQ ID NO 157. For example, the LCDR3 may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequences shown in SEQ ID NO:76, 83, 89, 100, 108, 115, 122, 132, 141, 149, 157.

a) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

b) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

c) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

d) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

e) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100;

f) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

g) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

h) LCDR1: SEQ ID NO. 114, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 115;

i) LCDR1: SEQ ID NO:121, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 122;

j) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

k) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

l) LCDR1: SEQ ID NO:140, LCDR2: SEQ ID NO. 28 and LCDR3: 141 of SEQ ID NO;

m) LCDR1: SEQ ID NO:147, LCDR2: SEQ ID NO 148 and LCDR3: 149 of SEQ ID NO;

n) LCDR1: SEQ ID NO:155, LCDR2: SEQ ID NO 156 and LCDR3: SEQ ID NO. 157.

In the present application, in the multispecific antibody, the first targeting moiety may be fused in-frame with the second targeting moiety.

In the present application, in the multispecific antibody, the first targeting moiety may be linked to the second targeting moiety by a hinge region.

In the present application, the hinge region may comprise a hinge region derived from: CD8, CD28 and 4-1BB.

In the present application, the hinge region may comprise SEQ ID NO:17, and a sequence of amino acids shown in seq id no.

Nucleic acids and vectors

In another aspect, the application also provides an isolated nucleic acid molecule or molecules which encode a fusion protein according to the application. For example, each of the one or more nucleic acid molecules may encode the entire fusion protein, or may encode a portion thereof (e.g., one or more of HCDR1-3, heavy chain variable regions).

The nucleic acid molecules of the application may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. For example, the isolated nucleic acid may be a nucleic acid molecule prepared by recombinant DNA techniques.

In the present application, nucleic acids encoding the fusion proteins may be prepared by a variety of methods known in the art, including, but not limited to, using reverse transcription PCR and PCR to obtain nucleic acid molecules of the fusion proteins of the present application.

In another aspect, the application provides one or more vectors comprising one or more nucleic acid molecules of the application. Each vector may comprise one or more of the nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter may be fused to a portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not include additional introns. One or more nucleic acid molecules of the application may be operably linked to the expression control element.

The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. The viral vector may be administered directly to the patient (in vivo) or the cells may be treated with the virus in an indirect form, e.g., in vitro, and then the treated cells are administered to the patient (ex vivo). Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors for gene transfer. In some cases, the gene transfer may be integrated into the host genome by retrovirus, lentivirus, and adeno-associated virus methods, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise a long terminal repeat 5'LTR and truncated 3' LTR, RRE, rev responsive element (cPPT), central Termination Sequence (CTS) and/or post-translational regulatory element (WPRE). The vectors of the application may be introduced into cells. For example, the vector of the application may be a lentiviral vector.

Modified cells

In another aspect, the application provides one or more modified cells that may comprise and/or express the fusion protein, one or more (e.g., 2 or more) of the nucleic acid molecules, and/or one or more (e.g., 2 or more) of the vectors.

In certain cases, the cells may further comprise and/or express a multispecific antibody of the application.

In some cases, the cells may include immune cells.

In some cases, the cells may include immune effector cells.

In some cases, the cells may include T cells, macrophagesNatural killer cells (NK cells) and/or NKT cells.

For example, the cells may include T cells. The T cells may include thymic cells, natural T cells, immature T cells, mature T cells, resting T cells, or activated T cells. The T cells may be helper T cells (Th), such as helper T cell 1 (Th 1) or helper T cell 2 (Th 2) cells. The T cells may be CD4+ helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), tumor infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+/CD8+ T cells, CD4-/CD8-T cells or any other T cell subtype. In certain instances, the modified T cell is a human T cell. Prior to expansion and genetic modification of the cells of the application, a cell source may be obtained from a subject, such as a patient, by various non-limiting methods. T cells can be obtained from a number of non-limiting sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of T cell lines available and known to those skilled in the art may be used. In other cases, the cells may be derived from healthy donors, from patients diagnosed with cancer, or obtained from patients diagnosed with infection. In other cases, the cells are part of a mixed population of cells that exhibit different phenotypic characteristics.

For example, when the modified cells use T cells, the method of treatment using the modified cells may be referred to as antigen-coupled antibody-directed T cell immunotherapy (Antigen Coupling Antibody Redirect T-cell Immunotherapy, agCAR-T).

In some cases, the cells may include NK cells. In some cases, the NK cells can include CD56bright and CD56dim. In certain cases, the NK cells may include NK1 and NK2. In some cases, the NK cells may include A-NK and NA-NK.

For example, when the modified cells use NK cells, the method of treatment using the modified cells may be referred to as antigen-coupled antibody-directed NK cell immunotherapy (Antigen Coupling Antibody Redirect NK-cell Immunotherapy, agCAR-NK).

In some cases, the cells may include NKT cells. For example, the NKT cells may include type 1 NKT cells, type 2 NKT cells, and NKT-like cells.

For example, when the modified cells use NKT cells, the method of treatment using the modified cells may be referred to as antigen-coupled antibody-directed NKT cell immunotherapy (Antigen Coupling Antibody Redirect NKT-cell Immunotherapy, agCAR-NKT).

In some cases, the cells may include macrophagesMacrophages are a substance that can engulf and digest cell debris, microorganisms, cancer cells, and all other substances lacking surface markers of normal cell surface expression, a process called phagocytosis. Macrophages are present in almost all tissues and are searched for possible pathogens by amoeba movements. In addition to their important role in non-specific innate immune responses, they can also help initiate acquired immunity by recruiting other immune cell types, such as lymphocytes.

For example, when the modified cells are usedIn the case of cells, methods of treatment using such modified cells may be referred to as antigen-coupled antibody redirectionCellular immunotherapy (Antigen Coupling Antibody Redirect)Immunotherapy, )。

In the present application, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody may be located on different vectors.

In the present application, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody may be on the same vector. In some cases, the nucleic acid molecule encoding the fusion protein may be linked to the nucleic acid molecule encoding the multispecific antibody by a sequence encoding a cleavage peptide.

In the present application, the nucleic acid sequence encoding the cleavage peptide may be selected from the group consisting of: P2A sequence, T2A sequence, F2A sequence, E2A sequence, bmCPV2A sequence and BmIFV2A sequence. For example, the nucleic acid sequence encoding the cleavage peptide may be a P2A sequence. For example, the nucleic acid sequence encoding the cleavage peptide may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 13-16.

In the present application, the fusion protein may be expressed in a different amount from the multispecific antibody. For example, the amount of expression of the fusion protein may differ from the amount of expression of the multispecific antibody by at least 1%. For example, the expression level of the fusion protein may differ from the expression level of the multispecific antibody by 1%,5%,6%,7%,10%,15%,20%,25%,30%,40%,50%,60%,70%,80%, or 90%.

In some cases, the vector may comprise, in addition to the general expression vector components, from the 5 'end to the 3' end, a nucleic acid sequence encoding an antigen or functionally active fragment thereof, a nucleic acid sequence encoding a co-stimulatory domain, a nucleic acid sequence encoding an intracellular signaling domain, a cleavage peptide sequence, a nucleic acid sequence encoding a first targeting moiety and a nucleic acid sequence encoding a second targeting moiety (as shown in FIG. 2).

In the present application, the modified cell may express the fusion protein and the multispecific antibody, wherein the fusion protein is located on the surface of the modified cell, the multispecific antibody is secreted outside the modified cell, the fusion protein may bind to a first targeting moiety of the multispecific antibody and activate the modified cell, and a second targeting moiety of the multispecific antibody may target a corresponding tumor cell, exert a cytotoxic effect to kill a tumor (e.g., acute myeloid leukemia, neuroblastoma, or solid tumor), as shown in fig. 3.

Pharmaceutical compositions, uses and methods

In another aspect, the application provides a pharmaceutical composition that may comprise the modified cell, and/or the one or more multispecific antibodies. In certain instances, the multispecific antibody may comprise a protein molecule comprising a first targeting moiety and a second targeting moiety purified using a bioengineering technique. In some cases, multispecific antibodies may be injected into a human. For example, the multispecific antibody may be infused into the body via a blood vessel. In some cases, the modified cells may be injected into the human body.

For example, in the pharmaceutical composition, the modified cell and the multispecific antibody may be co-administered. For example, in the pharmaceutical composition, the modified cell and the multispecific antibody may be administered separately. For example, the modified cells may be administered first, followed by the multispecific antibody. For example, the multispecific antibody may be administered first and then the modified cell. For example, the modified cell and the multispecific antibody may be administered in the same manner, e.g., the modified cell and the multispecific antibody may be administered in different manners.

For example, in the pharmaceutical composition, the modified cell and the multispecific antibody may be placed in the same container. For example, in the pharmaceutical composition, the modified cells and the multispecific antibody may be placed in different containers.

For example, in the pharmaceutical composition, the multispecific antibody may comprise a protein molecule comprising a first targeting moiety and a second targeting moiety that is purified using bioengineering techniques, and the multispecific antibody may be infused into a blood vessel and function with the fusion protein and/or the modified cell of the application.

In another aspect, the application provides a pharmaceutical composition comprising the fusion protein, one or more (e.g., 2 or more) of the isolated nucleic acid molecules, one or more (e.g., 2 or more) of the vectors, the modified cells, and/or pharmaceutically acceptable adjuvants and/or excipients.

In a further aspect, the application provides the use of said modified cells, and/or said one or more multispecific antibodies, in the manufacture of a medicament.

In another aspect, the application provides the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition, which is useful for treating a tumor.

In some cases, the tumor may comprise a hematological tumor.

In some cases, the tumor may comprise a solid tumor.

In another aspect, the application provides a fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition, which is useful for treating a disease or disorder associated with expression of CD 33.

In certain instances, the disease or disorder associated with expression of CD33 may include human myeloid leukemia.

In certain instances, the disease or disorder associated with expression of CD33 may include human Acute Myeloid Leukemia (AML).

In another aspect, the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions for use in treating a disease or disorder associated with DLL1 expression.

In certain embodiments, the disease or disorder associated with expression of CD33 comprises human myeloid leukemia.

In a further aspect, the present application provides the use of one or more of said fusion proteins, said isolated nucleic acid molecules, said vector, said modified cells, and/or said pharmaceutical composition for the preparation of a medicament for the treatment of a disease or disorder associated with the expression of GD 2.

Without intending to be limited by any theory, the following examples are presented in order to illustrate the fusion proteins, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

EXAMPLE 1 construction of genetically engineered vectors

1.1 first, as shown in FIGS. 4,5 and 6, the RRE sequence was inserted upstream of the multiple cloning site of the expression vector pWPXL-X, the WPRE sequence and cPPT sequence were inserted downstream of the multiple cloning site, and the EF1A promoter was inserted between the RRE sequence and the multiple cloning site.

1.2 the main components of the genetic engineering vector, except for the general expression vector components, are as follows, and the corresponding sequences are synthesized by a genetic synthesis method:

i-1: CD19 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ;

i-2: CD22 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ;

i-3: CD20 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ;

i-4: CD33 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ.

Wherein the amino acid sequence of the CD19 antigen is shown as SEQ ID NO. 2, the amino acid sequence of the CD22 antigen is shown as SEQ ID NO. 6, the amino acid sequence of the CD20 antigen is shown as SEQ ID NO. 4, the amino acid sequence of the CD33 antigen is shown as SEQ ID NO. 8, the amino acid sequence of the costimulatory molecule 4-1BB is shown as SEQ ID NO. 12, and the intracellular signaling domain CD3 zeta is shown as SEQ ID NO: 19.

1.3 design of primary antibodies CD19 scFv against CD19, CD20, CD22, CD33 (VH as set forth in SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:30, 34), CD20 scFv (VH shown in SEQ ID NO:38, 46, 51, VL shown in SEQ ID NO:42, 48, 53), CD22 scFv (VH shown in SEQ ID NO:54, 61, 58, 65), CD33scFv (VH shown in SEQ ID NO:69, 77, 84, 90, 93, 73, 81, 87, 91, 97), VL shown in SEQ ID NO: 33, B7H3, CD33, CD30, ERBB2, and VL 2 scFv (VH shown in SEQ ID NO:101, 123, 109, 116, 105, 124, 93, 73, 81, 87, 91, 97), CD33, B7H3, CD33, CD30, ERBB2, and VL (VL shown in SEQ ID NO:101, 123), VL ID NO:109, 116, 105, 124, 33, 125, and 43, and 33, and 39, and 33 91, 97), CD30 scFv (VH shown as SEQ ID NO:135, 142, VL shown as SEQ ID NO:139, 146), ERBB2 scFv (VH shown as SEQ ID NO:150, VL shown as SEQ ID NO: 154), the first antibody and the second antibody are connected by a hinge region (flexible amino acid hinge region), the amino acid sequence of the hinge region is shown as SEQ ID NO: 158. Synthesizing a corresponding sequence by a gene synthesis method, and assembling the sequence to obtain the following structure:

I-1-1：CD19 scFv-CD33 scFv；

I-1-2：CD19 scFv-GD2 scFv；

I-1-3：CD19 scFv-B7H3 scFv；

I-2-1：CD22 scFv-CD33 scFv；

I-2-2：CD22 scFv-GD2 scFv；

I-2-3：CD22 scFv-B7H3 scFv；

I-3-1：CD20 scFv-CD33 scFv；

I-3-2：CD20 scFv-GD2 scFv；

I-3-3：CD20 scFv-B7H3 scFv；

I-4-1：CD33 scFv-GD2 scFv；

I-4-2：CD33 scFv-B7H3 scFv；

1.4 As shown in FIG. 1, I-1 to I-4 and I-1-1 to I-1-3, I-2-1 to I-2-3, I-3-1 to I-3, I-4-1 to I-4-2 may be expressed on different expression vectors, respectively. As shown in FIG. 2, I-1 can be connected with I-1-1, I-1-2 and I-1-3 respectively through a cleavage peptide sequence (SEQ ID NO: 13), I-2 can be connected with I-2-1, I-2-2 and I-2-3 respectively through a cleavage peptide sequence, I-3 can be connected with I-3-1, I-3-2 and I-3-3 respectively through a cleavage peptide sequence, I-4 can be connected with I-4-1 and I-4-2 respectively through a cleavage peptide sequence, and corresponding sequences are synthesized through a gene synthesis method, so that the following structures are obtained:

II-1: CD19 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 19 scFv-CD33scFv;

II-2: CD19 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 19scFv-GD2scFv;

II-3: CD19 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 19scFv-B7H3scFv;

II-4: CD22 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 22scFv-CD33scFv;

II-5: CD22 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 22scFv-GD2scFv;

II-6: CD22 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 22scFv-B7H3scFv;

II-7: CD20 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3ζ -2A sequence-CD 20scFv-CD33scFv;

II-8: CD20 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3 zeta-2A sequence-CD 20scFv-GD2scFv;

II-9: CD20 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3 ζ -2A sequence-CD 20scFv-B7H3scFv;

II-10: CD33 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3 zeta-2A sequence-CD 33scFv-GD2scFv;

II-11: CD33 antigen sequence-costimulatory molecule 4-1 BB-intracellular signaling domain CD3 ζ -2A sequence-CD 33scFv-B7H3scFv;

1.5 by designing proper restriction enzyme sites, respectively double-restriction enzyme-cutting I-1 to I-4, I-1-1 to I-1-3, I-2-1 to I-2-3, I-3-1 to I-3-3, I-4-1 to I-4-2, II-1 to II-11 and expression vector pWPXL-x by using selected restriction endonucleases EcoRI and HindIII, respectively connecting the cut I-1 to I-4, I-1-1 to I-1-3, I-2-1 to I-2-3, I-3-1 to I-3, I-4-1 to I-4-2 and II-1 to II-11 with the expression vectors, transforming DH5 alpha-competent E.coli by using the connection products, and carrying out positive cloning overnight.

1.6 bacterial extraction of plasmids.

1.7 restriction enzyme identification and sequencing, the gene engineering vector CD19AgCAR backbond (I-1, shown in figure 4) is obtained after the expression vector is connected with the I-1 structure, the gene engineering vector CD19AgCAR Double antibody (I-1-1, the second antibody is CD33 scFv, shown in figure 5) is obtained after the expression vector is connected with the I-1 structure, and the gene engineering vector CD19AgCAR (II-1, shown in figure 6) is obtained after the expression vector is connected with the II-1 structure.

1.8 the expression vector was digested and ligated to the I-1 structure to obtain the genetic engineering vector CD19AgCAR backup (I-1, as shown in FIG. 4), the expression vector was digested and ligated to the I-1-2 structure to obtain the genetic engineering vector CD19AgCAR Double antibody (I-1-2, the second antibody was GD2 scFv), and the expression vector was digested and ligated to the II-2 structure to obtain the genetic engineering vector CD19AgCAR (II-2).

Example 2 preparation of a Virus with a genetically engineered vector

2.1 transfection of the genetically engineered vectors CD19AgCAR backbone, CD19AgCAR Double antibody, CD19AgCAR and viral packaging plasmid in a molar ratio of 1:1:1, respectively, total mass 6ug/10cm Dish, and collection of culture supernatants and virus concentration after lentiviral transfection of 293T cells for 72h and 96 h.

2.2 transfection of 1X10 by real-time quantitative PCR method at a concentration gradient of 1ul:2ul:4ul:8ul:16ul:32ul of lentivirus ⁶ Determination of viral titres by individual 293T cells, mostViruses with genetic engineering vectors CD19AgCAR backbone, CD19AgCAR Double antibody and CD19AgCAR are obtained respectively.

EXAMPLE 3 Virus transfection of immune cells

3.1 collecting human peripheral blood T lymphocytes, carrying out transfection of the virus, ensuring that the transfection efficiency is over 20%, and finding a proper MOI value for clinical treatment. It should be noted that each batch of viruses needs to be subjected to the above operation, so that the stable and reliable curative effect of the clinical test is ensured.

3.2, the transfection efficiency is detected by transfecting human peripheral blood T cells, a myc, flag, HA protein tag (tag) is added at the 5' -end of the scFv fragment of the first antibody or the scFv fragment of the second antibody, the transfection efficiency can be detected by adopting a corresponding fluorescent labeled antibody through a flow cytometry detection technology or by adopting a real-time quantitative PCR method, and the T cells after virus transfection are named AgCAR-T cells.

3.3 1:4 by expressing virus transfected ABC-T cells with the corresponding antigen of the secondary antibody; 1:10; and 1:20, and performing in vitro killing detection by flow cytometry after 0h, 24h and 48h, comparing the killing effect of cells at different incubation times and comparing with control viruses.

Example 4 acquisition and expansion of AgCAR-T cells

4.1, collecting peripheral blood by 0.5-2 ml/Kg, sorting magnetic beads to obtain CD3 positive T cells or CD56 to obtain NK cells, or sorting TCR gamma/delta to obtain gamma/delta T cells, adding CD3/CD28 immunomagnetic beads for stimulating activation and virus transfection, continuously giving CD3/CD28 immunomagnetic beads, interleukin-7 and interleukin-15 for in vitro amplification, wherein the amplification can be carried out for more than 100 times in general 7-8 days, and washing and reinfusion into human body after the proper cell number is reached. The number of the AgCAR-T cells of the reinfusion human body is 2 multiplied by 10 and is regulated according to the in vivo load ⁵ /Kg～2×10 ⁷ /Kg。

3.5 the virus prepared by the genetic engineering vector is generally transfected in vitro with the transfection efficiency of more than 20% and the vast majority of the virus is between 40 and 70%, as shown in figure 7. In fig. 7, the transfection efficiency after transfection of human T lymphocytes with the virus bearing CD19AgCAR, CD19AgCAR back and CD19AgCAR Double antibody is in that order.

EXAMPLE 5 Co-incubation with human myeloid leukemia cell line HL60

Viruses with CD19AgCAR (II-1), CD19AgCAR backup (I-1) and CD19AgCAR Double antibody (I-1-1) were transfected into human T lymphocytes and co-incubated with human myeloid leukemia cell line HL60, and CD19-CART cells and CD33-CART cells were co-incubated with human myeloid leukemia cell line HL60, respectively. After that, flow cytometry detection of HL60 cells remaining in the co-incubation system was performed at co-incubation times of 0h and 24 h. As shown in fig. 8: A. f is the result of co-incubation with HL60 cells for 0h and 24h after T cell transfection with lentivirus with CD19AgCAR (II-1), respectively; B. g is the result of co-incubation with HL60 cells for 0h and 24h after T cell transfection with lentivirus with CD19AgCAR backbone (I-1), respectively; C. results of H co-incubation with HL60 cells for 0H and 24H after T cell transfection with lentivirus carrying CD19AgCAR Double antibody (I-1-1), respectively; D. i is the result of co-incubation of CD19-CART cells with HL60 cells for 0h and 24h, respectively; e and J are the results of co-incubation of CD33-CART cells with HL60 cells for 0h and 24h, respectively. From the above results, it can be seen that 24 hours after T cell transfection with lentivirus with CD19AgCAR (II-1) can completely kill target cells, producing the same cytotoxic effect as CD33-CART cells, significantly enhanced over that of T cell transfection with lentivirus with CD19AgCAR back (I-1), T cell transfection with lentivirus with CD19AgCAR Double antibody (I-1-1) and CD19-CART cells.

Example 6 Co-incubation with human neuroblastoma cell line CHP134

Human T lymphocytes were transfected with viruses with CD19AgCAR (II-2), CD19AgCAR backup (I-1) and CD19AgCAR Double antibody (I-1-2), and co-incubated with human neuroblastoma cell line CHP134, and GD2-CART cells were co-incubated with human neuroblastoma cell line CHP134, and flow cytometry detection of residual CHP134 cells in the co-incubation system was performed at co-incubation for 0h, 24h and 48 h. As shown in fig. 9: A. b, C is the result of co-incubation with CHP134 cells for 0h, 24h and 48h after T cell transfection with lentivirus with CD19AgCAR (II-2); D. e, F is the result of co-incubation with CHP134 cells for 0h, 24h and 48h after T cell transfection with lentivirus with CD19AgCAR backup (I-1); G. h, I is the result of co-incubation with CHP134 cells for 0h, 24h and 48h after T cell transfection with lentivirus with CD19AgCAR Double antibody (I-1-2) in sequence; J. k, L is the result of incubation with CHP134 cells for 0h, 24h and 48h after GD2-CART cells in sequence; from the above results, it can be seen that the target fine GD2 positive neuroblastoma cell line CHP134 cells can be completely killed 24h and 48h after T cell transfection with the lentivirus of CD19AgCAR (II-2), resulting in better cytotoxicity than GD2-CART cells, and also significantly enhanced cytotoxicity on target cells than lentivirus transfected T cell with CD19AgCAR backbone (I-1) and lentivirus transfected T cell with CD19AgCAR Double antibody (I-1-2).

Example 7 and human acute lymphoblastic leukemia cell line MV4;11 co-incubation

Transfecting human T lymphocytes with viruses with CD22AgCAR (II-4), CD20AgCAR (II-7) and CD19AgCAR (II-2) and carrying out cell line MV4 of human acute lymphoblastic leukemia; 11 (MV 4;11 cell lines express CD33 and CD123, but do not express GD 2) and residual MV4 in the co-incubation system at 0, 24 and 48 h; flow cytometry detection of 11 cells. As shown in fig. 10: A. b, C is transfected with the lentivirus with CD22AgCAR (II-4) and then transfected with MV4;11 cells were co-incubated for 0h, 24h and 48 h; D. e, F is transfected with the lentivirus with CD20AgCAR (II-7) and then transfected with MV4;11 cells were co-incubated for 0h, 24h and 48 h; G. h, I is transfected with the lentivirus with CD19AgCAR (II-2) and MV4 after T cell transfection; 11 cells were incubated for 0h, 24h and 48 h. From the above results, it can be seen that target cells MV4 can be transfected 24h and 48h after T cells are transfected with lentiviruses bearing CD22AgCAR (II-4) and CD20AgCAR (II-7); 11 cells were completely killed (CD 123 positive cells were killed), yielding better cytotoxicity than CD19AgCAR (II-2) cells.

Example 8 therapeutic Effect of CD19AgCAR-33 (II-1) in humans

For one case of AML patient, the patient is whiteHematopathy cells specifically express CD33. Preparation of T lymphocytes from the patient himself transfected with CD19AgCAR-33 (II-1) virus 5X 10 ⁶ Per kg of CD19AgCAR-33T cells were injected peripherally and intravenously into the patient's blood vessel (day 0). The pre-treatment patient with bone marrow Minimal Residual Disease (MRD) was detected at 43.8% (illustration FIG. 11A), and at day 14 post-CD 19AgCAR-33T cell infusion, the patient's MRD was reduced to<0.1% (illustrative FIG. 11B) achieved molecular level remission, suggesting that CD19AgCAR-33T cells have clinical therapeutic value.

Example 9 therapeutic Effect of CD19AgCAR-B7H3 (II-3) in humans

For one example, neuroblastoma (NB) patient, the tumor cells of this patient specifically expressed B7H3. Preparation of T lymphocytes from the patient themselves transfected with CD19AgCAR-B7H3 (II-3) Virus to 6.3X10 ⁶ Per kg of CD19AgCAR-B7H3T cells were injected peripherally into the patient's blood vessel (day 0). Morphological visible 5% of neuroblastoma cells in the bone marrow of the pre-treatment patient, detected on day 25 after CD19AgCAR-B7H3T cell infusion, were free of morphological visible tumor cells in the bone marrow of the patient, while MRD of neuroblastoma was also reduced <0.01% and molecular level relief is achieved. Tumor cells were not seen subsequently, assessed 68 days and half a year after treatment. The above results suggest that CD19AgCAR-B7H3T cells have clinical therapeutic value.

Example 10 therapeutic Effect of CD20AgCAR-GD2 (II-8) in humans

For one Neuroblastoma (NB) patient, the tumor cells of this patient specifically expressed GD2. Preparation of T lymphocytes from the patient themselves transfected with CD20AgCAR-GD2 (II-8) Virus to 4.8X10 ⁶ Per kg of CD20AgCAR-GD2T cells were injected peripherally and intravenously into the patient's blood vessel (day 0). 2% of neuroblastoma cells were morphologically visible in the bone marrow of the patient prior to treatment, and no morphologically visible tumor cells were detected on day 30 after the infusion of CD20AgCAR-GD2T cells, and the bone marrow was still in remission when the patient was recently followed for 103 days. The above results suggest that CD20AgCAR-GD2T cells have clinical therapeutic value.

Example 11 Co-incubation of human neuroblastoma cell line SK-N-SH with 19AgCAR-ROR 1T cells, in vitro prepared double target antibody proteins

Human T lymphocytes were transfected with the virus bearing CD19AgCAR-ROR1 and co-incubated with human neuroblastoma cell line SK-N-SH (expressing ROR1 protein target), and CD19-ROR1 double-target antibody supernatants (shown in FIG. 12A) obtained by protein expression were added to the co-incubated culture system at different doses, and flow cytometry detection of residual SK-N-SH cells in the co-incubated system was performed at co-incubation times of 0h, 24h and 48 h. As shown in fig. 12B: with increasing amount of antibody expressing double targets, CD19AgCAR-ROR 1T cells exert the best target killing effect after 100ul of supernatant is added, and with increasing concentration of antibody, the killing activity is reduced instead, which suggests that there is an amount effect relationship between CD19AgCAR-ROR 1T cells and upper target antibodies, possibly related to blocking targets on CD19AgCAR-ROR 1T cells and SK-N-SH cells. This experiment further illustrates that the AgCAR structures mentioned in the present application for other targets also have a correspondingly specific targeted cytotoxic effect.

Claims

A fusion protein comprising: 1) An antigen or functionally active fragment thereof; 2) A costimulatory domain; and 3) an intracellular signaling domain.
The fusion protein of claim 1, wherein the antigen or functionally active fragment thereof comprises all relevant antigens or functional fragments thereof that, upon binding to the corresponding antibody or interacting molecule, can elicit immune cell activation.
The fusion protein of any one of claims 1-2, wherein the antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of: CD45, CD56 and CD58.
The fusion protein of any one of claims 1-3, wherein the antigen or functionally active fragment thereof comprises a tumor-associated antigen or functionally active fragment thereof.
The fusion protein of claim 4, wherein the tumor-associated antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3.
The fusion protein of any one of claims 4-5, wherein the tumor-associated antigen or functionally active fragment thereof comprises an antigen associated with B lymphocyte development.
The fusion protein of any one of claims 4-6, wherein the tumor-associated antigen or functionally active fragment thereof comprises a CD19 protein or functionally active fragment thereof.
The fusion protein of any one of claims 4-7, wherein the tumor-associated antigen or functionally active fragment thereof comprises a CD20 protein or functionally active fragment thereof.
The fusion protein of any one of claims 4-8, wherein the tumor-associated antigen or functionally active fragment thereof comprises a CD22 protein or functionally active fragment thereof.
The fusion protein of any one of claims 4-9, wherein the tumor-associated antigen or functionally active fragment thereof comprises a CD33 protein or functionally active fragment thereof.
The fusion protein of any one of claims 4-10, wherein the tumor-associated antigen or functionally active fragment thereof comprises a CD38 protein or functionally active fragment thereof.
The fusion protein according to any one of claims 4-11, wherein the tumor associated antigen or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 2, 4, 6, 8 and 10.
The fusion protein of any one of claims 1-12, wherein the co-stimulatory domain comprises a co-stimulatory domain derived from a group of proteins selected from the group consisting of: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88.
The fusion protein of any one of claims 1-13, wherein the costimulatory domain comprises a costimulatory domain derived from 4-1 BB.
The fusion protein of any one of claims 1-14, wherein the co-stimulatory domain comprises SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.
The fusion protein of any one of claims 1-15, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fceri γ, fceri β, fcgammaRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
The fusion protein of any one of claims 1-16, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from cd3ζ.
The fusion protein of any one of claims 1-17, wherein the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID No. 19.
The fusion protein of any one of claims 1-18, wherein the C-terminus of the antigen or functionally active fragment thereof is directly or indirectly linked to the N-terminus of the costimulatory domain.
The fusion protein of any one of claims 1-19, wherein the C-terminus of the costimulatory domain is directly or indirectly linked to the N-terminus of the intracellular signaling domain.
The fusion protein according to any one of claims 1-20, comprising the amino acid sequence shown in SEQ ID No. 20.
An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1-21.
The isolated nucleic acid molecule of claim 22, comprising the nucleic acid sequence set forth in SEQ ID No. 21.
A vector comprising the isolated nucleic acid molecule of any one of claims 22-23.
The vector of claim 24, comprising a viral vector.
The vector of any one of claims 24-25, comprising a lentiviral vector.
A modified cell comprising the fusion protein of any one of claims 1-21, the nucleic acid molecule of any one of claims 22-23 and/or the vector of any one of claims 24-26.
The modified cell of claim 27, wherein the cell comprises an immune cell.
The modified cell of any one of claims 27-28, wherein the cell comprises an immune effector cell.
The modified cell of claim 29, wherein the immune effector cell is selected from the group consisting of: t cells, macrophagesNatural killer cells (NK cells) and NKT cells.
The modified cell of any one of claims 27-30, comprising a T cell orAnd (3) cells.
The modified cell of any one of claims 27-31, comprising an NK cell or NKT cell.
The modified cell of any one of claims 27-32, further comprising and/or expressing one or more multispecific antibodies.
The modified cell of claim 33, wherein the multispecific antibody comprises a first targeting moiety and a second targeting moiety.
The modified cell of claim 34, wherein the first targeting moiety binds to the antigen or functionally active fragment thereof in the fusion protein.
The modified cell of any one of claims 34-35, wherein the first targeting moiety is capable of specifically binding to the tumor-associated antigen in the fusion protein.
The modified cell of any one of claims 34-36, wherein the tumor-associated antigen targeted by the first targeting moiety is selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3.
The modified cell of any one of claims 34-37, wherein the second targeting moiety is capable of specifically binding a tumor-associated antigen.
The modified cell of any one of claims 34-38, wherein the tumor-associated antigen targeted by the second targeting moiety is selected from the group consisting of: GD2, DLL1, DLL3, ROR1, GPC3, HER2, VEGFA, CD1a, CD33, B7H3, CD138, BCMA, EGFRVIII and ERBB2.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD19 protein or functionally active fragment thereof and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD19 protein or functionally active fragment thereof and the second targeting moiety recognizes a GD2 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD19 protein or functionally active fragment thereof and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD22 protein or functionally active fragment thereof and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD22 protein or functionally active fragment thereof and the second targeting moiety recognizes a GD2 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD22 protein or functionally active fragment thereof and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD20 protein or functionally active fragment thereof and the second targeting moiety recognizes a CD33 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD20 protein or functionally active fragment thereof and the second targeting moiety recognizes a GD2 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD20 protein or functionally active fragment thereof and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD33 protein or functionally active fragment thereof and the second targeting moiety recognizes a GD2 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-39, wherein the first targeting moiety recognizes a CD33 protein or functionally active fragment thereof and the second targeting moiety recognizes a B7H3 protein or functionally active fragment thereof.
The modified cell of any one of claims 34-50, wherein the first targeting moiety comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising an amino acid sequence of any one of SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 55, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 85 and SEQ ID No. 94.
The modified cell of claim 51, wherein the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, and SEQ ID NO: 95.
The modified cell of any one of claims 51-52, wherein the HCDR3 comprises an amino acid sequence of any one of SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 47, SEQ ID No. 52, SEQ ID No. 57, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 86 and SEQ ID No. 96.
The modified cell of any one of claims 51-53, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24 and HCDR3: SEQ ID NO. 25;

b) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

c) HCDR1: SEQ ID NO. 31, HCDR2: SEQ ID NO. 32 and HCDR3: SEQ ID NO. 33;

d) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 41;

e) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 47;

f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 40 and HCDR3: SEQ ID NO. 52;

g) HCDR1: SEQ ID NO. 55, HCDR2: SEQ ID NO. 56 and HCDR3: SEQ ID NO. 57;

h) HCDR1: SEQ ID NO. 62, HCDR2: SEQ ID NO. 63 and HCDR3: SEQ ID NO. 64;

i) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

j) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

k) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

l) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

m) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: SEQ ID NO. 96.
The modified cell of any one of claims 51-54, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 51, SEQ ID No. 54, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 84, SEQ ID No. 90 and SEQ ID No. 93.
The modified cell of any one of claims 34-55, wherein the first targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising an amino acid sequence of any one of SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 49, SEQ ID No. 59, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 88, SEQ ID No. 92 and SEQ ID No. 98.
The modified cell of claim 56, wherein said LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 44, SEQ ID NO. 28, SEQ ID NO. 67, SEQ ID NO. 75 and SEQ ID NO. 99.
The modified cell of any one of claims 56-57, wherein the LCDR3 comprises an amino acid sequence of any one of SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 50, SEQ ID No. 60, SEQ ID No. 68, SEQ ID No. 76, SEQ ID No. 83, SEQ ID No. 89 and SEQ ID No. 100.
The modified cell of any one of claims 56-58, wherein the LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) LCDR1: SEQ ID NO. 27, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 29;

b) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

c) LCDR1: SEQ ID NO. 35, LCDR2: SEQ ID NO 36 and LCDR3: SEQ ID NO. 37;

d) LCDR1: SEQ ID NO. 43, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

e) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 50;

f) LCDR1: SEQ ID NO. 49, LCDR2: SEQ ID NO. 44 and LCDR3: SEQ ID NO. 45;

g) LCDR1: SEQ ID NO 59, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 60;

h) LCDR1: SEQ ID NO:66, LCDR2: SEQ ID NO 67 and LCDR3: SEQ ID NO. 68;

i) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

j) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

k) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

l) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

m) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100.
The modified cell of any one of claims 56-59, wherein said VL comprises the amino acid sequence set forth in any one of SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 42, SEQ ID NO 48, SEQ ID NO 53, SEQ ID NO 58, SEQ ID NO 65, SEQ ID NO 73, SEQ ID NO 81, SEQ ID NO 87, SEQ ID NO 91 and SEQ ID NO 97.
The modified cell of any one of claims 34-60, wherein the second targeting moiety comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising an amino acid sequence of any one of SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 85, SEQ ID No. 94, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 117, SEQ ID No. 126, SEQ ID No. 136, SEQ ID No. 143 and SEQ ID No. 151.
The modified cell of claim 61, wherein the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:144, and SEQ ID NO: 152.
The modified cell of any one of claims 61-62, wherein the HCDR3 comprises an amino acid sequence of any one of SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 86, SEQ ID No. 96, SEQ ID No. 104, SEQ ID No. 112, SEQ ID No. 119, SEQ ID No. 128, SEQ ID No. 138, SEQ ID No. 145 and SEQ ID No. 153.
The modified cell of any one of claims 61-63, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR1: SEQ ID NO. 70, HCDR2: SEQ ID NO:71 and HCDR3: SEQ ID NO. 72;

b) HCDR1: SEQ ID NO:78, HCDR2: SEQ ID NO. 79 and HCDR3: 80 of SEQ ID NO;

c) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

d) HCDR1: SEQ ID NO. 85, HCDR2: SEQ ID NO. 79 and HCDR3: SEQ ID NO. 86;

e) HCDR1: SEQ ID NO. 94, HCDR2: SEQ ID NO. 95 and HCDR3: 96 of SEQ ID NO;

f) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

g) HCDR1: SEQ ID NO. 102, HCDR2: SEQ ID NO. 103 and HCDR3: SEQ ID NO. 104;

h) HCDR1: SEQ ID NO. 110, HCDR2: SEQ ID NO 111 and HCDR3: 112 of SEQ ID NO;

i) HCDR1: SEQ ID NO:117, HCDR2: SEQ ID NO. 118 and HCDR3: SEQ ID NO. 119;

j) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

k) HCDR1: SEQ ID NO. 126, HCDR2: SEQ ID NO:127 and HCDR3: SEQ ID NO. 128;

l) HCDR1: SEQ ID NO. 136, HCDR2: SEQ ID NO 137 and HCDR3: 138 of SEQ ID NO;

m) HCDR1: SEQ ID NO:143, HCDR2: SEQ ID NO. 144 and HCDR3: SEQ ID NO. 145;

n) HCDR1: SEQ ID NO:151, HCDR2: SEQ ID NO. 152 and HCDR3: SEQ ID NO. 153.
The modified cell of any one of claims 61-64, wherein the VH comprises an amino acid sequence set forth in any one of SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 84, SEQ ID No. 90, SEQ ID No. 93, SEQ ID No. 101, SEQ ID No. 123, SEQ ID No. 109, SEQ ID No. 116, SEQ ID No. 125, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 142 and SEQ ID No. 150.
The modified cell of any one of claims 34-65, wherein the second targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2, and LCDR3, the LCDR1 comprising an amino acid sequence of any one of SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 88, SEQ ID No. 92, SEQ ID No. 98, SEQ ID No. 106, SEQ ID No. 114, SEQ ID No. 121, SEQ ID No. 130, SEQ ID No. 140, SEQ ID No. 147, and SEQ ID No. 155.
A modified cell according to claim 66, wherein the LCDR2 comprises an amino acid sequence of any one of SEQ ID No. 75, SEQ ID No. 28, SEQ ID No. 99, SEQ ID No. 107, SEQ ID No. 131, SEQ ID No. 148 and SEQ ID No. 156.
The modified cell of any one of claims 66-67, wherein the LCDR3 comprises an amino acid sequence of any one of SEQ ID No. 76, SEQ ID No. 83, SEQ ID No. 89, SEQ ID No. 100, SEQ ID No. 108, SEQ ID No. 115, SEQ ID No. 122, SEQ ID No. 132, SEQ ID No. 141, SEQ ID No. 149 and SEQ ID No. 157.
The modified cell of any one of claims 66-68, wherein the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) LCDR1: SEQ ID NO. 74, LCDR2: SEQ ID NO 75 and LCDR3: SEQ ID NO. 76;

b) LCDR1: SEQ ID NO. 82, LCDR2: SEQ ID NO. 28 and LCDR3: 83 of SEQ ID NO;

c) LCDR1: SEQ ID NO. 88, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

d) LCDR1: SEQ ID NO. 92, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 89;

e) LCDR1: SEQ ID NO. 98, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 100;

f) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

g) LCDR1: SEQ ID NO. 106, LCDR2: SEQ ID NO 107 and LCDR3: SEQ ID NO. 108;

h) LCDR1: SEQ ID NO. 114, LCDR2: SEQ ID NO 99 and LCDR3: SEQ ID NO. 115;

i) LCDR1: SEQ ID NO:121, LCDR2: SEQ ID NO. 28 and LCDR3: SEQ ID NO. 122;

j) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

k) LCDR1: SEQ ID NO. 130, LCDR2: SEQ ID NO. 131 and LCDR3: SEQ ID NO. 132;

l) LCDR1: SEQ ID NO:140, LCDR2: SEQ ID NO. 28 and LCDR3: 141 of SEQ ID NO;

m) LCDR1: SEQ ID NO:147, LCDR2: SEQ ID NO 148 and LCDR3: 149 of SEQ ID NO;

n) LCDR1: SEQ ID NO:155, LCDR2: SEQ ID NO 156 and LCDR3: SEQ ID NO. 157.
The modified cell of any one of claims 66-69, wherein the VL comprises an amino acid sequence shown in any one of SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 87, SEQ ID No. 91, SEQ ID No. 97, SEQ ID No. 105, SEQ ID No. 124, SEQ ID No. 113, SEQ ID No. 120, SEQ ID No. 129, SEQ ID No. 134, SEQ ID No. 139, SEQ ID No. 146 and SEQ ID No. 154.
The modified cell of any one of claims 34-70, wherein the targeting moiety comprises VHH, scFv, fab, dAb and/or a derivative protein structure.
The modified cell of any one of claims 34-71, wherein the targeting moiety is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
The modified cell of any one of claims 34-72, wherein the first targeting moiety comprises an scFv.
The modified cell of any one of claims 34-73, wherein the second targeting moiety comprises an scFv.
The modified cell of any one of claims 34-74, wherein in the multispecific antibody, the first targeting moiety is fused in-frame with a second targeting moiety.
The modified cell of any one of claims 34-75, wherein in the multispecific antibody the first targeting moiety is linked to the second targeting moiety by a hinge region.
The modified cell of claim 76, wherein the hinge region comprises a hinge region derived from a histone: CD8, CD28, and 4-1BB.
The modified cell of any one of claims 76-77, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 17.
The modified cell according to any one of claims 27-78, comprising and/or expressing a nucleic acid molecule encoding the fusion protein of any one of claims 1-21 and/or a nucleic acid molecule encoding the multispecific antibody of any one of claims 33-78.
The modified cell of claim 79, wherein the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on separate vectors.
The modified cell of any one of claims 79-80, wherein the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on the same vector.
The modified cell of any one of claims 79-81, wherein the nucleic acid molecule encoding a fusion protein is linked to the nucleic acid molecule encoding a multispecific antibody by a sequence encoding a cleavage peptide.
The modified cell of claim 82, wherein the nucleic acid sequence encoding a cleavage peptide is selected from the group consisting of: P2A sequence, T2A sequence, F2A sequence, E2A sequence, bmCPV2A sequence and BmIFV2A sequence.
The modified cell of any one of claims 82-83, wherein the cleavage peptide sequence comprises a P2A sequence.
The modified cell of any one of claims 82-84, wherein the cleavage peptide sequence comprises a T2A sequence.
The modified cell of any one of claims 82-85, wherein the cleavage peptide sequence comprises the nucleic acid sequence of any one of SEQ ID NOs 13-16.
The modified cell of any one of claims 33-86, wherein the fusion protein is expressed in a different amount than the multispecific antibody.
A pharmaceutical composition comprising the modified cell of any one of claims 27-87, and/or one or more multispecific antibodies of any one of claims 33-78.
Use of the modified cell of any one of claims 27-87 and/or the one or more multispecific antibodies of any one of claims 33-78 in the manufacture of a medicament.
A pharmaceutical composition comprising the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or a pharmaceutically acceptable adjuvant and/or excipient.
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a tumor.
The use of claim 91, wherein the tumor comprises a hematological tumor.
The use of claim 91, wherein the tumor comprises a solid tumor.
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a disease or disorder associated with expression of CD 33.
The use of claim 94, wherein the disease or disorder associated with expression of CD33 comprises human Acute Myeloid Leukemia (AML).
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a disease or disorder associated with expression of GD 2.
The use of claim 96, wherein the disease or disorder associated with expression of GD2 comprises neuroblastoma.
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a disease or disorder associated with expression of B7H 3.
The use of claim 98, wherein the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a disease or disorder associated with expression of HER 2.
The use of claim 100, wherein the disease or disorder associated with expression of HER2 comprises breast cancer.
The fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 for use in treating a disease or disorder associated with expression of DLL 1.
The use of claim 102 wherein the disease or disorder associated with expression of DLL1 comprises lung cancer.
Use of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 in the manufacture of a medicament for treating a disease or disorder associated with expression of CD 33.
The use of claim 104, wherein the disease or disorder associated with expression of CD33 comprises human Acute Myeloid Leukemia (AML).
Use of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 in the manufacture of a medicament for treating a disease or disorder associated with GD2 expression.
The use of claim 106, wherein the disease or disorder associated with GD2 expression comprises neuroblastoma.
Use of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 in the manufacture of a medicament for treating a disease or disorder associated with expression of B7H 3.
The use of claim 108, wherein the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
Use of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 in the manufacture of a medicament for treating a disease or disorder associated with expression of HER 2.
The use of claim 110, wherein the disease or disorder associated with expression of HER2 comprises breast cancer.
Use of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90 in the manufacture of a medicament for treating a disease or disorder associated with expression of DLL 1.
The use of claim 112 wherein the disease or disorder associated with expression of DLL1 comprises lung cancer.
A method of preventing and/or treating a tumor comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
A method of preventing and/or treating a disease or disorder associated with expression of CD33 comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
The method of claim 115, wherein the disease or disorder associated with expression of CD33 comprises human Acute Myeloid Leukemia (AML).
A method of preventing and/or treating a disease or disorder associated with GD2 expression comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
The method of claim 117, wherein the disease or disorder associated with expression of GD2 comprises neuroblastoma.
A method of preventing and/or treating a disease or disorder associated with expression of B7H3 comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
The method of claim 119, wherein the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
A method of preventing and/or treating a disease or disorder associated with the expression of HER2 comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
The method of claim 121, wherein the disease or disorder associated with expression of HER2 comprises breast cancer.
A method of preventing and/or treating a disease or disorder associated with expression of DLL1 comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the modified cell of any one of claims 27-87, and/or the pharmaceutical composition of any one of claims 88 and 90.
The use of claim 123 wherein the disease or disorder associated with expression of DLL1 comprises lung cancer.
The pharmaceutical composition of claim 88, wherein the multispecific antibody comprises a protein molecule comprising a first targeting moiety and a second targeting moiety that is purified using a bioengineering technique.
The pharmaceutical composition of any one of claims 88 and 125, wherein the multispecific antibody is infused into a blood vessel.