WO2023145961A1 - Cells expressing pre-pro-precursor form chimeric antigen receptor targeting igf1r - Google Patents

Abstract

Provided is an IGF1R CAR-T cell that is expected to be effective against tumors that express IGF1R. Provided is a polynucleotide that encodes a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to the insulin-like growth factor-1 receptor (IGF1R), a transmembrane domain, and an intracellular signaling domain, wherein the target-binding domain is the pre-pro-precursor for insulin-like growth factor (IGF) or its E-domain-deleted fragment. Also provided are a vector containing this polynucleotide and genetically modified cells into which this polypeptide or vector has been introduced.

Description

Pre-pro progenitor-type chimeric antigen receptor-expressing cells targeting IGF1R

The present invention relates to a polynucleotide encoding a chimeric antigen receptor, a vector containing the same, a genetically modified cell expressing the chimeric antigen receptor, and a method for producing the same, which are useful in the field of adoptive immunotherapy.

Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that belongs to the insulin receptor (INSR) family and consists of two extracellular α subunits that constitute the ligand-binding domain and two kinase domains. It forms a heterotetramer composed of transmembrane β subunits. IGF1R binds its ligands (primarily IGF-1 and IGF-2) with varying affinities depending on the cell type and context in which it is expressed, and is responsible for Ras/Raf/MEK/ERK or PI3K/Akt/mTor signaling. It regulates proliferation, differentiation, survival, and metabolism of various cells through pathways. IGF1R can also form hybrid receptors with INSR-A or INSR-B (IGF1R/INSR-A, IGF1R/INSR-B, respectively), and these hybrid receptors are IGF-1, IGF-2 , can bind to insulin. Binding of these hybrid receptors to their ligands results in a signaling cascade similar to that of IGF1R binding of its ligands. Protein groups formed by IGF1R, INSR, their hybrid receptors (IGF1R/INSR-A, IGF1R/INSR-B), and their ligands (IGF-1, IGF-2, insulin), etc. is called

High expression of IGF1R and high levels of circulating IGF ligands have been confirmed in a wide range of cancer types, including sarcoma, breast cancer, prostate cancer, pancreatic cancer, and melanoma. have been reported to be correlated with Furthermore, signals from IGF1R have been shown to be essential for malignant transformation. In addition to IGF-1 and IGF1R, high expression of IGF-2 and INSR-A has also been reported in a wide range of cancer types, and the IGF axis is considered an ideal cancer therapeutic target (non-patent literature 1 and 2).

Various drugs targeting the IGF axis have been attempted to develop, and anti-IGF1R antibodies, IGF1R tyrosine kinase inhibitors, and anti-IGF-1/IGF-2 inhibitory antibodies have been reported to be effective in Ewing's sarcoma, osteosarcoma, and neuropathy. It has been evaluated in clinical trials for various cancer types such as endocrine tumors, small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, breast cancer, and ovarian cancer. is insufficient and has not yet been put to practical use (Non-Patent Documents 1 and 2). IGF1R, INSR, and IGF ligands are expressed at high levels in cancer cells, and it is thought that resistance to anti-IGF1R antibodies, etc. occurs because they act complementary to the suppression of IGF1R by anti-IGF1R antibodies, etc. It is In addition, anti-IGF1R antibodies, IGF1R tyrosine kinase inhibitors, etc. have been reported to cause severe and frequent hyperglycemia. (IGF1R/INSR-B) disorder may be a factor. Therefore, the emergence of new modalities targeting the IGF axis with excellent efficacy and safety is expected.

In recent years, chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells) have been expected as a promising next-generation modality against intractable cancers due to their remarkable cytotoxic activity. . Among them, research on CAR-T cells targeting IGF1R has been reported by Huang et al. (Non-Patent Document 3). Huang et al. showed that scFv-type CAR-T cells using a single-chain antibody (scFv) derived from an antibody against IGF1R as a target-binding domain exhibited specific immune responses and in vitro antitumor effects against IGF1R-high-expressing sarcoma. In addition, a xenograft mouse model in which sarcoma cells were injected intraperitoneally showed tumor shrinkage and survival effects, and a xenograft mouse model in which sarcoma cells were injected intravenously also showed tumor shrinkage. (Non-Patent Document 3). However, no IGF1R CAR-T cells have entered clinical trials to date.

On the other hand, as a method for producing CAR-T cells that target growth factor receptors, the present inventors developed CAR (ligand-type CAR) that uses receptor ligands instead of conventional scFV-type CAR target-binding domains. We have reported design techniques (Non-Patent Documents 4 and 5, Patent Document 1).

WO 2020/085480

Simpson A et al., Target Oncol., 2017, 12(5):571-597, doi: 10.1007/s11523-017-0514-5 Anastassios Philippou et al., Mutation Research. Reviews in Mutation Research, 2017, 772:105-122, doi: 10.1016/j.mrrev.2016.09.005 Huang X et al., PLoS One, 2015, 10(7), doi: 10.1371/journal.pone.0133152 Nakazawa Y et al., J Hematol Oncol., 2016, 9:27 Hasegawa A et al., Clin Transl Immunology., 2021, 10(5):e1282

As mentioned above, there are no IGF1R CAR-T cells that have reached clinical trials. Therefore, further development of IGF1R CAR-T cells expected to be effective against IGF1R-expressing tumors is desired.

The present inventors considered the possibility that CAR-T cells that use IGF1R ligands as target-binding domains instead of scFv could be applied as adoptive immunotherapeutic agents for IGF1R-expressing tumors, and conducted intensive studies. As a result, when CAR-T cells were prepared using the natural ligand mature IGF-1 as the target-binding domain, the CAR expression rate disappeared over time in the T cells transfected with the CAR gene, and CAR-T It was found that the cells could hardly kill IGF1R-expressing tumor cells. Therefore, the present inventors generated CAR-T cells using its precursor, pre-pro-IGF-1, as the target-binding domain instead of mature IGF-1. Surprisingly, the CAR expression rate was was maintained and the antitumor effect was improved. We also found that when a fragment lacking the E domain of pre-pro-IGF-1 was used as the target binding domain, the CAR expression rate was higher than when wild-type pre-pro-IGF-1 was used. and antitumor effect are shown, and further, when pre-pro-IGF-2 is used as a target binding domain, CAR expression rate is maintained and antitumor effect is shown, and to complete the present invention Arrived.

That is, the present invention includes the following.
[1] Encodes a chimeric antigen receptor (CAR) protein with a target-binding domain that binds to the insulin-like growth factor-1 receptor (IGF1R), a transmembrane domain, and an intracellular signaling domain A polynucleotide, wherein the target binding domain is the pre-pro precursor of insulin-like growth factor (IGF) or a fragment thereof lacking the E domain.
[2] The polynucleotide of [1], wherein the IGF is IGF-1.
[3] The poly of [1] or [2], wherein the target-binding domain consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 nucleotide.
[4] The polynucleotide of [1], wherein the IGF is IGF-2.
[5] The polynucleotide of [1] or [4], wherein the target-binding domain consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:14.
[6] A vector comprising the polynucleotide according to any one of [1] to [5].
[7] A genetically modified cell into which the polynucleotide of any one of [1] to [5] or the vector of [6] has been introduced.
[8] A method for producing a CAR protein-expressing cell, which comprises introducing the polynucleotide of any one of [1] to [5] or the vector of [6] into a cell.
[9] A therapeutic agent for diseases involving IGF1R-expressing cells, comprising the cells of [7].
[10] A pharmaceutical composition comprising the therapeutic agent of [9] and a pharmaceutically acceptable carrier.
[11] Diseases involving IGF1R-expressing cells include leukemia, multiple myeloma, lymphoma, lung cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, colorectal cancer, and colon. cancer, colon cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, renal cancer, adrenal cancer, melanoma, neuroendocrine tumors, retinoblastoma, and sarcoma The therapeutic agent of [9] or the composition of [10], which is selected from the group consisting of:
[12] A kit for producing CAR protein-expressing cells targeting IGF1R-expressing cells, comprising the vector of [6].
This specification includes the disclosure content of Japanese Patent Application No. 2022-012674, which is the basis of priority of this application.

The present invention provides genetically modified cells that bind to IGF1R-expressing target cells and exhibit antitumor effects.

FIG. 1 shows mature IGF-1, pre-pro-IGF-1 (IGF-1 Ea, IGF-1 Eb, IGF-1 Ec) and pre-pro, which were used as CAR target-binding domains in the Examples of the present application. - shows the structure of the E domain-deleted fragment of IGF-1 (IGF-1 w/oE). Figure 2 shows the vector map of the mature IGF-1 type CAR expression plasmid. FIG. 3 shows the vector map of the IGF-1 Ea-type CAR expression plasmid. FIG. 4 shows the vector map of the IGF-1 Eb-type CAR expression plasmid. FIG. 5 shows the vector map of the IGF-1 Ec-type CAR expression plasmid. FIG. 6 shows the vector map of the IGF-1 w/oE type CAR expression plasmid. Figure 7 shows the CAR expression rate in mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells on days 1, 3, 6 and 10 after gene transfer (Days 1, 3, 6 and 10). indicates Figure 8. Flow cytometry showing anti-tumor cell activity of control T cells when co-cultured with E:T ratios of 4:1, 2:1, 1:1 or 1:2 for 4 days. Shows the results of the metric. Figure 9 shows the IGF-1 Ea CAR when IGF-1 Ea CAR-T cells and tumor cells were co-cultured at an E:T ratio of 4:1, 2:1, 1:1 or 1:2 for 4 days. - shows flow cytometry results demonstrating the anti-tumor cell activity of T cells. FIG. 10 shows mature IGF-1 CAR when mature IGF-1 CAR-T cells and tumor cells were co-cultured at an E:T ratio of 4:1, 2:1, 1:1 or 1:2 for 4 days. - shows flow cytometry results demonstrating the anti-tumor cell activity of T cells. FIG. 11 shows the CAR expression rate in IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/oE type CAR-T cells 10 days after gene transfer (Day 10). FIG. 12 depicts IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/o E CAR-T cells or control T cells at an E:T ratio of 2:1, 1:1 or 1:1. 2 shows the number of tumor cells after 4 days of co-culture with tumor cells. FIG. 13 shows IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/o E CAR-T cells or control T cells at E:T ratios of 2:1, 1:1 or 1:1:1. 2 shows the number of T cells after co-culture with tumor cells for 4 days. Figure 14 shows the vector map of the pre-pro-IGF-2 type CAR expression plasmid. FIG. 15 shows the results of flow cytometry showing the results of CAR expression analysis of pre-pro-IGF-2 type CAR-T cells and control T cells on day 10 after gene transfer. Figure 16 shows flow cytometry results showing the anti-tumor cell activity of control T cells when control T cells and tumor cells were co-cultured at an E:T ratio of 2:1, 1:1 or 1:2 for 4 days. show. FIG. 17 shows pre-pro-IGF-2 levels when pre-pro-IGF-2 type CAR-T cells and tumor cells were co-cultured for 4 days at an E:T ratio of 2:1, 1:1 or 1:2. Fig. 2 shows flow cytometry results showing anti-tumor cell activity of type CAR-T cells. Figure 18 shows IGF-1 w/o E-type CAR-T cells or control T cells at an E:T ratio of 4:1, 2:1, 1:1, 1:2, or 1:4 for 4 days ( Tumor cell numbers after co-culture with A) breast cancer cell line MX-1, (B) lung adenocarcinoma cell line H1568, (C) endometrial cancer cell line ARK-1. FIG. 19 depicts IGF-1 w/o E-type CAR-T cells or control T cells at E:T ratios of 4:1, 2:1, or 1:1 for 4 days, (A) cervical cancer cell lines. HeLa, (B) Tumor cell numbers after co-culture with the ovarian cancer cell line RMG-1. Figure 20 shows IGF-1 w/o E CAR-T cells, IGF-1 w/o E des1-3 CAR-T cells, or control T cells at an E:T ratio of 1:1, 1:2, or Lysis rate of tumor cells after co-culture with lung adenocarcinoma cell line A549 at 1:4 for 4 days. FIG. 21 shows the IGFBP3 binding rate of IGF-1 w/o E type CAR-T cells and IGF-1 w/o E des1-3 type CAR-T cells. Figure 22 shows IGF-1 w/oE type CAR-T cells and IGF-1 w/oE des1-3 type CAR-T cells after stimulation with IGFBP3 or IGFBP3+IGF1R IFNγ concentration in the culture supernatant. .

The present invention encodes a chimeric antigen receptor (CAR) protein having a target binding domain that binds to insulin-like growth factor-1 receptor (IGF1R), a transmembrane domain, and an intracellular signaling domain. wherein the target binding domain is an insulin-like growth factor (IGF) pre-pro precursor or an E domain deleted fragment thereof.

As used herein, the term "chimeric antigen receptor (CAR)" refers to targeting specificity of T cells (e.g., naive T cells, stem cell memory T cells, central memory T cells, effector memory T cells, or combinations thereof). It refers to a modified receptor that can be transplanted into cells such as T cells). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors. As used herein, the term "CAR-T cell" means a T cell expressing a chimeric antigen receptor (CAR) on its cell surface.

As used herein, the term "domain" refers to a region within a polypeptide that folds into a specific structure independently of other regions.

As used herein, "polynucleotide" includes natural or synthetic DNA and RNA, such as genomic DNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), and/or tRNA.

As used herein, "encoding" means that a given nucleotide sequence encodes amino acid sequence information of a given protein or (poly)peptide, as commonly used in the art. In the text both the sense and antisense strands are used in the context of "encoding".

As used herein, the term "insulin-like growth factor-1 receptor (IGF1R)" refers to a receptor tyrosine kinase belonging to the insulin receptor family. It consists of two transmembrane β subunits with IGF1R binds to its ligands (mainly IGF-1 and IGF-2) and regulates proliferation, differentiation, survival, and metabolism of various cells via Ras/Raf/MEK/ERK or PI3K/Akt/mTor signaling pathways. to control.

　IGF1R is known to be expressed in a wide range of tumors. IGF1R-expressing tumors include, for example, hematological tumors such as leukemia, multiple myeloma, and lymphoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, e.g., lung adenocarcinoma, etc.), head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, colorectal cancer, colorectal cancer, colon cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, kidney Solid tumors such as cancer, adrenal carcinoma, melanoma, neuroendocrine tumors, and retinoblastoma, and sarcomas (eg, Ewing's sarcoma, osteosarcoma, etc.).

The amino acid sequence of the IGF1R protein has been registered, for example, as Uniprot accession number P08069.

As used herein, "insulin-like growth factor binding protein (IGFBP)" is a protein that binds to IGF-1 and IGF-2 and prolongs their blood half-lives. IGFBP encompasses six proteins (IGFBP1-6), of which IGFBP3 is the most abundant IGFBP in blood (L A Bach et al., Journal of Molecular Endocrinology, 2018, 61, T11- T28). The amino acid sequence of the human IGFBP3 protein is registered under Uniprot Accession No. P17936-1, for example. IGFBP is known to be secreted by various tumors. Tumors that secrete IGFBP3 include renal cancer, melanoma, pancreatic cancer, breast cancer, lung adenocarcinoma, head and neck squamous cell carcinoma, and the like.

As used herein, "insulin-like growth factor (IGF)" is a protein that exhibits growth-promoting effects by binding to and activating IGF1R. IGFs include two molecular species, IGF-1 and IGF-2. Both IGF-1 and IGF-2 have been reported to be overexpressed in a wide range of cancers and associated with poor prognosis. Amino acid sequences of naturally occurring mature IGF-1 include, for example, that shown in SEQ ID NO: 2, and the base sequences encoding it that are codon-optimized for expression in human cells include: For example, SEQ ID NO:1 can be mentioned. In addition, the amino acid sequence of naturally occurring mature IGF-2 includes, for example, that shown in SEQ ID NO: 12; , for example, SEQ ID NO: 11.

In vivo, IGF-1 and IGF-2 are known to become mature proteins by removing the N-terminal and C-terminal portions after being synthesized as precursor proteins. Specifically, IGF-1 is first translated from the N-terminal side as a signal peptide, mature IGF-1, and pre-pro-IGF-1, which is a precursor protein containing an E domain. It is known that pro-IGF-1 is formed by removal, and mature IGF-1 is formed by separation of the E domain by protease cleavage. Similarly, IGF-2 is translated from the N-terminal side as a signal peptide, mature IGF-2 peptide, and pre-pro-IGF-2, which is a precursor protein containing an E domain. It is known that it becomes IGF-2 and then mature IGF-2 by separating the E domain by protease cleavage (Brisson BK and Barton E, frontiers in ENDOCRINOLOGY, 2013, 4(42): 1-6) .

In the present invention, the CAR protein contains a target binding domain that binds to IGF1R. The target binding domain exhibits binding to IGF1R (preferably its extracellular ligand binding domain) and enables an immune response against target cells expressing IGF1R on the cell surface.

In the present invention, a pre-pro precursor of IGF, which is a ligand of IGF1R, or a fragment lacking the E domain thereof can be used as the target-binding domain.

As used herein, the term "pre-pro precursor of IGF (ligand precursor)" refers to an IGF precursor containing a signal peptide, mature IGF, and E domain before the N-terminal and C-terminal portions are removed. means. Pre-pro precursors of IGF can be used in humans, domestic animals (horses, cattle, sheep, goats, pigs, etc.), pets (dogs, cats, rabbits, etc.), experimental animals (mice, rats, monkeys, etc.), etc. can be derived from any mammal, but is preferably derived from humans. The pre-pro precursor of IGF includes the pre-pro precursor of IGF-1 (pre-pro-IGF-1) and the pre-pro precursor of IGF-2 (pre-pro-IGF-2). .

As used herein, an "E domain-deleted fragment" of the pre-pro precursor of IGF (also referred to herein as a "pre precursor") refers to a pre-pro precursor of IGF lacking the E domain. means a fragment obtained by depletion (removal). As used herein, the term "E domain" refers to the domain present at the C-terminus in the pre-pro precursor that is removed by the proprotein convertase. As used herein, the E domain of pre-pro-IGF-1 may start at the amino acid position corresponding to amino acid 119 of SEQ ID NO: 4, 6, or 8, for example. As used herein, the E domain of pre-pro-IGF-2 may start at the amino acid position corresponding to the 92nd amino acid of SEQ ID NO: 14, for example.

The IGF-1 gene contains six exons, and in humans, alternative splicing is known to give rise to three different types of mRNA variants termed IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec. (Candice G. T. Tahimic, et al., frontiers in ENDOCRINOLOGY, 2013, 4(6), p. 1-14). IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec mRNAs have in common that they contain exons 1 or 2 (encoding signal peptide) and exons 3 and 4 (encoding mature IGF). but differ in the C-terminal exon (encoding the E domain), IGF-1 Ea mRNA contains exon 6 but not exon 5, IGF-1 Eb mRNA contains exon 5 but not exon 6, IGF-1Ec mRNA contains both exons 5 and 6.

Pre-pro-IGF-1, as used herein, includes IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec (class 1 IGF-1 Ea, IGF-1 Eb), including the signal peptide encoded by exon 1 , and IGF-1 Ec) proteins, and IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec, including the signal peptide encoded by exon 2 (class 2 IGF-1 Ea, IGF-1 Ec). -1 Eb, and IGF-1 Ec.) proteins, preferably those selected from class 1 IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec proteins. .

In the present invention, pre-pro-IGF-1 may be naturally occurring full-length pre-pro-IGF-1 (IGF-1 Ea, IGF-1 Eb, and IGF-1 Ec). A naturally occurring full-length form of class 1 IGF-1 Ea includes, for example, one consisting of the amino acid sequence shown in SEQ ID NO: 4, which encodes codon-optimized for expression in human cells. Examples of base sequences include those shown in SEQ ID NO:3. A naturally occurring full-length class 1 IGF-1 Eb includes, for example, those consisting of the amino acid sequence shown in SEQ ID NO: 6, which encodes codon-optimized for expression in human cells. Examples of base sequences include those shown in SEQ ID NO:5. Naturally occurring full-length forms of class 1 IGF-1 Ec include, for example, those consisting of the amino acid sequence shown in SEQ ID NO: 8, which encodes codon-optimized for expression in human cells. Examples of base sequences include those shown in SEQ ID NO:7.

In the present invention, a fragment lacking the E domain of pre-pro-IGF-1 (also referred to herein as "IGF-1 w/oE" or "pre-IGF-1") is naturally occurring It may be full-length pre-pro-IGF-1 with the E domain deleted. Such fragments include, for example, those consisting of the amino acid sequence shown in SEQ ID NO: 10, and examples of the base sequence encoding it, codon-optimized for expression in human cells, include SEQ ID NO: 9 can be mentioned.

In the present invention, the target-binding domain includes the naturally occurring full-length pre-pro-IGF-1 or its E domain-deleted fragment, as well as variants thereof, such as SEQ ID NOS: 4, 6, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence shown in 8 or 10 A sequence consisting of amino acid sequences with % sequence identity can also be used.

The above-mentioned naturally occurring full-length pre-pro-IGF-1 or its E domain deleted fragment variant may have an amino acid mutation at any position of the mature IGF-1 portion, e.g. , may have 1-10 or 1-5 amino acid mutations at any position in the mature IGF-1 portion. As used herein, "amino acid mutation" means a change to a natural amino acid sequence, and includes insertion, deletion, substitution, addition, etc. of the amino acid sequence. Amino acid mutations in the mature IGF-1 portion that the above variants may have include, for example, 1, 2, 3, 4 or 5 amino acid mutations in the region corresponding to positions 1 to 5 of SEQ ID NO: 2. Deletion, insertion, and/or substitution, for example, deletion of 1, 2, or 3 amino acids in the region corresponding to positions 1 to 3 of SEQ ID NO: 2, preferably SEQ ID NO: 2 It may be a deletion of 3 amino acids in the region corresponding to positions 1-3 (particularly a deletion of 3 amino acids corresponding to amino acids 1-3 of SEQ ID NO:2). A fragment lacking the E domain of pre-pro-IGF-1 having a deletion of three amino acids in the region corresponding to positions 1 to 3 of SEQ ID NO: 2 has, for example, the amino acid sequence shown in SEQ ID NO: 27. can have. As used herein, the term “region corresponding to 1 to “x”th of SEQ ID NO: 2” refers to 1 to 1 of the amino acid sequence of SEQ ID NO: 2 in the amino acid sequence aligned with the amino acid sequence of SEQ ID NO: 2 Refers to the region aligned to the xth region.

As shown in Example 10 below, the present inventors used a fragment lacking the E domain of pre-pro-IGF-1 (IGF-1 w/oE) as a target binding domain in CAR-T cells ( IGF-1 w/oE-type CAR-T cells) and the mature IGF-1 portion of IGF-1 w/oE (SEQ ID NO: 2) with the first to third three amino acids (GPE) removed (IGF -1 w/oE des1-3) as a target binding domain (IGF-1 w/oE des1-3 type CAR-T cells) were generated, and the latter was more effective against IGFBP3-expressing tumors than the former. It was found to have high antitumor activity.

Therefore, CAR-T cells using pre-pro-IGF-1 having a deletion of 3 amino acids corresponding to the 1st to 3rd amino acids of SEQ ID NO: 2 or a fragment lacking the E domain thereof as a target binding domain may have higher antitumor activity in the presence of IGFBP3 compared to CAR-T cells using pre-pro-IGF-1 without the deletion or a fragment lacking its E domain as the target binding domain. Such CAR-T cells, which have high antitumor activity in the presence of IGFBP3, are used in cancers that secrete IGFBP3 (renal cancer, melanoma, pancreatic cancer, breast cancer, lung adenocarcinoma, head and neck squamous cell carcinoma, etc.). ) may be particularly useful in treating In addition, since IGFBP3 is present in blood, CAR-T cells with high anti-tumor activity in the presence of IGFBP3 may be particularly useful in the treatment of blood cancers and in the case of intravenous administration.

The present inventors also found that IGF-1 w/oE des1-3 with the above-described three amino acid deletions was transformed into IGF-1 w without the deletions, as shown in Example 11 below. It was found to have IGFBP3-binding ability more than three times that of /oE. A previous study reported that deletion of the above three amino acids markedly reduced the ability of IGF-1 to bind to IGFBP3 (Sara VR et al., Annals New York Academy of Sciences, 1993, 692 :183-91, Ballard F et al., Int. J. Biochem. Cell Biol., 1996, 28(10):1085-1087), so this result was unexpected.

The present inventors further demonstrated that IGF-1 w/oE des1-3 type CAR-T cells are not only activated by binding to IGF1R, but also bind to IGFBP3, as shown in Example 12 below. I found that it is also activated by

Therefore, without being bound by theory, pre-pro-IGF-1 having a deletion of three amino acids corresponding to amino acids 1 to 3 of SEQ ID NO: 2, or a fragment thereof lacking the E domain, is used in the target binding domain. CAR-T cells used as T cells are believed to be activated not only by binding to IGF1R but also by binding to IGFBP3, thereby exhibiting high antitumor activity in the presence of IGFBP3.

In the present invention, pre-pro-IGF-2 may be naturally occurring full-length pre-pro-IGF-2. A naturally occurring full-length form of pre-pro-IGF-2 includes, for example, one consisting of the amino acid sequence shown in SEQ ID NO: 14, which encodes codon-optimized for expression in human cells. Examples of such nucleotide sequences include those shown in SEQ ID NO: 13.

In the present invention, a fragment lacking the E domain of pre-pro-IGF-2 (also referred to herein as "IGF-2 w/oE" or "pre-IGF-2") is naturally occurring It may be full-length pre-pro-IGF-2 with the E domain deleted. Such fragments include, for example, those consisting of the 1st to 91st amino acid sequences of SEQ ID NO:14.

In the present invention, the target-binding domain includes the naturally occurring full-length pre-pro-IGF-2 or its E domain-deleted fragment, as well as variants thereof, such as the amino acid shown in SEQ ID NO: 14. have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence consisting of an amino acid sequence, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence shown in the 1st to 91st amino acid sequences of SEQ ID NO: 14 A sequence consisting of amino acid sequences having %, at least 97%, at least 98%, or at least 99% sequence identity can also be used.

The above-mentioned naturally occurring full-length pre-pro-IGF-2 or its E domain deleted fragment variant may have an amino acid mutation at any position of the mature IGF-2 portion, e.g. , may have 1-10 or 1-5 amino acid mutations at any position in the mature IGF-2 portion. Amino acid mutations in the mature IGF-2 portion that the above variants may have include, for example, 1, 2, 3, 4, 5, or 6 in the region corresponding to positions 1 to 6 of SEQ ID NO: 12. deletions, insertions, and/or substitutions of single amino acids. In the present specification, the “region corresponding to positions 1 to 6 of SEQ ID NO: 12” refers to regions 1 to 6 of the amino acid sequence of SEQ ID NO: 12 in the amino acid sequence aligned with the amino acid sequence of SEQ ID NO: 12. refers to the region that is aligned with the region of

When the CAR containing the above IGF pre-pro precursor or fragment thereof as a target binding domain is expressed in target cells, after the signal peptide in the IGF pre-pro precursor or fragment thereof is removed in the cell Although CAR may be displayed on the cell surface, for convenience herein the above pre-pro precursor of IGF or fragment thereof containing the signal peptide is referred to as the "target binding domain".

In the present invention, the target-binding domain itself may bind to IGF1R. The target binding domain may also bind IGF1R after the signal peptide has been removed from it.

The binding ability of the target-binding domain or the target-binding domain from which the signal peptide has been removed to IGF1R is intended to have a KD value of, for example, 100 nM or less, preferably 10 nM or less, more preferably 5 nM or less. This binding capacity may be a relatively weak binding compared to the binding capacity between the antigen and the antibody.

In the present invention, the target-binding domain can increase the CAR expression rate in a cell population into which a polynucleotide encoding a CAR containing it has been introduced, compared to when mature IGF is used as the target-binding domain. As used herein, the term "CAR expression rate" in a cell population means the ratio of specific cells (eg, T cells) expressing CAR on the cell surface in the cell population. The CAR expression rate can be, for example, the CAR expression rate 1 to 10 days (eg, 1, 3, 6, or 10 days) after gene (polynucleotide encoding CAR) introduction. The CAR expression rate is determined, for example, by staining a cell population with a fluorescently labeled antibody that detects a T cell marker and a fluorescently labeled antibody that detects a CAR, and subjecting the cell population to flow cytometry, and confirming that both the T cell marker and CAR are positive. It can be determined by measuring the cell rate as the CAR expression rate. A cell population transfected with a polynucleotide encoding a CAR having the same CAR expression rate in a cell population transfected with a polynucleotide encoding a CAR containing a certain target binding domain, except that mature IGF is used instead of the target binding domain When the CAR expression rate is increased (e.g., 10% or more, statistically significant increase) compared to the CAR expression rate in the cell population into which a polynucleotide encoding a CAR containing the target binding domain has been introduced It can be determined that the CAR expression rate in .

In the present invention, the CAR protein can optionally contain an "extracellular spacer domain" between the extracellular target-binding domain and the transmembrane domain. The extracellular spacer domain is desirably a sequence that promotes binding of CAR to an antigen and enhances intracellular signal transduction. For example, an antibody Fc fragment, or a fragment or derivative thereof, an antibody hinge region, or a fragment or derivative thereof, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof can be used.

In one aspect of the present invention, the extracellular spacer domain includes (i) IgG4 hinge, CH2, and CH3 regions, (ii) IgG4 hinge region, (iii) IgG4 hinge and CH2, and (iv) CD8a hinge region. , (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vii) the hinge and CH2 of IgG1, or combinations thereof. For example, as the hinge region of IgG1, one having the following amino acid sequence (SEQ ID NO: 15) can be preferably used, but it is not limited to this.

In addition, as the CH2 region of IgG1, one having the amino acid sequence shown in SEQ ID NO: 16, and as the CH3 region, one having the amino acid sequence shown in SEQ ID NO: 17 can be preferably used.

As a preferred embodiment, the extracellular spacer domain can use the hinge, CH2, and CH3 regions of human IgG1 or a portion thereof.

In a preferred embodiment, the extracellular spacer domain is composed of (i) human IgG1 hinge region alone (SEQ ID NO: 15), (ii) human IgG1 hinge region (SEQ ID NO: 15) and CH2 region (SEQ ID NO: 16) and CH3 region (SEQ ID NO: 17), (iii) hinge region (SEQ ID NO: 15) and CH3 region (SEQ ID NO: 17) of human IgG1, (iv) CH3 region alone (SEQ ID NO: 17). can.

As one aspect of the present invention, a spacer sequence represented by the formula (G4S)n can be used as an artificial spacer sequence for use in the extracellular spacer domain. In the formula, n is 1-10, preferably n=3. A spacer having such a spacer sequence is sometimes called a peptide linker. Peptide linkers suitably used in the art can be appropriately used in the present invention. In this case, the configuration and chain length of the peptide linker can be appropriately selected within the range that does not impair the function of the resulting CAR protein.

The extracellular spacer domain is not particularly limited, but can be appropriately selected from those exemplified above, or further modified based on the common technical knowledge in the field, and used for the present invention.

The extracellular spacer domain can be present between the target-binding domain and the transmembrane domain by ligating the base sequences encoding the amino acid sequences of the respective domains, inserting them into a vector, and expressing them in host cells. can be done. Alternatively, the extracellular spacer domain can be modified using a previously prepared polynucleotide encoding a plasmid CAR protein as a template.

Modification of the extracellular spacer domain, for example, improves the CAR gene expression rate in host cells of CAR-T cells introduced with a polynucleotide encoding CAR, signal transduction, cell senescence, distribution to tumors, antigen recognition or in It is useful when considering the effect on in vivo activity.

In the present invention, a CAR protein comprises an extracellular domain comprising a target binding domain and optionally an extracellular spacer domain, a transmembrane domain, an intracellular domain comprising an intracellular signaling domain and optionally a co-stimulatory domain. As is well known in the art, the "transmembrane domain" is a domain having affinity for the lipid bilayer that constitutes the cell membrane, whereas both the extracellular domain and the intracellular domain are hydrophilic domains. be.

The transmembrane domain is not particularly limited as long as the CAR protein can exist on the cell membrane and does not impair the functions of the target binding domain and the intracellular signaling domain, but it is a polypeptide derived from the same protein as the co-stimulatory domain described later. may serve as a transmembrane domain. Transmembrane domains such as CD28, CD3ε, CD8α, CD3, CD4 or 4-1BB can be used as transmembrane domains. For example, the transmembrane domain can be human CD28 (Uniprot No.: P10747 (153-179)). Specifically, those having the amino acid sequence encoded by the nucleotide sequence of NCBI Accession No.: NM_006139.3 (679-759) can be preferably used as the transmembrane domain.

CAR proteins can optionally contain a "co-stimulatory domain". A co-stimulatory domain specifically binds a co-stimulatory ligand, thereby triggering a co-stimulatory response by the cell, such as, but not limited to, CAR-T cell proliferation, cytokine production, functional differentiation, and target cell death. is mediated. Costimulatory domains include, for example, CD27, CD28, 4-1BB (CD137), CD134 (OX40), Dap10, CD27, CD2, CD5, CD30, CD40, PD-1, ICAM-1, LFA-1 (CD11a/ CD18), TNFR-1, TNFR-II, Fas, Lck can be used. For example, the co-stimulatory domain can be human CD28 (Uniprot No.: P10747 (180-220)) or 4-1BB (GenBank: U03397.1). Specifically, those having the amino acid sequence encoded by the nucleotide sequence of NCBI Accession No.: NM_006139.3 (760-882) can be preferably used as co-stimulatory domains.

When using both the transmembrane domain and the co-stimulatory domain derived from human CD28, for example, one having the amino acid sequence shown in SEQ ID NO: 18 can be used.

The CAR protein contains an "intracellular signaling domain". Intracellular signaling domains transmit the signals necessary for the exertion of immune cell effector functions. The intracellular signaling domain can be, for example, the human CD3 zeta chain, FcγRIII, FcεRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof. For example, the intracellular signaling domain can use the human CD3 zeta chain (eg nucleotides 299-637 of NCBI Accession No. NM_000734.3). Specifically, one having the amino acid sequence shown in SEQ ID NO: 19 can be preferably used as the intracellular signaling domain.

For the purpose of promoting the secretion of the CAR protein, the N-terminus of the protein appropriately contains a leader sequence that guides protein translocation during or after translation. Examples of useful leader sequences that can be suitably used in the present invention include, but are not limited to, human immunoglobulin (Ig) heavy chain signal peptide, CD8α signal peptide, or human GM-CSF receptor α signal peptide. be able to. Ig heavy chain signal peptides derived from, for example, IgG1, IgG2, IgG3, IgA1, and IgM can be preferably used.

In the present invention, the CAR protein can impart cytolytic activity against IGF1R-expressing cells to cells expressing the CAR protein.

The target polynucleotide can be easily produced according to a conventional method. It is possible to obtain the nucleotide sequence encoding the amino acid sequence from the NCBI RefSeq ID indicating the amino acid sequence of each domain or the GenBank Accession number, and the present invention uses standard molecular biological and / or chemical procedures. of polynucleotides can be generated. For example, based on these nucleotide sequences, nucleic acids can be synthesized, and DNA fragments obtained from a cDNA library using the polymerase chain reaction (PCR) are combined to produce the polynucleotide of the present invention. be able to.

Therefore, a polynucleotide encoding a CAR protein can be produced by ligating polynucleotides encoding each of the above domains, and introducing this polynucleotide into a suitable cell produces a genetically modified cell. be able to. A CAR protein can also be produced by using, as a template, a polynucleotide encoding an existing CAR protein having the same structural components other than the target-binding domain, and recombining the target-binding domain according to a conventional method.

Furthermore, depending on the purpose, one or more domains, such as the extracellular spacer domain, can be modified using the inverse PCR (iPCR) method, etc., using a polynucleotide encoding an existing CAR protein as a template. Techniques for modifying extracellular spacer domains are described, for example, in Oncoimmunology, 2016, Vol.5, No.12, e1253656.

The method of introducing polynucleotides for producing genetically modified cells may be any method that is commonly used, and is not particularly limited. When introducing using a vector, vectors that can be used include, but are not limited to, lentiviral vectors, retroviral vectors, foamy viral vectors, adeno-associated viral vectors, and the like. In addition, introduction of polynucleotides can be performed by non-viral methods such as transposon methods. For transposon methods, plasmid transposons can be used, and the sleeping beauty transposon system (e.g. Huang X, Guo H, et al. Mol Ther. 2008; 16: 580-9; Singh H, Manuri PR, et al. Cancer Res. 2008; 68: 2961-71; Deniger DC, Yu J, et al. PLoS One. 2015; 10: e0128151; Singh H, Moyes JS, et al. Cancer Gene Ther. Hou X, Du Y, et al. Cancer Biol Ther. 2015; 16: 8-16; Singh H, Huls H, et al. Immunol Rev. 2014; 257: 181-90; and Maiti SN, Huls H, et al 2013; 36: 112-23) or the piggyBac transposon system (Nakazawa Y, Huye LE, et al. J Immunother. 2009; 32: 826-36; Galvan DL, Nakazawa Y, et al. J Immunother 2009; 32: 837-44; Nakazawa Y, Huye LE, et al. Mol Ther. 2011; 19: 2133-43; Huye LE, Nakazawa Y, et al. S, Nakazawa Y, et al. J Vis Exp. 2012; (69): e4235; Nakazawa Y, Saha S, et al. J Immunother. 2013; 36: 3-10; Saito S, Nakazawa Y, et al. 2014; 16: 1257-69; and Nakazawa et al. Journal of Hematology & Oncology (2016) 9:27) can be preferably used.

When the piggyBac transposon system is used, typically, a plasmid carrying a gene encoding piggyBac transposase (referred to herein as a piggyBac plasmid) and a polynucleotide encoding a CAR protein contain the piggyBac inverted repeat sequence. A plasmid comprising a structure flanked by the two is introduced (transfected). Various methods such as electroporation, nucleofection, lipofection, and calcium phosphate method can be used for transfection. Both plasmids can contain poly-A added leader sequences, reporter genes, selectable marker genes, enhancer sequences and the like.

The device used for electroporation is not limited, but for example, 4D-Nucleofector (Lonza Japan Co., Ltd.), NEPA21 (Neppa Gene Co., Ltd.), Maxcyte GT (Maxcyte, Inc), etc. can be used. , can be operated according to the respective instructions for use.

By the above method, it is possible to introduce genes into 1×10 ⁶ to 2×10 ⁷ cells.

Cells into which the polynucleotide is introduced include mammals, for example, human-derived cells, or non-human mammal-derived T cells such as monkeys, mice, rats, pigs, cows, and dogs, or cell populations containing T cells. Cells that are available and release cytotoxic proteins (perforin, granzyme, etc.) are preferably used. Specifically, for example, a cell population containing T cells, T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), and NK-T cells can be used. Furthermore, cells capable of differentiating into these cells include various stem cells such as ES cells and iPS cells. T cells include CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells, or tumor infiltrating lymphocytes. Cell populations containing T cells and T cell progenitor cells include PBMCs. The above-mentioned cells may be those collected from living organisms, those obtained by expanding culture thereof, or those established as cell lines. When cells expressing the manufactured CAR or cells differentiated from the cells are transplanted into a living body, it is desirable to introduce the nucleic acid into the living body itself or cells collected from the same kind of living body.

As T cells into which a polynucleotide has been introduced for the genetically modified cells of the present invention and which are used for adoptive immunotherapy, T cells expected to have a sustained anti-tumor effect, such as stem cell memory T cells, should be used. can be done. Analysis of stem cell memory T cells can be easily confirmed according to a conventional method, for example, according to (Yang Xu, et al. Blood. 2014; 123:3750-3759).

In one embodiment, stem cell memory T cells can be, for example, CD45R0-, CD62L+, CD45RA+ and CCR7+ T cells.

The present invention also provides a vector comprising the above-described polynucleotide of the present invention.

The present invention also provides genetically modified cells into which the polynucleotide of the present invention or the vector of the present invention has been introduced. The genetically modified cells of the present invention express on the cell membrane a CAR protein that uses the pre-pro precursor of IGF or its E domain-deleted fragment as the target binding domain. This target-binding domain contains a signal peptide and mature IGF, and not only IGF1R, but also IGF1R/INSR-A and IGF1R/INSR-B hybrid receptors, before or after removal of the signal peptide. can combine. Therefore, the genetically modified cells of the present invention have a stronger anti-tumor effect than drugs that target only IGF1R-expressing tumor cells.

The present invention further provides a method for producing a CAR protein-expressing cell, comprising introducing the polynucleotide of the present invention or the vector of the present invention into a cell.

Cultivation and expansion of CAR protein-expressing cells are not particularly limited. Non-specific or CAR-specific stimuli can be applied to cells. Although the method of cell stimulation is not limited, for example, anti-CD3 antibody and/or anti-CD28 antibody stimulation can be used as non-specific cell stimulation, and K562 etc. can be used as CAR-specific stimulation. CAR-specific stimulation using artificial antigen-presenting cells (aAPCs) that express CAR-binding antigen molecules or co-stimulatory factors in tumor cell lines can be used. The culturing conditions here are not particularly limited, but for example, culturing at 37° C. under 5% CO ₂ for 1 to 10 days is suitable.

In the method of the present invention described above, introduction into cells is preferably performed by the transposon method, although this is not a limitation. In addition, as the transposon method, any of the transposon system described above and other systems suitable for the present invention may be used, and although not particularly limited, the method of the present invention is preferably performed using the piggyBac method. can be implemented.

In another embodiment of non-specific stimulation, a cell population containing T cells is stimulated with one or more types of viral peptide antigens, and then treated to inactivate virus proliferation by a conventional method as feeder cells. , can promote the activation of CAR-introduced cells. Viral peptide antigens used are, for example, AdV antigenic peptide mixtures, CMV antigenic peptide mixtures, EBV antigenic peptide mixtures, or combinations thereof.

In another embodiment of specific stimulation, as described in WO 2020/085480, PBMCs derived from the same individual from which the CAR-expressing cells are derived are cultured in a medium containing human IL-4 and GM-CSF. co-culture of immature dendritic cells and CAR-expressing cells produced by the method.

In addition, culture can be performed in the presence of one or more cytokines for the purpose of increasing cell viability/proliferation rate, and is preferably cultured in the presence of cytokines such as IL-7 and IL-15. be.

The present invention further provides a kit for producing CAR protein-expressing cells targeting IGF1R-expressing cells, which contains the vector of the present invention. The kit of the present invention can appropriately contain reagents, buffer solutions, reaction vessels, instructions for use, and the like necessary for producing CAR protein-expressing cells. The kit of the present invention can be suitably used for producing the above-described genetically modified cells of the present invention.

The cells of the present invention are activated by intracellular signal transmission by triggering a receptor-specific immune response against target cells expressing IGF1R on their surface. Activation of CAR-expressing cells varies depending on the host cell type and the intracellular domain of CAR. can be confirmed as an index. Release of cytotoxic proteins (perforin, granzyme, etc.) leads to destruction of cells expressing the receptor.

The genetically modified cells of the present invention can be used as therapeutic agents for diseases involving IGF1R-expressing cells. Therefore, the present invention provides therapeutic agents for diseases involving IGF1R-expressing cells, including the genetically modified cells of the present invention. The disease expected to be treated with the therapeutic agent of the present invention is not limited as long as it is a disease that shows sensitivity to the cells. For example, diseases associated with cells expressing IGF1R on the cell membrane, such as cancer , For example, hematological tumors such as leukemia, multiple myeloma, and lymphoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, e.g., lung adenocarcinoma, etc.), head and neck squamous cell carcinoma, liver cancer, hepatocytes cancer, pancreatic cancer, colorectal cancer, colorectal cancer, colon cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, kidney cancer, adrenal cancer , solid tumors such as melanoma, neuroendocrine tumors, and retinoblastoma, and sarcomas (eg, Ewing's sarcoma, osteosarcoma, etc.).

One aspect of the therapeutic agent of the present invention is an anticancer agent against IGF1R-expressing tumor cells such as the above tumors. The therapeutic agent or anticancer agent of the present invention can be used alone, but can also be used in combination with drugs and/or treatments with different mechanisms.

Therefore, the therapeutic agent of the present invention can be in the form of a pharmaceutical composition alone or in combination with other active ingredients. The therapeutic agents or pharmaceutical compositions of the invention can be administered locally or systemically. Specific administration modes include, but are not limited to, intravenous administration, intratumoral administration, and intrathecal administration. The preferred mode of administration is intravenous administration, for example for the treatment of leukemia. In addition to the therapeutic agent of the present invention and other active ingredients, pharmaceutical compositions may contain carriers, excipients, buffers, stabilizers, etc. commonly used in the art, depending on the mode of administration. can. In one aspect of the invention, a pharmaceutical composition can comprise, for example, a therapeutic agent of the invention and a pharmaceutically acceptable carrier.

The dose of the therapeutic agent of the present invention varies depending on the patient's body weight, ^age , severity of disease, etc., and ^is not particularly limited. It can be administered once to several times a day, every 2 days, every 3 days, every week, every 2 weeks, every month, every 2 months, every 3 months, within the range of /kg body weight.

Subjects to which the therapeutic agent or pharmaceutical composition of the present invention is administered include humans, domestic animals (horses, cows, sheep, goats, pigs, etc.), pets (dogs, cats, rabbits, etc.), experimental animals (mice, rats, etc.). Any mammals including monkeys, etc.) can be mentioned, but humans are preferred.

CD45RA + CD62L + cells (stem cell memory T cells) in CD4 + CAR-T cells and/or CD8 + CAR-T cells contained in the therapeutic agent or pharmaceutical composition of the present invention are, for example, 70% or more or 80% or more There may be.

The present invention further comprises administering to a patient a therapeutically effective amount of the genetically modified cells, therapeutic agents or pharmaceutical compositions of the present invention described above, diseases involving IGF1R-expressing cells, such as those expressing IGF1R on the cell membrane Diseases related to cells, e.g. hematological tumors such as leukemia, multiple myeloma, lymphoma, lung cancer (e.g. small cell lung cancer, non-small cell lung cancer, e.g. lung adenocarcinoma, etc.), head and neck squamous cell carcinoma, liver cancer , hepatocellular carcinoma, pancreatic cancer, colorectal cancer, colorectal cancer, colon cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, renal cancer , solid tumors such as adrenal cancer, melanoma, neuroendocrine tumors, and retinoblastoma, and cancers such as sarcomas (eg, Ewing's sarcoma, osteosarcoma, etc.). A therapeutically effective amount and dosage regimen can be determined as appropriate, taking into consideration various factors such as those described above.

Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1 Preparation of mature IGF-1, IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, or IGF-1 w/oE type CAR expression plasmids]
Mature IGF-1, pre-pro-IGF-1 (IGF-1 Ea, IGF-1 Eb, IGF-1 Ec), or a fragment lacking the E domain of pre-pro-IGF-1 (IGF-1 w /oE) as the target binding domain (mature IGF-1, IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/oE-type CARs, respectively) (Fig. 1).

Plasmids for expressing these (mature IGF-1, IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/o E-type CAR expression plasmids, respectively) were prepared in International Publication No. 2020/085480. It was prepared by the method described in .

　Figures 2 to 6 show the vector maps of each of the prepared plasmids.

The mature IGF-1 type CAR expression plasmid (SEQ ID NO: 20) comprises a leader sequence (30th to 86th of SEQ ID NO: 20), human mature IGF-1 (87th to 296th of SEQ ID NO: 20), a spacer (SEQ ID NO: 20) 297-1004), CD28 (1005-1208 of SEQ ID NO:20), and CD3ζ (1209-1547 of SEQ ID NO:20) (FIG. 2).

IGF-1 Ea, Eb, Ec, w/o E-type CAR expression plasmids (SEQ ID NOS: 21, 22, 23, 24, respectively) were replaced with human IGF-1 Ea, Eb, Ec, respectively, in place of human mature IGF-1. It is the same as the mature IGF-1 type CAR expression plasmid except that it contains a base sequence encoding w/oE (Figs. 3 to 6).

The base sequences of human mature IGF-1, IGF-1 Ea, Eb, Ec, and w/oE used in the above CAR expression plasmid are shown in SEQ ID NOs: 1, 3, 5, 7, and 9, respectively. Also, the amino acid sequences encoded by them are shown in SEQ ID NOs: 2, 4, 6, 8 and 10, respectively.

[Example 2 Culture and expansion of mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells]
Using the CAR expression plasmid prepared in Example 1, CAR-T cells using mature IGF-1 or IGF-1 Ea as the target binding domain (respectively, mature IGF-1 CAR-T cells and IGF-1 Ea CAR-T cells) were generated, cultured and expanded.

<2-1 Isolation of PBMC (Day 0)>
Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy volunteer donors using Ficoll-PaquePlus (GE Healthcare, Chicago, Ill.).

<2-2 Gene transfer and culture (Days 0-3)>
7.5 μg of the CAR expression plasmid (mature IGF-1 type CAR expression plasmid or IGF-1 Ea type CAR expression plasmid) prepared in Example 1 for 2×10 ⁷ PBMCs isolated in 2-1 above, Super PiggyBac 7.5 μg of transposase expression vector (Super PiggyBac Transposase Expression Vector; System Biosciences) and 100 μl of P3 Primary Cell Nucleofector ^TM solution (Lonza, Basel, Switzerland) supplemented with Supplement 1 were added, and 4D-Nucleofector apparatus (program FL-115) was used. electroporation was performed (Day 0).

Electroporated PBMCs were added to 10 ng/mL human IL-7 (Miltenyi Biotec, Bergisch Gladbach, Germany), 5 ng/mL human IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 5% artificial serum Animal-free; Cell Science & Technology Institute Inc., Japan) containing ALyS ^TM 705 culture medium (Cell Science and Technology Institute Inc., Japan), seeded in a 24-well plate, cultured at 37°C, 5% CO ₂ for 3 days (Days 0-3).

<2-3 Generation of immature dendritic cells (Days 0-3)>
3×10 ⁶ PBMCs isolated in 2-1 above were suspended in ALyS ^™ 705 culture medium containing 10 ng/mL human IL-4 and 10 ng/mL human GM-CSF, and plated on a 6-well G-REX ^R cell culture plate ( Wilson Wolf, Saint Paul, Minn.) and cultured at 37° C., 5% CO ₂ for 3 days (Days 0-3).

<2-4 Stimulation and culture of gene-introduced cells with immature dendritic cells (Days 3-10)>
Remove the supernatant from the G-REX ^R cell culture plate containing immature dendritic cells cultured for 3 days in 2-3 above, add 10 ng/mL human IL-7, 5 ng/mL human IL-15 and 5% artificial serum ( Artificial serum Animal-free) containing ALyS ^™ 705 medium was added. The transfected cells cultured for 3 days in 2-2 above were added to the G-REX ^R cell culture plate (Day 3), and the culture was continued for 7 days. During the culture period, half of the medium was replaced every 3 to 4 days, and IL-7 and IL-15 were added to final concentrations of 10 ng/mL and 5 ng/mL, respectively.

<2-5 Generation of control T cells (Days 0-10)>
1.5×10 ⁶ PBMCs isolated in 2-1 above were suspended in ALyS ^™ 705 culture medium containing 5 ng/mL IL-15 and 5% artificial serum, and anti-CD3 antibody and anti-CD28 antibody (Miltenyi Biotec, Auburn, CA) and seeded on a 24-well non-treating plate coated with 37°C and 5% CO ₂ (Day 0).

　On the second day after the start of culture (Day 2), the cells were transferred to a 24-well treat plate and cultured for another 8 days. During the culture period, the medium was changed every 3-4 days.

[Example 3 Evaluation of CAR expression rate in mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells (Days 1, 3, 6 and 10)]
The CAR expression rate in mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells obtained in Example 2 was evaluated by flow cytometry analysis.

Mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells cultured until days 1, 3, 6 and 10 after gene transfer in Example 2 (Days 1, 3, 6 and 10) were Stained with FITC (fluorescein isothiocyanate)-labeled anti-human IgG antibody and APC-labeled anti-CD3 antibody. Fluorescein (FITC) AffiniPure F(ab') ₂ Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch Inc. West Grove, PA, USA) was used as the FITC-labeled anti-human IgG antibody. CD3 Antibody, anti-human, APC (Miltenyi Biotec, Auburn, Calif.) was used as the APC-labeled anti-CD3 antibody. A FITC-labeled anti-human IgG antibody binds to the spacer portion of CAR and can detect CAR-expressing cells. APC-labeled anti-CD3 antibodies can detect T cells.

The stained cells were subjected to a flow cytometer BD Accuri ^™ C6 Plus (BD Biosciences San Jose, CA) and analyzed using Flowjo (BD Biosciences San Jose, CA), and the CAR positive CD3 positive cell rate was measured as the CAR expression rate. .

The results are shown in FIG.
In mature IGF-1 type CAR-T cells, a high CAR expression rate was observed the day after gene transfer (Day 1), but the CAR expression rate decreased stepwise during culture, and 10 days after gene transfer (Day 10). ) lost CAR expression.

On the other hand, IGF-1 Ea-type CAR-T cells showed a higher CAR expression rate than mature IGF-1-type CAR-T cells on the day after transfection (Day 1), and continued until 6 days after transfection (Day 6). Although a gradual decrease in the expression rate was observed, the CAR expression rate was maintained at 20% or more at 10 days after gene transfer (Day 10).

These results suggest that generating CAR-T cells with pre-pro-IGF-1 rather than mature IGF-1 as the target binding domain yields CAR-T cells with improved CAR expression. indicates

[Example 4 Comparison of antitumor cell activity between mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells]
In order to evaluate the anti-tumor cell activity of the mature IGF-1 type CAR-T cells and IGF-1 Ea type CAR-T cells prepared in Example 2, a co-culture test of CAR-T cells and tumor cells was performed. .

CAR-T cells and control T cells cultured until Day 10 in Example 2 were collected and cultured in cytokine-free 5% artificial serum-containing ALyS ^™ 705 medium.

Two days after the initiation of culture, 0.5×10 ⁶ human acute monocytic leukemia cell line THP-1 (American Type Culture Collection) was added to each well of a 24-well plate as tumor cells (target (T)). Then, the cultured CAR-T cells or control T cells (effector (E)) were treated with the 24 They were added to the wells of the well plate and co-cultured at 37°C under 5% CO ₂ . RPMI1640 medium supplemented with 10% FBS was used as the culture medium.

Cells were collected 4 days after the start of co-culture, and APC-labeled anti-CD3 antibody (detected T cells), FITC-labeled anti-CD33 antibody (detected tumor cells), and 7-Amino-Actinomycin D ; 7-AAD) (detects dead cells). CD33 Antibody, anti-human, FITC (Miltenyi Biotec, Auburn, CA) was used as the FITC-labeled anti-CD33 antibody, and CD3 Antibody, anti-human, APC (Miltenyi Biotec, Auburn, CA) was used as the APC-labeled anti-CD3 antibody. ) was used.

Stained cells were run on a flow cytometer BD Accuri ^™ C6 Plus (BD Biosciences San Jose, Calif.) and analyzed using Flowjo (BD Biosciences San Jose, Calif.).

The results are shown in Figures 8-10.
IGF-1 Ea-type CAR-T cells (Fig. 9) outperformed control T cells (Fig. 8) at all E:T ratios tested (expressed as percent CD3-negative CD33-positive cells). showed ability to kill tumor cells. On the other hand, mature IGF-1 type CAR-T cells (Fig. 10) did not reduce the tumor cell rate compared to control T cells (Fig. 8) at E:T ratios of 1:1 and 1:2, and killed tumor cells. showed no ability.

These results suggest that generating CAR-T cells with pre-pro-IGF-1 as the target binding domain rather than mature IGF-1 results in CAR-T cells with higher anti-tumor cell activity. indicates that

[Example 5 Culture and amplification of IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/oE type CAR-T cells, and evaluation of CAR expression rate and antitumor cell activity]
<5-1 Culture and amplification of CAR-T cells>
A CAR using IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/oE as a target binding domain is prepared in the same manner as in Example 2 using the CAR expression plasmid prepared in Example 1. -T cells (IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/oE type CAR-T cells, respectively) were generated, cultured and expanded. At the same time, control T cells (T cells activated with anti-CD3 antibody and anti-CD28 antibody) were cultured in the same manner as in Example 2.

<5-2 Evaluation of CAR expression rate>
CAR expression rate in IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, IGF-1 w/oE type CAR-T cells cultured until day 10 after gene transfer in 5-1 It was evaluated by flow cytometry analysis according to the method described in Example 3.

The results are shown in FIG.
IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/oE CAR-T cells all maintained a high CAR expression rate of over 15% on Day 10. In particular, IGF-1 w/oE CAR-T cells showed the highest CAR expression rate among the 4 types of CAR-T cells.

IGF-1 w/o E-type CAR-T cells target a peptide from pre-pro-IGF1 with the E domain deleted (i.e., a peptide containing only the signal peptide domain and the mature IGF-1 domain) to target CAR-T. It is used as a binding domain. Therefore, the experimental results of this example show that the CAR expression rate was improved by generating CAR-T cells using the signal peptide and mature IGF-1 portion of pre-pro-IGF-1 as the target binding domain. We show that CAR-T cells can be obtained and that the E domain is dispensable for improving CAR expression rates.

<5-3 Evaluation of antitumor cell activity>
In order to evaluate the anti-tumor cell activity of IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/oE type CAR-T cells prepared in 5-1, by the same method as in Example 4 , conducted a co-culture study of CAR-T cells and tumor cells.

CAR-T cells and control T cells cultured up to Day 10 on 5-1 were collected and cultured in cytokine-free 5% artificial serum-containing ALyS ^™ 705 medium.

Same as Example 4, except that CAR-T cells or control T cells were added to the tumor cells at an E:T ratio of 2:1, 1:1, or 1:2 two days after the start of culture. were co-cultured and immunostained.

To quantify the number of T cells and tumor cells, CountBright absolute Counting Beads (Invitrogen, Carlsbad, CA) were added to the stained cells followed by flow cytometer BD Accuri ^™ C6 Plus (BD Biosciences San Jose, CA). and analyzed using Flowjo (BD Biosciences San Jose, Calif.).

Results are shown in FIGS.
IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/o E CAR-T cells all showed significant tumor cell number reduction at all co-culture ratios tested. In particular, IGF-1 w/oE type CAR-T cells exhibited the strongest tumor suppressive effect among the four types of CAR-T cells (Fig. 12).

　IGF-1 Ea, IGF-1 Eb, IGF-1 Ec, and IGF-1 w/o E-type CAR-T cells all increased after co-culture. In particular, IGF-1 w/oE type CAR-T cells were significantly increased (Fig. 13).

These results suggest that generating CAR-T cells with the signal peptide of pre-pro-IGF-1 and the mature IGF-1 portion as the target binding domain results in CAR-T cells with higher anti-tumor cell activity. , indicating that the E domain is dispensable for the anti-tumor activity of CAR-T cells.

[Example 6 Preparation of pre-pro-IGF-2 type CAR expression plasmid]
A plasmid for expressing a CAR using pre-pro-IGF-2 as a target-binding domain (pre-pro-IGF-2 type CAR expression plasmid) was constructed by the method described in WO2020/085480.

A vector map of the constructed plasmid is shown in FIG.
The pre-pro-IGF-2 type CAR expression plasmid (SEQ ID NO: 25) has a leader sequence (30 to 86 of SEQ ID NO: 25), human pre-pro-IGF-2 (87 to 626 of SEQ ID NO: 25), a spacer (627th to 1334th of SEQ ID NO: 25), CD28 (1335th to 1538th of SEQ ID NO: 25), and CD3ζ (1539th to 1877th of SEQ ID NO: 25) (Fig. 14) .

　The base sequence of pre-pro-IGF-2 used in the above CAR expression plasmid is shown in SEQ ID NO: 13. Also, the amino acid sequence encoded by it is shown in SEQ ID NO:14.

[Example 7 Culture/amplification of pre-pro-IGF-2 type CAR-T cells and evaluation of CAR expression rate/antitumor cell activity]
<7-1 Culture and amplification of CAR-T cells>
Using the CAR expression plasmid prepared in Example 6, CAR-T cells using pre-pro-IGF-2 as a target binding domain (pre-pro-IGF-2 type CAR- T cells) were generated, cultured and expanded. At the same time, control T cells (T cells activated with anti-CD3 antibody and anti-CD28 antibody) were cultured in the same manner as in Example 2.

<7-2 Evaluation of CAR expression rate>
CAR expression rate in pre-pro-IGF-2 type CAR-T cells cultured until day 10 (Day 10) after gene transfer in 7-1 was evaluated by flow cytometry analysis according to the method described in Example 3. bottom.

The results are shown in FIG.
Pre-pro-IGF-2 type CAR-T cells showed a high CAR expression rate (14.5%) on Day 10. This result indicates that CAR-T cells with maintained CAR expression rate can be obtained by generating CAR-T cells using pre-pro-IGF-2 as the target binding domain.

<7-3 Evaluation of antitumor activity>
In order to evaluate the anti-tumor cell activity of the pre-pro-IGF-2 type CAR-T cells prepared in 7-1, a co-culture test of CAR-T cells and tumor cells was conducted in the same manner as in Example 4. gone.

CAR-T cells and control T cells cultured until Day 10 in 7-1 were collected and cultured in cytokine-free 5% artificial serum-containing ALyS ^™ 705 medium.

Same as Example 4, except that CAR-T cells or control T cells were added to the tumor cells at an E:T ratio of 2:1, 1:1, or 1:2 two days after the start of culture. were co-cultured and immunostained. At the same time, similar manipulations were performed without adding CAR-T cells or control T cells to the tumor cells.

To quantify the number of T cells and tumor cells, CountBright absolute Counting Beads (Invitrogen, Carlsbad, CA) were added to the stained cells followed by flow cytometer BD Accuri ^™ C6 Plus (BD Biosciences San Jose, CA). and analyzed using Flowjo (BD Biosciences San Jose, Calif.).

The results are shown in Figures 16 and 17.
Pre-pro-IGF-2-type CAR-T cells (Fig. 17) reduced the tumor cell percentage compared to control T cells (Fig. 16) at all E:T ratios tested, and demonstrated greater ability to kill tumor cells. Indicated. This result indicates that CAR-T cells with high anti-tumor cell activity can be obtained by generating CAR-T cells using pre-pro-IGF-2 as the target binding domain.

[Example 8 Evaluation of antitumor cell activity of IGF-1 w/oE type CAR-T cells]
Breast cancer cell line MX-1 (Cell Lines Service), lung adenocarcinoma cell line H1568 (American Type Culture Collection), endometrial cancer cell line ARK-1 (Department of Gynecology and Obstetrics, Kyoto University School of Medicine), cervical cancer IGF1R expression in cell line HeLa (JCRB cell bank) and ovarian cancer cell line RMG-1 (JCRB cell bank) was examined by flow cytometry, and all of these cancer cell lines expressed IGF1R. Therefore, these cell lines were used to evaluate the antitumor activity of IGF-1 w/oE CAR-T cells against breast cancer, lung adenocarcinoma, endometrial cancer, cervical cancer, and ovarian cancer. bottom.

Specifically, IGF-1 w/oE type CAR-T cells were prepared by the same method as in Example 2, cultured and expanded, and control T cells (activated with anti-CD3 antibody and anti-CD28 antibody). T cells) were cultured. By the same method as in Examples 4 and 5, the above IGF-1 w/oE type CAR-T cells or control T cells and tumor cells (breast cancer cell line MX-1, lung adenocarcinoma cell line H1568, or uterine body Cancer cell line ARK-1) (2 × 10 ⁵ cells) was co-cultured at an E:T ratio of 4:1, 2:1, 1:1, 1:2 or 1:4 for 5 days. of tumor cells were measured. Similarly, the above IGF-1 w/oE type CAR-T cells or control T cells and tumor cells (cervical cancer cell line HeLa, or ovarian cancer cell line RMG-1) (2 × 10 ⁵ cells) were co-cultured at an E:T ratio of 4:1, 2:1, or 1:1 for 4 days, and the number of tumor cells after co-culture was measured. However, B7-H3 was used instead of CD33 as a tumor cell marker. In addition, 24 hours after the start of co-culture, a portion of the culture supernatant was collected, and IFN-γ and IL-2 in the culture supernatant were analyzed using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). Concentration was measured.

Figures 18 and 19 show the results of measuring the number of tumor cells after co-culture. For all types of cancer cell lines tested, IGF-1 w/oE CAR-T cells showed a significant reduction in tumor cell numbers.

In addition, for all types of cancer cell lines tested, concentrations of IFN-γ and IL-2 in the culture supernatant after 24 h co-culture of IGF-1 w/oE CAR-T cells and tumor cells was significantly higher than the concentrations of IFN-γ and IL-2 in the culture supernatant after co-culture of control T cells and tumor cells, at least 24-fold higher.

These results demonstrate that the CAR-T cells of the present invention have antitumor effects against IGF1R-expressing tumors such as breast cancer, lung adenocarcinoma, endometrial cancer, cervical cancer, and ovarian cancer. show.

In addition, when the phenotype of IGF-1 w/oE-type CAR-T cells cultured and amplified for the study of anti-tumor cell activity against MX-1, H1568, and ARK-1 was examined by flow cytometry, CD4+ CAR The proportion of CD45RA+CD62L+ cells (stem cell memory T cells) was approximately 80% for both -T cells and CD8+ CAR-T cells.

[Example 9 Preparation of IGF-1 w/oE des1-3 type CAR expression plasmid]
We designed a CAR that uses the mature IGF-1 portion of IGF-1 w/oE with the N-terminal 3 amino acids (GPE) removed (IGF-1 w/oE des1-3) as the target binding domain. A plasmid for expression (IGF-1 w/oE des1-3 type CAR expression plasmid) was prepared by the method described in International Publication No. 2020/085480.

The IGF-1 w/oE des1-3 type CAR expression plasmid is similar to the IGF-1 w/oE type CAR except that the nucleotide sequence corresponding to the N-terminal three amino acids of the mature IGF-1 portion is deleted. Identical to the expression plasmid.

The nucleotide sequence of the IGF-1 w/oEdes1-3 type CAR expression plasmid is shown in SEQ ID NO: 26, and the amino acid sequence of IGF-1 w/oEdes1-3 is shown in SEQ ID NO:27.

[Example 10 Culture/amplification of IGF-1 w/oE type CAR-T cells and IGF-1 w/oE des1-3 type CAR-T cells, and comparison of CAR expression rate/antitumor cell activity]
<10-1. Culture and amplification of CAR-T cells>
Using the IGF-1 w/oE type CAR expression plasmid and the IGF-1 w/oE des1-3 type CAR expression plasmid prepared in Examples 1 and 9, in the same manner as in Example 2, IGF-1 w/ CAR-T cells using oE or IGF-1 w/oE des1-3 as the target binding domain (IGF-1 w/oE type CAR-T cells, IGF-1 w/oE des1-3 type CAR-T cells, respectively) ) were prepared, cultured and amplified. At the same time, control T cells (T cells activated with anti-CD3 antibody and anti-CD28 antibody) were cultured in the same manner as in Example 2.

<10-2. Comparison of CAR expression rate>
According to the method described in Example 3, the CAR expression rate (14 day) was examined. As a result, IGF-1 w/oE type CAR-T cells and IGF-1 w/oE des1-3 type CAR-T cells showed equivalent CAR expression rates.

<10-3. Comparison of antitumor activity>
Antitumor cell activity of IGF-1 w/oE type CAR-T cells and IGF-1 w/oE des1-3 type CAR-T cells prepared in 10-1 against lung adenocarcinoma cell line A549 (JCRB cell bank) examined.

Specifically, in the same manner as in Examples 4 and 5, the above IGF-1 w/oE CAR-T cells, IGF-1 w/oE des1-3 CAR-T cells, or control T cells Tumor cells (A549) (2×10 ⁵ cells) were co-cultured at an E:T ratio of 1:1, 1:2 or 1:4 for 4 days, and the number of tumor cells after co-culture was measured. However, in this experiment, tumor cells alone were also cultured in the same manner except that T cells were not added. Then, the lysis rate of tumor cells was calculated by the following formula.
Lysis rate (%) = {1 - (number of tumor cells when tumor cells and T cells were co-cultured) / (number of tumor cells when only tumor cells were cultured)} x 100

In addition, 24 hours after the start of co-culture, a portion of the culture supernatant was collected, and an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) was used to measure IFN-γ and IL-2 in the culture supernatant. Concentration was measured.

Fig. 20 shows the measurement results of the dissolution rate. IGF-1 w/oE CAR-T cells exhibited a higher lytic rate compared to control T cells, and IGF-1 w/oE des1-3 CAR-T cells showed IGF-1 w/oE CAR-T It showed an even higher lysis rate than T cells. Therefore, IGF-1 w/oE des1-3 CAR-T cells may have higher antitumor activity than IGF-1 w/oE CAR-T cells against lung adenocarcinoma cell line A549. shown.

In addition, when the IGFBP3 concentration in the culture supernatant after culturing the lung adenocarcinoma cell line A549 used in this example for 24 hours was measured by ELISA, it was about 2800 pg/ml. Therefore, A549 was secreting IGFBP3.

[Example 11 IGFBP3-binding ability of the target-binding domains of IGF-1 w/o E type CAR-T cells and IGF-1 w/o E des1-3 type CAR-T cells]

The ability of the target-binding domain of IGF-1 w/oE type CAR-T cells and IGF-1 w/oE des1-3 type CAR-T cells to bind to IGFBP3 was examined by flow cytometry.

Specific experimental procedures are as follows.
Step 1. His-tagged human IGFBP3 protein (ACROBiosystems, catalog number: IG3-H52H9, 1 μg/μl) was diluted to 0.25 μg/μl by adding PBS.
Step 2. Control T cells cultured and expanded in the same manner as in Example 10 (T cells activated with anti-CD3 antibody and anti-CD28 antibody), IGF-1 w/oE type CAR-T cells, or IGF- 1 w/oE des1-3 type CAR-T cells (1M) were collected in a test tube.
Step 3. PBS was added to the test tube from step 2 and centrifuged at 1500 rpm (or 400 g) for 10 minutes. This process was repeated twice.
Step 4. Remove the supernatant from the test tube in step 3 and add the 2 μl of the prepared His-tagged human IGFBP3 protein solution was added. All tubes were incubated for 1 hour at room temperature.
Step 5. Reagents for staining (reagents A to C) were prepared as follows.

Step 6. The tube incubated in step 4 was centrifuged at 1500 rpm (or 400 g) for 10 minutes and the supernatant was removed. This operation was repeated twice.
Step 7. Add reagent A to tubes containing control T cells, add reagent B or C to tubes containing IGF-1 w/oE CAR-T cells, and add IGF-1 w/oE des1 Reagent C was added to tubes containing type-3 CAR-T cells. All tubes were wrapped in aluminum foil and incubated at 4°C for 15 minutes.
Step 8. After washing the tubes from step 7 once, 300 μL of PBS was added to each tube.
Step 9. Cells in tube from step 8 were subjected to flow cytometry.
Step 10. IGFBP3 binding rate was calculated by the following formula.
IGFBP3 binding rate (%) = (number of CD3-positive CAR-positive His-tag positive cells) / (number of CD3-positive CAR-positive cells) x 100

The results are shown in FIG. IGF-1 w/o E type CAR-T cells and IGF-1 w/o E des1-3 type CAR-T cells showed IGFBP3 binding rates of about 20% and about 60%, respectively. Therefore, IGF-1 w/oE des1-3 showed higher IGFBP3 binding ability than IGF-1 w/oE.

Previous studies have shown that a variant of IGF-1 (Des1-3 IGF-1) with a deletion of three amino acids at the N-terminus has a markedly reduced ability to bind to IGFBP (Sara VR et al. J. Biochem. Cell Biol., 1996, 28(10):1085-1087). Therefore, it is expected that IGF-1 w/oE des1-3 having the deletion of the three amino acids showed higher IGFBP3-binding ability than IGF-1 w/oE without the deletion of the three amino acids. outside result.

[Example 12 Activation of IGF-1 w/oE des1-3 type CAR-T cells by IGFBP3 or IGFBP3 + IGF1R]
By examining the amount of IFNγ released from IGF-1 w/oE des1-3 type CAR-T cells after stimulation with IGFBP3 or IGFBP3 + IGF1R, IGF-1 w/oE des1-3 type CAR-T cells by IGFBP3 or IGFBP3 + IGF1R activation of

Specific experimental procedures are as follows.
Step 1. 500 μl of PBS with or without 1 μg of His-tagged human IGF1R protein (ACROBiosystems, catalog number: IGR-H5229) was added to the wells of a 24-well no-treat plate. This plate was incubated overnight in a refrigerator for immobilization.
Step 2. After washing the plate in step 1 twice, control T cells (T cells activated with anti-CD3 antibody and anti-CD28 antibody) cultured and expanded in the same manner as in Example 10 or IGF-1 w /oE des1-3 type CAR-T cells (10 ⁶ cells) and 1 μg of His-tagged human IGFBP3 protein (ACROBiosystems, catalog number: IG3-H52H9) were added to each well of the plate.
Step 3. RPMI was added to each well of the plate from step 2 to bring the total volume to 2 ml. The plate was incubated at 37°C for 24 hours.
Step 4. 1 mL of supernatant was collected from each well of the plate after incubation in step 3.
Step 5. The concentration of IFN-γ in the supernatant collected in step 4 was measured using an ELISA kit.

The results are shown in FIG.
Stimulation of IGF-1 w/oE des1-3 CAR-T cells with IGFBP3 resulted in higher levels of IFN-γ than control T cells stimulated with IGFBP3. This result indicates that IGF-1 w/oE des1-3 type CAR-T cells are activated not only by binding to IGF1R, but also by binding to IGFBP3.

In addition, the IFN-γ concentration in IGF-1 w/oE des1-3 type CAR-T cells stimulated with IGFBP3+IGF1R was higher than that in control T cells stimulated with IGFBP3+IGF1R. It was significantly higher than the IFN-γ concentration when IGF-1 w/oE des1-3 type CAR-T cells were stimulated with IGFBP3. These results indicate that IGF-1 w/oEdes1-3 type CAR-T cells are activated by binding to both IGFBP3 and IGF1R in the presence of IGFBP3 and IGF1R.

SEQ ID NO: 1 Nucleotide sequence encoding mature IGF-1
GGACCCGAAACACTGTGCGGAGCAGAGCTTGTCGATGCCTTGCAGTTCGTTTGTGGCGATCGAGGGTTCTACTTCAACAAACCAACGGGTTATGGCAGCAGCTCAAGGAGAGCACCTCAGACTGGAATCGTGGATGAGTGCTGCTTTCGCTCCTGTGACCTCAGACGCTTGGATGTACTGTGCTCCCCTGAAACCAGCCAAAAGCGCC

SEQ ID NO: 2 Amino acid sequence of mature IGF-1
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

SEQ ID NO: 3 Nucleotide sequence encoding IGF-1 Ea
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTTTGTGATTTCTTGAAGGTGAAGATGCACACCATGTCCTCCTCGCATCTCTTCTACCTGGCGCTGTGCCTGCTCACCTTCACCAGCTCTGCCACGGCTGGACCGGAGACGCTCTGCGGGGCTGAGCTGGTGGATGCTCTTCAGTTCGTGTGTGGAGACAGGGGCTTTTATTTCAACAAGCCCACAGGGTATGGCTCCAGCAGTCG GAGGGCGCCTCAGACAGGCATCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCTCGCTCTGTCCGTGCCCAGCGCCACACCGACATGCCCAAGACCCAGAAGGAAGTACATTTGAAGAACGCAAGTAGAGGGAGTGCAGGAAACAAGAACTACAGGATG

SEQ ID NO: 4 Amino acid sequence of IGF-1 Ea
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM

SEQ ID NO: 5 Nucleotide sequence encoding IGF-1 Eb
ATGGGGAAAATCTCCTCACTGCCTACACAGCTCTTCAAATGCTGCTTTGCGACTTTCTGAAGGTGAAGATGCACACGATGAGCTCTTCCCACCTCTTTTACCTGGCCCTTTGTCTGCTGACATTCACTTCCAGTGCTACTGCTGGACCCGAAACACTGTGCGGAGCAGAGCTTGTCGATGCCTTGCAGTTCGTTTGTGGCGATCGAGGGTTCTACTTCAACAAACCAACGGGTTATGGCAGCAGCTCAAGGAGA GCACCTCAGACTGGAATCGTGGATGAGTGCTGCTTTCGCTCCTGTGACCTCAGACGCTTGGAGATGTACTGTGCTCCCCTGAAACCAGCCAAAAGCGCCAGAAGCGTAAGGGCACAACGTCATACCGACATGCCCAAAACCCAGAAGTATCAGCCACCGTCAACCAACAAGAATACCAAGTCTCAACGGCGGAAAGGTTGGCCCAAGACCCATCCTGGAGGTGAACAGAAGGAAGGCACAGAAGCCTGCAGATACGG GGCAAGAAGAAGGAGCAAAGACGAGAGATTGGGTCTAGGAATGCGGAGTGTCGCGGCAAGAAAGGGAAA

SEQ ID NO: 6 Amino acid sequence of IGF-1 Eb
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGGEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK

SEQ ID NO: 7 Nucleotide sequence encoding IGF-1 Ec
ATGGGGAAAATCTCCTCACTGCCTACACAGCTCTTCAAATGCTGCTTTGCGACTTTCTGAAGGTGAAGATGCACACGATGAGCTCTTCCCACCTCTTTTACCTGGCCCTTTGTCTGCTGACATTCACTTCCAGTGCTACTGCTGGACCCGAAACACTGTGCGGAGCAGAGCTTGTCGATGCCTTGCAGTTCGTTTGTGGCGATCGAGGGTTCTACTTCAACAAACCAACGGGTTATGGCAGCAGCTCAAGGAGA GCACCTCAGACTGGAATCGTGGATGAGTGCTGCTTTCGCTCCTGTGACCTCAGACGCTTGGAGATGTACTGTGCTCCCCTGAAACCAGCCAAAAGCGCCAGAAGCGTAAGGGCACAACGTCATACCGACATGCCCAAAACCCAGAAGTATCAGCCACCGTCAACCAACAAGAATACCAAGTCTCAACGGCGGAAAGGTAGCACATTCGAGGAAAGGAAA

SEQ ID NO:8 Amino acid sequence of IGF-1 Ec
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGSTFEERK

SEQ ID NO: 9 Nucleotide sequence encoding IGF-1 w/oE
ATGGGGAAAATCTCCTCACTGCCTACACAGCTCTTCAAATGCTGCTTTGCGACTTTCTGAAGGTGAAGATGCACACGATGAGCTCTTCCCACCTCTTTTACCTGGCCCTTTGTCTGCTGACATTCACTTCCAGTGCTACTGCTGGACCCGAAACACTGTGCGGAGCAGAGCTTGTCGATGCCTTGCAGTTCGTTTGTGGCGATCGAGGGTTCTACTTCAACAAACCAACGGGTTATGGCAGCAGCTCAAGGAGA GCACCTCAGACTGGAATCGTGGATGAGTGCTGCTTTCGCTCCTGTGACCTCAGACGCTTGGAGATGTACTGTGCTCCCCTGAAACCAGCCAAAAGCGCC

SEQ ID NO: 10 Amino acid sequence of IGF-1 w/oE
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

SEQ ID NO: 11 Nucleotide sequence encoding mature IGF-2
GCGTATCGGCCTAGTGAGACCCTGTGTGGTGGTGAACTCTGGACACTCTCCAGTTCGTCTGTGGAGACAGAGGATTTTACTTCAGCAGGCCGGCCTCACGGGTAAGCCGCAGGAGCCGAGGCATTGTAGAAGAATGTTGCTTCCGGTCCTGCGACCTTGCTCTGCTGGAGACGTATTGTGCAACACCCGCAAAATCTGAG

SEQ ID NO: 12 amino acid sequence of mature IGF-2
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE

SEQ ID NO: 13 Nucleotide sequence encoding pre-pro-IGF-2
ATGGGGATTCCTATGGGGAAGTCCATGCTGGTGCTTCTGACGTTCCTCGCGTTTGCAAGCTGTTGTATAGCCGCCTACCGGCCATCTGAGACTCTGTGTGGAGGAGAACTGGTTGACACTTTGCAATTCGTGTGCGGGGATCGCGGCTTTTACTTTTCTCGCCCGGCTTCCCGGGTAAGCCGCAGATCCAGGGGCATCGTTGAAGAGTGTTGCTTTCGATCTTGCGACTTGGCACTCCTCGAAACCTATTGC GCCACGCCAGCTAAGTCAGAGAGGGACGTCTCCACCCCACCCACCGTCCTGCCTGACAACTTTCCACGGTACCCAGTCGGCAAATTCTTCCAGTATGATACCTGGAAGCAGAGTACACAGAGGCTGCGGCGGGGACTGCCAGCGTTGTTGCGGGCCAGAAGAGGCCACGTTCTTGCTAAGGAACTTGAAGCCTTCAGAGAAGCTAAACGCCACAGACCCCTTATCGCCCTGCCTACACAGGACCCAGCCCAT GGAGGCGCACCACCCGAGATGGCCTCCAATAGAAAA

SEQ ID NO: 14 amino acid sequence of pre-pro-IGF-2
MGIPMGKSMLVLLTFLAFASCCIAAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSERDVSTPPTVLPDNFPRYPVGKFFQYDTWKQSTQRLRRGLPALLRARRGHVLAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMASNRK

SEQ ID NO: 15 amino acid sequence of hinge region of IgG1 SEQ ID NO: 16 amino acid sequence of CH2 region of IgG1 SEQ ID NO: 17 amino acid sequence of CH3 region of IgG1 SEQ ID NO: 18 amino acid sequence of human CD28-derived transmembrane domain and co-stimulatory domain SEQ ID NO: 19 human Amino acid sequence of CD3 zeta chain-derived intracellular signaling domain SEQ ID NO: 20 Nucleotide sequence of mature IGF-1 type CAR expression plasmid SEQ ID NO: 21 Nucleotide sequence of IGF-1 Ea type CAR expression plasmid SEQ ID NO: 22 IGF-1 Eb type CAR expression plasmid Nucleotide sequence SEQ ID NO: 23 Nucleotide sequence of IGF-1 Ec type CAR expression plasmid SEQ ID NO: 24 Nucleotide sequence of IGF-1 w/oE type CAR expression plasmid SEQ ID NO: 26 IGF-1 w/oE des1-3 type CAR expression plasmid nucleotide sequence SEQ ID NO: 27 IGF-1 w/oE des1-3 amino acid sequence
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATATLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (12)

A polynucleotide encoding a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to insulin-like growth factor-1 receptor (IGF1R), a transmembrane domain, and an intracellular signaling domain A polynucleotide, wherein the target binding domain is an insulin-like growth factor (IGF) pre-pro precursor or an E-domain deleted fragment thereof. The polynucleotide according to claim 1, wherein the IGF is IGF-1. 3. The polynucleotide of claim 1 or 2, wherein the target binding domain consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10. The polynucleotide according to claim 1, wherein the IGF is IGF-2. The polynucleotide according to claim 1 or 4, wherein the target binding domain consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:14. A vector comprising the polynucleotide according to any one of claims 1 to 5. A genetically modified cell into which the polynucleotide according to any one of claims 1 to 5 or the vector according to claim 6 has been introduced. A method for producing a CAR protein-expressing cell, comprising introducing the polynucleotide according to any one of claims 1 to 5 or the vector according to claim 6 into a cell. A therapeutic agent for diseases involving IGF1R-expressing cells, comprising the cells according to claim 7. A pharmaceutical composition comprising the therapeutic agent according to claim 9 and a pharmaceutically acceptable carrier. Diseases involving IGF1R-expressing cells include leukemia, multiple myeloma, lymphoma, lung cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, colorectal cancer, colorectal cancer, and colon cancer. The group consisting of cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, renal cancer, adrenal cancer, melanoma, neuroendocrine tumor, retinoblastoma, and sarcoma 11. The therapeutic agent of claim 9 or the composition of claim 10, selected from: A kit for producing CAR protein-expressing cells targeting IGF1R-expressing cells, comprising the vector according to claim 6.

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