CN116715773B

CN116715773B - 5T4 targeting antibody and application thereof

Info

Publication number: CN116715773B
Application number: CN202310178303.1A
Authority: CN
Inventors: 王远; 张雷; 杨勇飞; 颜悦; 李惠琳
Original assignee: Xingyiang Shanghai Biotechnology Co ltd
Current assignee: Xingyiang Shanghai Biotechnology Co ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2024-03-01
Anticipated expiration: 2043-02-28
Also published as: WO2024179528A1; CN116715773A

Abstract

The invention provides an antibody targeting 5T4 and application thereof. The 5T4 antibody of the invention has excellent specificity and binding capacity, and the derived chimeric antigen receptor has excellent killing capacity to tumor cells, low cytotoxicity and low side effect.

Description

5T4 targeting antibody and application thereof

Technical Field

The present invention relates to the field of biotechnology. In particular to an antibody targeting 5T4 and application thereof.

Background

5T4 (also known as trophoblast glycoprotein, TPBG or Wnt activated inhibitor 1, WAIF1) is a human protein encoded by the TPBG gene. The molecular weight of the human TPBG protein is about 72kDa, comprising 420 amino acids, which contain 8 Leucine Rich Repeats (LRRs) and 7 potential N-glycosylation sites.

5T4 is widely expressed in various trophoblast cells during embryonic development. For normal adult tissues, 5T4 is expressed in only a limited number of epithelial cells. However, 5T4 is expressed in many cancer cells, such as uterine cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, oral cancer, prostate cancer, lung cancer or kidney cancer tissues, and there is evidence in colon cancer, stomach cancer or ovarian cancer that the expression level of 5T4 is associated with a low cure rate of cancer. Expression of 5T4 in tumor cells promotes tumor progression by promoting epithelial to mesenchymal transition (EMT), inhibiting canonical Wnt/beta-catenin signaling pathways, and activating non-canonical Wnt pathways. In tumors with high 5T4 expression, high concentrations of CXCL12 can chemotactic movement of tumor cells through CXCR 4; whereas in 5T 4-low expressing tumors, low concentrations of CXCL12 can provide anti-apoptotic effects for CXCR 7. In tissues of non-small cell lung, breast, kidney or pancreatic cancer, 5T4 expression is up to 90% or more. In addition, studies have shown that 5T4 is also widely expressed in acute myeloid lymphomas (AML, acute Myeloid Leukaemia). More importantly, 5T4 was also expressed higher in some tumor stem cells. Therefore, 5T4 is a tumor-associated antigen with great potential, and the targeting of 5T4 by means including antibodies, tumor vaccines, CAR-T, CAR-NK and the like has very wide application prospect. However, the biological properties of the prior art targeting 5T 4-related antibodies, CAR-T, CAR-NK, etc. remain unsatisfactory.

Thus, there is a need in the art to develop new 5T 4-targeting antibodies with excellent properties.

Disclosure of Invention

The invention aims to provide a 5T 4-targeted antibody and application thereof.

In a first aspect of the invention there is provided an antibody or antigen binding fragment thereof targeting 5T4, the antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising complementarity determining region CDRs selected from the group consisting of:

(1) The heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 31,

HCDR2 shown in SEQ ID NO. 32, and

HCDR3 shown in SEQ ID NO. 33;

and the light chain variable region comprises the following complementarity determining region CDRs:

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(2) The heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 22,

HCDR2 shown in SEQ ID NO. 23, and

HCDR3 shown in SEQ ID NO. 24;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(3) The heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 28,

HCDR2 shown in SEQ ID NO. 29, and

HCDR3 shown in SEQ ID NO. 30;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(4) The heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 16,

HCDR2 shown in SEQ ID NO. 17, and

HCDR3 shown in SEQ ID NO. 18;

LCDR1 shown in SEQ ID NO. 19,

LCDR2 as shown in SEQ ID NO. 20, and

LCDR3 as shown in SEQ ID NO. 21; wherein the CDR sequence is based on the numbering scheme of Kabat.

In another preferred embodiment, the heavy and light chain variable regions of the antibody or antigen binding fragment thereof comprise complementarity determining region CDRs selected from the group consisting of:

HCDR1 shown in SEQ ID NO. 40,

HCDR2 shown in SEQ ID NO. 41, and

HCDR3 shown in SEQ ID NO. 33;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 36,

HCDR2 shown in SEQ ID NO 37, and

HCDR3 shown in SEQ ID NO. 24;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 38,

HCDR2 shown in SEQ ID NO 39, and

HCDR3 shown in SEQ ID NO. 30;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 34,

HCDR2 shown in SEQ ID NO. 35, and

HCDR3 shown in SEQ ID NO. 18;

LCDR1 shown in SEQ ID NO. 19,

LCDR2 as shown in SEQ ID NO. 20, and

LCDR3 as shown in SEQ ID NO. 21; wherein the CDR sequence is based on the numbering scheme of Chothia.

In another preferred embodiment, the antibody comprises a heavy chain and a light chain, the heavy chain comprising the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; the light chain includes the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody comprises a monospecific, bispecific, or trispecific antibody.

In another preferred embodiment, the antibody specifically binds 5T4.

In another preferred embodiment, the antibody has an affinity for human 5T4 with a KD value (M) of 1.0E-7 to 2.0E-9.

In another preferred embodiment, the antibody has a heavy chain variable region as shown in SEQ ID NO. 7 and a light chain variable region as shown in SEQ ID NO. 8; or (b)

The antibody has a heavy chain variable region shown as SEQ ID NO. 3 and a light chain variable region shown as SEQ ID NO. 4; or (b)

The antibody has a heavy chain variable region shown as SEQ ID NO. 5 and a light chain variable region shown as SEQ ID NO. 6; or (b)

The antibody has a heavy chain variable region as shown in SEQ ID NO. 1 and a light chain variable region as shown in SEQ ID NO. 2.

In another preferred embodiment, the antibody has a heavy chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity (wherein the CDRs are unchanged or substantially unchanged) to the amino acid sequence set forth in SEQ ID NO. 7, 3, 5, 1.

In another preferred embodiment, the antibody has a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity (wherein the CDRs are unchanged or substantially unchanged) to the amino acid sequence set forth in SEQ ID NO. 8, 4, 6, 2.

In a second aspect of the present invention, there is provided a recombinant protein having:

(i) An antibody or antigen-binding fragment thereof according to the first aspect of the invention;

and (ii) optionally a tag sequence to aid expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to said element (i).

In a third aspect of the invention there is provided a chimeric antigen receptor CAR, the antigen binding domain of which comprises an antibody single chain variable region sequence scFv targeting 5T4, the heavy and light chain variable regions of the scFv comprising complementarity determining region CDRs selected from the group consisting of:

(1) The heavy chain variable region of the scFv comprises the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 31,

HCDR2 shown in SEQ ID NO. 32, and

HCDR3 shown in SEQ ID NO. 33;

and the light chain variable region of the scFv comprises the following complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(2) The heavy chain variable region of the scFv comprises the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 22,

HCDR2 shown in SEQ ID NO. 23, and

HCDR3 shown in SEQ ID NO. 24;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(3) The heavy chain variable region of the scFv comprises the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 28,

HCDR2 shown in SEQ ID NO. 29, and

HCDR3 shown in SEQ ID NO. 30;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

(4) The heavy chain variable region of the scFv comprises the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 16,

HCDR2 shown in SEQ ID NO. 17, and

HCDR3 shown in SEQ ID NO. 18;

LCDR1 shown in SEQ ID NO. 19,

LCDR2 as shown in SEQ ID NO. 20, and

LCDR3 as shown in SEQ ID NO. 21;

wherein the CDR sequence is based on the numbering scheme of Kabat.

In another preferred embodiment, the heavy and light chain variable regions of the scFv comprise complementarity determining region CDRs selected from the group consisting of:

HCDR1 shown in SEQ ID NO. 40,

HCDR2 shown in SEQ ID NO. 41, and

HCDR3 shown in SEQ ID NO. 33;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 36,

HCDR2 shown in SEQ ID NO 37, and

HCDR3 shown in SEQ ID NO. 24;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 38,

HCDR2 shown in SEQ ID NO 39, and

HCDR3 shown in SEQ ID NO. 30;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27; or (b)

HCDR1 shown in SEQ ID NO. 34,

HCDR2 shown in SEQ ID NO. 35, and

HCDR3 shown in SEQ ID NO. 18;

LCDR1 shown in SEQ ID NO. 19,

LCDR2 as shown in SEQ ID NO. 20, and

In another preferred embodiment, the scFv further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.

In another preferred embodiment, the linking peptide is (G4S) 3 or (G4S) 4.

In another preferred embodiment, the scFv is of formula a or formula B below:

V _H -V _L ,(A)；V _L -V _H ,(B)

wherein V is _H Is the antibody heavy chain variable region; v (V) _L Is the antibody light chain variable region; "-" is a connecting peptide or peptide bond;

and V is _H The amino acid sequence of (B) is shown as SEQ ID NO. 7, V _L The amino acid sequence of (2) is shown as SEQ ID NO. 8;

alternatively V _H The amino acid sequence of (2) is shown as SEQ ID NO. 3, V _L The amino acid sequence of (2) is shown as SEQ ID NO. 4;

alternatively V _H The amino acid sequence of (B) is shown as SEQ ID NO. 5, V _L Amino acids of (2)The sequence is shown as SEQ ID NO. 6;

alternatively V _H The amino acid sequence of (2) is shown as SEQ ID NO. 1, V _L The amino acid sequence of (2) is shown in SEQ ID NO.

In another preferred embodiment, the antibody single chain variable region comprises a human, murine, human murine chimeric antibody single chain variable region.

In another preferred embodiment, the scFv is of formula A (V _H -V _L ) As shown.

In another preferred embodiment, the antigen binding domain targets an extracellular region of 5T 4.

In another preferred embodiment, the chimeric antigen receptor has the structure of formula I:

L-scFv-H-TM-C-CD3ζ(I)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l is a none or signal peptide sequence;

scFv is a 5T 4-targeting scFv;

h is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling molecule;

cd3ζ is a cd3ζ cytoplasmic signaling sequence.

In another preferred embodiment, L is a signal peptide selected from the group consisting of: CD8, CD4, CD16, CD56, CD137, CSF2, DAP12, EF1, GM-CSF, IL-8, IL-21, or a combination thereof.

In another preferred embodiment, the L source is a CD8 derived signal peptide.

In another preferred embodiment, the scFv is of formula a or formula B below:

V _H -V _L ,(A)；V _L -V _H ,(B)

wherein V is _H Is the antibody heavy chain variable region; v (V) _L Is the antibody light chain variable region; "-" is a connecting peptide or peptide bond.

In another preferred embodiment, H is a hinge region selected from the group consisting of: CD8, CD28, CD137, fc, or a combination thereof.

In another preferred embodiment, the H is a CD28 derived hinge region.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD27, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM is a CD 28-derived transmembrane region.

In another preferred embodiment, C is a costimulatory signaling molecule of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred embodiment, C is a CD 28-derived costimulatory signaling molecule.

In another preferred embodiment, the amino acid sequence of the CAR is as set forth in any one of SEQ ID NOS.42-45.

In a fourth aspect of the invention there is provided a polynucleotide encoding an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or a chimeric antigen receptor CAR according to the third aspect of the invention.

In another preferred embodiment, the polynucleotide is isolated.

In a fifth aspect of the invention there is provided a vector comprising a polynucleotide according to the fourth aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

In another preferred embodiment, the vector is a retroviral vector.

In a sixth aspect of the invention there is provided a host cell comprising a vector according to the fifth aspect of the invention or a polynucleotide according to the fourth aspect of the invention integrated with an exogenous source in a chromosome.

In another preferred embodiment, the cell is an isolated cell and/or the cell is a genetically engineered cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is an NK cell or a T cell.

In another preferred embodiment, the host cell is an engineered immune cell.

In another preferred embodiment, the engineered immune cells comprise T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells), wherein the NK cell sources include peripheral blood, umbilical cord blood, embryonic Stem Cells (ESCs), induced pluripotent stem cells (ipscs), and the like.

In another preferred embodiment, the host cell is an immune cell expressing or exposed outside the cell membrane to an antibody according to the first aspect of the invention or to a chimeric antigen receptor according to the third aspect of the invention.

In another preferred embodiment, the immune cells include NK cells, T cells.

In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).

In a seventh aspect of the invention, there is provided a method of making a CAR-NK cell or CAR-T cell expressing a chimeric antigen receptor according to the third aspect of the invention, comprising the steps of:

transduction of the polynucleotide according to the fourth aspect of the invention or the vector according to the fifth aspect of the invention into an NK cell or a T cell, thereby obtaining the CAR-NK cell or CAR-T cell.

In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a polynucleotide according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, or a host cell according to the sixth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament or preparation for preventing and/or treating cancer or tumor.

In a ninth aspect of the invention, there is provided an immunoconjugate comprising:

(a) An antibody moiety selected from the group consisting of: an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or a combination thereof; and

(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.

In a tenth aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a polynucleotide according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, a host cell according to the sixth aspect of the invention, a pharmaceutical composition according to the eighth aspect of the invention or an immunoconjugate according to the ninth aspect of the invention,

(a) Preparing a detection reagent or a kit; and/or

(b) Preparing a medicament or a preparation for preventing and/or treating 5T4 related diseases.

In another preferred embodiment, the 5T 4-related disorder is cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or combinations thereof.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

In another preferred embodiment, the tumor is a 5T4 positive tumor; preferably selected from the group consisting of: uterine cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, oral cancer, prostate cancer, lung cancer, kidney cancer.

In another preferred embodiment, the detection is an immunoassay.

In another preferred embodiment, the immunoassay is an ELISA immunoassay, an immunochromatographic assay, an immunocytochemical staining assay or an immunohistochemical staining assay.

In another preferred embodiment, the diagnostic reagent is a test strip or a test plate.

In another preferred embodiment, the reagent comprises a chip, an immune microparticle coated with an antibody.

In an eleventh aspect of the invention, there is provided a method of detecting (including diagnostic or non-diagnostic) 5T4 protein in a sample in vitro, the method comprising the steps of:

(1) Contacting the sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of 5T4 protein in the sample.

In another preferred example, the method is a cell immunochemistry (Immunocytochemistry staning, ICC) assay, or an immunohistochemical (ihc) assay, or a whole cell (whole cell) ELISA assay, a cell lysate ELISA assay.

In a twelfth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) Culturing the host cell of the sixth aspect of the invention under conditions suitable for expression;

(b) Isolating from the culture a recombinant polypeptide which is an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention.

In a thirteenth aspect of the present invention, there is provided a detection plate comprising: a substrate (support) and a test strip comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immunoconjugate according to the ninth aspect of the invention, or a combination thereof.

In a fourteenth aspect of the present invention, there is provided a kit comprising:

(1) A first container comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention; and/or

(2) A second container containing a second antibody against the antibodies of the invention;

alternatively, the kit contains the detection plate according to the first aspect 3 of the present invention.

In a fifteenth aspect of the present invention there is provided a method of treating a disease comprising administering to a subject in need of treatment an amount of an antibody or antigen binding fragment thereof according to the first aspect of the present invention, a recombinant protein according to the second aspect of the present invention, a host cell according to the sixth aspect of the present invention or a pharmaceutical composition according to the eighth aspect of the present invention.

In another preferred embodiment, the disease is cancer or tumor.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the EC50 results of anti-5T 4 antibodies BC-B25A01 (Ab 01), BC-B33E03 (Ab 02), BC-B33E7A (Ab 03), BC-B33CJC (Ab 04) against different cells, with the A1 antibody (Ab 05) being a positive control.

FIG. 2 shows the affinity detection of the 5T4 antibodies BC-B25A01 (25A 01), BC-B33E03 (33E 03), BC-B33E7A (33E 7A), BC-B33CJC (33 CJC) for human (h 5T 4), cynomolgus monkey (fas 5T 4), mouse (m 5T 4) 5T4 antigen, with the 5T4-PF06263507 (A1) antibodies as positive controls.

Fig. 3 shows a schematic diagram of the 5t4CAR structure.

FIG. 4 shows the detection of flow-through cell surface 5T4 expression.

Figure 5 shows the expression of 5T4-CAR before and after flow detection sorting.

FIG. 6 shows the detection of the affinity of 5T4CAR-NK92 binding to target cells.

FIG. 7 shows the detection of the killing capacity of 5T4CAR-NK92 against various target cells, 5T4CAR-NK92 against SKOV3 (FIG. 7A), bxPC3 (FIG. 7B), A549 (FIG. 7C) and MDA-MB-468 (FIG. 7D) target cells at an effective target ratio of 1:1 and 3: a kill condition of 1.

FIG. 8 shows the isolated identification of primary NK cells.

FIG. 9 shows the expression rate of flow-detected primary NK cell 5T 4-CAR.

FIG. 10 shows the ability of 5T4-CAR NK to detect killing of SKOV3 and NCI-H460 target cells.

FIG. 11 shows the experimental results of the 5T4 antibody immunostaining method.

Detailed Description

The inventors have conducted extensive and intensive studies to conduct a large number of screens, and have obtained a targeted 5T antibody for the first time. Specifically, the invention screens and obtains four monoclonal antibody sequences of BC-B25A01, BC-B33E03 and BC-B33E7A, BC-B33CJC from more than 100 monoclonal antibody sequences. Based on this, a chimeric antigen receptor structure targeting 5T4 was further constructed, and NK cells expressing the chimeric antigen receptor exhibited superior killing ability against target cells. The present invention has been completed on the basis of this finding.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. VH and VL regions can be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

As used herein, the terms "heavy chain variable region" and "V _H "interchangeably used.

As used herein, the terms "light chain variable region" and "V _L "interchangeably used.

As used herein, the term "antigen binding domain" or the like includes any naturally occurring, enzymatically available, synthetic or genetically modified polypeptide or glycoprotein that specifically binds an antigen to form a complex. Any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally antibody constant domains may be used, for example, to derive antigen binding fragments of antibodies from whole antibody molecules. Such DNA is known and/or readily available or synthesizable from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries). The DNA may be sequenced and manipulated chemically or by using molecular biological techniques, for example, to arrange one or more variable and/or constant domains in a suitable arrangement, or to introduce codons, produce cysteine residues, modify, add or delete amino acids, and the like.

Non-limiting examples of antigen binding fragments or antigen binding domains, as used herein, include: (i) Fab fragments; (ii) F (ab') ₂ Fragments; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues mimicking an antibody hypervariable region (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide.

As used herein, an antigen binding fragment or antigen binding domainWill typically comprise at least one variable domain. The variable domain may have any size or amino acid composition and will typically comprise at least one CDR contiguous to or in-frame with one or more framework sequences. In the presence of V _L Domain associated V _H In the antigen binding fragment of the domain, V _H And V _L The domains may be disposed opposite each other in any suitable arrangement. For example, the variable region may be a dimer and contain V _H -V _H 、V _H -V _L Or V _L -V _L A dimer. Alternatively, the antigen binding domain may contain monomeric V _H Or V _L A domain.

In a given antibody light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well known antibody CDR assignment systems including, for example: chothia based on the three-dimensional structure of antibodies and topology of CDR loops, chothia definitions based on antibody sequence variability (Kabat, e., et al, U.S. Pat. No. of Health and Human Services, sequences of Proteins of Immunological Interest, (1983), abM (University of Bath), contact (University College London), international Immuno GeneTics database (IMGT), EU numbering system, and on loop structural positions.

It will be appreciated that the exact amino acid sequence boundaries of the CDRs in the present invention can optionally be defined using the different assignment systems mentioned above. Preferably, in the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues) reference is made to numbering positions according to the Kabat numbering system.

Antibodies to

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly 5 classes of immunoglobulins: igA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

Chimeric Antigen Receptor (CAR)

As used herein, the terms "chimeric antigen receptor of the invention", "CAR of the invention" are used interchangeably to refer to the chimeric antigen receptor of the third aspect of the invention.

The Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. Extracellular domains include target-specific binding elements (also referred to as antigen binding domains). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets 5T 4. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain, and the CD3 zeta signaling domain.

As used herein, "antigen binding domain" and "single chain antibody fragment" refer to Fab fragments, fab 'fragments, F (ab') ₂ Fragments, or single Fv fragments. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of an intact antibody. The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the scFv comprises an antibody specifically recognizing the extracellular region of 5T4, in particular an antibody specifically recognizing amino acid residues 24 to 41 of the 5T4 sequence, preferably a single chain antibody.

For the hinge and transmembrane regions (transmembrane domains), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

Preferably, the structure of the CAR of the invention comprises a signal peptide, an antigen recognition sequence (antigen binding domain), a linker region, a transmembrane region, a costimulatory factor signal region and a CD3zeta signaling region (zeta chain part), the order of attachment being as follows:

L-scFv-H-TM-C-CD3ζ(I)

NK cells

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. In recent years, NK cells have shown great application prospects in adoptive cellular immunotherapy. NK sources are wide ranging and include peripheral blood, cord blood, embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs), and the like.

NK-92 cells are an interleukin-2 (IL 2) -dependent NK cell line derived from peripheral blood mononuclear cells of a 50 year old male suffering from acute non-Hodgkin's lymphoma. NK-92 cells are the only NK cell line approved by the FDA at present in clinical test, the cell line has strong cytotoxicity, economy, off-the-shell, easy scale preparation, short survival time after killing tumor cells, easy in vitro expansion, no rejection to NK-92 cells by most of treated patients, no danger of graft versus host reaction, no expression of KIRs, in a constitutive activated state and good clinical safety up to now.

As used herein, the terms "CAR-NK cell", "CAR-NK", "calnk", "CAR-NK cell of the invention" all refer to CAR-NK cells expressing the chimeric antigen receptor CAR of the first aspect of the invention

Carrier body

Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The invention also provides vectors into which the expression cassettes of the invention are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are 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. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a retroviral vector.

Therapeutic applications

The invention includes cells (e.g., NK cells, T cells, etc.) transduced with a Retroviral Vector (RV) encoding an antibody, CAR of the invention. Transduced NK cells can elicit a CAR-mediated NK-cell or T cell response.

Accordingly, the present invention also provides a method of stimulating an NK cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administration to a mammal of NK cells expressing a CAR of the invention.

In one embodiment, the invention includes a class of cell therapies in which NK cells are genetically modified to express a CAR of the invention, and the CAR-NK cells are infused into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-NK cells are able to survive in vivo, producing long-term persistence that can lead to sustained tumor control.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or may include solid tumors. Types of cancers treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (indolent and high grade forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that do not normally contain cysts or fluid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named for the cell type that they are formed of (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic carcinoma ovarian carcinoma.

The CAR-modified NK cells of the invention can also be used as a vaccine type for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

Pharmaceutical composition

The antibodies, fusion proteins, or CAR-modified NK cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-15, IL-18, IL-21, or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an" antitumor effective amount "and,In the case of "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Individual cells/kg body weight doses (including all integer values within those ranges) are administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The main advantages of the invention include

(1) The chimeric antigen receptor disclosed by the invention has the advantages that the extracellular antigen binding domain is a specific anti-5T 4 scFv, the specific anti-5T 4 scFv binds to a specific hinge region and an intracellular domain, and the formed CAR shows great killing capacity to tumor cells, and is low in cytotoxicity and low in side effect.

(2) The CAR-NK cell has higher activation degree and excellent cytotoxicity on 5T4 positive target cells.

(3) The 5T4 antibody provided by the invention has excellent binding capacity, and can be used for performing immunostaining detection on a large number of tissue samples to ensure the specificity.

(4) The chimeric antigen receptor can be inserted into editable sites of iPSCs in a gene editing mode and the like, so that the edited iPSCs are further differentiated into NK, and the obtained NK carries editing characteristics and has corresponding functions.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 EC50 results of anti-5T 4 antibodies against different cells

The humanized mice are immunized by using a 5T4 antigen, single B cells are identified and sequenced by Beacon, corresponding sequences are transferred into CHO cells and are subjected to protein expression, and the obtained antibodies are purified, identified, subjected to flow screening and detection, so that 4 antibodies related to the invention, namely BC-B25A01 (Ab 01), BC-B33E03 (Ab 02), BC-B33E7A (Ab 03) and BC-B33CJC (Ab 04), are finally obtained. The antibodies have the characteristics of high affinity, strong specificity and the like. The half maximal effector concentrations EC50 of these antibodies on 5T4 positive tumor cells SKOV3 and NCI-H1975 were tested and used as positive controls with the A1 antibody (Ab 05,5T4-PF06263507, EP3130356 A1) as follows:

1. The antibody is diluted, firstly, the antibody is diluted 10 times, the antibody is diluted twice to 20 mug/mL 2 times, 10 gradients are used at 4 ℃.

2. Cells were plated, prepared, resuspended with PBS to a cell density of 1X 105/30. Mu.L after resuscitating, and transferred to 96-well round bottom plates at 30. Mu.L/well.

3. 30. Mu.L of antibody was added to the cells as planned, the plate edges were tapped, incubated at 4℃for 30min, and the maximum working concentration of antibody was 20ug/mL.

4. After incubation, 200. Mu.L of PBS was added to each well, and the supernatant was centrifuged off.

5. Add 50 μl 1 per well except NC well: 100 dilution of R-Phycoerythrin AffiniPure F (ab') 2Fragment Goat Anti-Human IgG, fcgamma fragment specific, incubated at 4℃for 15min.

6. After incubation, 200 μl of PBS was added to each well and washed twice, and the supernatant was discarded by centrifugation.

7. 50. Mu.L of PBS and 0.5. Mu.L of 7-AAD dead-living dye were added to each well and resuspended, incubated for 10min, and checked on the machine.

8. Based on raw data, MFI (geom. Mean) of different concentration gradient antibodies bound to cells was analyzed and derived using FlowJo V10. MFI was introduced into GraphPad Prism 7.00, and MFI and antibody concentrations were converted and fitted. The EC50 value is obtained by converting the X-axis (antibody concentration) into Log (X) using a Transfrom analysis, taking Log (antibody concentration) as the abscissa and MFI as the ordinate, and then using a Log (agonist) vs. response-Variable slope (four parameters) analysis to finally fit.

The results show that all 4 antibodies of the invention exhibit excellent binding properties (fig. 1), with BC-B33CJC performing optimally, and both the maximum MFI and EC50 being better than A1.

Example 2 5T4 antibody affinity detection

1. The open operating software confirms that the instrument (Biacore 8 k) is in good condition.

2. Preparing 10 XHBS-EP+ buffer solution, regulating pH to 7.4, preparing 1 XHBS-EP+ buffer solution with 10 XHBS-EP+ as mother liquor, regulating pH to 7.4, preparing Glycine regeneration solution, and regulating pH to 2.0.

3. The Protein A-47 chip immersed in the 1 XHBS-EP+7.4 buffer solution is taken out, washed by ultrapure water, air-dried, placed into an instrument and washed by a pipeline.

4. Purified antibody was diluted to 1ug/ml, antigen diluted to 200nM,0nM as reference.

5. The Protein A-47 chip was used to capture 1. Mu.g/mL of antibody at a flow rate of 10uL/min for 50s, the response value RU was recorded, and secondary dilution was performed according to the target capture amount to give an on-machine detection capture value (target capture value) of 50RU.

6. According to the established parameters, programming a loading program, putting a sample and a reagent into a designated hole site shown by software, capturing an antibody for 50s by using a Protein A-47 chip at a flow rate of 10uL/min, and then combining the antibody and an antigen for 180s at a flow rate of 30uL/min, and dissociating for 400s to obtain a combination curve.

7. Data analysis was performed by analysis software, and binding and dissociation curves were fitted using a 1:1binding model to obtain affinity kinetic data.

The results show that the 4 antibodies of the invention all show excellent binding characteristics to human 5T4 antigen, and KD value (M) is 1.0E-7-2.0E-9 (figure 2), wherein BC-B33CJC (33 CJC) and BC-B33E03 are significantly better than the control antibody A1.

Example 3 5T4-CAR Structure design

scFv of 5T4 CAR structure was designated BC-B25A01, BC-B33E03, BC-B33E7A, BC-B33CJC, respectively. The structure of the 5t4 CAR is shown in figure 3 and includes a signal peptide (scFv), scFv, range region, transmembrane (TM), costimulatory domain (costimulatory domain, CD) and CD3 zeta domain, the corresponding CAR structure being cloned onto pMSCV vector. Wherein the signal peptide may be selected from the following sequences (Table 1). In the present invention, the CD8 alpha SP signal peptide was used to construct a 5T4 CAR structure.

The data of the present invention are based on the 5T4 CAR structure of CD 8. Alpha. SP, CD28 range, CD28TM, and CD28 CD. Wherein, scFv can be composed of VL-Linker-VH or VH-Linker-VL, wherein Linker can be (G4S) 3 or (G4S) 4, etc.; VL, VH and corresponding CDR1, CDR2, CDR3 sequences are shown in table 2, table 3, table 4.

The complete CAR structure sequence is shown in table 2.

TABLE 1 Signal peptide sequences

Signal peptides	Sequence(s)	SEQ ID No:
			IL-21SP	MRSSPGNMERIVICLMVIFLGTLV	9
CD8αSP	MALPVTALLLPLALLLHAARP	10
			CSF2SP	MWLQSLLLLGTVACSIS	11
DAP12SP	MGGLEPCSRLLLLPLLLAVSG	12
			CD16SP	MWQLLLPTALLLLVSA	13
CD56SP	MLQTKDLIWTLFFLGTAVS	14
			IL-8SP	MTSKLAVALLAAFLISAALC	15

TABLE 2.5 VL and VH sequences of T4 CAR

/>

Note that: SEQ ID NO.

The BC-B33E03 light chain variable region (VL), the BC-B33E7A light chain variable region (VL), the BC-B33CJC light chain variable region (VL) are identical (i.e., SEQ ID NOS.4, 6, 8 are identical).

TABLE 3 Kabat-based CDR sequences

Note that: SEQ ID NO.

TABLE 4 Chothia-based CDR sequences

Note that: SEQ ID NO.

Example 4 detection of tumor cell surface 5T4 expression

The inventors examined the expression of NCI-H460, NCI-H1975, SKOV3, NCI-H226, A549, bxPC3, MDA-MB-231 and MDA-MB-468 tumor cell surface 5T4 by flow assay as follows.

2.1 taking 1E5-2E5 tumor cells to be detected, centrifuging at 200g-400g for 5min at 4 ℃, and discarding the supernatant.

2.2 200ul FACS Buffer (1% FBS in PBS), 200g-400g, centrifuged at 4℃for 5min, and the supernatant discarded.

2.3 100ul primary antibody (Invitrogen, MA 5-24229) was added, mixed well by blowing, and incubated at 4℃for 30min-60min.

2.4 after incubation, 200ul/well FACS Buffer,200g-400g, centrifugation at 4℃for 5min, and supernatant discarded.

2.5 repeat step4 once.

After 2.6 washing, 100ul secondary antibody (Jackson immunoresearch, 115-116-072) is added for blowing and mixing uniformly, and incubation is carried out at 4 ℃ for 30-60 min.

2.7 after incubation, 200ul/well FACS Buffer,200g-400g, centrifugation at 4℃for 5min, and supernatant discarded.

2.8 step4 was repeated twice.

2.9 after washing, 200ul/well FACS Buffer was added for resuspension and detection by an upflow instrument.

The results indicate that NCI-H460 does not or under-expressed 5T4, while several other cells all had varying degrees of 5T4 expression (fig. 4).

EXAMPLE 5 viral packaging and NK92 cell infection

The packaging vectors of the retrovirus are BaEV and pCMV-gag-pol, which are synthesized and extracted by Jin Weizhi after being designed by the laboratory. The virus packaging, cell infection and sorting processes are as follows:

5.1, after digestion, the HEK-293T cells in good condition are resuspended in the corresponding culture medium and inoculated in a culture dish of 10cm at a density of 7-8E 5/ml;

5.2, after the cells are placed in an incubator for culturing for 16 hours, observing the cell density, and starting plasmid transfection when the density is about 90%;

5.3 taking 2 1.5ml centrifuge tubes, adding 500ul Opti-MEM, respectively ^TM I serum-reduced medium (Gibco, 31985062), 7.5ug BaEV, 10ug pCMV-gag-pol and 20ug of the corresponding pMSCV vector expressing CAR were added to one of the centrifuge tubes; adding 40ul of PEIPro solution (polyplus, 115-010) to another centrifuge tube;

5.4, lightly mixing the solutions of the two centrifuge tubes, and standing at room temperature for 15-20min to form a transfection compound;

5.5, lightly dripping the transfection complex into HEK-293T culture supernatant, and placing the culture supernatant in a culture medium for continuous culture for 4-6h;

5.6, replacing the preheated fresh culture medium, and placing the cells in an incubator for continuous culture for 48 hours;

5.7 cell culture supernatants were collected and virus concentrated using a Lenti-X Concentrator (Takara, 631232);

5.8 resuspension of the virus with 100ul of medium, taking 4E5 NK92 cells in 6 well plates, adding 10ul of virus concentrate, respectively, and adding polybrene with a final concentration of 5 ug/ml;

5.9 cells were centrifuged at 32℃and 800g for 1h, and then placed in an incubator overnight for culture;

5.10 the cells were replaced with fresh medium and cultured for 3-5 days, followed by flow detection.

5.11 5T4-CAR NK92 cells in culture were blow-aspirated several times to disperse them into individual cells, 200. Mu.L to 1.5ml centrifuge tubes were used for NC-200 cell counting.

5.12 taking 1E7 cells to be sorted in a centrifuge tube, centrifuging at 300g for 5min, and discarding the supernatant.

5.13 cells were resuspended in 10mL MACS Buffer (or PBS), centrifuged at 300g for 5min and the supernatant discarded.

5.14 with 2mL of 3ug/mL Unconjugated Human TPBG/5T4 Protein,His,Avitag ^TM (ACRO, TPG-H52E 5) dilutions (diluted in PBS) resuspended cells and incubated at 4℃for 30min.

5.15 after incubation, an appropriate amount of MACS Buffer was added, and the supernatant was discarded after centrifugation at 300g for 5 min.

5.16 Add 5mL MACS Buffer and wash once more.

5.17 80uL (80 uL/1E7 cells) MACS Buffer was added to resuspend cells.

mu.L (20. Mu.L/1E 7 cells) Anti-Biotin MicroBeads (Order No.: 130-090-485) was added to 5.18.

5.19 incubated at 4℃for 15min after mixing.

5.20 after incubation was completed, 2mL (2 mL/1E7 cells) MACS Buffer was added and washed once.

5.21 500uL MACS Buffer resuspension (500uL Buffer per 1E8 cells).

5.22 adsorption column LS was loaded on a magnet rack and the column was rinsed with 3mL MACS Buffer.

5.23 the treated cells were loaded into the column.

5.24 and 3mL MACS Buffer in the column, the cells that were not magnetically attracted were eluted.

Step3 was repeated twice at 5.25.

5.26 After 3 times of elution are completed, a 15mL centrifuge tube is taken, the column is taken off the magnetic rack and placed on the centrifuge tube.

5.27 5mL MACS Buffer was added to the column, the plunger was pushed into the column, and the magnetic bead labeled cells were washed out.

5.28 appropriate amount of the sorted cells were counted in NC-200.

5.29 cells from the sorting were centrifuged at 300g for 5min and the supernatant discarded.

5.30 resuspended in an appropriate volume of complete Medium at 37℃with 5% CO ₂ Culturing in an incubator.

The flow type result shows (figure 5) that after virus infection, the expression of NK92 cells 5T4-CAR in each group is between 50% and 85%, and the positive rate of each group is basically consistent after magnetic bead sorting, and reaches more than 98%.

Example 6 detection of affinity of 5T4-CAR NK92 binding to target cells

The affinity (intensity) of 5T4-CAR NK92 binding to target cells was examined using a z-Movi cell affinity analyzer, the procedure was as follows,

6.1 target cells were HEK293T cells overexpressing human 5T4 (HEK 293T-h5T 4), which were ensured to be in a healthy state prior to use.

6.2Z-Movi detection the day before, poly-L-Lysine (Poly L-Lysine, sigma-Aldrich, P4707, 0.01%) was diluted with PBS 1:5. 200uL of diluted Poly-L-Lysine was added, the syringe was pulled to fill the viewing chamber and the air bubbles were vented and left at room temperature for 15 minutes. Subsequently, all polylysine solution was withdrawn and the air bubbles in the viewing chamber were removed by pushing the syringe quickly and the chip was oven dried overnight at 37 ℃.

6.3Z-Movi on the day of the run, the required media for the Z-Movi experiments (DMEM+10% FBS) was removed, the chip was fully warmed up, 100uL of warmed up PBS solution was added to the sample chamber, the syringe was pulled to leave 10uL of liquid in the sample chamber, the valve was closed, and no air bubbles in the observation chamber were confirmed. 100uL of preheated target cell culture medium was added to the sample chamber, the syringe was pulled to leave 10uL of liquid in the sample chamber, the valve was closed, and no bubbles were confirmed in the observation chamber.

6.4 removal of target cells Using TrypLE ^TM The cells were digested with Select Enzyme (Gibco, 12563029), and after counting, cell viability was confirmed to be 95% or higher, and resuspended in 1.5mL centrifuge tubes at 6E7/mL with medium.

6.5 mounting the chip on a z-Movi instrument, opening the software, entering a monolayer test mode, fully blowing and sucking target cells to avoid cell aggregation, then adding 20uL of cell suspension into a sample injection chamber, and slowly opening a valve. At this point, it was observed that cells flowed into the observation chamber. After closing the valve, the focal plane is adjusted, and the coverage condition of the cells after adhering is observed, wherein the coverage rate is required to be between 90 and 105 percent.

6.6 after closing the valve, cells in the sample chamber were slowly aspirated to a remaining about 5uL and washed with 3X200uL target cell culture medium. 300uL of target cell culture medium was added to the sample chamber, and the chip was placed in a 37℃oven for cell attachment for 3 hours.

6.7 effector cell (5T 4CAR NK92) staining was started at 2 hours of target cell attachment. Effector cell staining Using CellTrace ^TM Far infrared cell proliferation kit (Invitrogen, C34564). Effector cells were counted and at a density of 6E6/mL, 20uL of effector cells were used per run for effector cell access.

6.8 cells were centrifuged and washed once with an equal volume of PBS solution at 1:1000 preparation of Far Red in PBS staining buffer, using staining buffer to re-suspend the cells 1E6/mL,37 ℃ light shielding staining for 20 minutes. After staining, 5 volumes of medium were added and incubated for 5 minutes. After centrifugation, cells were resuspended at a density of 6E 6/mL.

6.9 after the target cell adherence is finished, a detection chip is installed, and a monolayer test module is used for detecting the monolayer cell adherence effect. After the force is applied, the valve is opened to wash away the target cells which are not firmly combined

6.10 adding stained effector cells, slowly opening the valve, and filling the effector cells. The fluorescent channel was opened, the effector cell density was observed, the valve was closed after appropriate, and incubation was started in a click procedure. Cells in the sample-in chamber were slowly aspirated to a remaining about 5uL and washed with 3x200uL of target cell culture medium. Closing the upper cover, and waiting for the program to automatically complete incubation, stress application and data recording. After recording was completed, the force was again applied according to the machine instructions and the valve was opened to flush with medium, ensuring that about 50-100uL of medium was flowed. The next run is started.

The detection results show that NK92 cells rapidly fall off from target cells after the application of force (FIG. 6), and the intensity of NK92 cells expressing 5T4CAR combined with target cells is obviously stronger than that of NK92 cells, wherein the CJC, E03, E7A, A01T 4CAR and target cells related to the invention have higher affinity than that of the control group A1. The overall performance is compared as follows: 5T4 CAR-CJC > 5T4 CAR-E03,5T4 CAR-E7A > 5T4 CAR-A01.

Example 7 detection of killing ability of 5T4-CAR NK92 against multiple target cells

The killing capacity of the 5T4-CAR NK92 to the SKOV3 target cells is detected by using a real-time label-free dynamic cell analysis technology (RTCA, real Time Cellular Analysis), and the specific operation steps are as follows,

7.1 IL-2 in NK92 medium was removed 24 hours in advance.

7.2 taking out RTCA E96 Plate, adding 50uL of corresponding culture medium into each hole, newly constructing experiment in RTCA program, completing relevant setting of experiment, locking after putting E-Plate in, automatically carrying out baseline measurement by the instrument, and completing the baseline measurement in one minute.

7.3 digestion of SKOV3 target cells, inoculated in E-plate at 2E 4/well.

7.4 after inoculation, the cells were left at room temperature for 30 minutes, the E-plate was placed in a recording tank and the growth of the cells was recorded once for 15 minutes.

7.5 observing the target cell resistance status, and supplementing 5T4-CAR NK92 effector cells before and after reaching the plateau.

7.6 target cells were added according to the experimental set effective target ratio (E: T), the E-plate was placed on the plate, and after 10 minutes the killing step was initiated.

7.7 preparation of multiple rounds of target cell stimulation after 5-8 hours of round 1 killing, treatment of SKOV3 target cells, addition of 2E4 target cells, placement of E-plate on plate, recording of killing step started after 10 minutes.

The results showed (fig. 7A) that the effective target ratio was 1:1 and 3:1, each group of 5T4-CAR NK92 exhibited more pronounced cytotoxicity against SKOV3 target cells than NK92 cells. After the first round of killing was completed, each group of 5T4-CAR NK92 still exhibited superior cytotoxicity to SKOV3 target cells that were added continuously.

The killing ability of 5T4-CAR NK92 to BxPC3 (FIG. 7B), A549 (FIG. 7C), MDA-MB-468 (FIG. 7D) and other target cells was also tested by a similar method, and the result shows that 5T4-CAR NK92 can show strong killing ability to the target cells positive to the 5T 4.

Example 8 isolation and identification of Primary NK cells

To further verify the function of 5T4-CAR on primary NK, NK cells were isolated from human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) using NK cell isolation kit (NK Cell Isolation Kit human, cat:130-092-657, miltenyi) as follows,

8.1 resuspension of cells with MACS Buffer every 10 ⁷ PBMC corresponded to 40. Mu.L MACS Buffer.

8.2 every 10 ⁷ mu.L of NK Cell Biotin-Antibody Cocktail was added to each PBMC.

8.3, and incubating for 5min in a refrigerator (2-8 ℃).

8.4 every 10 ⁷ mu.L of MACS Buffer was added to each PBMC.

8.5 every 10 ⁷ Mu. L NK Cell MicroBead Cocktail were added to each PBMC.

8.6, and incubating for 10min in a refrigerator (2-8 ℃).

8.7 the column was loaded on a magnetic rack and the column was rinsed with 3mL MACS Buffer.

8.8 transfer the incubated cells to an adsorption column and collect the shed cells.

8.9 the column was washed once with 3mL MACS Buffer and the outgoing cells were also collected.

8.10 the collected cells were centrifuged at 300g for 5min and the supernatant was discarded. Cells were resuspended in 3mL of medium.

After culture expansion, the obtained NK cells were identified. The results of the identification are shown in FIG. 8. The results show that CD3 can be obtained by isolation and amplification culture ⁺ CD56 ⁺ Greater than 95% of NK cell populations.

EXAMPLE 9 viral infection of primary NK cells

The virus production process and primary NK infection steps were as described before, and after 48 hours of infection, each group of 5T4-CAR expression was examined by flow, and the results in FIG. 9 indicate that each group of 5T4-CAR had an infection efficiency of 53% -85%.

Example 10 detection of 5T4-CAR NK killing ability against target cells

The ability of 5T4-CAR NK to kill SKOV3 and NCI-H460 target cells was tested using real-time label-free dynamic cell analysis (RTCA, real Time Cellular Analysis), the 5T4-CAR positive rates of each group were adjusted to be consistent (53.659%) using blank NK cells, and the remaining steps were referred to above.

The results showed (fig. 10) that the effective target ratio was 1:1 and 3:1, each group of 5T4-CAR NK showed more pronounced cytotoxicity against SKOV3 target cells than NK cells. Because NCI-H460 is under-expressed 5T4, each group of 5T4-CAR NK can only exhibit weak cytotoxicity against NK cells.

Example 11.5T4 development of antibody immunostaining methods

In view of the excellent performance of BC-B33CJC, further development of immunohistochemical methods was performed with BC-B33 CJC. To maximize similarity to the CAR structure of the present invention, the performance is reflected more accurately. Antibodies in the form of CJC-scFv-mouse IgG1-Fc were also constructed and 54T positive and negative sections were immunohistochemically stained by the following procedure, respectively.

11.1 4 μm sections were left at 60℃overnight, dewaxed and hydrated: sequentially soaking xylene I-xylene II-100% ethanol I-100% ethanol II-95% ethanol-85% ethanol-75% ethanol-deionized water for 5min each;

11.2 antigen retrieval: preparing 1x antigen retrieval liquid, incubating in Pt module at 98 ℃ for 20min, and cooling at room temperature for 30min; PBST wash for 3min 3;

11.3 placing the sections in 100ul serum blocking solution, standing at room temperature for incubation for 10min, and washing with PBST for 3min x 3;

11.4 plus primary antibody (1.48 mg/ml,1:25 dilution) at 4 ℃ overnight, standing for incubation, PBST wash 3min 3;

11.5 rewarming for 30min, incubating the sections in 100ul peroxidase blocker for 15min, pbst washing for 3min 3;

11.6 adding secondary antibody (dilution ratio 1:2000), standing and incubating for 1h at room temperature, and washing with PBST for 3 min;

11.7 adding 30ul DAB chromagen to 1.5mL DAB subbate, mixing, adding onto tissue slice, standing at room temperature for 5min, and washing with running water;

counterstaining with hematoxylin for 3min, differentiating with 1% ethanol hydrochloride for 1-3s, and returning blue liquid for 5min;

11.9 gradient alcohol dehydration, transparent xylene, neutral resin sealing piece.

The results indicated (FIG. 11), CJC-scFv-mouse IgG1-Fc exhibited a specific membrane positive staining signal for the 5T4 positive PDX sample LD1-0025-200636 (RNA TPM: 147); whereas for the 5T4 negative PDX samples LD1-0032-361649 (RNA TPM: 0), CJC-scFv-mouse IgG1-Fc did not generate staining signals. Meanwhile, with mIgG as a negative control of the system, no staining signal was generated for the 5T4 positive PDX samples LD1-0025-200636 (RNA TPM: 147). The above data demonstrate that CJC-scFv-mouse IgG1-Fc can specifically detect the 5T4 antigen on the surface of tumor tissue in immunohistochemical staining.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof that targets 5T4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

the heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 31,

HCDR2 shown in SEQ ID NO. 32, and

HCDR3 shown in SEQ ID NO. 33;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27;

wherein the CDR sequence is based on the numbering scheme of Kabat.

2. A recombinant protein, said recombinant protein comprising:

(i) The antibody or antigen-binding fragment thereof of claim 1;

and (ii) tag sequences that facilitate expression and/or purification.

3. A chimeric antigen receptor CAR, characterized in that the antigen binding domain of said chimeric antigen receptor comprises an antibody single chain variable region sequence scFv targeting 5T4,

the heavy chain variable region of the scFv comprises the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 31,

HCDR2 shown in SEQ ID NO. 32, and

HCDR3 shown in SEQ ID NO. 33;

LCDR1 shown in SEQ ID NO. 25,

LCDR2 as shown in SEQ ID NO 26, and

LCDR3 as shown in SEQ ID NO. 27;

wherein the CDR sequence is based on the numbering scheme of Kabat.

4. A polynucleotide encoding the antibody or antigen binding fragment thereof of claim 1, the recombinant protein of claim 2, or the chimeric antigen receptor CAR of claim 3.

5. A vector comprising the polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5 or the polynucleotide of claim 4 integrated into a chromosome.

7. A method of making a CAR-NK cell or CAR-T cell expressing the chimeric antigen receptor of claim 3, comprising the steps of:

introducing the polynucleotide of claim 4 or the vector of claim 5 into an NK cell or a T cell, thereby obtaining the CAR-NK cell or CAR-T cell.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, the chimeric antigen receptor of claim 3, the polynucleotide of claim 4, the vector of claim 5, or the host cell of claim 6, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. An immunoconjugate, said immunoconjugate comprising:

(a) An antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or a combination thereof; and

(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a radionuclide, or a combination thereof.

10. The use of an antibody or antigen binding fragment thereof according to claim 1, a recombinant protein according to claim 2, a chimeric antigen receptor according to claim 3, a polynucleotide according to claim 4, a vector according to claim 5, a host cell according to claim 6, a pharmaceutical composition according to claim 8 or an immunoconjugate according to claim 9,

(a) Preparing a detection reagent or a kit for detecting 5T4 related diseases; and/or

(b) Preparing a medicament or preparation for preventing and/or treating 5T4 related diseases;

the 5T4 related diseases are breast cancer, ovarian cancer, lung cancer and pancreatic cancer.

11. The use according to claim 10, wherein the 5T 4-related disorder is lung cancer.

12. A method for producing a recombinant polypeptide, comprising:

(a) Culturing the host cell of claim 6 under conditions suitable for expression;

(b) Isolating from the culture a recombinant polypeptide which is the antibody or antigen-binding fragment thereof of claim 1, or the recombinant protein of claim 2.

13. A test plate, said test plate comprising: a substrate and a test strip comprising the antibody or antigen binding fragment thereof of claim 1, the recombinant protein of claim 2, the immunoconjugate of claim 9, or a combination thereof.

14. A kit, comprising:

(1) A first container comprising the antibody or antigen-binding fragment thereof of claim 1; and/or

(2) A second container containing a second antibody against the antibody of claim 1;

alternatively, the kit contains the assay plate of claim 13.