CN116496397B

CN116496397B - Humanized antibodies targeting CD19CAR-T cells

Info

Publication number: CN116496397B
Application number: CN202310346252.9A
Authority: CN
Inventors: 吴国祥; 梅双; 臧超琦; 王文靓; 吴莉莉; 李春莲
Original assignee: Keyi Zhejiang Pharmaceutical Technology Co ltd
Current assignee: Keyi Zhejiang Pharmaceutical Technology Co ltd
Priority date: 2023-04-03
Filing date: 2023-04-03
Publication date: 2024-03-01
Anticipated expiration: 2043-04-03
Also published as: CN116496397A

Abstract

The present invention relates to humanized antibodies targeting CD19CAR-T cells. The chimeric antigen receptor includes a signal peptide, a single chain variable region fragment targeting CD19, a transmembrane region, a co-stimulatory factor, and an intracellular signaling domain. The targeted CD19 humanized antibody provided by the invention can be combined with a CD19 antigen with high specificity, has higher affinity and biological activity, low immunogenicity, stable structure and good patentability, and has a remarkable killing effect on CD19 positive tumor cells.

Description

Humanized antibodies targeting CD19CAR-T cells

Technical Field

The present invention relates to the field of biopharmaceuticals, in particular to humanized antibodies targeting CD19CAR-T cells.

Background

Malignant tumors are one of diseases that seriously threaten human health. Adoptive cellular immunotherapy (adoptive cell therapy, ACT), which is a new approach to highly personalized cancer treatment by infusing immune cells with anti-tumor activity into cancer patients, often relies on the surface compatible antigens of T cells themselves, limiting their therapeutic scope.

To address this problem, chimeric antigen receptor T cell (chimeric antigen receptor gene-modifedt cell, CAR-T) technology has evolved. CAR-T cells are constructed by introducing nucleic acid fused with a CAR gene into the genome of an autologous or allogeneic T lymphocyte, and the antigen binding domain is used to recognize the tumor cell of interest, typically using scFv regions of targeted tumor antigen antibodies, which include the light chain variable region (light chainvariable, VL) and the heavy chain variable region (heavy chain variable, VH) of the antibody, and Linker linkage is used between the two.

CD19 is a CD molecule expressed by B cells (i.e., leukocyte differentiation antigen). All B cell lines except plasma cells, malignant B cells and FDC (follicular dendritic cells) express this molecule. It is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and also promotes BCR signaling. CD19 plays a role as a co-receptor in B cell activation and signaling, regulates B cell activation and proliferation, participates in the signaling function of B cells, and mediates T cell killing of target cells.

B-cell malignancies mainly include various types of leukemia and lymphoma, and most adult chronic lymphocytic leukemia and mantle lymphomas patients are not cured by the currently clinically usual therapies except for a few patients who receive allogeneic hematopoietic stem cell transplantation, so development of new therapies, especially biological immunotherapy, is extremely important for B-cell malignancies.

Disclosure of Invention

It is an object of the present invention to provide a humanized antibody targeting CD 19.

It is another object of the invention to provide the use of humanized antibodies targeting CD 19.

In a first aspect of the invention there is provided a heavy chain variable region of an antibody targeting CD19, said heavy chain variable region being selected from the group consisting of:

(1a) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 4, 5, 2 or 3;

(1b) A heavy chain variable region having the function of the heavy chain variable region of (1 a) and formed by substitution, deletion, modification and/or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues of the amino acid sequence shown in SEQ ID NO. 4, 5, 2 or 3.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is as set forth in SEQ ID NO: 4. 5, 2 or 3.

In a second aspect of the invention there is provided a heavy chain of an antibody targeting CD19, said heavy chain having a heavy chain variable region according to the first aspect of the invention.

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

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

In a third aspect of the invention, there is provided a light chain variable region of an antibody targeting CD19, said light chain variable region being selected from the group consisting of:

(2a) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 7, 8, 9 or 10;

(2b) The light chain variable region having the light chain variable region function of (1 a) and formed by substitution, deletion, modification and/or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues of the amino acid sequence shown in SEQ ID NO. 7, 8, 9 or 10.

In another preferred embodiment, the light chain variable region sequence of the antibody is set forth in SEQ ID NO. 7, 8, 9 or 10.

In a fourth aspect of the invention there is provided a light chain of an antibody targeting CD19, said light chain having a light chain variable region according to the third aspect of the invention.

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

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

In a fifth aspect of the invention, there is provided an antibody targeting CD19, the antibody having:

(1) A heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention;

alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody comprises a single chain antibody (scfv) or a diabody.

In another preferred embodiment, the single chain antibody comprises, or comprises, in sequence, a light chain variable region, a linker and a heavy chain variable region.

In another preferred embodiment, the linker is (G4S) n, where n is an integer from 1 to 5.

In another preferred embodiment, the sequence of the linker is shown in SEQ ID NO. 19.

In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 4; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 7; or (b)

The heavy chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 7.

In another preferred embodiment, the sequence of the single chain antibody is shown in SEQ ID NO. 12 or 13.

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

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

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

In a sixth aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus:

(i) The antibody according to the fifth aspect of the invention,

(ii) A transmembrane domain comprising a transmembrane domain,

(iii) At least one co-stimulatory domain, and

(iv) An activation domain.

In another preferred embodiment, the chimeric antigen receptor fusion protein has a structure according to formula I:

L-scFv-H-TM-C-CD3ζ(I)

In the method, in the process of the invention,

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

scFv is an antibody according to the fifth aspect of the invention;

h is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

l is an optional signal peptide sequence.

In another preferred embodiment, the scFv comprises the amino acid sequence shown in SEQ ID NO.12 or 13.

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

In another preferred embodiment, the H is a CD8 derived hinge region.

In another preferred embodiment, said H comprises the amino acid sequence shown in SEQ ID NO. 15.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM comprises a CD 8-derived transmembrane region.

In another preferred embodiment, the TM comprises the amino acid sequence shown as SEQ ID NO. 16.

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

In another preferred embodiment, said C comprises a costimulatory signaling molecule of 4-1BB origin.

In another preferred embodiment, said C comprises the amino acid sequence shown in SEQ ID NO. 17.

In another preferred embodiment, said CD3 zeta comprises the amino acid sequence shown as SEQ ID NO. 18.

In another preferred embodiment, the signal peptide of L is CD8.

In another preferred embodiment, L comprises the amino acid sequence shown in SEQ ID NO. 14.

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

(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and; and

(ii) Optionally a tag sequence to assist expression and/or purification.

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

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

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

In an eighth aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) An antibody according to the fifth aspect of the invention, a CAR fusion protein according to the sixth aspect of the invention; and

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

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

In a ninth aspect of the invention there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or (b)

(2) A chimeric antigen receptor fusion protein according to the sixth aspect of the invention;

(3) The recombinant protein according to the seventh aspect of the invention.

In a tenth aspect of the invention there is provided a vector comprising a polynucleotide according to the ninth aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In an eleventh aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the tenth aspect of the invention incorporating a polynucleotide according to the ninth aspect of the invention or expressing an antibody according to the fifth aspect of the invention, a CAR fusion protein according to the sixth aspect of the invention.

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

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

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

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

In another preferred embodiment, the engineered immune cells comprise T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

In a twelfth aspect of the invention, there is provided a method of preparing an engineered immune cell expressing a CAR fusion protein according to the sixth aspect of the invention, comprising the steps of: transduction of a nucleic acid molecule according to the ninth aspect of the invention or a vector according to the tenth aspect of the invention into a T cell or NK cell, thereby obtaining said engineered immune cell.

In another preferred embodiment, the method further comprises the step of performing functional and validity assays on the obtained engineered immune cells.

In a thirteenth aspect of the invention, there is provided a formulation comprising an antibody according to the fifth aspect of the invention, a CAR fusion protein according to the sixth aspect of the invention, or a vector according to the tenth aspect of the invention, or a host cell according to the eleventh aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In a fourteenth aspect of the invention there is provided the use of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a CAR fusion protein according to the sixth aspect of the invention, or a cell according to the eleventh aspect of the invention, for the preparation of a medicament or formulation for the prevention and/or treatment of a CD 19-associated cancer or tumour.

In another preferred embodiment, the CD 19-associated cancer or tumor is a solid tumor.

In another preferred embodiment, the CD 19-associated cancer or tumor is selected from the group consisting of: b-cell lymphoma, B-cell leukemia, non-hodgkin lymphoma, multiple myeloma, and the like.

In a fifteenth aspect of the present invention there is provided a kit for preparing a cell according to the eleventh aspect of the present invention, the kit comprising a container and within the container a nucleic acid molecule according to the ninth aspect of the present invention, or a vector according to the tenth aspect of the present invention.

In a sixteenth aspect of the invention there is provided a method of treating a condition associated with a CD19 molecule comprising administering to a subject in need thereof an appropriate cell according to the eighth aspect of the invention, or a formulation according to the thirteenth aspect of the invention.

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

Drawings

FIGS. 1A-1E are ELISA binding EC50 and FACS binding assays of different VL-Linker-VH protein combinations after humanization of murine CD19 antibodies.

FIG. 2 shows the protein equilibrium dissociation constants (KD values) of the sequences of SEQ ID NO:13 (KQ-1-1) and SEQ ID NO:12 (KQ-1-2) after humanization.

FIG. 3 is a graph showing the change in positive rate of T after detection of CD19-CAR-T on days 8 and 11 after virus infection with CD19-CAR lentivirus.

FIG. 4 shows the results of killing H929-CD19-Luc cells by KQ-1, KQ-1-2 after humanization and CD19 (FMC 63) -CART WT before humanization at effective target ratios of 2:1 and 1:2.

FIG. 5 shows the release levels of interleukin 2 by KQ-1, KQ-1-2 after humanization and CD19 (FMC 63) -CART WT at an effective target ratio of 1:1 for H929-CD19-Luc cells and K562 cells before humanization.

FIG. 6 shows the level of interferon gamma release by KQ-1, KQ-1-2 after humanization and CD19 (FMC 63) -CART WT on H929-CD19-Luc cells and K562 cells at an effective target ratio of 1:1.

FIG. 7 shows the in vitro proliferation of CAR-T cells after 2 rounds of H929-CD19 tumor cell stimulation.

FIG. 8 is a plasmid map of the KQ-1-1-CAR structure.

FIG. 9 is a plasmid map of the KQ-1-2-CAR structure.

FIG. 10 is a schematic diagram of the CD19-CAR structure.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, have unexpectedly obtained highly specific and high affinity CD19 humanized antibodies. Experiments show that the CD19 humanized antibody can prolong the survival time of the CAR-T cells, reduce the immune response generated by the CD19 antigen antibody and improve the curative effect of the CD 19-CAR-T. On this basis, the present inventors have completed the present invention.

Terminology

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

CD19

CD19 is a CD molecule expressed by B cells (i.e., leukocyte differentiation antigen) belonging to the Ig superfamily. All B cell lines except plasma cells, malignant B cells and FDC express this molecule. It is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and also promotes BCR signaling.

Chimeric Antigen Receptor (CAR)

As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes also referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

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

The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.

As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids. Preferably, the linker is a flexible linker, for example, the linker is (G4S) n, where n is 1-4.

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 41BB signaling domain, and the cd3ζ signaling domain.

As used herein, an "antigen binding domain" or "single chain antibody fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of an intact antibody. The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the scFv comprises an antibody, preferably a single chain antibody, specifically recognizing the tumor highly expressed antigen CD 19.

In the present invention, scFv of the present invention also includes conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature as compared to the amino acid sequence of scFv of the present invention to form a polypeptide.

In the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids of the original amino acid sequence.

In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.

For the hinge and transmembrane regions (transmembrane domains), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.

Carrier body

The invention also provides DNA constructs encoding the CAR sequences of the invention.

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

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

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

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

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

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning: A laboratory Manual, cold spring harbor laboratory, N.Y.), and other virology and molecular biology handbooks. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

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

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

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

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

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

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

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

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

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

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

In the case of non-viral delivery systems, genome editing techniques, such as CRISPR-Cas9, ZFNs, or TALENs, are illustratively used to accomplish the present invention.

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

The DNA construct further comprises a signal peptide coding sequence. Preferably, the signal peptide sequence is linked upstream of the nucleic acid sequence of the antigen binding domain.

Therapeutic applications

The invention includes cells transduced with a Lentiviral Vector (LV) encoding an expression cassette of the invention. The transduced T cells can induce a CAR-mediated T cell response.

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

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

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can 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.

Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-CD 19 scFv, hinge and transmembrane regions, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of changes to each of the construct components.

Suitable diseases that can be treated include CD 19-associated cancers or tumors, such as CD19 positive tumors or cancers. The CD 19-associated cancer or tumor may include solid tumors, in particular hepatocellular carcinoma, melanoma, ovarian cancer, lung squamous cell carcinoma, gastric cancer, breast cancer, or a combination thereof.

Types of cancers treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain benign and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

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

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

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

In general, activated and expanded cells as described herein can be used to treat and prevent diseases that result in immunocompromised individuals. In particular, the CAR modified T cells of the invention are useful for the treatment of CCL. In certain embodiments, the cells of the invention are used to treat a patient at risk of CCL. Accordingly, the invention provides a method of treating or preventing CCL comprising administering to a subject in need thereof a therapeutically effective amount of a CAR modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-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.

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

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

Administration of the subject compositions may be performed 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.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment ⁶ Up to 1X 10 ¹⁰ The modified T cells of the invention (e.g., GC76BB ζ cells) are administered to a patient by, for example, intravenous infusion.

The main advantages of the invention include:

(a) The targeted CD19 humanized antibody can be combined with a CD19 antigen with high specificity, has higher affinity and biological activity, has stable structure, has obvious killing effect on CD19 positive tumor cells, and improves the treatment effect of CD 19-CAR-T.

(b) The invention adopts the CD19 humanized antibody, has low immunogenicity, reduces the immune response generated by the CD19 antigen antibody, reduces the rejection reaction, can prolong the CAR-T cell duration and improves the curative effect of the CD 19-CAR-T.

(c) The invention adopts the CD19 humanized high-affinity antibody, has good pharmaceutical property, can reduce the recovery amount of CAR-T cells, reduce the occurrence of side effects such as cytokine release and the like, and improves the safety.

(d) The invention can obviously increase the cell factor necessary for the CAR-T cell to release in vivo, keep the CAR-T cell to continuously expand in vivo and further improve the treatment effect.

(e) The product of the invention reduces the recovery quantity of the CAR-T cells, shortens the production and preparation time of the CAR-T cells and shortens the waiting time of patients.

(f) The product of the invention reduces the recovery quantity of the CAR-T cells, shortens the production and preparation time of the CAR-T cells and reduces the production cost.

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

Example 1CD19 humanization

The murine CD19 antibody sequence (SEQ ID NO: 11) was delegated to Shanghai Biotechnology Co., ltd, wherein the VL humanized sequence was shown as SEQ ID NO:7-10 and the VH humanized sequence was shown as SEQ ID NO: 2-5. The humanized antibody sequences were expressed in combination and the binding capacity of the recombinant CD19 antibodies to CD19 antigen was tested.

Results: as shown in FIGS. 1A-1E, ELISA binding experimental detection results of the humanized CD19 antibody show that the humanized CD19 antibody with the sequences of SEQ ID NO. 12 and SEQ ID NO. 13 is equivalent to the EC50 of the parent murine CD19 antibody SEQ ID NO. 11, and the affinity of the antibody is not significantly reduced. Further, the sequence proteins of SEQ ID NO. 12, SEQ ID NO. 13 and parent SEQ ID NO. 11 are tested for KD values, which shows that the affinity is equivalent to that of the parent.

Example 2CD19-CAR plasmid construction

The humanized sequences SEQ ID NO. 12 and SEQ ID NO. 13 are selected and combined with the sequences SEQ ID NO. 14-19 respectively, the arrangement sequence is shown in FIG. 8 and FIG. 9, and are named KQ-1-2 and KQ-1-1, and the sequence SEQ ID NO. 11 before the humanization is named CD19 (FMC 63) -CART WT. KQ-1-1-CART, KQ-1-2-CART and CD19 (FMC 63) -CART WT sequence synthesis and lentiviral vector plasmid preparation were completed by Suzhou Jin Weizhi Biotechnology Co. The structure of the CD19-CART plasmid is shown in FIG. 10.

Example 3T cell activation

The water bath was opened and the temperature was set at 37 ℃. Taking out frozen PBMC from the liquid nitrogen tank, rapidly placing into a water bath kettle, and rapidly shaking to completely dissolve the cell solution within 1 min; taking 1 piece of 15ml centrifuge tube, adding 5ml of 1 XPBS, adding the cell freezing suspension into the centrifuge tube, and centrifuging at 1500rpm for 3min; the cells were resuspended in 5ml of 1 XPBS, centrifuged at 1500rpm for 5min, the supernatant discarded and the wash repeated once; the cells were resuspended in 3ml of X-VIVO-15 medium (X-VIVO-15+5% FBS+1% P/S+200IU/ml IL-2) and counted using a Count Star cell counter. The required amount of beads was calculated (beads: T cell=2:1), 600 μl of activated beads were taken in a 1.5ml EP tube and placed on a magnetic rack. After the magnetic beads are all adsorbed by magnetic force, the solvent is sucked away, 1ml of 1 XPBS is added, the magnetic beads are uniformly mixed by leaving the magnetic frame, and the magnetic beads are placed on the magnetic frame again after being uniformly mixed, and the washing is repeated for 3 times. Diluting the T cells to 2e 6/ml, and adding the washed magnetic beads into the T cells for blowing and mixing uniformly. A12-well plate was used, 500. Mu.l of the mixture was added to each well, and T-cell plating density was 1e 6/ml. 500 μl of medium was added to each well, and the 12-well plate was gently shaken to mix the solutions well, and incubated at 37deg.C with 5% CO2 for 24 hours.

Experimental example 4 viral infection

The 12-well plate, on which cells were plated the day before, was removed, and 5. Mu.l of Polybrene (working concentration: 5. Mu.g/ml), a pro-sense agent, was added to each well; calculating the amount of virus used according to the virus titer and moi=3, setting a control well (no virus added), and adding the required KQ-1-1-CAR, KQ-1-2-CAR and CD19 (FMC 63) -CART WT virus to each well; each hole is mixed evenly in sequence, a 12-hole plate is wrapped by tin foil paper and protected from light, and 500g is centrifuged for 30min. Placing the centrifuged 12-hole plate into 37 ℃ and incubating with 5% CO 2; after 24h of incubation, the culture solution is replaced every hole, and the volume of the fresh culture solution is 2ml; carrying out transfer fluid with a complete culture medium (culture medium components: X-VIVO-15+5% FBS+1% P/S+200U/ml IL-2) every 48-72 h, wherein the CAR-T cell subculture density is kept between 5e 5/ml and 1e 6/ml; CAR T cells were collected in cultures day5, day7, day9, and day12, respectively, and counted by a cytometer.

Experimental example 5CAR-T Positive Rate detection

Collecting CAR-T cells on the 7 th day of culture, counting by a cell counter, and calculating the growth speed of the CAR-T cells; 200. Mu.L of CAR-T cells per well were added to a 1.5ml EP tube, mixed with 1ml of 1 XPBS, and centrifuged at 1500rpm for 5min; the supernatant was discarded, resuspended in 100. Mu.L of 1 XPBS, and each tube was separately added with antibody and incubated at 4℃for 30min in the absence of light; cells were washed with 1ml of 1 XPBS, centrifuged at 1500rpm for 5min, the supernatant discarded, resuspended with 200. Mu.L of 1 XPBS and detected on-line by flow cytometry.

The results are shown in FIG. 3. Humanized KQ-1-2 and KQ-1-2 CAR-T cells were prepared and CAR positive rates were measured on day 5, day 8, day 12 and day 15. KQ-1-2 was 44.58%, 36.45%, 34.50% and 39.60%, respectively, as shown in FIG. 3. KQ-2-2 was 48.58%, 44.40%, 39.06% and 35.95%, respectively. It was demonstrated that humanized KQ-2-1 and KQ-2-2 exhibited excellent T cell transfection efficiency and stability CAR positive rate, and were not affected by cell culture time. The T cell expansion capacity of transduced CARs remained excellent.

Experimental example 6CAR-T cytokine release Capacity assay

1. Effector cell treatment

The untransduced T cells, KQ-1-1-CART, KQ-1-2-CART, CD19 (FMC 63) -CART WT cells are respectively placed in 15ml centrifuge tubes, 5ml of 1 XPBS are respectively added, and the mixture is centrifuged at 1500rpm for 3min; the cells were resuspended in 5ml of 1 XPBS, centrifuged at 1500rpm for 5min and the supernatant discarded; repeating the previous step and washing twice; cells were resuspended in 3ml of X-VIVO-15 medium containing 1% FBS, counted with a Count Star counter and the cell density was adjusted to 1e6 cells/ml for use.

2. Target cell preparation

Collecting target cells and non-target cells with good growth state in a 15ml centrifuge tube, centrifuging at 1000rpm for 3min, and discarding the supernatant; washing the cells twice with dilution buffer 1 XPBS, centrifuging at 1000rpm for 3min, and discarding the supernatant; cells were resuspended in X-VIVO-15 containing 1% FBS, counted with Count Star and the cell viability was checked and the cells were diluted to a concentration of 1e 6/ml for use.

3. Efficient target cell mixing

Co-culturing the treated effector cells with target cells and non-target cells, respectively: mu.L of target cells and 100. Mu.L of CAR-T cells were 1:1 mixed into 96-well plates; according to CAR-T cells: CO-culturing target cells at a ratio of 1:1 (2 replicates per sample), and CO-culturing in a 5% CO2 incubator at 37 ℃ for 20-24 hours; a proper amount of 1.5ml EP tube was taken, 50. Mu.L of each corresponding cell co-culture supernatant was added to each tube, and the mixture was centrifuged at 1000rpm for 5min to collect 30. Mu.L of the supernatant for cytokine release detection.

4. Cytokine detection

According to the requirements of the CBA detection kit specification, determining the number of cytokines and samples to be detected, and preparing capture beads: taking out the cytokine capturing bead bottles to be detected, mixing the cytokine capturing bead bottles with force, taking out the capturing bead amount of x10 mu l/6 of each bead (the number of samples to be detected plus the number of negative controls), and carrying out turbine vibration mixing on each bead; n 1.5ml EP tubes (n=number of samples+number of controls) were taken and 10 μl of the mixed beads were added to each tube (sample, negative control); adding 10 μl of the corresponding test reagent (sample, control) to each tube; adding 10 mu l PE detection reagent into each tube, fully and uniformly mixing, and incubating for 3 hours at room temperature in a dark place; after incubation, adding 500 μl of washing buffer to each sample, mixing thoroughly, centrifuging 500g for 5min, and discarding the supernatant; resuspension with 200 μl wash buffer, centrifugation at 500g for 5min, washing, discarding supernatant, suspending the sample with 150 μl wash buffer; and (5) detecting on-line in a streaming mode.

The results are shown in fig. 5 and 6. As shown in FIG. 5, after incubation of the CAR-T cells with tumor cells H929-CD19, there was sufficient IL-2 cytokine production, indicating that an adequate immune response occurred between the CAR-T cells and the tumor cells. Humanized KQ-1-1 and KQ-1-2CAR-T cells produced more IL-2 released compared to the positive control FMC-63 CAR-T. IL-2 is necessary for the continuous expansion of CAR-T cells in vivo, demonstrating that the humanized KQ-1-1 and KQ-1-2CAR-T cells have more excellent continuous expansion capability.

As shown in fig. 6, the CAR-T cells had a significant ability to release IFN- γ after incubation with tumor cells H929-CD 19. IFN-gamma is a cytokine necessary for CAR-T cells to achieve tumor cell clearance, confirming that humanized KQ-1-1 and KQ-1-2CAR-T cells can effectively clear tumor cells through IFN-gamma.

Experimental example 7CAR-T cell killing Capacity assay

1. Effector cell preparation

The untransduced T cells, KQ-1-1-CART, KQ-1-2-CART, CD19 (FMC 63) -CART WT cells are respectively placed in 15ml centrifuge tubes, 5ml of 1 XPBS are respectively added, and the mixture is centrifuged at 1500rpm for 3min; the cells were resuspended in 5ml of 1 XPBS, centrifuged at 1500rpm for 5min and the supernatant discarded; repeating the step (2), and washing twice; cells were resuspended in 3ml of X-VIVO-15 medium containing 1% FBS, counted with Count star, and the CAR-T cell densities were adjusted to 4e 5/ml, 1e 5/ml, and the effective target ratios were 2:1, 1:2 (calculated from CAR-T positive cells) for use.

2. Target cell preparation

Collecting target cells H929-CD19-Luc with good growth state in a 15ml centrifuge tube, centrifuging at 1000rpm for 3min, and discarding the supernatant; washing the cells twice with dilution buffer 1 XPBS, centrifuging at 1000rpm for 3min, and discarding the supernatant; cells were resuspended in X-VIVO-15 medium containing 1% FBS, counted and examined for cell viability using a Count star cytometer, and finally the cell density was diluted to 2e5 cells/ml for later use.

3. Efficient target cell mixing

Co-culturing the treated effector cells with target cells and non-target cells, respectively: mix 50 μl target cells and 50 μl CAR-T cells into a 96-well plate; co-culturing 5% CO2 at 37 ℃ for 18 hours; after the completion of the culture, 100. Mu.l of ONE-Glo TM luciferase Assay System detection substrate was added to each well, and the mixture was left standing for 5 minutes. And (3) detecting the TECAN enzyme label on-machine (wavelength 560 nm).

The kill rate was calculated from the OD values.

Results: as shown in FIG. 4, both humanized CD19 CAR-T cells and FMC63-CART have significant killing ability. Effective target ratio E, t=1: 1 shows that the cell killing ability of the humanized KQ-1-1 and KQ-1-2 cells is better than that of FMC63-CART cells. When the effective target ratio E, t=1: 2, namely when the CAR-T cells cope with double tumor cells, the cell killing capacity of the humanized KQ-1-1 and KQ-1-2 cells is obviously better than that of FMC63-CART cells, which proves that the humanized KQ-1-1 and KQ-1-2 cells can effectively clear a large number of tumor cells.

Experimental example 8CAR-T cell targeted proliferation

1. Effector cell preparation

The untransduced T cells, KQ-1-1-CART, KQ-1-2-CART, CD19 (FMC 63) -CART WT cells are respectively placed in 15ml centrifuge tubes, 5ml of 1 XPBS are respectively added, and the mixture is centrifuged at 1500rpm for 3min; the cells were resuspended in 5ml of 1 XPBS, centrifuged at 1500rpm for 5min and the supernatant discarded; repeating the step (2), and washing twice; cells were resuspended in 3ml of X-VIVO-15 medium containing 1% FBS and counted with a Count star; cell density was adjusted to 2e5 cells/ml with an effective target ratio of 1:1 (calculated on CAR-T positive cells) for use.

2. Target cell preparation

Collecting target cells with good growth state in a 15ml centrifuge tube, centrifuging at 1000rpm for 3min, and discarding the supernatant; washing the cells twice with dilution buffer 1 XPBS, centrifuging at 1000rpm for 3min, and discarding the supernatant; cells were resuspended in X-VIVO-15 medium containing 1% FBS, counted and examined for cell viability using a Count star cytometer, and finally the cell density was diluted to a concentration of 2e5 cells/ml for later use.

3. Coculture of effector cells and target cells

Adding the treated effector cells and target cells into a 12-well plate according to the set effective target ratio, and adding 500 μl of target cells and 500 μl of CAR-T cells into each well; after the sample is added, the 12-hole plate is gently shaken to uniformly mix the cells; culturing the cells in a 5% CO2 incubator at 37 ℃;

4. Multiple rounds of tumor cell stimulation proliferation

After the tumor cells are completely lysed, respectively harvesting CAR-T cells, centrifuging for 5min at 500g, and re-suspending the cells in 1ml of X-VIVO-15 culture medium containing 1% FBS, and counting the CAR-T cells; taking 500 μl of the re-suspended CAR-T cells, co-culturing with 1e5 tumor cells for second round of stimulated proliferation, and finally observing the residual tumor cells and counting the final proliferation times of the CAR-T cells.

The results are shown in FIG. 7. After the first co-culture with tumor cells, the expansion capacity of KQ-1-1 and KQ-1-2CART cells and FMC63-CART cells was similar, indicating that it has excellent tumor cell removal capacity and sustained expansion capacity.

After the second co-culture with tumor cells, KQ-1-1 and KQ-1-2CART cells have significantly better proliferation capacity in vitro than FMC63-CART cells. This is in addition to the proof that KQ-1-1 and KQ-1-2CART cells in FIG. 5 can release more IL-2 cytokines, which indicates that KQ-1-1 and KQ-1-2CART cells have a durable cell expansion capacity, and that CAR-T cells have a good proliferation capacity while eliminating tumor cells, which indicates that they have a durable capability of killing tumor cells.

Sequence listing

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

Claims

1. An antibody targeting CD19, said antibody having a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 4 or 5; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7.

2. The antibody of claim 1, wherein the antibody is a single chain antibody.

3. The antibody of claim 2, wherein the antibody has a sequence as set forth in SEQ ID No.12 or 13.

4. A Chimeric Antigen Receptor (CAR) fusion protein, comprising, from N-terminus to C-terminus:

(i) The antibody according to claim 2,

(ii) A transmembrane domain comprising a transmembrane domain,

(iii) At least one co-stimulatory domain, and

(iv) An activation domain.

5. The chimeric antigen receptor fusion protein of claim 4, wherein the scFv amino acid sequence is set forth in SEQ ID No.12 or 13.

6. A recombinant protein, said recombinant protein comprising:

(i) The antibody of claim 1; and; and

(ii) Optionally a tag sequence to assist expression and/or purification.

7. An antibody drug conjugate, comprising:

(a) The antibody of claim 1, the CAR fusion protein of claim 4; and

8. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody of claim 1; or (b)

(2) The chimeric antigen receptor fusion protein of claim 4;

(3) The recombinant protein according to claim 6.

9. A vector comprising the polynucleotide of claim 8.

10. A genetically engineered host cell comprising the vector or genome of claim 9 integrated with the polynucleotide of claim 8, or expressing the antibody of claim 1 or the CAR fusion protein of claim 4.

11. The host cell of claim 10, wherein the cell is an engineered immune cell.

12. The host cell of claim 11, wherein the engineered immune cell is a T cell or an NK cell.

13. A method of making an engineered immune cell that expresses the CAR fusion protein of claim 4, comprising the steps of: transduction of the nucleic acid molecule according to claim 8 or the vector according to claim 9 into T cells or NK cells, thereby obtaining said engineered immune cells.

14. A formulation comprising the antibody of claim 1, the CAR fusion protein of claim 4, or the vector of claim 9, or the host cell of claim 10, and a pharmaceutically acceptable carrier, diluent, or excipient.

15. Use of the antibody of claim 1, the CAR fusion protein of claim 4, or the host cell of claim 10 for the preparation of a medicament for the prevention and/or treatment of CD 19-associated cancers or tumors selected from the group consisting of: b cell lymphoma, B cell leukemia, and multiple myeloma.