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CN116284389A - anti-AFP/HLA 02 TCR-like antibodies and uses thereof - Google Patents

anti-AFP/HLA 02 TCR-like antibodies and uses thereof Download PDF

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CN116284389A
CN116284389A CN202211117275.4A CN202211117275A CN116284389A CN 116284389 A CN116284389 A CN 116284389A CN 202211117275 A CN202211117275 A CN 202211117275A CN 116284389 A CN116284389 A CN 116284389A
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amino acid
acid sequence
hcdr1
hcdr3
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王华菁
成超
何晓文
谢二敏
李霄培
陈晓锐
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Oricell Therapeutics Co Ltd
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Abstract

The present application relates to an anti-AFP/HLA 02 TCR-like antibody and uses thereof, which antibody is capable of a K of about 3.1E-09M or less D Value and human AFP 158‑166 HLA-A02 x 01 x complex specific binding. The application also relates to chimeric antigen receptors comprising said antibodies, immunoconjugates and cells comprising said chimeric antigen receptors.

Description

anti-AFP/HLA 02 TCR-like antibodies and uses thereof
Technical Field
The application relates to the field of biological medicine, in particular to an anti-AFP/HLA 02 TCR-like antibody and application thereof.
Background
Alpha-fetoprotein (AFP) is a glycoprotein, and in adults, AFP can be increased in about 80% of serum of liver cancer patients, the positive rate of AFP in germ cell tumors is 50%, and the AFP can also be increased to different degrees in pancreatic cancer, lung cancer, liver cirrhosis and other patients. Therefore, AFP is expected to be a target for treating various solid tumors.
Intracellular tumor-specific antigens can be processed into peptides and presented to the surface of tumor cells via class I Major Histocompatibility Complex (MHC), and TCR-like antibodies can bind to the polypeptide/MHC complex, thereby inducing tumor cell death. TCR-like antibodies can be converted to CAR structures, mediating specific tumor lysis by T cells.
At present, CART technology has achieved great success in hematological tumors, but has not yet been significantly altered 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.
Thus, there is a need to develop new antibodies and CAR structures to treat solid tumors.
Disclosure of Invention
The present application provides an isolated antigen binding protein having one or more of the following properties: 1) At a K of about 3.1E-09M or less D Value and human AFP 158-166 HLA-A02 x 01 x complex specific binding; and 2) can be matched with mouse AFP 158 Complex binding is/HLA-A 02.
In one aspect, the present application provides an isolated antigen binding protein comprising HCDR3, the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO: 56.
In certain embodiments, the antigen binding protein comprises HCDR2, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55.
In certain embodiments, the antigen binding protein comprises HCDR1, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region set forth in any one of SEQ ID NOs 72 through 84.
In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 19, SEQ ID NO. 25, SEQ ID NO. 30, SEQ ID NO. 35, SEQ ID NO. 46, SEQ ID NO. 52, and SEQ ID NO. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
In certain embodiments, 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. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
In certain embodiments, the antigen binding protein comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 58 through SEQ ID NO 65.
In certain embodiments, the antigen binding protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 66.
In certain embodiments, the antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 67-69.
In certain embodiments, the antigen binding protein comprises H-FR4, the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO 70 through SEQ ID NO 71.
In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in any one of SEQ ID NO. 58 to SEQ ID NO. 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 in said antigen binding protein comprises any one of the amino acid sequences selected from the group consisting of:
a) H-FR1: SEQ ID NO. 58, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
b) H-FR1: SEQ ID NO 59, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
c) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
d) H-FR1: SEQ ID NO. 61, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
e) H-FR1: SEQ ID NO. 62, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
f) H-FR1: SEQ ID NO. 63, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
g) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
h) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 68 and H-FR4: SEQ ID NO. 71; and
i) H-FR1: SEQ ID NO. 65, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO 69 and H-FR4: SEQ ID NO. 70.
In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs 72 to 84.
In certain embodiments, the antigen binding protein comprises an antibody or antigen binding fragment thereof.
In certain embodiments, the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and dAb.
In certain embodiments, the antigen binding protein comprises a VHH or antigen binding fragment thereof.
In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, and fully human antibodies.
In certain embodiments, the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 72 to 84.
In another aspect, the present application provides a chimeric antigen receptor comprising a targeting moiety comprising HCDR3, the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO: 56.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR2, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region set forth in any one of SEQ ID NOs 72 through 84.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, HCDR2, HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 30, SEQ ID No. 35, SEQ ID No. 46, SEQ ID No. 52 and SEQ ID No. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
In certain embodiments, the HCDR1, HCDR2 and HCDR3 in the chimeric antigen receptor comprise an amino acid sequence selected from any one of the group consisting of:
a) HCDR1: SEQ ID NO. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 58 through SEQ ID NO 65.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 66.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:67 to SEQ ID NO: 69.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO 70 through SEQ ID NO 71.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in any one of SEQ ID NO. 58 to SEQ ID NO. 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 in said chimeric antigen receptor comprises any one of the amino acid sequences selected from the group consisting of:
a) H-FR1: SEQ ID NO. 58, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
b) H-FR1: SEQ ID NO 59, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
c) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
d) H-FR1: SEQ ID NO. 61, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
e) H-FR1: SEQ ID NO. 62, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
f) H-FR1: SEQ ID NO. 63, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
g) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
h) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 68 and H-FR4: SEQ ID NO. 71; and
i) H-FR1: SEQ ID NO. 65, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO 69 and H-FR4: SEQ ID NO. 70.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises an antibody or antigen binding fragment.
In certain embodiments, the antigen binding fragment in the chimeric antigen receptor is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, VHH and/or dAb.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises a VHH.
In certain embodiments, the VHH in the chimeric antigen receptor targets human AFP 158-166 HLA-A02 x 01 x complex.
In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NO 72 through SEQ ID NO 84.
In certain embodiments, the chimeric antigen receptor comprises a hinge region.
In certain embodiments, the hinge region in the chimeric antigen receptor comprises a hinge region derived from: igG4, igG1 and CD8.
In certain embodiments, the hinge region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO. 147.
In certain embodiments, the chimeric antigen receptor comprises a transmembrane domain.
In certain embodiments, the transmembrane domain in the chimeric antigen receptor comprises a transmembrane domain derived from a protein selected from the group consisting of: CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, fcepsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154 and SLAM.
In certain embodiments, the transmembrane domain in the chimeric antigen receptor comprises a transmembrane domain derived from CD 8.
In certain embodiments, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 149.
In certain embodiments, the N-terminus of the transmembrane domain in the chimeric antigen receptor is linked to the C-terminus of the hinge region.
In certain embodiments, the chimeric antigen receptor comprises a costimulatory signaling domain.
In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from a protein selected from the group consisting of: CD28, 4-1BB, 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 ligand, CD40 and MyD88.
In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from 4-1 BB.
In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain that is derived from CD 28.
In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises the amino acid sequence as depicted in SEQ ID NO. 132 or SEQ ID NO. 130.
In certain embodiments, the N-terminus of the costimulatory signaling domain is linked to the C-terminus of the transmembrane domain in the chimeric antigen receptor.
In certain embodiments, the chimeric antigen receptor comprises an intracellular signaling domain.
In certain embodiments, the intracellular signaling domain in the chimeric antigen receptor 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 in the chimeric antigen receptor comprises an intracellular signaling domain derived from cd3ζ.
In certain embodiments, the intracellular signaling domain in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 134.
In certain embodiments, the N-terminus of the intracellular signaling domain in the chimeric antigen receptor is linked to the C-terminus of the costimulatory signaling domain.
In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NO 112 through SEQ ID NO 124.
In another aspect, the present application provides a polypeptide comprising the isolated antigen binding protein and/or the chimeric antigen receptor.
In another aspect, the present application provides an immunoconjugate comprising the isolated antigen binding protein.
In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.
In another aspect, the present application provides one or more vectors comprising the isolated nucleic acid molecules.
In another aspect, the present application provides one or more modified cells comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule and/or the vector.
In certain embodiments, the modification comprises upregulating the expression of a low density lipoprotein receptor-related protein or fragment thereof in the cell.
In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of: low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof is of human origin.
In certain embodiments, the functional fragment comprises a fragment or a truncate of the low density lipoprotein receptor-related protein having activity of the low density lipoprotein receptor-related protein.
In certain embodiments, the low density lipoprotein receptor-related protein comprises low density lipoprotein receptor-related protein 6 and truncations thereof, and/or low density lipoprotein receptor-related protein 5 and truncations thereof.
In certain embodiments, the truncations of the low density lipoprotein receptor-related protein 6 comprise the intracellular region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 comprise the intracellular region of the low density lipoprotein receptor-related protein 5.
In certain embodiments, the truncations of the low density lipoprotein receptor-related protein 6 comprise a transmembrane region of the low density lipoprotein receptor-related protein 6 and an LDLR region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 comprise the transmembrane region of the low density lipoprotein receptor-related protein 5 and the LDLR region of the low density lipoprotein receptor-related protein 5.
In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, and SEQ ID NO: 144.
In certain embodiments, the modification comprises introducing into the modified cell a vector that up-regulates the expression of the low density lipoprotein receptor-related protein or fragment thereof.
In certain embodiments, the modified cell comprises an immune cell.
In certain embodiments, the immune cell is 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.
In certain embodiments, the modified cell comprises a T cell.
In another aspect, the present application provides a method of making the isolated antigen binding protein, the chimeric antigen receptor, and/or the polypeptide, the method comprising culturing the modified cell under conditions such that the isolated antigen binding protein and/or the polypeptide is expressed.
In another aspect, the present application provides one or more pharmaceutical compositions comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the modified cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.
In another aspect, the present application provides a method for detecting an AFP protein comprising:
administering said isolated antigen binding protein, said polypeptide or said immunoconjugate.
In another aspect, the present application provides a kit for detecting an AFP protein, comprising said isolated antigen binding protein, said polypeptide or said immunoconjugate.
In another aspect, the present application provides the use of said isolated antigen binding protein, said polypeptide or said immunoconjugate in the manufacture of a kit for detecting the presence and/or amount of an AFP protein.
In another aspect, the present application provides the use of said isolated antigen binding protein, said chimeric antigen receptor, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, and/or said modified cell in the manufacture of a medicament for the prevention and/or treatment of a tumor.
In certain embodiments, the tumor in the use comprises a solid tumor.
In certain embodiments, the tumor in the use comprises a non-solid tumor.
In certain embodiments, the tumor in the use comprises a tumor associated with the expression of AFP.
In certain embodiments, the tumor in the use comprises liver cancer.
In another aspect, the present application provides the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the modified cell for use in preventing and/or treating a tumor.
In certain embodiments, the tumor in the use comprises a solid tumor.
In certain embodiments, the tumor in the use comprises a non-solid tumor.
In certain embodiments, the tumor in the use comprises a tumor associated with the expression of AFP.
In certain embodiments, the tumor in the use comprises liver cancer.
In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the modified cell.
In certain embodiments, the tumor in the method comprises a solid tumor.
In certain embodiments, the tumor in the method comprises a non-solid tumor.
In certain embodiments, the tumor in the method comprises a tumor associated with the expression of AFP.
In certain embodiments, the tumor in the method comprises liver cancer.
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 invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:
FIG. 1 shows the results of the 5 rounds of panning ELISA assays described herein.
FIG. 2 shows the sequence reproducibility of the single clones picked after panning as described in this application.
FIG. 3 shows the results of PCR amplification of the candidate VHH antibody nucleotide sequences described herein.
FIG. 4 shows SDS-PAGE results of the expressed purified VHH antibodies described in the present application.
FIG. 5 shows eukaryotic expression vectors (exemplified by 1C 11) for a candidate VHH-Fc antibody described herein.
FIG. 6 shows the affinity of the candidate VHH-Fc antibodies described herein.
FIG. 7A shows the results of a flow assay of a candidate VHH-Fc antibody described herein with human TERT540/T2 cells.
FIG. 7B shows the results of a flow assay for a candidate VHH-Fc antibody and a polypeptide/T2 cell described herein.
FIG. 7C shows the results of a flow assay of a candidate VHH-Fc antibody described herein with mouse AFP158/T2 cells.
FIG. 8 shows a map of a core plasmid vector described herein.
FIG. 9 shows a block diagram of the CAR core plasmids described herein.
FIG. 10A shows the results of PCR amplification of the human AFP158-166 nucleotide sequence described herein.
FIG. 10B shows the results of the vector cleavage described herein.
FIG. 11 shows the results of a flow screen assay monoclonal as described herein.
FIG. 12 shows the results of the flow assay screening for HEPG2-MiniG and SK-HEP-1-MiniG positives described herein.
FIGS. 13A-13B show the results of a CAR-T cell repeat-stimulus expansion assay as described herein.
Fig. 13C and 13F show the effective target ratio 1 described herein: results of in vitro cell killing experiments with 1CAR-T cells.
Fig. 13D and 13G show the effective target ratio 1 described herein: results of 1 IFN-gamma cytokine secretion.
Fig. 13E and 13H show the effective target ratio 1 described herein: results of 1IL-2 cytokine secretion.
Fig. 14 shows a flow chart of an animal experiment described in the present application.
Fig. 15A shows a graph of tumor growth as described herein.
Figure 15B shows a graph of animal body weight as described herein.
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In the present application, the term "isolated antigen binding protein" generally refers to a polymer of polypeptides capable of specifically recognizing and/or neutralizing a particular antigen. For example, an isolated antigen binding protein may comprise a portion of a heavy chain. For example, an isolated antigen binding protein may comprise a heavy chain variable region. The term "isolated antigen binding protein" may include single domain antibodies. For example, an isolated antigen binding protein may include, but is not limited to, a human single domain antibody.
In this application, the term "single domain antibody" or "sdAb" or "VHH" generally refers to a class of antibodies that lack the antibody light chain but only the heavy chain variable region. In some cases, the single domain antibody may be from a Bactrian camel, droctrian camel, alpaca, llama, nurse shark, dairy shark or ray (see, e.g., kang Xiaozhen et al, bioengineering journal 2018,34 (12): 1974-1984). For example, the single domain antibody may be from alpaca. Single domain antibodies may be composed of heavy chain variable regions (VH). The term "heavy chain variable region" generally refers to the amino terminal domain of the heavy chain of an antigen binding fragment. The heavy chain variable region can be further divided into hypervariable regions called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved as Framework Regions (FR). Each heavy chain variable region 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 heavy chain variable region contains a binding domain that interacts with an antigen (e.g., AFP).
In the present application, the term "TCR-like antibody" generally refers to an antibody capable of recognizing a peptide/MHC complex on the surface of a tumor cell. These TCR-like antibodies share functional identity with TCRs in terms of target recognition. TCR-like antibodies can be produced technically by conventional hybridoma techniques or by in vitro antibody library techniques known to those skilled in the art. In the present application, the "TCR-like antibody" can recognize AFP +.HLA02. For example, the TCR-like antibody can recognize human AFP 158-166 HLA-A02 x 01 x complex.
In this application, the term "transmembrane domain" generally refers to a sequence of a cell surface protein that spans the cell membrane, which may comprise a hydrophobic alpha helix. The transmembrane domain may be linked to an intracellular signaling domain, which serves to transmit signals. In the present application, the transmembrane domain may be derived from any type I, type II or type III transmembrane protein. In the present application, the transmembrane domain may comprise a transmembrane domain derived from a protein selected from the group consisting of: CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, fcepsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154 and SLAM. For example, the transmembrane domain may comprise a transmembrane domain derived from the CD 8.
In the present application, the term "chimeric antigen receptor (Chimeric Antigen Receptor, CAR)" generally refers to a fusion protein comprising a targeting moiety capable of binding an antigen and at least one intracellular domain. CARs are core components of chimeric antigen receptor T cells (CAR-T), which can include a targeting moiety (e.g., targeting a tumor-specific antigen and/or a tumor-associated antigen), a signal peptide, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In this application, the CAR can be based on an antigen of an antibody (e.g., AFP 158-166 HLA-A02 x 01 x complex) specificity is combined with the T cell receptor activating intracellular domain. The genetically modified CAR-expressing T cells can specifically recognize and eliminate malignant cells that express the target antigen. For a description of CARs and CAR-T cells, see, e.g., sadelain M, brentjens R, rive' ere i.the basicprinciples of chimeric antigen receptor design.cancer discovery.2013; 3 (4) 388-398; turtle CJ, hudecek M, jensen MC, riddell SR.engineered T cells for anti-cancer therapy.Curr Opin immunol.2012;24 633-639; dotti G, gottschalk S, savoldo B, brenner MK. Design and development of therapi es using chimeric antigen receptor-expressing T cells. Immunol rev.2014;257 (1) 107-126; and WO2013154760, WO2016014789.
In this 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, 4-1BB, 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 ligand, 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 this 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: cd3ζ, cd3δ, cd3γ, cd3ε, CD79a, CD79b, fceriγ, fceriβ, fcyriia, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi sarcoma herpes virus (HSKV), DAP10, DAP-12, and domains comprising at least one ITAM. 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 this application, the term "antigen binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind an antigen. The antigen binding function of an antibody may be achieved by a full-length fragment of the antibody. The antigen binding function of an antibody can also be achieved by: a heavy chain comprising a fragment of Fv, scFv, dsFv, fab, fab 'or F (ab') 2, or a light chain comprising a fragment of Fv, scFv, dsFv, fab, fab 'or F (ab') 2. (1) Fab fragments, monovalent fragments typically consisting of VL, VH, CL and CH domains; (2) F (ab') 2 fragments, may comprise a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of VH and CH domains; (4) Fv fragments consisting of the VL and VH domains of the antibody single arm; (5) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341:544-546); (6) The isolated Complementarity Determining Regions (CDRs) and (7) may optionally be a combination of two or more isolated CDRs joined by a linker. In addition, monovalent single chain molecules Fv (scFv) formed by the pairing of VL and VH (see Bird et al (1988) Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci.85:5879-5883) may be included. The "antigen binding fragment" may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) A binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) An immunoglobulin heavy chain CH2 constant region fused to a hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. For example, the antigen binding fragment may also include a single domain antibody.
In the present application, the term "monoclonal antibody" generally refers to a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that occur in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. For example, the monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells by using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described by Clackson et al, nature,352:624-628 (1991) and Marks et al, mol. Biol.,222:581-597 (1991).
In the present application, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to a corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portion is homologous to the antibody sequences from another species (e.g., human). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, and the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region with which it is combined. Meanwhile, since the constant region of a chimeric antibody may be derived from human, the chimeric antibody may be less likely to elicit an immune response upon injection than an antibody using a constant region of non-human origin.
In the present application, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing immunogenicity of a xenogeneic antibody when introduced into humans, while maintaining the full antigen binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al, nature 321:522 (1986)) and variants thereof may be used; non-human binding domains are humanised by technical means including "remodeling" (reshaping), (Verhoeyen, et al, 1988Science 239:1534-1536;Riechmann,et al, 1988Nature 332:323-337;Tempest,et al), "high addition" (hyperchimerisation), "Queen, et al, 1989Proc Natl Acad Sci USA 86:10029-10033; co, et al, 1991Proc Natl Acad Sci USA 88:2869-2873; co, et al, 1992J Immunol 148:1149-1154) and" veneering "(Mark, et al," "Derivation of therapeutically active humanized and veneered anti-CD18 ntibodies," In: metcalf B W, dalton B J, eds.cell adhesion: molecular definition to therapeutic potential New York: plasma Press, 1994:291-312), surface reconstruction (US patent US 5639641). If other regions, such as hinge and constant region domains, are also derived from non-human sources, these regions may also be humanized.
In the present application, the term "ribosome jump site", also called internal ribosome entry site (Internal ribosome entry site, IRES), generally refers to a nucleotide sequence located in the middle of an mRNA sequence for translation initiation. The ribosome jump site can allow translation to be initiated in a cap-independent manner. IRES is typically located in the 5' UTR. In this application, the ribosome jump site can include the sequences from position 1 to position 578 of SEQ ID NO. 145.
In this application, the term "tumor" generally refers to a neoplasm formed by local tissue cell proliferation. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in the tumor as compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression amount of AFP is up-regulated in the tumor microenvironment or in the tumor as compared to a normal cell. The tumor associated with protein expression of AFP may be an AFP-positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than in normal cells.
In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other therapeutic agent to the isolated antigen binding protein, which can deliver the other therapeutic agent to a target cell (e.g., a tumor cell) through specific binding of the isolated antigen binding protein to an antigen on the target cell. In addition, the antigen may also be secreted by the target cell and located in the extracellular space of the target cell.
In the present application, the term "K D "(likewise," K) D "or" K D ") generally refers to an" affinity constant "or" equilibrium dissociation constant "and refers to a value obtained at equilibrium in a titration measurement, or by dividing the dissociation rate constant (kd) by the association rate constant (ka). Using the binding rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K) D ) Represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) to an antigen (e.g., an AFP protein). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by Biacore (analysis of biomolecular interactions) (e.g., an instrument available from BIAcoreInternationalAB, aGEHealthcarecompany, uppsala, sweden) D Values, other experimental pathways and instruments such as Octet detection may also be used. In addition, kinExA (dynamic) available from SapidyneInstruents (Boise, idaho) may also be usedExclusion assay (KineticExclusionAssay) the K was determined D Value, or determination of the K using a Surface Plasmon Resonance (SPR) D Values.
In this application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof, either isolated from the natural environment or synthesized.
In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host. The vector may transfer the inserted nucleic acid molecule into and/or between cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable cell. Typically, the vector will produce the desired expression product by culturing a suitable cell containing the vector. In this application, the vector may comprise a lentiviral vector.
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 progeny cells may not necessarily be identical in morphology or in genome to the original parent cell due to natural, accidental or deliberate mutation, but are capable of expressing the chimeric antigen receptor or antigen binding protein described herein. The cells may be obtained by transfecting the cells in vitro using the vectors described herein. 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 this 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 this 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 this 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 addition to the specific proteins and nucleotides mentioned herein, the present application may also include functional variants, derivatives, analogs, homologs and fragments thereof.
The term "functional variant" refers to a polypeptide having or encoded by a substantially identical amino acid sequence as a naturally occurring sequence 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 "derivative" generally refers to any substitution, variation, modification, substitution, deletion, and/or addition of one (or more) amino acid residues from/to a sequence as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
In this application, the term "analog" generally refers to a polypeptide or polynucleotide, and includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.
Generally, amino acid substitutions can be made, for example, at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.
Proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
In the present application, the term "AFP antigen" generally refers to a glycoprotein, which belongs to the albumin family. The amino acid sequence of human AFP can be found in UniProt/Swiss-Prot accession number P02771.
In this application, the term "T cell", also known as T lymphocyte, is a subtype of white blood cell that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of T cell receptors on the cell surface. In the present application, the T cells may include memory stem cell-like T cells (TSCM) and central memory T Cells (TCM).
In this application, the term "T cell receptor" is often also referred to as "TCR", and generally refers to the molecular structure of a T cell that specifically recognizes and binds to an antigen peptide-MHC molecule. The T cell receptor may be present on the surface of the T cell in a complex with a CD3 molecule. The TCR may be a heterodimer immobilized on the cell membrane, mostly consisting of highly variable alpha and beta subunits linked by disulfide bonds; the minority consists of gamma and delta peptide chains. The TCR may comprise a variable region and a constant region, wherein the constant region may be adjacent to the cell membrane, linked to a transmembrane region and an intracellular terminal end, and the variable region is responsible for recognition of the polypeptide/MHC complex.
In the present application, the term "low density lipoprotein receptor-related protein" (Low density lipoprotein receptor-related protein, LRP) generally refers to a mosaic protein comprising 839 amino acids (after removal of the 21 amino acid signal peptide). Embedding in the outer phospholipid layer of LDL (Low density lipoprotein) particles is an endocytic receptor that can mediate endocytosis of cholesterol-rich LDL, a member of the low density lipoprotein receptor (Low density lipoprotein receptor, LDLR) gene family. The LRP is most significantly expressed in bronchial epithelial cells and adrenal and cortical tissue. In the present application, the low density lipoprotein receptor-related protein may comprise one or more selected from the group consisting of: low density lipoprotein receptor-related proteins 1-12 or truncations.
In the present application, the terms "low density lipoprotein receptor-related protein 6" (LRP-6) and "low density lipoprotein receptor-related protein 5" (LRP-5) generally refer to unique subgroups of the Low Density Lipoprotein Receptor (LDLR) family. Human LRP-6 has an accession number of O75581 in UniProt. Human LRP-5 has an accession number of O75197 in UniProt.
In the present application, the term "truncate" (Truncated protein) generally refers to a truncated protein. The truncations may be obtained by proteolytic or operative structural gene elimination of the N-or C-terminal part of the protein. Alternatively, the truncations may be obtained by prematurely terminating translation by nonsense mutations that result in the presence of a stop codon in the structural gene.
In this application, the term "and/or" is understood to mean either one of the selectable items or both of the selectable items.
In this application, the term "comprising" is generally intended to include the features specifically recited, but does not exclude other elements.
In this 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 this application, the terms "comprises," "comprising," and "includes" are used in their plain, inclusive, and open-ended meaning. In some cases, the meaning of "as", "consisting of … …" is also indicated.
Detailed Description
Isolated antigen binding proteins as described herein
In one aspect, the present application provides an isolated antigen binding protein that can be used in a ForteBio AHC sensor assay at or below K of 3.1E-09M D Value (e.g. the K D Not higher than about 3.1E-09M, not higher than about 3.0E-09M, not higher than about 2.9E-09M, not higher than about 2.8E-09M, not higher than about 2.7E-09M, not higher than about 2.6E-09M, not higher than about 2.3E-09M, not higher than about 2.0E-09M, not higher than about 1.5E-09M, not higher than about 1E-09M, or not higher than 5E-10M or below) to human AFP 158-166 HLA-A02 x 01 x complex specific binding.
In one aspect, the present application provides an isolated antigen binding protein that may comprise at least one CDR in an antibody heavy chain variable region VH that may comprise the amino acid sequences shown in SEQ ID NO 72 through SEQ ID NO 84.
In the present application, the HCDR of the isolated antigen binding protein may be divided in any form, so long as VH is identical to the amino acid sequences shown in SEQ ID NO:72 to SEQ ID NO:84, and the HCDR divided in any form falls within the scope of the present application.
The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, north's, a combination of Kabat/Chothia et al. These coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In this application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme; variants thereof are also contemplated, including amino acid substitutions, deletions and/or additions to the amino acid sequence of the CDRs. Such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; homologues thereof are also contemplated, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequences of the CDRs.
In the present application, the antigen binding protein may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.
In this application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49 and SEQ ID NO. 54. For example, the HCDR3 sequence of the antigen binding protein may be defined according to the IMGT numbering system.
In this application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO: 56. For example, the HCDR3 sequence of the antigen binding protein may be defined according to the Kabat numbering system.
In this application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48 and SEQ ID NO. 53. For example, the HCDR2 sequence of the antigen binding protein may be defined according to the IMGT numbering system.
In this application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the antigen binding protein may be defined according to the Kabat numbering system.
In this application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47 and SEQ ID NO. 57. For example, the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT numbering system.
In this application, the HCDR1 of the antigen binding protein may comprise an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the antigen binding protein may be defined according to the Kabat numbering system.
For example, the HCDR1 of the antigen binding protein may comprise an amino acid sequence set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47, and SEQ ID NO. 57; the HCDR2 may comprise an amino acid sequence shown in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48, and SEQ ID NO. 53; and the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49, and SEQ ID NO. 54. For example, the HCDR1, HCDR2, HCDR3 sequences of the antigen binding protein may be defined according to the IMGT numbering system.
In the present application, the antigen binding protein comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 19, SEQ ID NO. 25, SEQ ID NO. 30, SEQ ID NO. 35, SEQ ID NO. 46, SEQ ID NO. 52 and SEQ ID NO. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1, HCDR2 and HCDR3 sequences of the antigen binding protein may be defined according to the Kabat numbering system.
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 18; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 19. For example, the antigen binding protein may comprise antibody 1B3 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the antigen binding protein may comprise antibody 1C4 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the antigen binding protein may comprise antibody 1C11 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the antigen binding protein may comprise antibody 1D12 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 34; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 35. For example, the antigen binding protein may comprise antibody 2F9 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the antigen binding protein may comprise antibody number 3 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the antigen binding protein may comprise antibody number 4 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the antigen binding protein may comprise antibody number 7 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the antigen binding protein may comprise antibody number 8 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 44; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 45; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 46. For example, the antigen binding protein may comprise antibody number 15 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 50; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 51; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 52. For example, the antigen binding protein may comprise antibody number 17 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 55; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 56. For example, the antigen binding protein may comprise antibody number 24 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the antigen binding protein may comprise antibody number 29 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the VH of the antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO 58 to SEQ ID NO 65.
In this application, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 66.
In the present application, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO: 69.
In the present application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 70 to SEQ ID NO. 71.
In the present application, said H-FR1 of said antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO. 58 to SEQ ID NO. 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 58; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody 1B3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody 1C4 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody 1C11 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody 1D12 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody 2F9 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 62; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 4 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 7 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 63; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 8 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 15 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 68; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 71. For example, the antigen binding protein may comprise antibody number 17 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 24 or an antigen binding fragment thereof having the same H-FR1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 65; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 69; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the antigen binding protein may comprise antibody number 29 or an antigen binding fragment thereof having the same H-FR1-4.
In the present application, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO 72 to SEQ ID NO 84.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 18; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 19. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 58; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 72. For example, the antigen binding protein may comprise antibody 1B3 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 73. For example, the antigen binding protein may comprise antibody 1C4 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 74. For example, the antigen binding protein may comprise antibody 1C11 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 75. For example, the antigen binding protein may comprise antibody 1D12 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 34; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 35. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 76. For example, the antigen binding protein may comprise antibody 2F9 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 77. For example, the antigen binding protein may comprise antibody number 3 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 62; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 78. For example, the antigen binding protein may comprise antibody number 4 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 79. For example, the antigen binding protein may comprise antibody number 7 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 63; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 80. For example, the antigen binding protein may comprise antibody number 8 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 44; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 45; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 46. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 81. For example, the antigen binding protein may comprise antibody number 15 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 50; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 51; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 52. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 68; the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 71. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 82. For example, the antigen binding protein may comprise antibody number 17 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 55; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 56. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 83. For example, the antigen binding protein may comprise antibody number 24 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 65; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 69; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 84. For example, the antigen binding protein may comprise antibody number 29 or an antigen binding protein having the same heavy chain variable region as it.
In the present application, the isolated antigen binding protein may compete with the reference antibody for binding to human AFP 158-166 HLA-A02 x 01 x complex.
In the present application, the reference antibody may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.
In this application, the HCDR3 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49 and SEQ ID NO. 54. For example, the HCDR3 sequence of the reference antibody may be defined according to the IMGT numbering system.
In this application, the HCDR3 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO: 56. For example, the HCDR3 sequence of the reference antibody may be defined according to the Kabat numbering system.
In this application, the HCDR2 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48 and SEQ ID NO. 53. For example, the HCDR2 sequence of the reference antibody may be defined according to the IMGT numbering system.
In this application, the HCDR2 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the reference antibody may be defined according to the Kabat numbering system.
In this application, the HCDR1 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47 and SEQ ID NO. 57. For example, the HCDR1 sequence of the reference antibody may be defined according to the IMGT numbering system.
In this application, the HCDR1 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the reference antibody may be defined according to the Kabat numbering system.
For example, the HCDR1 of the reference antibody can comprise an amino acid sequence set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47, and SEQ ID NO. 57; the HCDR2 may comprise an amino acid sequence shown in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48, and SEQ ID NO. 53; and the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49, and SEQ ID NO. 54. For example, the HCDR1, HCDR2, HCDR3 sequences of the reference antibody may be defined according to the IMGT numbering system.
In the present application, the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO. 19, SEQ ID NO. 25, SEQ ID NO. 30, SEQ ID NO. 35, SEQ ID NO. 46, SEQ ID NO. 52 and SEQ ID NO. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1, HCDR2 and HCDR3 sequences of the reference antibody may be defined according to the Kabat numbering system.
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 18; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 19. For example, the reference antibody may comprise antibody 1B3 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the reference antibody may comprise antibody 1C4 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the reference antibody may comprise antibody 1C11 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the reference antibody may comprise antibody 1D12 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 34; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 35. For example, the reference antibody may comprise antibody 2F9 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the reference antibody may comprise antibody number 3 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the reference antibody may comprise antibody number 4 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the reference antibody may comprise antibody number 7 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the reference antibody may comprise antibody number 8 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 44; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 45; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 46. For example, the reference antibody may comprise antibody number 15 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO. 50; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 51; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 52. For example, the reference antibody may comprise antibody number 17 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 55; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 56. For example, the reference antibody may comprise antibody number 24 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the reference antibody may comprise antibody number 29 or an antigen-binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
Chimeric antigen receptor and modified cells
In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.
In this application, HCDR3 of the targeting moiety of the chimeric antigen receptor may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49 and SEQ ID NO. 54. For example, the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.
In this application, HCDR3 of the targeting portion of the chimeric antigen receptor may comprise an amino acid sequence set forth in any one of SEQ ID NO. 19, SEQ ID NO. 25, SEQ ID NO. 30, SEQ ID NO. 35, SEQ ID NO. 46, SEQ ID NO. 52, and SEQ ID NO. 56. For example, the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the Kabat numbering system.
In this application, HCDR2 of the targeting portion of the chimeric antigen receptor may comprise an amino acid sequence set forth in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48, and SEQ ID NO. 53. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.
In this application, HCDR2 of the targeting portion of the chimeric antigen receptor may comprise an amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the Kabat numbering system.
In this application, HCDR1 of the targeting moiety of the chimeric antigen receptor may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47 and SEQ ID NO. 57. For example, the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.
In this application, HCDR1 of the targeting moiety of the chimeric antigen receptor can comprise an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the Kabat numbering system.
For example, HCDR1 of the targeting moiety of the chimeric antigen receptor can comprise an amino acid sequence as set forth in any one of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 47, and SEQ ID NO. 57; the HCDR2 may comprise an amino acid sequence shown in any one of SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 48, and SEQ ID NO. 53; and the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 49, and SEQ ID NO. 54. For example, the HCDR1, HCDR2, HCDR3 sequences of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.
In this application, the targeting moiety of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52 and SEQ ID NO: 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50. For example, the HCDR1, HCDR2 and HCDR3 sequences of the targeting portion of the chimeric antigen receptor can be defined according to the Kabat numbering system.
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 18; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 19. For example, the targeting moiety of the chimeric antigen receptor can include antibody 1B3 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the targeting moiety of the chimeric antigen receptor can include antibody 1C4 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the targeting moiety of the chimeric antigen receptor can include antibody 1C11 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the targeting moiety of the chimeric antigen receptor can include antibody 1D12 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 34; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 35. For example, the targeting moiety of the chimeric antigen receptor can include antibody 2F9 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 3 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 4 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 7 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 8 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 44; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 45; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 46. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 15 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 50; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 51; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 52. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 17 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 55; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 56. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 24 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, HCDR1 of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 29 or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 as it).
For example, the VH of the targeting portion of the chimeric antigen receptor can comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO: 65.
In this application, the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 66.
In the present application, the H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO: 69.
In the present application, the H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO. 70 to SEQ ID NO. 71.
In the present application, said H-FR1 of the targeting moiety of said chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO. 58 to SEQ ID NO. 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 58; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1B3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1C4 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1C11 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1D12 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody 2F9 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody number 3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 62; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 4 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 7 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 63; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 8 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 15 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 68; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 71. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 17 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 24 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown as SEQ ID NO. 65; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 69; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 70. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 29 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the heavy chain variable region of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in any one of SEQ ID NO:72 to SEQ ID NO: 84.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 18; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 19. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 58; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 72. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1B3 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown as SEQ ID NO. 73. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1C4 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 74. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1C11 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 75. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 1D12 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 34; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 35. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 76. For example, the targeting moiety of the chimeric antigen receptor can comprise antibody 2F9 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 77. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 3 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 62; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 78. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 4 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 79. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 7 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 63; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 80. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 8 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 44; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 45; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 46. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO. 81. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 15 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 50; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 51; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 52. For example, the H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 64; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 68; the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 71. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence set forth in SEQ ID NO. 82. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 17 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 23; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 55; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 56. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 61; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 67; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown as SEQ ID NO. 83. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 24 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the targeting portion of the chimeric antigen receptor can comprise a heavy chain variable region, which can comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 24; the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 30. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 65; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 66; the H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 69; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the heavy chain variable region of the targeting portion of the chimeric antigen receptor can comprise the amino acid sequence shown as SEQ ID NO. 84. For example, the targeting moiety of the chimeric antigen receptor can include antibody number 29 or a targeting moiety of a chimeric antigen receptor having the same heavy chain variable region as it.
In the present application, the chimeric antigen receptor can comprise a hinge region. For example, the hinge region may comprise a hinge region derived from: igG4, igG1 and CD8. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO. 147. For example, the hinge region 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 147. For example, the nucleotide sequence encoding the hinge region may comprise the nucleotide sequence set forth in SEQ ID NO. 148.
In the present application, the chimeric antigen receptor may comprise a transmembrane domain. For example, the transmembrane domain may include, but is not limited to, a transmembrane domain derived from a protein selected from the group consisting of: CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, fcepsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154 and SLAM. For example, the transmembrane domain may include a transmembrane domain derived from CD 8. For example, the transmembrane domain may comprise the amino acid sequence shown as SEQ ID NO. 149. For example, the transmembrane domain 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 sequence set forth in SEQ ID NO: 149. For example, the nucleotide sequence encoding the transmembrane domain may comprise the nucleotide sequence shown as SEQ ID NO. 150.
For example, the hinge and transmembrane regions may comprise the amino acid sequence shown in SEQ ID NO. 128. For example, the hinge and transmembrane regions 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. 128. For example, the hinge and transmembrane regions may comprise the nucleotide sequence shown as SEQ ID NO. 127.
In the present application, the chimeric antigen receptor can comprise a costimulatory signaling domain. For example, the costimulatory signaling domain may comprise, but is not limited to, a costimulatory signaling domain derived from a protein selected from the group consisting of: CD28, 4-1BB, 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 ligand, CD40 and MyD88. For example, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from 4-1 BB. For example, the costimulatory signaling domain may comprise the amino acid sequence depicted as SEQ ID NO. 132. For example, the costimulatory signaling 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. 132. For example, the coding co-stimulatory signaling domain may comprise the nucleotide sequence of SEQ ID NO. 131.
For example, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from CD 28. For example, the costimulatory signaling domain may comprise the amino acid sequence depicted as SEQ ID NO. 130. For example, the costimulatory signaling 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. 130. For example, the coding co-stimulatory signaling domain may comprise the nucleotide sequence of SEQ ID NO. 129.
In the present application, the chimeric antigen receptor can comprise an intracellular signaling domain. For example, 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 the amino acid sequence shown as SEQ ID NO. 134. For example, 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. 134. For example, the coding intracellular signaling domain may comprise the nucleotide sequence of SEQ ID NO. 133.
For example, the N-terminus of the transmembrane domain may be linked to the C-terminus of the targeting moiety. For example, the C-terminal of the transmembrane domain may be linked to the N-terminal of the costimulatory signaling domain. For example, the C-terminal end of the costimulatory signaling domain may be linked to the N-terminal end of the intracellular signaling domain.
For example, the chimeric antigen receptor may comprise the following domains in order from N-terminus to C-terminus: a targeting moiety, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.
For example, the chimeric antigen receptor may comprise the following domains in order from N-terminus to C-terminus: the antigen binding proteins of the present application (e.g., VHH), transmembrane domains derived from CD8, costimulatory signaling domains derived from CD137, and intracellular signaling domains derived from CD3 zeta.
In this application, the chimeric antigen receptor may comprise the amino acid sequence set forth in any one of SEQ ID NO 112 through SEQ ID NO 124. For example, the chimeric antigen receptor 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 112 through SEQ ID NO 124.
In another aspect, the present application provides a modified cell that may comprise the chimeric antigen receptor described above. In the present application, the modification may include up-regulating the expression level of the low density lipoprotein receptor-related protein or a fragment thereof in the cell.
Low density lipoprotein receptor-related proteins or fragments thereof
In the present application, the low density lipoprotein receptor-related protein may comprise one or more selected from the group consisting of: low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
In the present application, the low density lipoprotein receptor-related protein or fragment thereof may be of mammalian origin. For example, it may be derived from humans, macaques, rats and mice. For example, the low density lipoprotein receptor-related protein or fragment thereof may be of human origin.
In the present application, the functional fragment may comprise a fragment or a truncate of the low density lipoprotein receptor-related protein having the activity of the low density lipoprotein receptor-related protein. For example, the low density lipoprotein receptor-related protein may comprise low density lipoprotein receptor-related protein 6 and truncations thereof, and/or low density lipoprotein receptor-related protein 5 and truncations thereof.
In the present application, the truncations of the low density lipoprotein receptor-related protein 6 may comprise the intracellular region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 may comprise the intracellular region of the low density lipoprotein receptor-related protein 5. In this application, the term "intracellular region" generally refers to a domain of a protein located within a cell membrane. In the present application, the intracellular domain may refer to a domain within the cell membrane of the low density lipoprotein receptor-related protein. In this application, the intracellular region may comprise the sequence from position 24 to position 243 of SEQ ID NO. 140 or an amino acid sequence having at least 80% homology thereto, or alternatively, comprise the sequence from position 24 to position 231 of SEQ ID NO. 144 or an amino acid sequence having at least 80% homology thereto. For another example, the truncations of the low density lipoprotein receptor-related protein 6 may comprise a transmembrane region of the low density lipoprotein receptor-related protein 6 and an LDLR region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 may comprise the transmembrane region of the low density lipoprotein receptor-related protein 5 and the LDLR region of the low density lipoprotein receptor-related protein 5. In the present application, the term "LDLR region" generally refers to the domain of the low density lipoprotein receptor-related protein that is near the N-piece outside the transmembrane region. The domain may have the function of enhancing Wnt signaling. In this application, the LDLR region may include or have at least 80% homology to amino acid sequences from position 5 to 119 of SEQ ID NO. 138, or from position 4 to 119 of SEQ ID NO. 142.
In the present application, the low density lipoprotein receptor-related protein or fragment thereof comprises an amino acid sequence shown in any one of the following or an amino acid sequence having at least 80% homology thereto: 138, 140, 142 and 144.
In the present application, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or fragment thereof comprises a nucleic acid sequence as set forth in any one of the following or a nucleic acid sequence having at least 80% homology thereto: 137, 139, 141 and 143.
In the present application, the modified cells may comprise immune cells (e.g., lymphocytes). In the present application, the modified cells may include modified T cells. In the present application, the modified cells may comprise modified memory stem cell-like T cells (TSCM) and/or genetically modified central memory T Cells (TCM). In this application, the TSCM may be comprised as ccr7+ and/or cd62l+. In this application, the TSCM further comprises a composition having one or more properties selected from the group consisting of: cd45ra+ or cd45ra-, cd45ro+ or cd45ro-, cd27+, cd28+, cd127+, cd122+, cd3+, cd4+ and cd8+.
In the present application, the modified cell may express the Chimeric Antigen Receptor (CAR). For example, the modified cell may comprise a vector capable of expressing the Chimeric Antigen Receptor (CAR). The vector may comprise a nucleotide molecule encoding the Chimeric Antigen Receptor (CAR). For another example, the carrier may be selected from the group consisting of: retroviral vectors, lentiviral vectors and/or transposon plasmids.
In the present application, the vector capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof and the vector capable of expressing the Chimeric Antigen Receptor (CAR) may be the same vector or may be different vectors, as long as the one or more vectors can express the low density lipoprotein receptor-related protein or a fragment thereof and the Chimeric Antigen Receptor (CAR), thereby providing the genetically modified immune cell with both the low density lipoprotein receptor-related protein or a fragment thereof and the Chimeric Antigen Receptor (CAR).
For example, the vector capable of expressing the low density lipoprotein receptor-related protein or fragment thereof and the vector capable of expressing the Chimeric Antigen Receptor (CAR) may be the same vector. In this vector, the nucleotide molecule encoding the low density lipoprotein receptor-related protein or fragment thereof and the nucleotide molecule encoding the Chimeric Antigen Receptor (CAR) may be in the same expression cassette. For example, the nucleotide molecule encoding the low density lipoprotein receptor-related protein or fragment thereof may be located at the 3' end of the nucleotide molecule encoding the Chimeric Antigen Receptor (CAR).
In the present application, the nucleotide molecule encoding the low density lipoprotein receptor-related protein or fragment thereof may be directly or indirectly linked to the nucleotide molecule encoding the Chimeric Antigen Receptor (CAR). For example, the indirect connection may be through a connection sequence. The 5 'end of the linker sequence may be linked to the 3' end of the nucleotide molecule encoding the low density lipoprotein receptor-related protein or fragment thereof, and the 3 'end of the linker sequence may be linked to the 5' end of the nucleotide molecule encoding the Chimeric Antigen Receptor (CAR).
In the present application, the low density lipoprotein receptor-related protein or fragment thereof expressed by the modified immune cell and the Chimeric Antigen Receptor (CAR) may be two independent proteins. That is, the two do not have any interconnecting relationship to form any form of di (poly) mer or protein complex. However, the low density lipoprotein receptor-related protein or fragment thereof expressed by the genetically modified immune cell and the Chimeric Antigen Receptor (CAR) may also be linked to each other. For example, in some cases, the two proteins formed by translation are not cleaved completely, and the low density lipoprotein receptor-related protein or fragment thereof and the chimeric antigen receptor form a complex.
The chimeric antigen receptor can comprise a 2A sequence. The term "2A sequence" generally refers to a self-cleaving amino acid sequence that is independent of protease. The 2A sequence may facilitate transcription to produce two proteins. In this application, the 2A sequence may include the sequence from position 1 to position 54 in SEQ ID NO. 136. For example, the nucleotide sequence encoding the 2A sequence may comprise the nucleotide sequence of SEQ ID NO: 135.
In the present application, the chimeric antigen receptor may comprise a leader sequence. For example, the nucleotide sequence encoding the leader sequence may comprise the nucleotide sequence of SEQ ID NO. 146.
Polypeptides and immunoconjugates
In another aspect, the present application provides one or more polypeptides, which may comprise an isolated antigen binding protein of the present application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide can include a multispecific antibody (e.g., a bispecific antibody).
In another aspect, the present application provides one or more immunoconjugates that may comprise the isolated antigen binding proteins of the present application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, a label, and/or a detection agent.
Nucleic acids, vectors and cells
In another aspect, the present application also provides an isolated one or more nucleic acid molecules that may encode an isolated antigen binding protein or polypeptide described herein. For example, each of the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode a portion thereof (e.g., one or more of HCDR1-3, heavy chain variable regions).
For example, when nucleic acid molecules encode a portion of the antigen binding protein or polypeptide, respectively, the products encoded by the nucleic acid molecules together may form an isolated antigen binding protein of the present application that is functional (e.g., can bind AFP).
The nucleic acid molecules described herein 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 isolated antigen binding proteins can 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 isolated antigen binding proteins or polypeptides described herein.
In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. 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 this application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, the human U6RNA promoter, the 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 comprise additional introns. One or more nucleic acid molecules described herein 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 described herein may be introduced into cells.
In another aspect, the present application provides a cell. The cell may comprise an isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules, and/or one or more vectors described herein. For example, each or each cell may comprise one or more nucleic acid molecules or vectors described herein. For example, each or each cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cell, such as a prokaryotic cell (e.g., a bacterial cell), CHO cell, NS/0 cell, HEK293T cell, 293F cell, or HEK293A cell, or other eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, or the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like. For example, the cells may include yeast cells. For example, the cells may include E.coli cells. For example, the cells may include mammalian cells. For example, the cells may include immune cells.
The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells may include T cells, B cells, natural Killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells. For example, the cells may include T cells.
Pharmaceutical composition
In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise an isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient described herein. In this application, the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surfactants. Any conventional medium or agent is contemplated for use in the pharmaceutical compositions of the present application unless incompatible with the cells described herein. In this application, the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical formulation, which may also be referred to as excipients. For example, the excipients may include binders, fillers, disintegrants, lubricants in the tablet. For example, the excipients may include wine, vinegar, juices, etc. in a traditional Chinese medicine pill. For example, the excipient may comprise a base portion of a semi-solid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
Kit, use and method
In another aspect, the present application provides a method for detecting or assaying AFP, which may comprise using the isolated antigen binding protein or the polypeptide.
In this application, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic purpose.
For example, the method may comprise a method of detecting the presence and/or amount of AFP for non-diagnostic purposes, which may comprise the steps of:
1) Contacting the sample with an antigen binding protein of the present application; and
2) Detecting the presence and/or amount of said antigen binding protein bound by the sample to determine the presence and/or expression level of AFP in a sample obtained from the subject.
In another aspect, the present application provides a kit of AFP, which may comprise the use of said isolated antigen binding protein or said polypeptide.
In the present application, the kit may further comprise instructions describing a method for detecting the presence and/or amount of AFP. For example, the method may comprise an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides the use of said isolated antigen binding protein or said polypeptide in the preparation of a kit useful in a method of detecting the presence and/or amount of AFP. For example, the method may comprise an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides an isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said pharmaceutical composition for use in preventing, alleviating and/or treating a disease or disorder.
In another aspect, in the present application, the kit and/or the pharmaceutical combination is for use in preventing, alleviating and/or treating a disease or condition.
For example, the disease or condition may include a tumor. For example, the tumor may comprise a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells as compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression amount of AFP is up-regulated in the tumor microenvironment or in the tumor cells as compared to normal cells. The tumor associated with protein expression of AFP may be an AFP-positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than in normal cells. For example, the tumor may comprise a solid tumor. For example, the tumor may include liver cancer.
In another aspect, the present application provides the use of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease or disorder.
In another aspect, the present application provides the use of a pharmaceutical combination for the manufacture of a medicament for the prevention, alleviation and/or treatment of a disease or condition.
For example, the disease or condition may include a tumor. For example, the tumor may comprise a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells as compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression amount of AFP is up-regulated in the tumor microenvironment or in the tumor cells as compared to normal cells. The tumor associated with AFP protein expression may be an AFP positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than in normal cells.
For example, the tumor may comprise a solid tumor. For example, the tumor may include liver cancer.
In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof said isolated antigen binding protein, said isolated nucleic acid molecule, said vector, said cell, said pharmaceutical composition.
For example, the disease or condition may include a tumor. For example, the tumor may comprise a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells as compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression amount of AFP is up-regulated in the tumor microenvironment or in the tumor cells as compared to normal cells. The tumor associated with AFP protein expression may be an AFP positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than in normal cells.
For example, the tumor may comprise a solid tumor. For example, the tumor may include liver cancer.
The pharmaceutical compositions and methods described herein may be used in conjunction with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, and the like. The pharmaceutical compositions and methods described herein are useful for other immune response dependent disease conditions, such as inflammation, immune disorders, and infectious diseases.
In this application, the subject may include a human or non-human animal. For example, the non-human animal may be selected from the group consisting of: monkey, chicken, goose, cat, dog, mouse and rat. Furthermore, the non-human animal may also include any animal species other than human, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The person may be caucasian, african, asian, sphaleid, or other race, or a hybrid of various races. As another example, the person may be an elderly, adult, adolescent, child, or infant.
The effective amount in humans can be presumed from the effective amount in experimental animals. For example, freireich et al describe the relationship of animal and human dose (milligrams per square meter of body surface) (Freireich et al, cancer chemther. Rep.50, 219 (1966)). Body surface area may be approximately determined from the height and weight of the patient. See, e.g., scientific Tables, geigy Pharmaceuticals, ardsley, n.y.,537 (1970).
The present application also provides the following embodiments:
1. an isolated antigen binding protein having one or more of the following properties:
1) Specifically binds to the human AFP158-166/HLA-A02 x 01 x complex with a KD of about 3.1E-09M or less;
2) Can bind to the mouse AFP158// HLA-A02 x 01 x complex.
2. The isolated antigen binding protein of embodiment 1, comprising HCDR3, said HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO: 56.
3. The isolated antigen binding protein of any of embodiments 1-2, comprising HCDR2, the HCDR2 comprising an amino acid sequence of any one of SEQ ID No. 18, SEQ ID No. 24, SEQ ID No. 34, SEQ ID No. 45, SEQ ID No. 51 and SEQ ID No. 55.
4. The isolated antigen binding protein of any of embodiments 1-3, comprising HCDR1, the HCDR1 comprising an amino acid sequence of any one of SEQ ID No. 17, SEQ ID No. 23, SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 44 and SEQ ID No. 50.
5. The isolated antigen binding protein of any of embodiments 1-4, comprising HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of any one of SEQ ID NOs 72 to 84.
6. The isolated antigen binding protein of any one of embodiments 1-5, comprising HCDR1, HCDR2 and HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 30, SEQ ID No. 35, SEQ ID No. 46, SEQ ID No. 52 and SEQ ID No. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
7. The isolated antigen binding protein of embodiment 6, 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. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
8. The isolated antigen binding protein of any one of embodiments 1-7, comprising H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of any one of SEQ ID NOs 58 to 65.
9. The isolated antigen binding protein of any of embodiments 1-8, comprising H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence of SEQ ID No. 66.
10. The isolated antigen binding protein of any one of embodiments 1-9, comprising H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising the amino acid sequence of any one of SEQ ID NOs 67 to 69.
11. The isolated antigen binding protein of any one of embodiments 1-10, comprising H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 70 to 71.
12. The isolated antigen binding protein of any one of embodiments 1-11, comprising H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 58 to 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
13. The isolated antigen binding protein of embodiment 12, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise any one of the group of amino acid sequences selected from the group consisting of seq id nos:
a) H-FR1: SEQ ID NO. 58, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
b) H-FR1: SEQ ID NO 59, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
c) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
d) H-FR1: SEQ ID NO. 61, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
e) H-FR1: SEQ ID NO. 62, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
f) H-FR1: SEQ ID NO. 63, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
g) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
h) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 68 and H-FR4: SEQ ID NO. 71; and
i) H-FR1: SEQ ID NO. 65, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO 69 and H-FR4: SEQ ID NO. 70.
14. The isolated antigen binding protein of any one of embodiments 1-13, comprising a heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs 72 to 84.
15. The isolated antigen binding protein of any of embodiments 1-14, comprising an antibody or antigen binding fragment thereof.
16. The isolated antigen binding protein of embodiment 15, wherein said antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and dAb.
17. The isolated antigen binding protein of any one of embodiments 1-16, comprising a VHH, or antigen binding fragment thereof.
18. The isolated antigen binding protein of any one of embodiments 15-17, wherein the antibody is selected from the group consisting of: monoclonal antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, and fully human antibodies.
19. The isolated antigen binding protein of any of embodiments 1-18, comprising an amino acid sequence set forth in any one of SEQ ID NOs 72 to 84.
20. A chimeric antigen receptor comprising a targeting moiety comprising HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 30, SEQ ID No. 35, SEQ ID No. 46, SEQ ID No. 52 and SEQ ID No. 56.
21. The chimeric antigen receptor according to embodiment 20, wherein the targeting moiety comprises HCDR2, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO: 55.
22. The chimeric antigen receptor according to any one of embodiments 20-21, wherein the targeting moiety comprises HCDR1, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
23. The chimeric antigen receptor according to any one of embodiments 20-22, wherein the targeting moiety comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of any one of SEQ ID NOs 72 to 84.
24. The chimeric antigen receptor according to any one of embodiments 20-23, wherein the targeting moiety comprises HCDR1, HCDR2, HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 30, SEQ ID No. 35, SEQ ID No. 46, SEQ ID No. 52 and SEQ ID No. 56; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 45, SEQ ID NO. 51 and SEQ ID NO. 55; and the HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO: 50.
25. The chimeric antigen receptor according to embodiment 24, 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. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
26. The chimeric antigen receptor according to any one of embodiments 20-25, wherein the targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of any one of SEQ ID NOs 58 to 65.
27. The chimeric antigen receptor according to any one of embodiments 20-26, wherein the targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO: 66.
28. The chimeric antigen receptor according to any one of embodiments 20-27, wherein the targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of any one of SEQ ID NOs 67 to 69.
29. The chimeric antigen receptor according to any one of embodiments 20-28, wherein the targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 70 to 71.
30. The chimeric antigen receptor according to any one of embodiments 20-29, wherein the targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 58 to 65; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 66; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 67 to SEQ ID NO 69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO: 71.
31. The chimeric antigen receptor according to embodiment 30, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise any one of the group of amino acid sequences selected from the group consisting of:
a) H-FR1: SEQ ID NO. 58, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
b) H-FR1: SEQ ID NO 59, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
c) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
d) H-FR1: SEQ ID NO. 61, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
e) H-FR1: SEQ ID NO. 62, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
f) H-FR1: SEQ ID NO. 63, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
g) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 67 and H-FR4: SEQ ID NO. 70;
h) H-FR1: SEQ ID NO. 64, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO. 68 and H-FR4: SEQ ID NO. 71; and
i) H-FR1: SEQ ID NO. 65, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO 69 and H-FR4: SEQ ID NO. 70.
32. The chimeric antigen receptor according to any of embodiments 20-31, wherein the targeting moiety comprises an antibody or antigen binding fragment.
33. The chimeric antigen receptor of embodiment 32, wherein the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, VHH and/or dAb.
34. The chimeric antigen receptor of any one of embodiments 20-33, wherein the targeting moiety comprises a VHH.
35. The chimeric antigen receptor of embodiment 34, wherein the VHH targets human AFP158-166/HLA-A02 x 01 x complex.
36. The chimeric antigen receptor according to any of embodiments 20-35, wherein the targeting moiety comprises an amino acid sequence set forth in any of SEQ ID NOs 72 to 84.
37. The chimeric antigen receptor according to any of embodiments 20-36, comprising a hinge region.
38. The chimeric antigen receptor of embodiment 37, wherein the hinge region comprises a hinge region derived from: igG4, igG1 and CD8.
39. The chimeric antigen receptor according to any one of embodiments 37-38, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO 147.
40. The chimeric antigen receptor according to any of embodiments 20-39, comprising a transmembrane domain.
41. The chimeric antigen receptor according to embodiment 40, wherein the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of: CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, fcepsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154 and SLAM.
42. The chimeric antigen receptor according to any one of embodiments 40-41, wherein the transmembrane domain comprises a transmembrane domain derived from CD 8.
43. The chimeric antigen receptor according to any of embodiments 40-42, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 149.
44. The chimeric antigen receptor according to any of embodiments 40-43, wherein the N-terminus of the transmembrane domain is linked to the C-terminus of the hinge region.
45. The chimeric antigen receptor according to any of embodiments 20-44, comprising a costimulatory signaling domain.
46. The chimeric antigen receptor of embodiment 45, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from a protein selected from the group consisting of: CD28, 4-1BB, 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 ligand, CD40 and MyD88.
47. The chimeric antigen receptor according to any of embodiments 45-46, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from 4-1 BB.
48. The chimeric antigen receptor according to any of embodiments 45-47, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from CD 28.
49. The chimeric antigen receptor according to any of embodiments 45-48, wherein the costimulatory signaling domain comprises the amino acid sequence depicted in SEQ ID NO. 132 or SEQ ID NO. 130.
50. The chimeric antigen receptor according to any of embodiments 45-49, wherein the N-terminus of the costimulatory signaling domain is linked to the C-terminus of the transmembrane domain.
51. The chimeric antigen receptor according to any of embodiments 20-50, comprising an intracellular signaling domain.
52. The chimeric antigen receptor of embodiment 51, 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 PBj14Nef, kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
53. The chimeric antigen receptor according to any of embodiments 51-52, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from cd3ζ.
54. The chimeric antigen receptor according to any of embodiments 51-53, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO 134.
55. The chimeric antigen receptor according to any of embodiments 51-54, wherein the N-terminus of the intracellular signaling domain is linked to the C-terminus of the costimulatory signaling domain.
56. The chimeric antigen receptor according to any one of embodiments 20-55, comprising an amino acid sequence set forth in any one of SEQ ID NO 112 through SEQ ID NO 124.
57. A polypeptide comprising the isolated antigen binding protein of any one of embodiments 1-19 and/or the chimeric antigen receptor of any one of embodiments 20-56.
58. An immunoconjugate comprising the isolated antigen binding protein of any one of embodiments 1-19.
59. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, or the polypeptide of embodiment 57.
60. A vector comprising the isolated nucleic acid molecule of embodiment 59.
61. A modified cell comprising the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, the polypeptide of embodiment 57, the immunoconjugate of embodiment 58, the isolated nucleic acid molecule of embodiment 59, and/or the vector of embodiment 60.
62. The modified cell of embodiment 61, wherein the modification comprises upregulating the expression of a low density lipoprotein receptor-related protein or fragment thereof in the cell.
63. The modified cell of embodiment 62, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises one or more of the following selected from the group consisting of: low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
64. The modified cell of any one of embodiments 62-63, wherein the low density lipoprotein receptor-related protein or fragment thereof is of human origin.
65. The modified cell of any one of embodiments 63-64, wherein the functional fragment comprises a fragment or a truncate of the low density lipoprotein receptor-related protein having the activity of the low density lipoprotein receptor-related protein.
66. The modified cell of any one of embodiments 62-65, wherein the low density lipoprotein receptor-related protein comprises low density lipoprotein receptor-related protein 6 and truncations thereof, and/or low density lipoprotein receptor-related protein 5 and truncations thereof.
67. The modified cell of any one of embodiments 62-66, wherein the truncations of the low density lipoprotein receptor-related protein 6 comprise the intracellular region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 comprise the intracellular region of the low density lipoprotein receptor-related protein 5.
68. The modified cell of any one of embodiments 62-67, wherein the truncations of the low density lipoprotein receptor-related protein 6 comprise the transmembrane region of the low density lipoprotein receptor-related protein 6 and the LDLR region of the low density lipoprotein receptor-related protein 6; and/or, the truncations of the low density lipoprotein receptor-related protein 5 comprise the transmembrane region of the low density lipoprotein receptor-related protein 5 and the LDLR region of the low density lipoprotein receptor-related protein 5.
69. The modified cell of any one of embodiments 62-68, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence of any one of SEQ ID No. 138, SEQ ID No. 140, SEQ ID No. 142 and SEQ ID No. 144.
70. The modified cell of any one of embodiments 61-69, wherein the modification comprises introducing into the modified cell a vector that up-regulates the expression level of the low density lipoprotein receptor-related protein or fragment thereof.
71. The modified cell of any one of embodiments 61-70, comprising an immune cell.
72. The modified cell of embodiment 71, wherein the immune cell is 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.
73. The modified cell of any one of embodiments 61-72, wherein the cell comprises a T cell.
74. A method of making the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, and/or the polypeptide of embodiment 57, comprising culturing the modified cell of any one of embodiments 61-73 under conditions such that the isolated antigen binding protein and/or the polypeptide are expressed.
75. A pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, the polypeptide of embodiment 57, the immunoconjugate of embodiment 58, the isolated nucleic acid molecule of embodiment 59, the vector of embodiment 60, the modified cell of any one of embodiments 61-73, and/or a pharmaceutically acceptable adjuvant and/or excipient.
76. A method for detecting AFP protein, comprising:
administering the isolated antigen binding protein of any of embodiments 1-19, the polypeptide of embodiment 57 or the immunoconjugate of embodiment 58.
77. A kit for the detection of AFP protein comprising the isolated antigen binding protein of any one of embodiments 1-19, the polypeptide of embodiment 57, or the immunoconjugate of embodiment 58.
78. Use of the isolated antigen binding protein of any one of embodiments 1-19, the polypeptide of embodiment 57 or the immunoconjugate of embodiment 58 in the preparation of a kit for detecting the presence and/or amount of an AFP protein.
79. Use of the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, the polypeptide of embodiment 57, the immunoconjugate of embodiment 58, the isolated nucleic acid molecule of embodiment 59, the vector of embodiment 60, and/or the modified cell of any one of embodiments 61-73, in the manufacture of a medicament for the prevention and/or treatment of a tumor.
80. The use of embodiment 79, wherein the tumor comprises a solid tumor.
81. The use of any one of embodiments 79-80, wherein the tumor comprises a non-solid tumor.
82. The use of any one of embodiments 79-81, wherein the tumor comprises a tumor associated with the expression of AFP.
83. The use according to any one of embodiments 79-82, wherein the tumor comprises liver cancer.
84. The isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, the polypeptide of embodiment 57, the immunoconjugate of embodiment 58, the isolated nucleic acid molecule of embodiment 59, the vector of embodiment 60, and/or the modified cell of any one of embodiments 61-73 for use in the prevention and/or treatment of a tumor.
85. The use of embodiment 84, wherein the tumor comprises a solid tumor.
86. The use of any one of embodiments 84-85, wherein the tumor comprises a non-solid tumor.
87. The use of any one of embodiments 84-86, wherein the tumor comprises a tumor associated with expression of AFP.
88. The use according to any one of embodiments 84-87, wherein the tumor comprises liver cancer.
89. A method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein of any one of embodiments 1-19, the chimeric antigen receptor of any one of embodiments 20-56, the polypeptide of embodiment 57, the immunoconjugate of embodiment 58, the isolated nucleic acid molecule of embodiment 59, the vector of embodiment 60, and/or the modified cell of any one of embodiments 61-73.
90. The method of embodiment 89, wherein the tumor comprises a solid tumor.
91. The method of any one of embodiments 89-90, wherein the tumor comprises a non-solid tumor.
92. The method of any one of embodiments 89-91, wherein the tumor comprises a tumor associated with expression of AFP.
93. The method of any one of embodiments 89-92, wherein the tumor comprises liver cancer.
Without intending to be limited by any theory, the following examples are presented merely to illustrate the antigen binding proteins, chimeric antigen receptors, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the invention of the present application.
Examples
EXAMPLE 1 construction of an artificially synthesized nanobody (VHH antibody) library and screening for anti-AFP/HLA 02 TCR-like antibodies
1.1 construction of a library of artificially synthesized nanobodies (VHH antibodies)
Determining mutation strategy of CDRs of artificially synthesized nano antibody library by comparing 296 nano antibody (VHH antibody) nucleotide sequences without repetitive sequences in PDB (Protein Data Bank) database and protein structure, wherein the length of CDR3 is three: 14. 17 and 21 amino acids. Selecting nanometer antibody Capacizumab (publication No. NL300966I 2) on the market as skeleton, Synthesizing the complete nucleotide sequence of the nano antibody Capacizumab. After cloning the artificially synthesized Capacizumab complete nucleotide sequence to an HP153 phage vector, extracting single-stranded DNA of the HP153 phage vector, taking the extracted single-stranded DNA as a template, mutating nucleotides of CDRs region of the nano-antibody Capacizumab by using a Kunkel Mutagenesis method, and obtaining a double-stranded DNA library of the mutated nano-antibody. Electrotransformation of double-stranded DNA into M13KO7 pre-infected competent E.coli SS320, overnight culture and phage supernatant collection, the final construction of diversity of 1.44X10 10 The artificial synthesis of nanobody library nanoori_1.0 (original biosciences (Shanghai) Co., ltd.) was used as a seed library for antibody sequence screening.
1.2 liquid phase panning of Artificial synthetic nanobody libraries
TG1 e.coli competent cells were plated on antibiotic-free bacterial culture plates, and 15ml of antibiotic-free 2 xyt medium was inoculated with TG1 single colonies the next day and cultured overnight at 37 ℃. 200 μl of the TG1 strain solution cultured overnight was inoculated into a new antibiotic-free 2 XYT medium, and the TG1 strain solution was cultured until the OD600 = 0.8 at 37℃and a rotation speed of 250rpm for use. Taking 900 μl of magnetic beads with streptavidin, adding into 3 EP tubes of 1.5ml on average, placing 300 μl of each tube on a magnetic rack, and washing with PBS for 3 times; wherein 30 μg of biotin-labeled human TERT540 polypeptide is added to two EP tubes respectively, which are marked as 540-1 and 540-2, 30 μg of biotin-labeled human AFP polypeptide is added to a third tube, which is marked as hAFP,3 EP tubes of 1.5ml are incubated for 1h at room temperature, placed on a magnetic rack, and washed 3 times with PBS; adding 700 μl phage library into 540-1 tube, incubating at room temperature for 1 hr, placing on magnetic rack, and collecting supernatant; the supernatant was added to the tubes of hAFP, incubated for 1h at room temperature, placed on a magnetic rack, and washed 10 times with PBST (Tween 0.05%); adding 500 μl of Gly-HCl (pH 2.2) to elute phage, standing at room temperature for 15min, placing on a magnetic rack, collecting supernatant, and adding 250 μl of Tris-HCl (pH 8.0) to the supernatant for neutralization; adding the neutralized supernatant into a tube of 540-2, incubating for 1h at room temperature, placing on a magnetic rack, and taking the supernatant; obtaining a secondary library; adding 20ml of the previously prepared TG1 bacterial liquid (OD 600 = 0.8) into a secondary stock, standing at 37 ℃ for 30min, taking 1ml, and counting; adding 40. Mu.l of helper phage M13K07, standing at 37deg.C for 30min; adding antibiotics ampicillin and kanamycin, adding 20ml of 2 XYT culture medium, and culturing at 37deg.C and 220rpm overnight; centrifuging the overnight grown bacterial liquid at 8000rpm, taking the supernatant, adding 1/5 volume of PEG-NaCl sediment of the supernatant, ice-bathing for 1h, centrifuging at 8000rpm for 30min, removing the supernatant, re-suspending the sediment with 1ml of PBS to obtain the first round of panning result, and then performing the second round of panning with the phage. The desired objective can be achieved by adjusting the screening pressure during each subsequent round of panning, for a total of 5 rounds of panning, with the specific conditions shown in table 1. The panning results are shown in table 1, with significant enrichment beginning at the third round and the 5 rounds of panning ELISA results are shown in figure 1. The secondary antibody was selected from M13 phage antibody (HRP) (manufacturer: sino Biological).
TABLE 1 nanobody panning conditions and results
Figure BDA0003846075070000601
Selecting a monoclonal to carry out sequencing identification analysis; the sequence alignment was performed using the software BioEdit, and the statistical results are shown in FIG. 2, wherein 13 repeated VHH antibody 1B3 nucleotide sequences (nucleotide sequence shown as SEQ ID NO: 1), 11 repeated VHH antibody 1C4 nucleotide sequences (nucleotide sequence shown as SEQ ID NO: 2), 5 repeated VHH antibody 1C11 nucleotide sequences (nucleotide sequence shown as SEQ ID NO: 3), 4 repeated VHH antibody 1D12 nucleotide sequences (nucleotide sequence shown as SEQ ID NO: 4) and 4 repeated VHH antibody 2F9 nucleotide sequences (nucleotide sequence shown as SEQ ID NO: 5) were counted in the picked monoclonal antibodies, and 8 non-repeated VHH antibody single nucleotide sequences were counted.
Example 2 expression and characterization of candidate VHH-Fc antibodies
2.1 construction of eukaryotic expression vectors for candidate VHH-Fc antibodies and antibody purification
The nucleotide sequence of the candidate VHH antibody obtained in example 1 (the nucleotide sequence is shown in any one of SEQ ID NO:1 to SEQ ID NO: 13) was designed and subjected to PCR amplification, and the nucleotide sequence was cloned into a pcDNA3.4 vector with ampicillin resistance, which was digested with the restriction endonucleases SfiI and NotI, by homologous recombination, respectively, and the 3 '-end of the nucleotide sequence of the VHH antibody was ligated with the 5' -end of the Fc portion (SEQ ID NO: 125) of the antibody, thereby constructing a eukaryotic expression vector of the candidate VHH-Fc antibody, and the constructed vector map is shown in FIG. 5. The recombinant plasmid with correct sequence is transfected into an Expi293F cell for transient expression, and purified by a ProteinA column to obtain different candidate VHH-Fc antibodies, and PCR results are shown as figure 3, wherein the PCR results are 100bp marker,1B3 (VHH antibody 1B3 nucleotide), 1C4 (VHH antibody 1C4 nucleotide), 1C11 (VHH antibody 1C11 nucleotide), 1D12 (VHH antibody 1D12 nucleotide) and 2F9 (VHH antibody 2F9 nucleotide) (about 400 bp) bands in sequence from left to right; the electrophoresis diagram of the purified protein is shown in FIG. 4, and the Marker, the reductive 1B3 (VHH-Fc antibody containing the nucleotide sequence of the VHH antibody 1B 3) and the non-reductive 1B3 are sequentially arranged from left to right, and the molecular weight of the purified 1B3 is correct (about 41kDa after reduction).
2.2 identification of affinity of candidate VHH-Fc antibodies
Binding to 100nM candidate VHH-Fc antibody using a ForteBio AHC sensor, binding to human AFP158-166/HLA-A02 x 01 or human TERT540/HLA-A02 x 01 complex, 150nM start, 2-fold dilution, 7 concentration gradients, binding for 5 min, dissociation for 10 min, and Analysis of affinity Data using Data Analysis 9.0, data results are shown in Table 2, affinity results are shown in FIG. 6, wherein number 17 (VHH-Fc antibody comprising the nucleotide sequence of VHH antibody number 17, antibody nucleotide sequence shown in SEQ ID NO: 11), no. 24 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 24, the antibody nucleotide sequence of which is shown as SEQ ID No. 12) is substantially not dissociated, no. 3 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 3, the antibody nucleotide sequence of which is shown as SEQ ID No. 6) has affinity higher than that of the positive control antibody Ab61 of the prior art (VHH-Fc antibody containing the nucleotide sequence of SEQ ID No. 86, the nucleotide sequence of which is shown as SEQ ID No. 85, the amino acid sequence of which is shown as SEQ ID No. 88, the nucleotide sequence of which is shown as SEQ ID No. 87), no. 4 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 4, the antibody nucleotide sequence of which is shown as SEQ ID No. 7), no. 29 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 29, the antibody nucleotide sequence of which is shown as SEQ ID No. 13), no. 1B3 (VHH-Fc antibody containing the nucleotide sequence of which is shown as SEQ ID No. 1B 3) and No. 1C (VHH antibody containing the nucleotide sequence of which is shown as SEQ ID No. 1) and No. 4 VHH antibody (VHH-Fc antibody containing the nucleotide sequence of which is shown as SEQ ID No. 4), the nucleotide sequence of the antibody is shown in SEQ ID NO: 2) is also superior to the euruike positive control antibody Ab61.
TABLE 2 nanobody affinity assay results
Figure BDA0003846075070000611
Figure BDA0003846075070000621
2.3 identification of non-specific binding Activity of candidate VHH-Fc antibodies to human different endogenous polypeptides
The binding activity of the antibodies was detected using an iQue Screener flow cytometer, specifically as follows, T2 cells were HLA-A02 x 01 positive cells, each carrying human AFP 158 Polypeptide (amino acid sequence shown in SEQ ID NO:90, the nucleic acid sequence is shown as SEQ ID NO: 89), human NY-Eso-1 157 polypeptide (amino acid sequence as set forth in SEQ ID NO: 91) and IFI30 (amino acid sequence shown in any one of SEQ ID NO: 92-95), BTG2 (amino acid sequence shown in SEQ ID NO: 96), BCR (amino acid sequence shown in any one of SEQ ID NO: 97), SSR1 (amino acid sequence shown in any one of SEQ ID NO: 107), PPP2R1B (amino acid sequence shown in SEQ ID NO: 100), DDX5 (amino acid sequence shown in SEQ ID NO: 101), CTSG (amino acid sequence shown in SEQ ID NO: 102), CD247 (amino acid sequence shown in SEQ ID NO: 103), DMTN (amino acid sequence shown in SEQ ID NO: 104), CALR (amino acid sequence shown in SEQ ID NO: 105), PIM1 (amino acid sequence shown in SEQ ID NO: 106), HLA-E (amino acid sequence shown in SEQ ID NO: 107), RPS6KB1 (amino acid sequence shown in SEQ ID NO: 108), CSF2RA (amino acid sequence shown in SEQ ID NO: 109), IL7 (amino acid sequence shown in SEQ ID NO: 101), TERT540, human polypeptide (human polypeptide shown in SEQ ID NO: 110) and human cell type cell type human cell polypeptide human cell type polypeptide human cell polypeptide human, nucleoprotein p68 and analogs thereof. Human AFP158/T2 is positive binding cell, human TERT540/T2, human NY-Eso-1 157/T2 and other 19 human endogenous mixed polypeptides/T2 are non-specific control cells, and buffer solution is used for preparing the concentration of 1X 10 6 Each cell/ml of cells was added to a 96 Kong Jian bottom plate at 30. Mu.l per well to 3X 10 per well 4 Is a cell number of (a); the detection antibody was prepared using a buffer, starting at 10 μg/ml, 3-fold dilution of the antibody, setting 7 concentration gradients, and detecting with a flow instrument to further identify candidate VHH antibody specific binding activity. As a result, as shown in FIG. 7A, 1D12 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1D 12), 2F9 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 2F 9), 1B3 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1B 3) had no nonspecific binding activity to human TERT540/T2 cells, whereas 1C11 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1C 11), 1C4 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1C 4) and Uarelix positive control antibody Ab61 had nonspecific binding activity to human TERT540/T2 cells at a high concentration of 10. Mu.g/ml. As shown in FIG. 7B, 1C11, 1C4, 2F9, 1D12, 1B3 were all able to bind to human AFP158/T2 cells, and all 5 candidate VHH-Fc antibodies did not have nonspecific binding activity to human NY-Eso-1 157/T2 control polypeptide and human endogenous mixed polypeptide/T2.
2.4 identification of Cross-reactivity of candidate VHH-Fc antibodies with mouse AFP158 peptide (amino acid sequence shown as SEQ ID NO: 126)
Further identification of cross-reactivity against the mouse AFP158 peptide/HLA-A 02:01 complex on the surface of living cells. The mouse AFP158 peptide differs from the human AFP158 peptide at two amino acid positions 4 and 9. Antibodies cross-reactive with both human and mouse AFP158 peptide/MHC complex are suitable for assessing antibody drug toxicity in HLA-A02 x 01 x transgenic mice. As a result, as shown in FIG. 7C, all of the 5 candidate VHH-Fc antibodies (VHH-Fc antibodies containing the nucleotide sequences of VHH antibodies 1C11, 1C4, 2F9, 1B3, 1D 12) bound to mouse AFP158/T2 cells, while the Youruike positive control antibody Ab61 was a weaker binding activity.
Example 3 construction of CAR core plasmid containing candidate VHH antibody nucleotide sequence, lentiviral packaging and CAR-T cell preparation
3.1 construction of CAR core plasmids containing VHH antibody nucleotide sequences
Double digestion is carried out on the core plasmid by using restriction endonucleases SphI and NotI, the core plasmid is respectively mixed with candidate VHH antibody nucleotide sequences according to the proportion of 1:3 (molar proportion) for homologous recombination, sequencing is carried out to verify the homologous recombination core plasmid nucleotide sequence, amplified plasmids are preserved for standby, the core plasmid map is shown in figure 8, the structure of the CAR core plasmid nucleotide sequence is shown in figure 9, and 41BB and CD3 zeta are selected as co-stimulatory domains.
3.2 lentiviral packaging and titre determination
The vector system used to construct the lentiviral plasmids of the present invention belongs to the third generation lentiviral vector system, which has three plasmids in total, namely 1: packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein; 2: a PMD2.G plasmid encoding envelope protein VSV-G; 3:3.1 construction of a CAR core plasmid containing the nucleotide sequence encoding the VHH antibody. In the core plasmid based on the BBz platform plasmid, the gene encoding the CAR was expressed regulated by the elongation factor-1α (EF-1α) promoter. The packaging process of lentiviruses is as follows: 1X 10 6 Is suspended in 2ml of DMEM medium containing 10% FBS, spread on a single well of a 6-well plate, and cultured overnight; 144ul of medium (50 times the plasmid mass) was aspirated, 144ul of Opti-MEM medium was mixed in, containing 2.88. Mu.g of packaging plasmid (psPAX 2: PMD2.G: core plasmid=3:2:4) and 8.64. Mu.l FuGENEHD transfection reagent, gently mixed and then incubated in CO 2 Culturing for 12 hours at 37 ℃ in an incubator; removing the culture medium containing the plasmid, washing once with PBS, replacing the culture medium with 2ml of DMEM culture medium containing 5% FBS, and culturing for 48 hours; 2.5ml of virus supernatant was collected, centrifuged at 3000rpm for 5 minutes, and the supernatant was frozen at-80℃until use, and its titer was measured.
3.3 preparation of VHH antibody-containing CAR-T cells
The production method of CAR-T cells containing VHH antibodies is as follows: obtaining human peripheral blood mononuclear cells by a density gradient centrifugation method; resuspension of peripheral blood mononuclear cells with medium containing 200U/ml interleukin 2 to a cell density of 2X 10 6 Adding CD3/CD28 magnetic beads according to the ratio of 1:3 (cells: magnetic beads) to activate T cells; activated peripheral blood mononuclear cells were allowed to stand at 37℃CO 2 Culturing in an incubator for 24 hours; adding the obtained lentivirus supernatant according to the ratio of the virus infection complex (MOI) of 3, adding Polybrene to the final concentration of 5 mug/ml, placing the cell suspension in a pore plate, and centrifuging for 1 hour by using a horizontal centrifuge at 1200 rpm; the well plate is put back into a CO2 incubator at 37 ℃ for culturing for 24 hours; centrifugation at 300g for 5 min, removal of supernatant, and resuspension of cells to a cell density of 0.6X10 with fresh X-VIVO medium (manufacturer: LONZA) containing 500U/ml interleukin 2 6 /ml, placed at 37℃CO 2 Culturing in an incubator; cells were counted every 2 days and fresh X-VIVO medium (manufacturer: LONZA) containing 500U/ml interleukin 2 was supplemented to adjust the cell density back to 0.6X10 6 /ml; cell positive rate of CAR-T cells cultured for 9-14 days: GFP is carried by lentivirus used for infecting cells, and GFP positive rate is detected by a flow cytometry after the lentivirus infects cells so as to obtain CAR expression positive rate and positive rate >20% of the cells can be used for tumor killing experiments.
EXAMPLE 4 construction and detection of overexpressing cell lines HepG2-MiniG cells and SK-HEP-1-MiniG cells
Human AFP is packaged by slow virus and infected by slow virus 158-166 The genes (SEQ ID NO: 89) were introduced into HepG2-MiniG and SK-HEP-1 cells, respectively, to prepare HepG2-MiniG and SK-HEP-1-MiniG cells. Taking SK-HEP-1-MiniG cell construction as an example, pLV-C-GFPS is selected as a vector, a human AFP158-166 nucleotide sequence is inserted, a recombinant plasmid pLV-C-GFPS-AFP-short (SEQ ID NO:) is constructed, the PCR and digestion results are shown in FIG. 10, and the amplification PCR results of the human AFP158-166 nucleotide sequence are as follows (A): from left to right, the sequence is 100bp Marker, human AFP158-166 band (about 150 bp); the vector enzyme digestion results are as follows (B): marker IV is adopted in sequence, and the carrier is cut into strips (about 7500 bp); after lentiviral infection, the recombinant plasmid pLV-C-GFPS-AFP-short is transferred into SK-HEP-1 cells, and monoclonal SK-HEP-1-MiniG cells are selected for flow binding activity detection, and the specific result is shown in FIG. 11; the average fluorescence intensity of the monoclonal SK-HEP-1-MiniG-7 (SK-HEP-1-MiniG cell monoclonal No. 7) is highest, and the amplified culture is used as a target cell, and the construction method of the HepG2-MiniG cell is the same as that of the SK-HEP-1-MiniG cell. Monoclonal cell expansion culture for flow detection of HepG2-MiniG cells and SK-HEP-1-MiniG cells The positive rate after that is shown in FIG. 12, and the positive rate of the HepG2-MiniG cells reaches 87.5%, and the positive rate of the SK-HEP-1-MiniG cells reaches 93.9%.
Example 5 in vitro killing of CAR-T cells and factor detection with candidate VHH antibodies
5.1 CAR-T cell in vitro repetitive stimulation experiments containing candidate VHH antibodies
CAR-T cells were prepared according to example 3, positive rates were detected on expansion to days 9-12, and CAR-T cells were resuspended to 4X 10 with serum-free X-VIVO medium (manufacturer: LONZA) 5 Density/ml as effector cells; hepG2-MiniG cells prepared in example 4 were resuspended to 4X 10 with serum-free X-VIVO medium (manufacturer: LONZA) 5 Density/ml as target cells; after mixing the effective target ratio of 1:1, the mixture is mixed with 5 percent CO at 37 DEG C 2 The culture was allowed to stand in an incubator, and the color of the medium was observed every 2 days, and 1-fold volume of the medium was supplemented when the medium turned from orange to yellow. Cell counts were performed on days 4 to 5, and fold expansion was counted. Counting and taking 5 multiplied by 10 5 CAR-T cells were again subjected to second, third and fourth rounds of expansion, the method being as described previously, the first round of expansion being multiplied by the second, third and fourth rounds to calculate the total cumulative expansion.
The results of the repeated stimulation are shown in FIG. 13A, and the cumulative expansion ratio of CAR-T cell 1B3 after three rounds of repeated stimulation is equal to that of CAR-T cell Ab61, and the cumulative expansion ratio of CAR-T cell Ab61 after four rounds of repeated stimulation of CAR-T cell No. 3, no. 4, no. 7, no. 8, no. 17 and No. 29 containing the candidate VHH antibody is higher than that of CAR-T cell Ab61.
5.2 in vitro killing experiments of CAR-T cells containing candidate VHH antibodies
The SK-HEP-1-MiniG cells prepared in example 4 were used as target cells, the CAR-T cells containing the candidate VHH antibodies prepared in example 3 were used as effector cells, and the T cells not infected with lentivirus were used as control effector cells. The specific experimental procedure is as follows, the infection efficiency of the packaged CAR core plasmid lentivirus prepared in example 3 is detected, and the infection proportion is adjusted to be consistent with each group by using T cells which are not infected with the lentivirus; according to effector cells: target cells (effective target ratio) =1:1 were added to 200 μ l X-VIVO medium, target cell numberIs 1X 10 4 Holes, as experimental group; wells containing only the same amount of effector cells as the experimental group as the effector cell self-releasing background group; wells containing only the same amount of target cells as the experimental group as the target cell self-releasing background group; the resulting cells were subjected to CO at 37 ℃ 2 Culturing in an incubator for 18 hours; to a portion of the wells containing only target cells, 20. Mu.l of 10 Xlysate was added and reacted for 45 minutes as maximum release of target cells. The resulting cell culture well plates were centrifuged at 300g for 5 minutes, respectively, and 50. Mu.l of the supernatant was collected for detection of the release amount of lactate dehydrogenase LDH, by referring to the instructions of the Cytotox96 non-radioactive cytotoxicity kit (manufacturer: promega). The released LDH was in the culture supernatant and was detected by a coupled enzymatic reaction. The cell killing activity is calculated as:
Killing toxicity% = 100× (experimental group-effector cell self-release-target cell self-release + medium background value)/(target cell maximum release-target cell self-release).
According to a cell killing formula, the killing effect of each CAR-T cell containing the candidate VHH antibody on target cells is analyzed, and the candidate VHH antibody with obvious killing effect is selected to enter the in vivo functional evaluation.
The killing results are shown in fig. 13C, which shows that 5 candidate VHH antibodies have little difference in killing effect on HLA-A02 x 01 +/afp+ positive cells SK-HEP-1-MiniG cells, and that CAR-T cells containing candidate VHH antibody 1C11 have better killing effect on target cells than CAR-T cells containing positive control antibody Ab61, while CAR-T cells containing candidate VHH antibody 2F9 have better killing effect on HLA-A02 x 01 +/afp+ positive cells HepG2 at low expression level than CAR-T cells containing positive control antibody Ab 61. As shown in fig. 13F, the results show that CAR-T cells containing candidate VHH antibodies 3, 4, 7, 8, 15, 17, 24, 29 all had comparable cell killing effects on HLA-A02 x 01 +/afp+ positive cells SK-HEP-1-MiniG as CAR-T cells containing positive control antibody Ab61, whereas in the cell killing results with HepG2-MiniG as target cells, CAR-T cells containing candidate VHH antibodies 3, 4 were significantly better than CAR-T cells containing positive control antibody Ab 61.
5.3 CAR-T cell in vitro cytokine secretion experiments with candidate VHH antibodies
HepG2-MiniG or SK-HEP-1-MiniG cells prepared in example 4 were used as target cells, CAR-T cells containing the candidate VHH antibodies prepared in example 3 were used as effector cells, and T cells not infected with lentivirus were used as control effector cells. The specific experimental procedure is as follows, the infection efficiency of the packaged CAR core plasmid lentivirus prepared in example 3 is detected, and the proportion is adjusted to be consistent with each group by using T cells of uninfected virus; according to effector cells: target cells (effective target ratio) =1:1 were added to 200 μl x-VIVO medium, the number of target cells was 2×10 4 Holes, as experimental group; wells containing only effector cells equivalent to the experimental group as background group; the resulting cells were subjected to CO at 37 ℃ 2 Culturing in an incubator for 18 hours; the obtained cell culture well plates were centrifuged at 300g for 5 minutes, respectively, and 50. Mu.l of the supernatant was collected for detection of IL2 and IFN-gamma expression levels by referring to R&D DuoSet ELISA kit (manufacturer: R)&Dsystem) instruction.
As a result of the detection, as shown in FIG. 13D, IFN-. Gamma.factor secretion was increased in the CAR-T cells containing the candidate VHH antibodies 1B3, 1C4, and 1C11 when the target cells were HepG2-MiniG or SK-HEP-1-MiniG; as shown in fig. 13E, all CAR-T cells containing candidate VHH antibodies had a low background of IL2 cytokines for HepG2-MiniG cells and non-virus infected T cells. As shown in FIG. 13G, IFN-gamma factor release is better for CAR-T cells containing candidate VHH antibodies 3, 4, 7, 8, 15, 17, 1B3 when the target cell is SK-HEP-1-MiniG. As shown in fig. 13H, when the target cells were HepG2-MiniG cells or SK-HEP-1-MiniG cells, the IL2 cytokines of CAR-T cells containing candidate VHH antibodies 15, 17, 1B3 were secreted better, and the IL2 background of CAR-T cells of all candidate VHH antibodies was lower for HepG2-MiniG cells and non-virus infected T cells.
EXAMPLE 6 in vivo efficacy validation of VHH antibody CAR-T cells
CAR-T cells containing candidate VHH antibody 17 and CAR-T cells containing candidate VHH antibody 1B3 were arbitrarily selected for in vivo efficacy evaluation. 6X 10 injections were given to 6 mice, respectively 6 K-HEP-1-MiniG cells, tumor is carried subcutaneously in the back of the right upper limb until the tumor reaches 100mm 3 When NDG mice were divided into groupsGroup 7, each route of administration was intratumoral injection or tail vein: the T cell group (control group I.V), the 2 CAR-T cell group (Ab 61-I.T is intratumoral injection group, ab61-I.V is tail vein injection group) containing positive control antibody Ab61, the 2 CAR-T cell group (1B 3-I.T is intratumoral injection group, 1B3-I.V is tail vein injection group) containing candidate VHH antibody 1B3, the 2 CAR-T cell group (17-I.T is intratumoral injection group, 17-I.V is tail vein injection group) containing candidate VHH antibody 17, 5 each of them is returned 1X 10 respectively 7 Respective CAR-T cells. The respective CAR-T cells were returned once every 2 weeks, the body weight of each group of mice after administration was recorded, the tumor size was changed, and various biological responses were observed.
The tumor volume and tumor inhibition ratio of each group in PG (D52) are shown in Table 3, and the average tumor volume of the T cell group not infected with virus is 973.98 + -136.05 mm 3 The method comprises the steps of carrying out a first treatment on the surface of the Compared to the non-virus infected T cell group (control group i.v), the CAR-T cell intratumoral injection group (Ab 61-I.T) containing positive control antibody Ab61, the CAR-T cell intratumoral injection group (1B 3-I.T) containing candidate VHH antibody 1B3, the CAR-T cell intratumoral injection group (17-I.T) containing candidate VHH antibody 17, the CAR-T cell tail intravenous injection group (Ab 61-i.v) containing positive control antibody Ab61, the CAR-T cell tail intravenous injection group (1B 3-i.v) containing candidate VHH antibody 1B3, the CAR-T cell tail intravenous injection group (17-i.v) containing candidate VHH antibody 17 had average tumor volumes of 2.73±1.71mm, respectively 3 、3.38±2.11mm 3 、9.58±1.12mm 3 、19.72±2.08mm 3 、24.8±1.08mm 3 、38.32±1.33mm 3 The method comprises the steps of carrying out a first treatment on the surface of the There were significant differences compared to the uninfected virus T cell group (control group i.v) (P<0.0001 A) is provided; tumor volume inhibition rates were 109.68%, 109.57%, 108.88%, 107.75%, 107.19%, 105.67%, respectively.
Table 3 statistics of tumor volumes and inhibition rates in PG (D52) mice and animals
Figure BDA0003846075070000671
Animal experimental procedure as shown in fig. 14, and the graph of fig. 15A shows that the tumor of each group of mice starts to continuously regress on day 14 after the respective CAR-T cells are infused back in the first dose, the average tumor volumes of the mice in each experimental group are significantly different (0.0001 < p < 0.001) compared with the control group, the tumor volume inhibition ratio (TGI) is more than 100%, the tumor inhibition effect of the intratumoral infusion back infusion group is obviously better than that of the tail intravenous infusion back infusion group, but no statistical difference exists, and the CAR-T cell group containing the candidate VHH antibodies 1B3, 17 is not equal to that of the positive control group, and no statistical difference exists. FIG. 15B is a graph of animal body weight showing that one mouse in each of Ab61-I.T, 1B3-I.T groups had more than 20% weight loss, and that no abnormalities were found in the anatomical observation of the vital organs after euthanasia. The mice of the rest of the experimental groups were relatively stable in body weight. Experimental results show that the CAR-T cells are safe and effective for an animal model of SK-HEP-1-MiniG cell tumor-bearing.

Claims (10)

1. An isolated antigen binding protein having one or more of the following properties:
1) At a K of about 3.1E-09M or less D Value and human AFP 158-166 HLA-A02 x 01 x complex specific binding;
2) Can be matched with mouse AFP 158 Complex binding is/HLA-A 02.
2. The isolated antigen binding protein of claim 1, comprising HCDR1, HCDR2 and HCDR3, the HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from any one of the group consisting of:
a) HCDR1: SEQ ID NO. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
3. The isolated antigen binding protein of any one of claims 1-2, comprising a heavy chain variable region VH comprising the amino acid sequence set forth in any one of SEQ ID NOs 72 to 84.
4. A chimeric antigen receptor comprising a targeting moiety comprising HCDR1, HCDR2 and HCDR3, the HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from any one of the group consisting of:
a) HCDR1: SEQ ID NO. 17, HCDR2: SEQ ID NO. 18, and HCDR3: SEQ ID NO. 19;
b) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
c) HCDR1: SEQ ID NO. 29, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
d) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 34, and HCDR3: SEQ ID NO. 35;
e) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
f) HCDR1: SEQ ID NO. 39, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 25;
g) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30;
h) HCDR1: SEQ ID NO. 44, HCDR2: SEQ ID NO. 45, and HCDR3: SEQ ID NO. 46;
i) HCDR1: SEQ ID NO. 50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO. 52;
j) HCDR1: SEQ ID NO. 23, HCDR2: SEQ ID NO:55, and HCDR3: SEQ ID NO. 56;
and
k) HCDR1: SEQ ID NO. 37, HCDR2: SEQ ID NO. 24, and HCDR3: SEQ ID NO. 30.
5. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-3.
6. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-3, or the chimeric antigen receptor of claim 4.
7. A vector comprising the isolated nucleic acid molecule of claim 6.
8. A modified cell comprising the isolated antigen binding protein of any one of claims 1-3, the chimeric antigen receptor of claim 4, the immunoconjugate of claim 5, the isolated nucleic acid molecule of claim 6 and/or the vector of claim 7.
9. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4, the immunoconjugate of claim 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the modified cell of claim 8, and/or a pharmaceutically acceptable adjuvant and/or excipient.
10. Use of the isolated antigen binding protein of any one of claims 1-3, the chimeric antigen receptor of claim 4, the immunoconjugate of claim 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, and/or the modified cell of claim 8 in the preparation of a medicament for the prevention and/or treatment of a tumor.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024103251A1 (en) * 2022-11-15 2024-05-23 原启生物科技(上海)有限责任公司 Anti-afp/hla02 tcr-like antibody and use thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024103251A1 (en) * 2022-11-15 2024-05-23 原启生物科技(上海)有限责任公司 Anti-afp/hla02 tcr-like antibody and use thereof

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