CN116284419A

CN116284419A - Monoclonal antibody targeting human GUCY2C protein and application thereof

Info

Publication number: CN116284419A
Application number: CN202310122249.9A
Authority: CN
Inventors: 马丽雅; 谢海涛; 薛卫巍; 文明
Original assignee: Shenzhen Xiankangda Life Science Co ltd
Current assignee: Shenzhen Xiankangda Life Science Co ltd
Priority date: 2023-02-07
Filing date: 2023-02-07
Publication date: 2023-06-23

Abstract

The invention discloses a monoclonal antibody targeting human GUCY2C protein and application thereof; the monoclonal antibody comprises four groups of heavy chain variable regions and four groups of light chain variable regions, and the amino acid sequences of the four groups of heavy chain variable regions can be respectively combined with the amino acid sequences of the four groups of light chain variable regions to form double chains for use. The monoclonal antibody can specifically identify human GUCY2C antigen, has good specificity in normal tissues of human, only binds GUCY2C positive tissues, does not identify other tissues and organs, and has no off-target risk; meanwhile, the monoclonal antibody has good targeting to both natural configuration state and recombinant configuration state, and has good application prospect of antibody drug, multi-drug and recombinant antibody protein.

Description

Monoclonal antibody targeting human GUCY2C protein and application thereof

Technical Field

The invention relates to the technical field of biological cells, in particular to a monoclonal antibody targeting human GUCY2C protein and application thereof, such as application in biological materials and biological preparations.

Background

Guanylate cyclase C (guanylyl cyclase C, GUCY 2C) is a transmembrane protein belonging to the family of receptor guanylate cyclases, and when activated by escherichia coli thermostable enterotoxins (STa), guanosine and uridine, transmits extracellular information into the cell, participating in the regulation of intestinal function. GUCY2C is expressed in primary colorectal cancer cells, in which GUCY2C is expressed 2-10 times that of normal intestinal epithelial cells, and in lymph node and liver tissues with colon cancer metastasis. In addition, GUCY2C was also expressed in pancreatic, gastric and esophageal cancers (up to 60%) and the above information suggests that GUCY2C can be a target for these diseases.

Disclosure of Invention

In view of the above, it is necessary to solve the above problems by providing a monoclonal antibody targeting human GUCY2C CAR and its use, namely a specific antibody which specifically recognizes GUCY2C.

The technical scheme of the invention is as follows:

a monoclonal antibody targeting a human GUCY2C CAR, comprising a heavy chain variable region and a light chain variable region; wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively selected from any one of the following groups:

group 1: the amino acid sequence of the heavy chain variable region HCDR1 is GFTFSTYA, the amino acid sequence of the heavy chain variable region HCDR2 is ISSGGST, and the amino acid sequence of the heavy chain variable region HCDR3 is TRGADY; the amino acid sequence of the light chain variable region LCDR1 is QSLLYSSNQMNY, the amino acid sequence of the light chain variable region LCDR2 is WAS, and the amino acid sequence of the light chain variable region LCDR3 is QQYSSYPLT; or (b)

Group 2: the amino acid sequence of the heavy chain variable region HCDR1 is GYRFTSSW, the amino acid sequence of the heavy chain variable region HCDR2 is IHPDRGII, and the amino acid sequence of the heavy chain variable region HCDR3 is ARWGQLGLRYAMDY; the amino acid sequence of the light chain variable region LCDR1 is ESVDKYGISF, the amino acid sequence of the light chain variable region LCDR2 is DAS, and the amino acid sequence of the light chain variable region LCDR3 is QQSKEVPLT; or (b)

Group 3: the amino acid sequence of the heavy chain variable region HCDR1 is GFSLTSFG, the amino acid sequence of the heavy chain variable region HCDR2 is IWSGGRK, and the amino acid sequence of the heavy chain variable region HCDR3 is VRHGTRPYWYFEV; the amino acid sequence of the light chain variable region LCDR1 is QDISNY, the amino acid sequence of the light chain variable region LCDR2 is YTS, and the amino acid sequence of the light chain variable region LCDR3 is QQGNSLPWT; or (b)

Group 4: the amino acid sequence of the heavy chain variable region HCDR1 is GYTFTSYW, the amino acid sequence of the heavy chain variable region HCDR2 is IYPGGDGDT, and the amino acid sequence of the heavy chain variable region HCDR3 is AREDGYYDY; the amino acid sequence of the light chain variable region LCDR1 is QSLLDSDGKTY, the amino acid sequence of the light chain variable region LCDR2 is LVS, and the amino acid sequence of the light chain variable region LCDR3 is WQGTHFPQT.

In the monoclonal antibody, the amino acid sequences corresponding to the heavy chain variable region and the light chain variable region are as follows:

in the 1 st group, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; or (b)

In group 2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4; or (b)

In group 3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6; or (b)

In group 4, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

The invention also provides a nucleic acid molecule containing the monoclonal antibody, and a biological preparation such as an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line containing the nucleic acid molecule, or a biological preparation containing the monoclonal antibody; wherein, the recombinant vector also comprises a gene recombinant expression vector and a chimeric antigen receptor.

Preferably, the biological agent is a reagent for detecting the concentration of human GUCY2C protein, a reagent for detecting the expression level of human GUCY2C protein on the surface of tumor cells or a complement-dependent or cell-dependent cytotoxic reaction reagent for carrying out antibody mediation on human GUCY2C positive cells; the biological reagent contains antibody coupled toxin, kills human GUCY2C positive cells, and is prepared into targeted human GUCY2C by coupling other antibodies with the antibody and is prepared into functional recombinant protein of targeted human GUCY2C by coupling other proteins with the antibody.

The monoclonal antibody provided by the invention has the following advantages:

1. has the specificity of recognizing human GUCY2C protein, and partial combination has the specificity of human and mouse double species;

2. the antibody has good tissue specificity, only recognizes GUCY 2C-expressing tissues, does not recognize other tissues and organs, and has good detection application prospect;

3. the CAR-T prepared by the antibody has a specific killing function on GUCY2C positive target cells, and has a good application prospect of cell immunotherapy;

4. the antibody has good targeting property in both a natural configuration state and a recombinant configuration state, and has good application prospects of Antibody Drugs (ADC), multi-drug and recombinant antibody proteins.

Drawings

FIGS. 1a, 1b, 1C and 1d show, respectively, a negative pattern of HGC27 cells GUCY2C expression, a positive pattern of HGC27-hGUCY2C cells expression hGUCY2C, a negative pattern of K562 cells expression mGUCY2C, and a positive pattern of K562-mGUCY2C cells expression in example 1;

FIG. 2 shows the results of murine monoclonal antibody affinity assay;

FIGS. 3a, 3b, 3c, 3d are results of species-specific detection of murine monoclonal antibodies; FIG. 3a is a graph showing the binding activity of murine monoclonal antibody to HGC27 cells; FIG. 3b is a graph showing the binding activity of murine monoclonal antibody to HGC 27-hUCY 2C cells; FIG. 3c is a graph showing the binding activity of murine mab to K562 cells; FIG. 3d is a graph of the binding activity of murine mab to K562-mGluY 2C cells;

FIG. 4 shows the results of tissue-specific detection of murine monoclonal antibodies;

FIG. 5 is a CAR-GUCY2C structure;

FIG. 6 is an amplification curve of immune cell culture;

FIGS. 7a, 7b, 7c, 7d, 7e, 7f are the expression profiles of CAR, respectively; wherein, fig. 7a is a negative profile of NT cell (control cell) CAR expression; FIG. 7b is a graph of the positive rate of CAR expression in CAR-081 cells; FIG. 7c is a graph of the positive rate of CAR expression in CAR-1H8 cells; FIG. 7d is a graph of the positive rate of CAR expression in CAR-3F7 cells; FIG. 7e is a graph of the positive rate of CAR expression in CAR-4D9 cells; FIG. 7f is a graph of the positive rate of CAR expression in CAR-4E5 cells;

FIG. 8a is a graph of the killing of CAR-T against HGC27 cells;

FIG. 8b is a graph of the killing of HGC 27-hUCY 2C cells by CAR-T.

Detailed Description

In the present invention, monoclonal antibodies (also abbreviated as monoclonal antibodies) are derived from murine sources, and the technical scheme is illustrated and described.

The invention provides a monoclonal antibody targeting a human GUCY2C CAR, comprising a heavy chain variable region (HV) and a light chain variable region (LV); wherein, in the heavy chain variable region and the light chain variable region which correspond to the amino acid sequence and the nucleic acid sequence respectively, CDR1, CDR2 and CDR3 are three complementarity determining regions of the corresponding amino acid sequence and nucleic acid sequence respectively; in the present invention, HCDR1, HCDR2 and HCDR3 represent three complementarity determining regions of the heavy chain variable region; LCDR1, LCDR2 and LCDR3 represent three complementarity determining regions of the light chain variable region;

in the present invention, the amino acid sequences and the nucleic acid sequences of the heavy chain variable region and the light chain variable region are any one of the following groups:

group 1

A) HV nucleic acid sequence is shown as SEQ ID NO. 11:

ATGAACTTCGGGTTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAACTGAAACTGGTGGAGTCTGGGGGAGGCTTAATGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTC AGTACCTATGCCATGTCTTGGGTTCGTCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTG GCAGCACCTACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTTTCTGCAAATGATCAGTCTGAGGTCTGAGGACACGGCCATGTATTATTGTACAAGAGGTGCGGACTACTGGGGGCAAGGAACCTCAGTCACCGTCTCCTCA; wherein:

a-HCDR1 represents GGATTCACTTTCAGTACCTATGCC;

a-HCDR2 represents ATTAGTAGTGGTGGCAGCACC;

a-HCDR3 represents ACAAGAGGTGCGGACTAC;

a) The HV amino acid sequence is shown as SEQ ID NO. 1:

MNFGFSLIFLVLVLKGVQCELKLVESGGGLMKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGSTYYSDSVKGRFTISRDNARNILFLQMISLRSEDTAMYYCTRGADYWGQGTSVTVSS; wherein:

a-HCDR1 represents GFTFSTYA;

b-HCDR2 represents ISSGGST;

c-HCDR3 represents TRGADY;

b) LV nucleic acid sequence is shown in SEQ ID NO. 12:

ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGC CTTTTATATAGTAGCAATCAAATGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTTCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCATCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTCTAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA; wherein:

B-LCDR1 represents CAGAGCCTTTTATATAGTAGCAATCAAATGAACTAC;

B-LCDR2 represents TGGGCATCC;

B-LCDR3 represents CAGCAATATTCTAGCTATCCGCTCACG;

b) LV amino acid sequence is shown as SEQ ID NO. 2:

MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQMNYLAWYQQKPGQSPKLLIFWASTRESGVPDRFTGSGSGTDFTLIISSVKAEDLAVYYCQQYSSYPLTFGAGTKLELK; wherein:

b-LCDR1 represents QSLLYSSNQMNY;

b-LCDR2 represents WAS;

b-LCDR3 represents QQYSSYPLT.

Group 2

C) HV nucleic acid sequence is shown as SEQ ID NO. 13:

ATGAGCACTGAACACAGACACCTCACCATGAACTTCGGGTTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTCAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGGATCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACAGGTTCACCAGCTCCTGGATGCACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTGATAGAGGTATTATAAACTATAATGAGAAATTCAAGGACAAGGCCACACTGACTGTAGACACATCCTCCAACACGGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATGGGGACAACTCGGACTACGGTATGCTATGGACTACTGGGGTCGAGGAACCTCAGTCACCGTCTCCTCA; wherein:

C-HCDR1 represents GGCTACAGGTTCACCAGCTCCTGG;

C-HCDR2 represents ATTCATCCTGATAGAGGTATTATA;

C-HCDR3 represents GCAAGATGGGGACAACTCGGACTACGGTATGCTATGGACTAC;

c) The HV amino acid sequence is shown as SEQ ID NO: 3:

MSTEHRHLTMNFGFSLIFLVLVLKGVQCQVQLQQPGSVLVRPGGSVKLSCKASGYRFTSSWMHW AKQRPGQGLEWIGEIHPDRGIINYNEKFKDKATLTVDTSSNTAYVDLSSLTSEDSAVYYCARWGQLGL RYAMDYWGRGTSVTVSS; wherein:

c-HCDR1 representsGYRFTSSW；

c-HCDR2 represents IHPDRGII;

c-HCDR3 represents ARWGQLGLRYAMDY;

d) LV nucleic acid sequence is shown in SEQ ID NO. 14:

ATGGAGAAAGACACACTCCTGCTATGGGTCCTGCTTCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCCAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGT GTTGATAAATATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTTTG ATGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGGGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCACCCTATGGAGGAAGATGACAGTGGAATGTACTTCTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGTGCTGGGACCAACCTGGAGCTGAAA; wherein:

D-LCDR1 represents GAAAGTGTTGATAAATATGGCATTAGTTTT;

D-LCDR2 represents GATGCATCC;

D-LCDR3 represents CAGCAAAGTAAGGAGGTTCCGCTCACG;

d) LV amino acid sequence is shown as SEQ ID NO. 14:

MEKDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSPGQRATISCRASESVDKYGISFMNWFQQKP GQPPKLLIFDASNQGSGVPARFRGSGSGTDFSLNIHPMEEDDSGMYFCQQSKEVPLTFGAGTNLELK；

wherein:

d-LCDR1 represents ESVDKYGISF;

d-LCDR2 represents DAS;

d-LCDR3 represents QQSKEVPLT.

Group 3

E) HV nucleic acid sequence is shown as SEQ ID NO. 15:

ATGGCTGTCTTGGGGCTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTATCCCAGGTGCAGCTGAAGCAGTCGGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTA ACTAGCTTTGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATTTGGAGTGGTG GAAGGAAAGACTATAATGCAGTTTTCGTATCCAGGCTGAGCATCAGTAAGGACAATTCCAAGAGCCGAGTTTTCTTTAAAATGAACAGTCTGCAAGCTAATGACACAGCCATATATTACTGTGTCAGACACGGTACTAGACCCTACTGGTAC TTCGAAGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA; wherein:

E-HCDR1 represents GGTTTCTCATTAACTAGCTTTGGT;

E-HCDR2 represents ATTTGGAGTGGTGGAAGGAAA;

E-HCDR3 represents GTCAGACACGGTACTAGACCCTACTGGTACTTCGAAGTC;

e) The HV amino acid sequence is shown as SEQ ID NO. 5:

MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTSFGVHWVRQSPGKGL EWLGVIWSGGRKDYNAVFVSRLSISKDNSKSRVFFKMNSLQANDTAIYYCVRHGTRPYWYFEVWGAGT TVTVSS; wherein:

e-LCDR1 represents GFSLTSFG;

e-LCDR2 represents IWSGGRK;

e-LCDR3 represents VRHGTRPYWYFEV;

f) LV nucleic acid sequence as shown in SEQ ID NO. 16:

ATGATTGCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGACATTGTGATGACCCAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGAC ATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCACGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGGCACTTACTTTTGCCAACAGGGTAATTCGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA; wherein:

F-HCDR1 represents CAGGACATTAGCAATTAT;

F-HCDR2 represents TACACATCA;

F-HCDR3 represents CAACAGGGTAATTCGCTTCCGTGGACG;

f) LV amino acid sequence is shown as SEQ ID NO. 6:

MIASAQFLGLLLLCFQGTRCDIVMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTV KLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIGTYFCQQGNSLPWTFGGGTKLEIK; wherein:

f-LCDR1 represents QDISNY;

f-LCDR2 represents YTS;

f-LCDR3 represents QQGNSLPWT.

Group 4

G) HV nucleic acid sequence is shown as SEQ ID NO. 17:

ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCAGTAACTTCAGGTGTCTACTCACAGGTTCAGCTCCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTT ACTAGCTACTGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAG ATGGTGATACTAGGTACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAAGATGGTTACTACGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA; wherein:

G-HCDR1 represents GGCTACACCTTTACTAGCTACTGG;

G-HCDR2 represents ATTTATCCTGGAGATGGTGATACT;

G-HCDR3 represents GCAAGAGAAGATGGTTACTACGATTAC;

g) The HV amino acid sequence is shown as SEQ ID NO: 7:

MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGL EWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCAREDGYYDYWGQGTTLT VSS; wherein:

g-HCDR1 represents GYTFTSYW;

g-HCDR2 represents IYPGGDT;

g-HCDR3 represents AREDGYYDY;

h) LV nucleic acid sequence is shown as SEQ ID NO. 18:

ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATATCATGATGACCCAAACTCCACTCTCTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGC CTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA; wherein:

H-LCDR1 represents CAGAGCCTCTTAGATAGTGATGGAAAGACATAT;

H-LCDR2 represents CTGGTGTCT;

H-LCDR3 represents TGGCAAGGTACACATTTTCCTCAGACG;

h) LV amino acid sequence is shown as SEQ ID NO. 8:

MMSPAQFLFLLVLWIRETNGDIMMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQR PGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK；

wherein:

h-LCDR1 represents QSLLDSDGKTY;

h-LCDR2 represents LVS;

h-LCDR3 represents WQGTHFPQT.

In the present invention, the letters A, B, C, D, E, F, G, H, a, b, c, d, e, f, g, h and the like corresponding to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are used for the sake of distinction, and are not used in any other way.

In the present invention, the amino acid sequences of the monoclonal antibodies can be selected by the following combinations when used.

For example, any of the amino acid sequences a), c), e), g) of the heavy chain variable region HV may be double-stranded with any combination of the amino acid sequences b), d), f), h) of the light chain variable region LV, respectively; or any single strand of the amino acid sequences a), b), c), d), e), f), g), h).

Specifically, for the use of double-stranded combinations of amino acids, for example, when the amino acid sequence of the heavy chain variable region HV is a), the amino acid sequence of the light chain variable region LV is then the sequence b); alternatively, when the amino acid sequence of the heavy chain variable region HV is c), the amino acid sequence of the light chain variable region LV is the sequence d); alternatively, when the amino acid sequence of the heavy chain variable region HV is e), the amino acid sequence of the light chain variable region LV is the sequence f); alternatively, when the amino acid sequence of the heavy chain variable region HV is g), the amino acid sequence of the light chain variable region LV is the sequence h) or the like.

Accordingly, the nucleic acid sequences of the monoclonal antibodies can also be selected in combination as follows: any of the nucleic acid sequences a), C), E), G) of the heavy chain variable region HV may be double-stranded for use with any combination of the nucleic acid sequences B), D), F), H) of the light chain variable region LV, respectively; or any single strand of the nucleic acid sequences A), B), C), D), E), F), G), H). Specifically, for the use of a combination of nucleic acid strands, for example, when the nucleic acid sequence of the heavy chain variable region HV is a), the nucleic acid sequence of the light chain variable region LV is then the sequence B); alternatively, when the nucleic acid sequence of the heavy chain variable region HV is C), the nucleic acid sequence of the light chain variable region LV is sequence D); alternatively, when the nucleic acid sequence of the heavy chain variable region HV is E), the nucleic acid sequence of the light chain variable region LV is sequence F); alternatively, when the nucleic acid sequence of the heavy chain variable region HV is G), the nucleic acid sequence of the light chain variable region LV is H), or the like.

In one embodiment, when the amino acid sequences in the light and heavy chain variable regions of the monoclonal antibody are paired for use and expressed as murine monoclonal antibody, it is preferred to express any one of the following paired groups:

1) The amino acid sequence a) and the amino acid sequence b) are paired, and the expression and purification murine monoclonal antibody is named 1H8;

2) Pairing the amino acid sequence c) and the amino acid sequence d), and expressing and purifying the mouse monoclonal antibody to be named as 3F7;

3) The amino acid sequence e) and the amino acid sequence f) are paired, and the expression and purification murine monoclonal antibody is named as 4D9;

4) Amino acid sequence g) and amino acid sequence h) were paired and the expression purified murine monoclonal antibody was designated 4E5.

In one embodiment of the invention, after constructing a plasmid expression cassette for the murine mab gene, the expression cassette is transferred into 293T cells by a delivery system for antibody expression; wherein the delivery system may be one of lentivirus, retrovirus, common plasmid vector, episomal vector, nano-delivery system, electrotransduction and transposon.

In another example, the fermented purified murine mab can be co-incubated with GUCY2C positive cells to verify affinity and specificity of murine mab; alternatively, the fermented and purified murine mab was subjected to immunohistochemical detection with tissue sections and normal tissue chips to verify the tissue cross-reaction specificity of murine mab.

In one embodiment, the murine mab light and heavy chain variable regions can be concatenated into scFv sequences to construct chimeric antigen receptors targeting human GUCY 2C; the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain; wherein:

1) The extracellular domain of the chimeric antigen receptor includes an antigen binding domain; the antigen binding domain comprises an scFv, and the scFv is the light-heavy chain variable region of 1H8, 3F7, 4D9, 4E 5;

2) The transmembrane domain of the chimeric antigen receptor includes the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, the hinge region of CD8 and the transmembrane domain of ICOS, and the like; wherein the transmembrane domain of the chimeric antigen receptor is the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB or the costimulatory domain of 4-1BB or ICOS;

3) The intracellular domain of the chimeric antigen receptor includes a costimulatory signaling region, a cytokine receptor intracellular region, and a CD3 zeta chain moiety; wherein the costimulatory signaling region refers to a portion of an intracellular domain comprising a costimulatory molecule; costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens; the intracellular domain may be any one of the co-stimulatory domains of CD28, 4-1BB, ICOS in combination with the intracellular activation signal CD3ζ.

In the invention, when the monoclonal antibody is humanized or modified from other animal sources, the amino acid sequence and/or the nucleic acid sequence in three complementary determining regions CDR1, CDR2 and CDR3 are changed by not more than 20 percent; alternatively, the amino acid sequence and/or nucleic acid sequence of the three complementary determining regions of CDR1, CDR2 and CDR3 of the monoclonal antibody are changed to 0 when humanized or other animal-derived engineering is performed.

In the present invention, the recombinant cell line is an immune cell, and may be any immune cell including T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell. Specifically, when the immune cells express the chimeric antigen receptor CAR, NK cells, NKT cells, TIL, gamma-delta T cells are equivalent to T cells (or T cells may replace NK cells).

The targeted human GUCY2C monoclonal antibody can be prepared into a pharmaceutically acceptable carrier, diluent or excipient, has good forming effect, and simultaneously maintains good drug effect; such as in biological materials and/or biological agents.

When the monoclonal antibody is used in a biological preparation, the biological preparation comprises an expression cassette (e.g., a gene expression cassette), a recombinant vector (e.g., a plasmid), a recombinant protein (e.g., a fusion protein, an antibody protein, etc.), a recombinant microorganism (e.g., E.coli, phage, etc.), or a recombinant cell line (e.g., an immune cell, CHO cell, etc.) constructed from the above-described nucleic acid sequences or amino acid sequences derived from the above-described monoclonal antibody; wherein the recombinant vector comprises a gene recombinant expression vector and a chimeric antigen receptor.

When the monoclonal antibody is applied to biological agents, the monoclonal antibody exists as a biological agent preparation component, and the biological agent component also comprises a reagent for detecting the concentration of human GUCY2C protein, a reagent for detecting the expression degree of the human GUCY2C protein on the surface of tumor cells, an antibody coupled toxin for killing human GUCY2C positive cells, other antibodies coupled with the antibody for preparing targeted human GUCY2C and other antigen polyclonal antibodies, and other proteins coupled with the antibody for preparing functional recombinant proteins for targeting human GUCY2C.

In summary, compared with the prior art, the monoclonal antibody provided by the invention has the following advantages:

1. the specific recognition target human GUCY2C protein is provided, and partial combination has the specificity of human and mouse double species;

3. the CAR-T prepared by the antibody has a specific killing function on GUCY2C positive target cells, and has good application prospect of cell immunotherapy;

The monoclonal antibody expansion application of the present invention will be described in detail below, typically using CAR-T cells as an example.

Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. Wherein the extracellular domain comprises an antigen binding domain, the intracellular domain comprises a costimulatory signaling region, which refers to a portion of the intracellular domain comprising a costimulatory molecule, which is a cell surface molecule required for the efficient response of lymphocytes to an antigen, a cytokine receptor intracellular region, and a CD3 zeta chain moiety.

In a preferred embodiment, the extracellular domain of a CAR provided by the invention comprises a murine monoclonal antibody light heavy chain variable region antigen binding domain that targets GUCY2C. When expressed in T cells, the CARs of the invention are capable of antigen recognition based on antigen binding specificity or protein receptor binding. The antigen binding domain is preferably fused to an intracellular domain from a costimulatory molecule and a cd3ζ chain. Preferably, the antigen binding domain is fused to the intracellular domains of the combination of the CD28, 4-1BB, ICOS signaling domain and CD3 zeta signaling domain, respectively.

In the CAR of the invention, the intracellular domains include the signaling domains of CD28, 4-1BB, ICOS and CD3 zeta.

The vector of the expression cassette is selected from: one or more of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, retrovirus vector, transposon, and other gene transfer system. Preferably, the vector is a viral vector.

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

The present invention also provides vectors inserted into the expression cassette, derived from a retrovirus, such as a lentivirus, characterized by long-term, stable integration of the gene of interest into the cell; transduction of non-proliferating cells, such as hepatocytes; low immunogenicity; the safety is high. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression vector may be provided to the cell as a viral vector. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence, another example is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

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

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. A preferred method of introducing the polynucleotide into a host cell is by liposome transfection. The nucleic acid may be associated with a lipid, the nucleic acid associated with a lipid may be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, which is a fatty substance, which may be a naturally occurring or synthetic lipid. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

The invention provides CAR-T cells containing a GUCY 2C-targeting antibody as described above, and a pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 ³ ～1×10 ⁸ The concentration of CAR-T cells per ml, more preferably, is 1X 10 ⁴ ～1×10 ⁷ And each ml. In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for use in silenceIntravenous administration.

The invention includes therapeutic applications using cells transduced with lentiviral vectors (e.g., T cells) encoding the nucleic acid constructs of the antibodies of the invention. Transduced T cells can elicit a CAR-mediated T-cell response. The injected cells are capable of killing the recipient's tumor cells and the CAR-T cells are capable of replication in vivo, resulting in long-term persistence that can lead to sustained tumor control. And the CAR-T cell membrane can express the murine monoclonal antibody scFv structural antigen chimeric receptor targeting GUCY2C, specifically kills GUCY2C positive target cells and has no off-target risk.

In one embodiment, the immune cells of the invention may undergo robust in vivo T cell expansion and may last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified T cells induce an immune response specific for an antigen binding domain in the CAR. For example, GUCY2C CAR-T cells elicit a specific immune response against cells expressing GUCY2C.

Treatable symptoms include tumor cells that are positive for GUCY2C expression. Symptoms that may be caused by tumor cells include, but are not limited to, carcinoma, blastoma, sarcoma, and the like.

CAR immune cells using the antibodies of the invention, ex vivo procedures to modify T cells, at least one of the following occurs in vitro prior to administration of the cells into a human: i) Amplifying the cells, ii) introducing into the cells a nucleic acid encoding a CAR and a nucleic acid encoding an immune checkpoint antibody protein expressed on the cell membrane fused to a cytokine receptor, and/or iii) cryopreserving the cells. Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells isolated in human peripheral blood are used to express the CARs disclosed herein and immune checkpoint antibody proteins expressed on the cell membrane fused to cytokine receptors. T-modifying cells can be administered to a recipient to provide therapeutic benefit. Second, the immune cells may be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

Immune cells using antibodies of the invention CAR modified T cells can be administered alone or in combination with other drugs, pharmaceutical compositions, diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

Pharmaceutical compositions made using immune cells of the antibodies of the invention may be administered in a manner appropriate for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and by the clinical regimen. When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be 1 x 10 ⁴ ～1×10 ⁹ A dose of individual per kg body weight, preferably 1X 10 ⁵ ～1×10 ⁶ A dose of individual per kg body weight. T cell compositions may also be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular patient can be determined by one of skill in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the formulations of the present invention may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

The following is a detailed description of specific examples.

EXAMPLE 1 preparation of monoclonal antibodies

The monoclonal antibody was prepared as follows:

s10: mouse immunization and murine monoclonal antibody sequence acquisition

Balbc mice were immunized with self-produced human GUCY2C protein, and mice were immunized by subcutaneous multiple injections at 20 ug/dose, once a week. After 4 weeks of immunization, mice with blood antibody titers above 1k were sacrificed and spleen cells were fused with SP2.0 cells. And (3) performing titer detection on the fusion cells, screening positive fusion cells, selecting monoclonal cells by using a limiting dilution method, performing antibody fermentation and function evaluation, extracting genes from the finally screened mouse monoclonal antibodies, constructing antibody light and heavy chain expression plasmid fermentation purified antibodies, and constructing CAR plasmids.

S20: murine monoclonal antibody fermentation purification

Pairing the constructed light and heavy chain expression plasmids of the antibody, using a PEI reagent as a transfection reagent, simultaneously transiently transfecting 293T cells with a pair of light and heavy chain expression plasmids for antibody protein fermentation, naturally pairing the light and heavy chain expression plasmids of the antibody in the fermentation process, and collecting culture solution. The culture broth was filtered using a 0.22 μm sterile filter to obtain a culture supernatant, which was then subjected to affinity chromatography purification.

The purification method comprises the following steps: (1) 20mM PB, pH7.0 equilibrated with 1ml-Protein A affinity column, 1.0ml/min; (2) 50ml of the culture medium is loaded, 1ml/min is used for balancing; (3) Eluting with 0.1M glycine-HCl, pH2.7, 1ml/min, and collecting 280nm absorption peak; (4) 20-50mM PB,150mM NaCl,pH6.7 equilibrate Superdex 200Increase 10/300GL column, 1ml/min; and (5) loading, and collecting 2ml of 280nm absorption peak.

HPLC analysis is performed on the purified sample, the received peak is detected, the concentration of the detected peak is detected, and the amount of protein is calculated.

EXAMPLE 2 cell line culture

Cell lines that do not express GUCY 2C: HGC27 (human gastric cancer cell), K562 (human chronic myelogenous leukemia cell), purchased from ATCC.

Cell line expressing human GUCY 2C: HGC27-hGUCY2C, self-produced lentivirus infection construction.

Cells expressing murine GUCY 2C: K562-mGUCY2C, and self-produced lentivirus infection construction.

Packaging cell: 293T (human embryonic kidney cell line), purchased from ATCC.

Establishment of cell lines overexpressing human GUCY2C and murine GUCY 2C: cloning the base sequences for expressing hGUCY2C and mGUCY2C into a PHBLV lentiviral vector skeleton, placing under a promoter of EF1 alpha (EF-1 alpha) to form PHBLV-EF1 alpha-hGUCY 2C and PHBLV-EF1 alpha-mGUCY 2C, and transferring three plasmids of PHBLV-EF1 alpha-hGUCY 2C and PHBLV-EF1 alpha-mGUCY 2C, lentiviral envelope Plasmid pMD2.G (Addgene, plasmid# 12259) and lentiviral packaging Plasmid psPAX2 (Addgene plasmid#12260) into 293T by using Lipofectamine3000 to prepare a lentiviral complete expression vector; virus supernatants were collected at 48h and 72h, and concentrated by super-isolation (Merck Millipore); the concentrated virus can be used for infecting HGC27 and K562 cells, and finally the HGC27 monoclonal cell line which overexpresses hGUCY2C and the K562 mixed cell line which overexpresses mGUCY2C are obtained, and the mixed cell line is named HGC27-hGUCY2C and K562-mGUCY2C.

All of the above cell culture media were cultured: adherent cells were cultured using DMEM medium, and suspension cells were cultured using 1640 medium. All media were supplemented with 10% (v/v) fetal bovine serum.

FIG. 1a, 1b, 1C and 1d are graphs showing the expression of human or murine GUCY2C by HGC27, HGC27-hGUCY2C and K562, K562-mGUCY2C cells, respectively; wherein:

FIG. 1a shows the results of an anti-human GUCY2C antibody assay for HGC27 cells, with the peak position to the left of the vertical line, indicating that the assay is GUCY2C negative;

FIG. 1b shows the results of the detection of HGC27-hGUCY2C cells using anti-human GUCY2C antibodies, wherein the peak positions are on the right side of the vertical line, and the detection results are positive for hGUCY 2C;

FIG. 1C shows the detection results of K562 cells using anti-murine GUCY2C antibodies, with peak locations to the left of the vertical line, indicating that the detection results are mUCY 2C negative;

FIG. 1d shows the results of detection of K562-mUCY 2C cells using anti-murine GUCY2C antibodies.

The K562-mGluY 2C cells are not selected for monoclonal after construction, the expression is not completely positive, the peak position in the figure is mGluY 2C positive on the right side of the vertical line, the peak position is mGluY 2C negative on the left side of the vertical line, and the detection result shows that the positive rate of the K562-mGluY 2C cells is 68.5%.

EXAMPLE 3 evaluation of murine monoclonal antibody affinity and species specificity

In this example, the cells of example 2 were used for affinity and species-specific evaluation of the monoclonal antibodies of example 1.

The EC50 affinities of the GUCY2C murine mab 1H8, 3F7, 4D9, 4E5 were tested using the HGC27-hGUCY2C cells of example 2 as positive cells and 081 antibodies as positive controls, and the results of the tests are shown in fig. 2. The results showed that the EC50 affinities of 1H8, 3F7, 4D9, 4E5 were all close to or higher than control antibody 081.

The species specificity of GUCY2C murine monoclonal antibodies 1H8, 3F7, 4D9, 4E5 was examined using HGC27, HGC27-hGUCY2C, K, K562-mGUCY2C cells of example 2 as target cells, and the results are shown in FIGS. 3a, 3b, 3C, 3D.

In FIG. 3a, the binding peaks of murine monoclonal antibodies 1H8, 3F7, 4D9, 4E5, respectively, with HGC27 cells are all on the left side of the vertical line, indicating that the binding is negative; the binding peaks of 1H8, 3F7, 4D9, 4E5 and HGC 27-hUCY 2C cells in FIG. 3b are all on the right side of the vertical line, indicating positive binding; the peaks of 1H8, 3F7, 4D9, 4E5 binding to K562 cells in fig. 3c are to the left of the vertical line, indicating that their binding is negative; in FIG. 3D, the binding peaks of 1H8, 3F7, 4D9, 4E5 and K562-mGluY 2C cells are shown in part on the left side of the vertical line as negative binding, and in part on the right side of the vertical line as positive binding, the positive rate matches the positive rate of K562-mGluY 2C cells, and the detected 3F7 antibody binding cells were positive K562-mGluY 2C cells, indicating that the 3F7 antibody can bind to mGluY 2C.

The above results indicate that the murine mab 1H8, 3F7, 4D9, 4E5 antibodies can specifically bind to human GUCY2C and that some antibodies can bind to murine GUCY2C. The antibody has two species specificities of human and mouse, and can be used for preliminarily evaluating pathological toxicology characteristics in a subsequent experiment in a mouse animal experiment.

Example 4 evaluation of tissue-specific detection of murine monoclonal antibody

Immunohistochemical evaluation of tissue specificity of murine mab in example 1 was performed using a human normal tissue chip comprising 37 normal tissue types: esophageal tissue, gastric tissue, small intestine tissue, colon tissue, liver tissue, pancreatic tissue, appendices tissue, tongue tissue, salivary gland tissue, pharyngeal mucosa, lung tissue, testicular tissue, prostate tissue, breast tissue, ovarian tissue, endometrial tissue, cervical canal tissue, cervical tissue, renal cortex tissue, renal medullary tissue, bladder tissue, tonsil tissue, lymph node tissue, thymus tissue, spleen tissue, skin tissue, skeletal muscle tissue, arterial tissue, pleural mesothelial tissue, peripheral nerve tissue, cerebellar tissue, white matter of the brain, grey matter of the brain, adrenal tissue, thyroid tissue, myocardial tissue. A total of 102 tissue points. The detection result is shown in fig. 4, and the result shows that the dye has a dyeing effect only on the cell membrane surface of esophagus and stomach tissues (GUCY 2C positive expression), and no cell membrane surface is dyed on the other normal tissues, so that the tissue specificity is good.

EXAMPLE 5 isolation of peripheral blood PBMC and culture of T cells

Mononuclear cells were isolated from human donor peripheral blood, density gradient centrifuged using ficol, and T cells were enriched with T cell sorting kit (CD 3 microblades, human-lyophilized, 130-097-043), cultured and expanded using anti-CD3/anti-CD28 coupled magnetic beads; the medium was TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) containing 10% FBS,2mM L-glutamine,100IU/ml rhIL2, all cells were placed at 37℃and 5% CO ₂ Culturing in a constant temperature incubator to obtain T cells.

EXAMPLE 6CAR structural design and lentiviral packaging

GUCY2C-CAR constructs, i.e., GUCY 2C-targeted CAR constructs:

in this example, four pairs of target GUCY2C murine monoclonal antibody light and heavy chain variable region scFv sequences designated 1H8, 3F7, 4D9, 4E5 were respectively constructed on one expression cassette of the second generation CAR. The core structure of the CAR includes a secretion signal peptide sequence; a CD8 transmembrane region; the intracellular segment stimulation signals 4-1BB-CD3 zeta are respectively named as CAR-1H8, CAR-3F7, CAR-4D9 and CAR-4E5, the expression frame of the CAR is constructed by contrast with the light and heavy chain variable region scFv of the GUCY2C antibody, and the expression frame is named as CAR-081, and as shown in figure 5, the identification numbers 1#, 2#, 3#, 4#, and 5# respectively represent 5 GUCY2C-CAR structural schematic diagrams.

As shown in FIG. 5, GUCY2C scFv/TM/4-1BB/CD3 zeta represents a chimeric antigen receptor expressed on the cell membrane of an immune cell.

Cloning the expression cassette into a PHBLV lentiviral vector backbone, placing under a promoter of EF 1a (EF-1 a), forming three plasmids PHBLV-EF1 a-CAR-1H 8, PHBLV-EF1 a-CAR-3F 7, PHBLV-EF1 a-CAR-4D 9, PHBLV-EF1 a-CAR-4E 5 and PHBLV-EF1 a-CAR-081, transferring PHBLV-EF1 a-CAR-1H 8, PHBLV-EF1 a-CAR-3F 7, PHBLV-EF1 a-CAR-4D 9, PHBLV-EF1 a-CAR-4E 5 and PHBLV-EF1 a-CAR-081, lentiviral envelope Plasmid pmd2.g (adedge, plasmid # 12259) and lentiviral packaging Plasmid psPAX2 (adedge Plasmid # 12260) into a lentiviral vector 293T using lipoctamine 3000 to prepare a complete expression vector; after culturing the cells for 48 hours and 72 hours, transferring the cells into a centrifuge tube for centrifugation, collecting virus supernatant after centrifugation is stopped, and performing ultracentrifugation concentration (Merck Millipore) on the supernatant; the concentrated virus can be used to infect T cells.

Example 7CAR-T cell preparation

1. Lentiviral infection

After the primary T cells isolated and purified in example 5 were activated for 1 day, 5 lentiviruses, respectively, lv-CAR-1H8, lv-CAR-3F7, lv-CAR-4D9, lv-CAR-4E5, lv-CAR-081, packaged in example 6 were used. Lentiviral vector infection was performed at MOI (1-10) and virus-infected T cells were transferred to cell culture flasks and placed at 37℃in 5% CO ₂ Culturing in a constant temperature incubator.

2. Cell proliferation and CAR positive rate detection

The number of cells was sampled every day and examined on

days

6, 9, 11 and 13 after T cell infection, and the CAR positive rate of T cells was examined on day 6, and medium was fed every 1-2 days.

Using the 5 lentiviral vectors of example 6, 5 CAR-T cells were successfully constructed, designated CAR-T-1H8, CAR-T-3F7, CAR-T-4D9, CAR-T-4E5, CAR-T-081, respectively, with non-lentiviral infected T cells as controls (NT).

As shown in FIG. 6, the proliferation rates of 5 cells were not significantly different from each other under the same culture conditions.

As shown in fig. 7a, 7b, 7c, 7D, 7E, 7F, respectively, represent expression of NT cells, CAR-T-081, CAR-T-1H8, CAR-T-3F7, CAR-T-4D9, CAR-T-4E5, tested CAR on day 6, as shown by the CAR positive ratios to the right of the vertical line in fig. 7a, 7b, 7c, 7D, 7E, 7F, it can be seen that different scFv have no significant effect on CAR positive ratios.

Example 8 detection experiment of killing HGC 27-hUCY 2C cells by CAR-T

In vitro killing experiments were performed on 5 kinds of CAR-T cells obtained in example 7, killing effect of CAR-T cells was detected by using RTCA equipment, target cells were incubated with effector cells for 24 hours, killing efficiency was obvious, and the results are shown in fig. 8a and 8 b. In FIG. 8a, there is shown no killing effect of each of CAR-T-081, CAR-T-1H8, CAR-T-3F7, CAR-T-4D9, and CAR-T-4E5 on HGC27 cells, indicating no killing activity on non-targeted cells; in FIG. 8b, it is shown that CAR-T-1H8, CAR-T-3F7, CAR-T-4D9, and CAR-T-4E5 all have substantially comparable killing effects on HGC 27-hUCY 2C cells compared to control CAR-T-081.

The above examples illustrate only one embodiment of the invention, which is described in more detail and is not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A monoclonal antibody targeting the human GUCY2C protein, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively selected from any one of the following groups:

2. The monoclonal antibody according to claim 1, wherein in group 1, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; or (b)

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. An expression cassette comprising the nucleic acid molecule of claim 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 3.

6. The recombinant vector according to claim 5, wherein the recombinant vector comprises a gene recombinant expression vector.

7. A recombinant microorganism comprising the nucleic acid molecule of claim 3.

8. A recombinant cell line comprising the nucleic acid molecule of claim 3.

9. A biologic comprising the monoclonal antibody of claim 1 or 2.

10. The biological agent according to claim 9, wherein the biological agent is an agent for detecting the concentration of human GUCY2C protein, or an agent for detecting the expression level of human GUCY2C protein on the surface of tumor cells, or an agent for antibody-mediated complement-dependent or cell-dependent cellular cytotoxicity on human GUCY2C positive cells.