CN109912718B

CN109912718B - Isolated binding proteins of the B7-H3 antigen binding domain, nucleic acids, vectors, CAR-T cells and uses thereof

Info

Publication number: CN109912718B
Application number: CN201910210679.XA
Authority: CN
Inventors: 陈模江; 王刚; 刘金超
Original assignee: Beijing Shanke Bio Tech Co ltd
Current assignee: Beijing Shanke Bio Tech Co ltd
Priority date: 2019-03-20
Filing date: 2019-03-20
Publication date: 2020-12-11
Anticipated expiration: 2039-03-20
Also published as: CN109912718A

Abstract

The present invention relates to the field of CAR-T cells, in particular, to isolated binding proteins, nucleic acids, vectors, CAR-T cells of the B7-H3 antigen binding domain and uses thereof. The invention aims at scFV fragments obtained by B7-H3 antigen, designs and constructs second generation B7-H3-CAR taking CD28 or 4-1BB as co-stimulatory molecules, and verifies the capability of the scFV fragments to kill cancer cells in B7-H3 positive non-small cell lung cancer, and the result shows that when the ratio of T cells to cancer cells is 1:5, the B7-H3-CAR-T can still kill B7-H3 positive non-small cell lung cancer cells very efficiently, and release a large amount of type I interferon, such as IFN gamma and TNF alpha.

Description

Isolated binding proteins of the B7-H3 antigen binding domain, nucleic acids, vectors, CAR-T cells and uses thereof

Technical Field

The present invention relates to the field of CAR-T cells, in particular, to isolated binding proteins, nucleic acids, vectors, CAR-T cells of the B7-H3 antigen binding domain and uses thereof.

Background

Lung cancer is the first tumor of cancer mortality worldwide, and can increase by about 130 ten thousand new cases each year, and about 80-85% of newly diagnosed lung cancers are non-small cell lung cancers, such as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like. In 2017, about 80 ten thousand new lung cancer cases and 70 ten thousand deaths of lung cancer cases exist in China, and the morbidity and the mortality of the lung cancer cases account for about 40 percent of the lung cancer in the world and are far higher than those of other countries. In China, lung cancer is also the first cancer with both morbidity and mortality, and with the increasing air pollution problem in recent years, the number of lung cancer patients is expected to increase further in the future, which seriously affects the health and life of people in China.

Although early stage lung cancer can be cured by surgical removal of the tumor, some patients can be cured, in most cases, patients are diagnosed with advanced stage cancer, which is an unresectable lesion or metastasis, and is an incurable disease state. Non-small cell lung cancer patients in the local progressive stage can receive concurrent chemoradiotherapy and combined or not combined with surgical treatment, so that the disease-progression-free survival of 8 months can be averagely achieved, but the 5-year overall survival rate is still less than 15 percent. Patients who have been diagnosed with advanced lung cancer at the time of diagnosis may receive novel cytotoxic therapeutic agents, such as pemetrexed and the like, with a median survival time of about 17 months.

CAR-T (English abbreviation of Chimeric Antigen Receptor T Cells) is a genetically engineered autologous T cell-based immunotherapy. The therapy is to express a specific protein, namely a Chimeric Antigen Receptor (CAR), on the surface of T cells by genetic modification after extracting the T cells of a patient, thereby recognizing specific antigens on the surface of cancer cells and killing the cancer cells through the antigen-removing ability of the T cells. CAR-T therapy has met with great success in several clinical trials currently in progress, particularly CAR-T targeting CD19 and BCMA. Although CAR-T has achieved very good clinical efficacy in treating hematological tumors such as lymphomas, leukemias, etc., CAR-T has been studied relatively slowly in solid tumors, an important reason for this being the lack of suitable targets, since antigens that are normally highly expressed in solid tumors are also expressed in some normal tissues.

CAR-T has achieved surprising therapeutic effects in leukemia as the latest revolutionary immunotherapeutic approach, and further development is needed for how it can be applied to solid tumors.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims at scFV fragments obtained by B7-H3 antigen, designs and constructs second generation B7-H3-CAR taking CD28 or 4-1BB as co-stimulatory molecules, and verifies the capability of the scFV fragments to kill cancer cells in B7-H3 positive non-small cell lung cancer, and the result shows that when the ratio of T cells to cancer cells is 1:5, the B7-H3-CAR-T can still kill B7-H3 positive non-small cell lung cancer cells very efficiently, and release a large amount of type I interferon, such as IFN gamma and TNF alpha.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

in one aspect, the invention provides an isolated binding protein comprising a B7-H3 antigen binding domain, wherein the amino acid sequence of the light chain variable region and/or the heavy chain variable region is set forth in SEQ ID NO.1-2, and wherein the amino acid sequence has at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO. 1-2.

It should be understood that the amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.1-2 can be deduced by those skilled in the art that the amino acid sequence still has good binding effect with the B7-H3 antigen by one or more amino acid sequence changes. As practical, at least 95% sequence identity may be 95%, 96%, 97%, 98%, 99%, etc., and the number of amino acid changes may be 1, 2, 3, 4, 5, 6, etc.

Further, the binding protein is a sc FV fragment.

Further, the light chain variable region and the heavy chain variable region are connected by a connecting region, and the amino acid sequence of the connecting region is shown as SEQ ID NO. 3.

B7-H3 is a type I membrane protein, is highly expressed in various solid tumors, such as 93.7% of pancreatic cancer, 90.6% of breast cancer, 83% of ovarian cancer, 86% of rectal cancer, 93.8% of liver cancer and 70% of non-small cell lung cancer, and is a new target point for tumor immunotherapy. However, B7-H3 is not expressed in normal tissues, such as nerve cells of the heart, lung, kidney, liver and brain. In recent years, a plurality of antibody drugs targeting B7-H3, including Fc enhanced antibodies, isotope labeled antibodies, antibody-drug conjugates and the like, show better anti-tumor effects in a plurality of phase I clinical trials and preclinical mouse tumor model researches. These findings further indicate that it is reasonable and feasible to design CARs targeting B7-H3 for the treatment of solid tumors.

The anti-B7-H3 monoclonal antibody 2D6 is obtained by screening, and can stably secrete an anti-B7-H3 antibody. The heavy chain variable region and the light chain variable region of the monoclonal antibody and the connection region between the heavy chain variable region and the light chain variable region are cloned by a molecular biological means, the nucleic acid sequences of the monoclonal antibody are sequentially shown as SEQ ID NO.4-6, and the amino acid sequences of the monoclonal antibody are sequentially shown as SEQ ID NO. 1-3.

In a second aspect, the invention provides an isolated nucleic acid encoding a binding protein as described above.

Further, the nucleic acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

Further, the nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5.

Further, the nucleic acid sequence of the connecting region is shown as SEQ ID NO. 6.

Further, the nucleic acid is also linked to a signal peptide nucleic acid sequence.

Further, the nucleic acid sequence of the signal peptide is shown as SEQ ID NO. 7. The corresponding amino acid sequence of the signal peptide is shown as SEQ ID NO. 8.

Further, the signal peptide is located at the nitrogen terminus of the light chain variable region.

The signal peptide sequence of the present invention is included in the N-terminal primer used in PCR, and the sequence thus amplified contains the signal peptide.

In a third aspect, the present invention provides a vector comprising a nucleic acid as described above.

Further, the vector contains the hinge region of CD8 a, the transmembrane domain of CD8 a, and the intracellular domain of CD3 ζ, and comprises CD28 or 4-1BB co-stimulatory domains.

Further, the nucleic acid sequences of the hinge region of CD8 alpha and the transmembrane domain of CD8 alpha are shown as SEQ ID NO.9, and the corresponding amino acid sequences are shown as SEQ ID NO. 10.

Further, the nucleic acid sequence of the intracellular domain of CD3 ζ is shown as SEQ ID NO.11, and the corresponding amino acid sequence is shown as SEQ ID NO. 12.

Further, the nucleic acid sequence of CD28 is shown as SEQ ID NO.13, and the corresponding amino acid sequence is shown as SEQ ID NO. 14.

Furthermore, the nucleic acid sequence of 4-1BB is shown as SEQ ID NO.15, and the corresponding amino acid sequence is shown as SEQ ID NO. 16.

Further, the vector is a lentiviral vector; the lentiviral vector is preferably pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB.

Further, the connection mode of each nucleic acid sequence in the vector is as follows:

pCDH-CMV-B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB;

wherein the nucleic acid sequence of B7-H3-CAR-28 is sequentially signal peptide-VL-Linker-VH-CD 8 alpha-CD 28-CD3 zeta;

preferably, the nucleic acid sequence of B7-H3-CAR-28 is shown as SEQ ID NO.17, and the corresponding amino acid sequence is shown as SEQ ID NO. 18.

The nucleic acid sequence of B7-H3-CAR-BB is sequentially signal peptide-VL-Linker-VH-CD 8 alpha-4-1 BB-CD3 zeta.

Preferably, the nucleic acid sequence of B7-H3-CAR-BB is shown as SEQ ID NO.19, and the corresponding amino acid sequence is shown as SEQ ID NO. 20.

In the vector provided by the invention, a second generation B7-H3-CAR taking CD28 or 4-1BB as a costimulatory molecule is constructed, the capability of the vector in killing cancer cells is verified in B7-H3 positive non-small cell lung cancer, and B7-H3 positive non-small cell lung cancer cells can be killed very efficiently.

In a fourth aspect, the present invention provides a host cell comprising a nucleic acid as described above or a vector as described above.

Further, the host cell is used for expressing the nucleic acid or vector. Namely, a cell line in which the host cell is used for expressing a foreign gene.

Further, the host cell is 293 cell.

The 293 cell is a human kidney epithelial cell line, and has various derivatives, such as HEK293, 293T/17 and the like.

The host cell provided by the invention is used for amplifying and enriching lentivirus or plasmids. For subsequent CAR-T cell preparation.

In practice, after transfection of host cells with plasmids, the culture supernatants containing lentiviruses were collected, filtered through a 0.45 μm filter and frozen until use.

In a fifth aspect, the invention provides a method for producing a CAR-T cell, comprising the steps of:

the supernatant containing the virus prepared by the host cell is infected with T cells, and the CAR-T cells are obtained by separation.

T cells were prepared using the following method: separating PBMC lymphocytes from peripheral blood, adding into a culture plate coated with CD3 and CD28 antibodies, adding IL2(10 μ g/mL) after 20-30 hours, stimulating for 40-60 hours, and collecting T cells.

Further, PBMC lymphocytes were added to CD3 and CD28 antibody coated 24-well plates at 1X 10⁶Each cell per well.

Peripheral blood was obtained from healthy volunteers.

The stimulated T cells were used for the recombinant lentivirus infection described above.

Specifically, the use of T cells for the above recombinant lentivirus infection was performed using the following method:

add 5X 10 to 24 well cell culture plates⁵And adding 1mL of lentivirus expressing B7-H3-CAR-28 or B7-H3-CAR-BB to the stimulated T cells, centrifuging for 1.5 hours at 2000g, culturing the cells in a cell culture box at 37 ℃ after centrifugation is finished, removing culture medium containing the lentivirus after 24 hours, replacing the culture medium with fresh culture medium every two days, collecting CAR-T cells after 12 days, and detecting the killing function of the CAR-T cells.

In a sixth aspect, the invention provides a CAR-T cell prepared by the above-described preparation method.

In a seventh aspect, the invention provides the use of a CAR-T cell as described above in the preparation of a formulation for the treatment of a tumour.

Further, the tumors include all B7-H3 positive tumors, including hematological and solid tumors, such as, but not limited to, any of lung cancer, pancreatic cancer, gastric cancer, breast cancer, ovarian cancer, cervical cancer, brain tumor, head and neck cancer, colorectal cancer, melanoma, liver cancer, gastric cancer, esophageal cancer, testicular cancer, prostate cancer, renal cancer, glioma, neuroblastoma, myeloma, lymphoma.

The invention verifies that the provided CAR-T cell has high killing power on non-small cell lung cancer. Obviously, the medicine can be inferred to have high killing power on other solid tumors such as pancreatic cancer, breast cancer, ovarian cancer, rectal cancer and liver cancer.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention designs and constructs a second generation B7-H3-CAR taking CD28 or 4-1BB as co-stimulatory molecules aiming at scFV fragments obtained by B7-H3 antigen, and verifies the capability of the scFV fragments to kill cancer cells in B7-H3 positive non-small cell lung cancer.

(2) When the ratio of the CAR-T cells to the cancer cells is 1:5, the CAR-T cells can still kill B7-H3 positive non-small cell lung cancer cells very efficiently, and release a large amount of type I interferons such as IFN gamma and TNF alpha, so that a good basis is provided for treating solid tumors.

(3) The CAR-T cell provided by the invention can be used for treating solid tumors, wherein the solid tumors comprise non-small cell lung cancer, pancreatic cancer, breast cancer, ovarian cancer, rectal cancer, liver cancer and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic structural view of a carrier in example 1 of the present invention;

FIG. 2 is a graph showing the expression of B7-H3-CAR-28 and B7-H3-CAR-BB in CAR-T cells prepared by lentivirus infection of activated T cells with plasmid DNA of B7-H3-CAR-28 or B7-H3CAR-BB in example 1 of the present invention;

FIG. 3 is a graph showing flow cytometry detection of T cells (CD 3) after cocultivation of different cells with different non-small cell lung cancer cell lines in example 1 of the present invention⁺) And the ratio of remaining cancer cells (B7-H3)⁺) A graph of results of (1);

FIG. 4 is a graph showing the expression profiles of B7 to H3 after infection of different non-small cell lung cancer cell lines with the B7 to H3 antibody in example 1 of the present invention;

FIG. 5 is a bar graph showing the amount of IFN γ and TNF α secreted by different cells in example 1 of the present invention when they act on non-small cell lung cancer cells;

FIG. 6 is a graph of the clearance of B7-H3-CAR-T from lung cancer tumors in mice in example 2 of the invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1. B7-H3 antibody screening

BALB/c mice were immunized by subcutaneous injection with purified B7-H3-His protein mixed with Freund's adjuvant once every two weeks for a total of three immunizations. On day 35, blood was collected and serum was assayed for anti-B7-H3 antibodies by ELISA, indicating a higher concentration of anti-B7-H3 antibodies in the serum when the OD value after 4000-fold dilution was greater than 1.0. On day 39, mice were re-immunized, and mouse spleen cells were collected on day 42, single cell suspensions were prepared, and fused with myeloma cells F0 by PEG. After 7 days of culture, supernatants of the fused hybridoma clones were screened by ELISA to test their specificity and sensitivity, and after rescreening and subcloning, monoclonal 2D6 was obtained that was able to stably secrete antibodies against B7-H3.

2. Plasmid construction

To construct a CAR targeting B7-H3, we selected a 2D6 monoclonal antibody against B7-H3, cloned a DNA sequence (scFv) encoding the antibody variable region into a lentiviral vector to make CAR-T cells infected with activated T with a lentivirus comprising plasmid DNA encoding B7-H3-CAR-28 or B7-H3 CAR-BB.

The lentiviral vector was either pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB, both of which contained the hinge and transmembrane domains of CD8 α and the intracellular domain of CD3 ζ and CD28 or 4-1BB costimulatory domain, respectively, to construct B7-H3-CAR-28 and B7-H3-CAR-BB (FIG. 1).

The variable region fragments of the light and heavy chains of the anti-B7-H3 monoclonal antibody 2D6 (VL and VH) were amplified by PCR, the Signal Peptide (SP) sequence was included in the N-terminal primers used in PCR, and then cloned into the lentiviral vector pCDH-CMV-CAR-28(pCDH-CMV-B7-H3-CAR-28) or pCDH-CMV-CAR-BB (pCDH-CMV-B7-H3-CAR-BB) by T4 ligase after cleavage with XbaI and BamHI restriction enzymes.

Wherein, the DNA sequence (scFv) of the variable region of the antibody coded by the 2D6 monoclonal antibody comprises a light chain variable region (the nucleic acid sequence is shown as SEQ ID NO. 4) and a heavy chain variable region (the nucleic acid sequence is shown as SEQ ID NO. 5), and a connecting region (the nucleic acid sequence is shown as SEQ ID NO. 6) between the light chain variable region and the heavy chain variable region; the nucleic acid sequence of the signal peptide is shown as SEQ ID NO.7, and the corresponding amino acid sequence is shown as SEQ ID NO. 8; the nucleic acid sequence of the CD8 alpha is shown as SEQ ID NO.9, and the corresponding amino acid sequence is shown as SEQ ID NO. 10; the nucleic acid sequence of CD3 zeta is shown in SEQ ID NO.11, and the corresponding amino acid sequence is shown in SEQ ID NO. 12; the nucleic acid sequence of the CD28 is shown as SEQ ID NO.13, and the corresponding amino acid sequence is shown as SEQ ID NO. 14; the nucleic acid sequence of 4-1BB is shown as SEQ ID NO.15, and the corresponding amino acid sequence is shown as SEQ ID NO. 16; B7-H3-CAR-28, B7-H3-CAR-CD8 alpha-CD 28-CD3 zeta complete sequence is shown in SEQ ID NO.17, and its corresponding amino acid sequence is shown in SEQ ID NO. 18; the complete sequence of B7-H3-CAR-BB, namely B7-H3-CAR-CD8 alpha-4-1 BB-CD3 zeta is shown in SEQ ID NO.19, and the corresponding amino acid sequence is shown in SEQ ID NO. 20.

3. Lentivirus preparation expressing B7-H3-CAR

Will be 7X 10⁶293T cell engraftmentAfter 16 hours in a 10cm cell culture dish, 4. mu.g of pCDH-CMV-B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB plasmid, 4. mu.g of psPAX2 plasmid and 2. mu.g of pMD2G plasmid were mixed, and the plasmid mixture was mixed with GeneJuice transfection reagent (Merck Millipore), left for 10 minutes and then transfected into 293T cells. 48 hours after transfection, the lentivirus-containing culture supernatants were collected, filtered through a 0.45 μm filter, and frozen until use.

4. Preparation and in vitro expansion of CAR-T cells

PBMC lymphocytes were isolated from peripheral blood of healthy volunteers and added to CD3 and CD28 antibody-coated 24-well plates at 1X 10⁶Each cell per well. After 24 hours, IL2 (10. mu.g/mL) was added, and T cells were collected two days after stimulation and counted for use.

And (3) infecting the stimulated T cells with the recombinant lentivirus, wherein the specific infection steps are as follows: add 5X 10 to 24 well cell culture plates⁵And adding 1mL of lentivirus expressing B7-H3-CAR-28 or B7-H3-CAR-BB to the stimulated T cells, centrifuging for 1.5 hours at 2000g, culturing the cells in a cell culture box at 37 ℃ after centrifugation is finished, removing culture medium containing the lentivirus after 24 hours, replacing the culture medium with fresh culture medium every two days, collecting CAR-T cells after 12 days, and detecting the killing function of the CAR-T cells. After 4 days of infection, 5X 10 of the above-mentioned extracts were taken⁵Cells, CAR on CAR-T cell surface stained with anti-Fab antibody, and B7-H3-CAR expression was detected by flow cytometry, as shown in figure 2.

That is, activated T cells were infected with lentiviruses containing plasmid DNA encoding B7-H3-CAR-28 or B7-H3CAR-BB to make CAR-T cells, indicating that B7-H3-CAR-28 and B7-H3-CAR-BB can be expressed efficiently on the CAR-T cell surface, with over 95% of CAR-T cells being B7-H3-CAR positive (fig. 2).

5. Flow assay

T cells and cancer cells were labeled with antibodies to CD3-APC-H7 and B7-H3-APC (BD bioscience), respectively, expression of B7-H3-CAR was labeled with antibodies to Anti-Fab (Jackson ImmunoResearch Laboratories INC.), labeled samples were examined by BD FACSCAnto II flow cytometer with BD Diva software (BD Biosciences), a minimum of 10,000 viable cells were collected per sample, and the results of the collection were analyzed by Flowjo 10 software.

NT, B7-H3-CAR-28-T or B7-H3-CAR-BB-T cells were co-cultured with three non-small cell lung cancer cell lines HCC827, NCI-H522 and NCI-H1299 at a ratio of T cells to cancer cells of 1:5, and after 5 days of co-culture, T cells were detected by flow cytometry (CD 3)⁺) And the ratio of remaining cancer cells (B7-H3)⁺)。

Detection of B7-H3-CAR-28 and B7-H3-CAR-BB killing B7-H3⁺The capacity of cancer cells, three B7-H3 of HCC827, NCI-H522 and NCI-H1299⁺Lung cancer cells (fig. 3) were co-cultured with control T cells (T cells not infected with lentivirus, NT), B7-H3-CAR-28-T or B7-H3-CAR-BB-T cells at a ratio of T cells to cancer cells of 1:5, after 5 days of co-culture, suspended T cells and adherent cancer cells were collected, the two cells were combined, the T cells and cancer cells were labeled with antibodies to CD3 and B7-H3, respectively, and then the ratio of residual cancer cells was examined by flow cytometry. The results are shown in FIG. 3.

As can be seen in FIG. 3, after 5 days of co-culture, both B7-H3-CAR-28-T and B7-H3-CAR-BB-T cells completely cleared the three lung cancer cells. It was demonstrated that B7-H3-CAR-28-T and B7-H3-CAR-BB-T cells were able to recognize and target kill these three lung cancer cells.

Non-small cell lung cancer cell lines HCC827, NCI-H522 and NCI-H1299 were infected with the antibody to B7-H3, and then the expression of B7-H3 was examined by flow cytometry, the results of which are shown in FIG. 4.

As can be seen from FIG. 4, the non-small cell lung cancers all highly expressed B7-H3.

6. ELISA detection

B7-H3-CAR-T secretes large amounts of IFN gamma and TNF alpha when killing non-small cell lung cancer cells. The amounts of IFN γ and TNF α were measured by ELISA. The method comprises the following specific steps:

non-small cell lung cancer cells are treated at 5 × 10⁵Add 24 wells per well to a cell culture plate and after 16 hours add 5X 10⁵NT, B7-H3-CAR-28 or B7-H3-CAR-BB CAR-T cells at a ratio of T cells to cancer cells of 1:5, 2mL of cell culture medium per well. After 24 hours of co-cultivation, 1mL of the suspension was collectedELISA kits using IFN γ and TNF α (R)&D system) to determine the amount of these two type I interferons in the co-culture supernatant. The results are shown in FIG. 5.

As can be seen from FIG. 5, B7-H3-CAR-28-T and B7-H3-CAR-BB-T cells released large amounts of type I interferon-IFN γ and TNF α.

Example 2

1. Co-culture killing experiment

Non-small cell lung cancer cells are treated at 5 × 10⁵Add 24 wells per well to a cell culture plate and after 16 hours add 5X 10⁵NT, B7-H3-CAR-28 or B7-H3-CAR-BB CAR-T cells at a ratio of T cells to cancer cells of 1: 5. After 5 days of co-culture, the suspended T cells and the adherent cancer cells were collected in their entirety, labeled with antibodies against CD3 and B7-H3, respectively, and then the ratio of remaining cancer cells was examined by flow cytometry, and dead cells were removed by Zombie Aqua Dye (Biolegend).

2. Evaluation of the tumor killing Effect of B7-H3-CAR-T in mice Using H522 Lung cancer metastasis model

Luciferase-overexpressing H522 lung cancer cells were injected via tail vein into NSG mice, each mouse injected 2X 10⁶H522 cells are used for constructing a mouse lung cancer metastasis model. 14 days later, tumor-bearing mice were treated with NT, B7-H3-CAR-28-T or B7-H3-CAR-BB-T cells, and each mouse was injected with 10X 10⁶Individual T cells were monitored weekly for changes in tumor size by IVIS instruments.

As shown in FIG. 6, after one week of treatment, cancer cells in the lung of mice completely disappeared in the B7-H3-CAR-28-T or B7-H3-CAR-BB-T cell-treated group, but some cancer cells remained alive near the tail of the mice, and after two weeks, cancer cells in the whole body of the mice were completely cleared by B7-H3-CAR-28-T or B7-H3-CAR-BB-T cells, and no recurrence of lung cancer was observed in all the B7-H3-CAR-T treated mice in the follow-up monitoring for 60 days. The above results indicate that B7-H3-CAR-28-T and B7-H3-CAR-BB-T cells can kill B7-H3 positive lung cancer cells efficiently in mice and prevent relapse.

As B7-H3 is almost highly expressed in all solid tumor cancer cellsCells, such as 93.7% pancreatic cancer, 90.6% breast cancer, 83% ovarian cancer, 86% rectal cancer, 93.8% liver cancer and 70% non-small cell lung cancer, etc. Therefore, the B7-H3-targeted CAR-T can be widely applied to the targeted therapy of all B7-H3⁺A solid tumor or myeloma.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Beijing Shanke Biotechnology Co., Ltd

<120> isolated binding proteins of B7-H3 antigen binding domain, nucleic acids, vectors, CAR-T cells and uses thereof

<130> 2019

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<213> Homo sapiens

<400> 9

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 10

<211> 69

<212> PRT

<213> Homo sapiens

<400> 10

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 11

<211> 339

<212> DNA

<213> Homo sapiens

<400> 11

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 12

<211> 112

<212> PRT

<213> Homo sapiens

<400> 12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 13

<211> 123

<212> DNA

<213> Homo sapiens

<400> 13

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 14

<211> 41

<212> PRT

<213> Homo sapiens

<400> 14

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 15

<211> 126

<212> DNA

<213> Homo sapiens

<400> 15

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 16

<211> 42

<212> PRT

<213> Homo sapiens

<400> 16

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 17

<211> 1443

<212> DNA

<213> Artificial sequence

<400> 17

atggaattcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgccaa 60

attgttctca cccagtctcc agcaatcatg tctgcatctc caggggagaa ggtcaccatg 120

acctgcagtg ccagctcaag tgtaatttac atgcactggt accagcagaa gtcaggcacc 180

tcctccaaaa gatggatcta tgacacatcc aaactggctt ctggagtccc tgctcgcttc 240

agtggcagtg cgtctggcgc ctcttactct ctctcaatca gcagcatgga ggctgaagat 300

gctgccactt attactgcca gcagtgggaa attaacccgc tcacgttcgg tgctgggacc 360

aagctggagc tgaaaggcgg cggaggatct ggcggaggcg gaagtggcgg agggggctct 420

caggtgcagc tgcaggagtc aggacctggc ctggtggtgc actcacagag cctgtccatc 480

acatgcaccg tttcagggtt ctcattaatc ggctatggtg taaactgggt tcgccagcct 540

ccaggaaaga gtctggagtg gctcggaatg atatggtgtg atggaaggac agactataat 600

tcaggtctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 660

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt actgtgccag agggtatggt 720

aaccacgcct ggcttgctta ctggtgccaa gggactctgg tcagtgtctc tgcaaccacg 780

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 840

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 900

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 960

ctggttatca ccctttactg caggagtaag aggagcaggc tcctgcacag tgactacatg 1020

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1080

cgcgacttcg cagcctatcg ctccagagtg aagttcagca ggagcgcaga cgcccccgcg 1140

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1260

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1320

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1380

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1440

taa 1443

<210> 18

<211> 480

<212> PRT

<213> Artificial sequence

<400> 18

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

20 25 30

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val

35 40 45

Ile Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Ser Lys Arg

50 55 60

Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe

65 70 75 80

Ser Gly Ser Ala Ser Gly Ala Ser Tyr Ser Leu Ser Ile Ser Ser Met

85 90 95

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Glu Ile Asn

100 105 110

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

130 135 140

Gln Glu Ser Gly Pro Gly Leu Val Val His Ser Gln Ser Leu Ser Ile

145 150 155 160

Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr Gly Val Asn Trp

165 170 175

Val Arg Gln Pro Pro Gly Lys Ser Leu Glu Trp Leu Gly Met Ile Trp

180 185 190

Cys Asp Gly Arg Thr Asp Tyr Asn Ser Gly Leu Lys Ser Arg Leu Ser

195 200 205

Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser

210 215 220

Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Gly Tyr Gly

225 230 235 240

Asn His Ala Trp Leu Ala Tyr Trp Cys Gln Gly Thr Leu Val Ser Val

245 250 255

Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

260 265 270

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

275 280 285

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

290 295 300

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

305 310 315 320

Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu His

325 330 335

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

340 345 350

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

355 360 365

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

370 375 380

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

385 390 395 400

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

405 410 415

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

420 425 430

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

435 440 445

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

450 455 460

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475 480

<210> 19

<211> 1446

<212> DNA

<213> Artificial sequence

<400> 19

atggaattcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgccaa 60

attgttctca cccagtctcc agcaatcatg tctgcatctc caggggagaa ggtcaccatg 120

acctgcagtg ccagctcaag tgtaatttac atgcactggt accagcagaa gtcaggcacc 180

tcctccaaaa gatggatcta tgacacatcc aaactggctt ctggagtccc tgctcgcttc 240

agtggcagtg cgtctggcgc ctcttactct ctctcaatca gcagcatgga ggctgaagat 300

gctgccactt attactgcca gcagtgggaa attaacccgc tcacgttcgg tgctgggacc 360

aagctggagc tgaaaggcgg cggaggatct ggcggaggcg gaagtggcgg agggggctct 420

caggtgcagc tgcaggagtc aggacctggc ctggtggtgc actcacagag cctgtccatc 480

acatgcaccg tttcagggtt ctcattaatc ggctatggtg taaactgggt tcgccagcct 540

ccaggaaaga gtctggagtg gctcggaatg atatggtgtg atggaaggac agactataat 600

tcaggtctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 660

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt actgtgccag agggtatggt 720

aaccacgcct ggcttgctta ctggtgccaa gggactctgg tcagtgtctc tgcaaccacg 780

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 840

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 900

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 960

ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1020

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1080

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1140

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1200

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgctaa 1446

<210> 20

<211> 481

<212> PRT

<213> Artificial sequence

<400> 20

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

20 25 30

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val

35 40 45

Ile Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Ser Lys Arg

50 55 60

Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe

65 70 75 80

Ser Gly Ser Ala Ser Gly Ala Ser Tyr Ser Leu Ser Ile Ser Ser Met

85 90 95

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Glu Ile Asn

100 105 110

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

130 135 140

Gln Glu Ser Gly Pro Gly Leu Val Val His Ser Gln Ser Leu Ser Ile

145 150 155 160

Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr Gly Val Asn Trp

165 170 175

Val Arg Gln Pro Pro Gly Lys Ser Leu Glu Trp Leu Gly Met Ile Trp

180 185 190

Cys Asp Gly Arg Thr Asp Tyr Asn Ser Gly Leu Lys Ser Arg Leu Ser

195 200 205

Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser

210 215 220

Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Gly Tyr Gly

225 230 235 240

Asn His Ala Trp Leu Ala Tyr Trp Cys Gln Gly Thr Leu Val Ser Val

245 250 255

Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

260 265 270

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

275 280 285

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

290 295 300

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

305 310 315 320

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

325 330 335

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

340 345 350

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

355 360 365

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

370 375 380

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

385 390 395 400

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

405 410 415

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

420 425 430

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

435 440 445

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

450 455 460

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

465 470 475 480

Arg

Claims

1. An isolated binding protein comprising the antigen binding domain of B7-H3, wherein the amino acid sequences of the light chain variable region and the heavy chain variable region are in sequence as shown in SEQ ID nos. 1-2.

2. The binding protein according to claim 1, wherein said binding protein is an scFv.

3. The binding protein according to claim 1, wherein said light chain variable region is linked to said heavy chain variable region by a linking region, said linking region having an amino acid sequence as set forth in SEQ ID No. 3.

4. An isolated nucleic acid encoding the binding protein of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein the variable region of the light chain has the nucleic acid sequence set forth in SEQ ID No. 4.

6. The nucleic acid of claim 4, wherein the heavy chain variable region has the nucleic acid sequence set forth in SEQ ID No. 5.

7. The nucleic acid of claim 4, wherein the nucleic acid sequence of the linker region is as set forth in SEQ ID No. 6.

8. The nucleic acid of claim 4, further linked to a signal peptide nucleic acid sequence.

9. The nucleic acid of claim 8, wherein the nucleic acid sequence of the signal peptide is set forth in SEQ ID No. 7.

10. The nucleic acid of claim 8, wherein the signal peptide is located at the nitrogen terminus of the light chain variable region.

11. A vector, characterized in that it comprises a nucleic acid according to any one of claims 4 to 10.

12. The vector of claim 11, wherein the vector comprises a hinge region of CD8 a, a transmembrane domain of CD8 a, and an intracellular domain of CD3 ζ and comprises a CD28 or 4-1BB co-stimulatory domain; the nucleic acid sequence of the vector is signal peptide-VL-Linker-VH-CD 8 alpha-CD 28-CD3 zeta or signal peptide-VL-Linker-VH-CD 8 alpha-4-BB-CD 3 zeta in sequence.

13. The vector according to claim 12, wherein the nucleic acid sequence of the hinge region of CD8 a, the transmembrane domain of CD8 a is as set forth in SEQ ID No. 9.

14. The vector of claim 12, wherein the nucleic acid sequence of the intracellular domain of CD3 ζ is set forth as SEQ ID No. 11.

15. The vector of claim 12, wherein the nucleic acid sequence of CD28 is set forth in SEQ ID No. 13.

16. The vector of claim 12, wherein the nucleic acid sequence of 4-1BB is set forth in SEQ ID No. 15.

17. The vector of any one of claims 11-16, wherein the vector is a lentiviral vector.

18. The vector of claim 17, wherein the lentiviral vector is pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB.

19. The vector of claim 18, wherein the nucleic acid sequences are linked in the following manner:

pCDH-CMV-B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB;

20. The vector of claim 19, wherein the nucleic acid sequence of B7-H3-CAR-28 is as set forth in SEQ ID No. 17.

21. The vector of claim 19, wherein the nucleic acid sequence of B7-H3-CAR-BB is set forth in SEQ ID No. 19.

22. A host cell comprising the nucleic acid of any one of claims 4 to 10 or the vector of any one of claims 11 to 21.

23. The host cell of claim 22 for expressing the nucleic acid or vector.

24. The host cell of claim 23, which is a 293 cell.

25. A method for producing a CAR-T cell, comprising the steps of:

the host cell of any of claims 22-24, wherein the virus-containing supernatant is infected with a T cell and isolated to obtain a CAR-T cell.

26. CAR-T cells produced by the method of claim 25.

27. Use of a CAR-T cell according to claim 26 in the manufacture of a formulation for the treatment of lung cancer.