CN110396129A

CN110396129A - Humanization CD19 antigen binding single-chain antibody and its Chimeric antigen receptor, immunocyte and application

Info

Publication number: CN110396129A
Application number: CN201910621505.2A
Authority: CN
Inventors: 梅恒; 周棠怡; 陈雄波; 熊巍; 刘玉芝
Original assignee: Wuhan Think Medical Technology Co Ltd
Current assignee: Wuhan Think Medical Technology Co Ltd
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2019-11-01
Anticipated expiration: 2039-07-10
Also published as: CN110396129B

Abstract

The present invention provides humanization CD19 antigen binding single-chain antibody and its Chimeric antigen receptor, immunocyte and applications.Humanization CD19 antigen binding single-chain antibody carries out humanization to light chain framework area and heavy chain framework area based on the FMC63 antibody of mouse anti human CD 19 antigen.The Chimeric antigen receptor of Humanized anti-human CD19 antigen successively includes leader peptide, the humanization CD19 antigen binding single-chain antibody, hinge area, transmembrane region and intracellular region.The Chimeric antigen receptor sequence of Humanized anti-human CD19 antigen is transduceed to immunocyte, application of the immunocyte prepared in the drug of preparation treatment people CD19 positive tumor disease, the immunocyte has lower immunogenicity, it can survive in vivo more long, safer, more efficient feature, while there is stronger lethal effect to people's CD19 positive tumor cell.

Description

Humanized CD19 antigen-binding single-chain antibody and chimeric antigen receptor, immune cell and application thereof

Technical Field

The invention belongs to the technical field of immunotherapy, and particularly relates to a humanized CD19 antigen-binding single-chain antibody, a chimeric antigen receptor thereof, an immune cell and application thereof.

Background

Immunotherapy is a new approach to the treatment of neoplastic diseases following surgery, radiotherapy, chemotherapy. Tumor cells can adopt various strategies to carry out immune evasion, so that the human immune system can not identify the tumor cells, and simultaneously, the functions of the immune system are inhibited, so that the human immune cells can not effectively kill the tumor cells.

Chimeric Antigen Receptors (CARs) are cell surface receptors that recognize specific proteins (antigens) that are composed of parts of other receptors (chimeras). In the immune system, B cells and T cells have surface receptors, called B Cell Receptors (BCRs) and T Cell Receptors (TCRs), respectively, that recognize specific proteins associated with a disease or pathogen. Both receptors have their own advantages and disadvantages. Without the help of antigen presenting cells, TCRs are unable to recognize antigens. However, a TCR may signal its T cells to kill directly the cells it finds, or may signal to recruit more cells. On the other hand, BCRs can recognize antigens without any help. However, the signals from BCR can only be used to recruit other cells and do not directly kill the target. CARs contain portions of multiple immune receptors with the goal of designing a system that recognizes an antigen (e.g., BCR) without any assistance, and then kills the recognized cells directly.

CD19 is expressed on B lineage cells (excluding mature plasma cells) and follicular dendritic cells, but not in other normal tissues. CD19 is an important signaling molecule that regulates growth activation and activation of B lymphocytes, plays an important role in regulating the signal threshold of B lymphocyte antigen receptors or other surface receptors, is an important membrane antigen involved in B lymphocyte differentiation, activation, proliferation and antibody production, and thus it is the best marker for clinical diagnosis of B lymphocyte tumor lines and identification of B lymphocytes.

Most of the current CAR-T technologies targeting CD19 have antibody recognition sequences derived from murine sources, and although murine antibodies have been successfully applied to clinical application and show good effects, some patients have strong immunogenicity after reinfusion, which causes human anti-mouse antibody (HAMA) reaction and anti-antibody reaction (AAR) due to individual differences, so that CAR-T cells after reinfusion are easily recognized and eliminated by the autoimmune system, the long-term efficacy of CAR-T is affected, and the risk of relapse may exist. It was reported that more than half of the patients relapse within one year after receiving murine CAR-T reinfusion therapy, although the short-term (3 months) remission rate was significant.

In view of the above, there is an urgent need in the art to develop binding single chain antibodies against the CD19 antigen with low immunogenicity and high activity, as well as corresponding Chimeric Antigen Receptors (CARs) and immune cells.

Disclosure of Invention

The invention aims to provide a humanized CD19 antigen-binding single-chain antibody (scFV), a CAR containing a humanized anti-human CD19 antigen and application thereof.

In a first aspect of the invention there is provided a humanized CD19 antigen-binding single chain antibody which is based on the FMC63 antibody of murine anti-human CD19 antigen and which humanizes the light chain framework region VL FR1, VL FR2, VL FR3, VL FR4 and the heavy chain framework region VH FR1, VH FR2, VH FR3, VH FR 4;

preferably, the amino acid sequence of humanized VL FR1 is shown in SEQ ID No. 1;

the amino acid sequence of humanized VL FR2 is shown in SEQ ID No. 2;

the amino acid sequence of humanized VL FR3 is shown in SEQ ID No. 3;

the amino acid sequence of humanized VL FR4 is shown in SEQ ID No. 4;

the amino acid sequence of humanized VH FR1 is shown as SEQ ID No. 5;

the amino acid sequence of humanized VH FR2 is shown as SEQ ID No. 6;

the amino acid sequence of humanized VH FR3 is shown as SEQ ID No. 7; and

the amino acid sequence of humanized VH FR4 is shown in SEQ ID No. 8.

In another preferred embodiment, the heavy chain variable region of the humanized CD19 antigen-binding single chain antibody comprises three complementarity determining regions CDRs: VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region of the single chain antibody comprises the following three complementarity determining regions CDRs: VL CDR1, VL CDR2 and VL CDR3, wherein the 6 CDRs are identical to the 6 CDRs of the FMC63 antibody against murine CD19 antigen.

In another preferred embodiment, the humanized CD19 antigen binds to the 6 CDRs in a single chain antibody as follows:

VL CDR1, shown in SEQ ID No. 9;

VL CDR2, shown in SEQ ID No: 10;

VL CDR3, shown in SEQ ID No. 11;

VH CDR1, shown as SEQ ID No: 12;

VH CDR2, shown as SEQ ID No. 13; and

VH CDR3, shown in SEQ ID No: 14.

In another preferred embodiment, the humanized CD19 antigen-binding single chain antibody comprises, in order, a light chain, a linking region, and a heavy chain, or comprises, in order, a heavy chain, a linking region, and a light chain;

wherein the light chain comprises 3 light chain complementarity determining region VL CDRs and 4 humanized light chain framework region VL FRs; the heavy chain comprises 3 heavy chain complementarity determining region VH CDRs and 4 humanized heavy chain framework region VH FRs.

In another preferred embodiment, the humanized CD19 antigen binds to a single chain antibody, wherein the segments of the light chain are linked in the following order: humanized VL FR1-VL CDR 1-humanized VL FR2-VL CDR 2-humanized VL FR3-VL CDR 3-humanized VL FR 4;

in another preferred embodiment, the sequence of joining the segments in the heavy chain is: humanized VH FR1-VH CDR 1-humanized VH FR2-VH CDR 2-humanized VH FR3-VH CDR 3-humanized VH FR 4.

In another preferred embodiment, the humanized CD19 antigen binds to a single chain antibody, the light chain variable region has the sequence shown in SEQ ID No. 15 and the heavy chain variable region has the sequence shown in SEQ ID No. 16.

In another preferred embodiment, the humanized CD19 antigen binds to a single chain antibody, and the amino acid sequence of the humanized CD19 antigen binding single chain antibody is shown as SEQ ID No. 19.

In another preferred embodiment, the humanized CD19 antigen binding single chain antibody is encoded by a single nucleotide having the nucleotide sequence shown in SEQ ID No. 20.

In a second aspect of the invention there is provided a humanized anti-human CD19 antigen Chimeric Antigen Receptor (CAR) comprising in order an optional leader peptide, a humanized CD19 antigen binding single chain antibody of the first aspect of the invention, a hinge region, a transmembrane region and an intracellular region.

In another preferred embodiment, the leader peptide of the humanized anti-human chimeric antigen receptor of CD19 antigen is a CD8leader chimeric receptor signal peptide; preferably, the amino acid sequence of the CD8leader chimeric receptor signal peptide is shown as 1-21 in SEQ ID No. 21;

the hinge region is a CD28hinge chimeric receptor hinge; preferably, the amino acid sequence of the hinge of the CD28hinge chimeric receptor is shown as 266-310 in SEQ ID No. 21;

the transmembrane region is a CD28TM transmembrane region; preferably, the amino acid sequence of the CD28TM transmembrane region is shown as position 311-334 in SEQ ID No. 21; and/or

The intracellular region comprises 41-BB and CD3zeta in sequence;

preferably, the amino acid sequence of 41-BB is shown as position 335 and 376 in SEQ ID No. 21; and the amino acid sequence of the CD3zeta is shown as position 377-488 in SEQ ID No. 21.

Preferably, in another preferred embodiment, the coding sequence of each element is selected from the group consisting of:

the nucleotide sequence of the CD8leader chimeric receptor signal peptide is shown in SEQ ID No: positions 1-63 in 22;

the nucleotide sequence of the CD28hinge chimeric receptor hinge is shown in SEQ ID No.: 22 at position 796-930;

the nucleotide sequence of the CD28TM transmembrane region is shown in SEQ ID No.: 22 at positions 931 and 1002;

the nucleotide sequence of the 41-BB is shown in SEQ ID No:22 at position 1003-1128; and/or

The nucleotide sequence of the CD3zeta (zeta) is shown in SEQ ID No: 1129-1464 in 22.

In another preferred embodiment, the amino acid sequence of the Chimeric Antigen Receptor (CAR) is shown as SEQ ID No. 21.

In a third aspect, the present invention provides a polynucleotide encoding a Chimeric Antigen Receptor (CAR) of a humanized CD19 antigen-binding single chain antibody according to the first aspect of the invention or a humanized anti-human CD19 antigen according to the second aspect of the invention.

In another preferred embodiment, the variable region of the light chain has the nucleotide sequence shown as SEQ ID No. 17, and the variable region of the heavy chain has the nucleotide sequence shown as SEQ ID No. 18.

In another preferred embodiment, the nucleic acid sequence of the humanized CD19 antigen binding single chain antibody is shown in SEQ ID No. 20.

In another preferred embodiment, the nucleotide sequence of the Chimeric Antigen Receptor (CAR) is shown as SEQ ID No: 22.

In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide of the third aspect of the present invention.

In another preferred embodiment, the expression vector is selected from the group consisting of: a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, an oncolytic viral vector, or a combination thereof.

In another preferred embodiment, the vector is a viral vector (e.g., a lentiviral vector).

In another preferred embodiment, the expression vector is a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of: an AAV vector, a lentiviral vector, or a combination thereof.

In a fifth aspect of the invention, there is provided a host cell comprising an expression vector or genome according to the fourth aspect of the invention into which a polynucleotide according to the third aspect of the invention has been integrated.

In another preferred embodiment, the host cell comprises a eukaryotic cell.

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

In another preferred embodiment, the immune cell is selected from the group consisting of: t cells, NK cells, or a combination thereof.

In a sixth aspect of the invention, there is provided an immune cell expressing an exogenous humanized CD19 antigen binding single chain antibody according to the first aspect of the invention or a chimeric antigen receptor of a humanized anti-human CD19 antigen according to the second aspect of the invention.

In another preferred embodiment, the genome of said immune cell comprises a gene sequence encoding a humanized CD19 antigen-binding single chain antibody according to the first aspect of the invention or a gene sequence of a chimeric antigen receptor of a humanized anti-human CD19 antigen according to any of the second aspect of the invention.

In another preferred embodiment, the immune cells comprise T cells or natural killer cells.

In a seventh aspect of the invention, there is provided a use of the humanized CD19 antigen binding single chain antibody of the first aspect of the invention, or the humanized chimeric antigen receptor of anti-human CD19 antigen of the second aspect of the invention, or the immune cell of the sixth aspect of the invention, in the preparation of a medicament for the treatment of a human CD 19-positive neoplastic disease, a CD 19-positive immune disease, or a non-CD 19-positive neoplastic disease.

In another preferred example, the human CD 19-positive neoplastic disease includes CD 19-positive acute lymphoblastic leukemia, CD 19-positive acute myelocytic leukemia, CD 19-positive chronic lymphocytic leukemia, or CD 19-positive acute lymphoma.

An eighth aspect of the present invention provides a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: the humanized CD19 antigen of the first aspect of the present invention binds to a single chain antibody or an Antibody Drug Conjugate (ADC) thereof, or a chimeric antigen receptor of the humanized anti-human CD19 antigen of the second aspect, or an immune cell of the sixth aspect, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, in another preferred embodiment, the pharmaceutical composition is a liquid preparation.

Preferably, in another preferred embodiment, the pharmaceutical composition is an injection.

Preferably, in another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 × 10³-1×10⁹Individual cells/ml, preferably 1X 10⁵-1×10⁸Individual cells/ml.

Preferably, in another preferred embodiment, the pharmaceutical composition further comprises other drugs (such as nucleic acid drugs, antibody drugs, targeting drugs, other immune cell drugs, other CAR-T drugs, chemotherapeutic drugs, or a combination thereof) for selectively killing tumor cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a schematic diagram showing the connection of a chimeric antigen receptor of the humanized anti-human CD19 antigen provided by the present invention, wherein LTR is a long terminal repeat;

FIG. 2 is a graph of the results of flow cytometry analysis of cells for antibodies specific to the Fab'2 portion of human IgG;

FIG. 3A shows the results of the measurement of the killing power of HeLa-CD19 cells in different treatment groups;

FIG. 3B shows the cytotoxicity test results of HeLa-CD19 cells in different treatment groups;

FIG. 4 is a graph showing the results of the INF-gamma release by HeLa-CD19 cells according to different treatment groups;

FIG. 5 is an image of tumorigenic mice injected with luciferase-expressing Raji cells from different treatment groups;

FIG. 6 is a graph of the effect of different treatment groups on the change in body weight of tumorigenic mice after treatment;

FIG. 7 is a graph of the effect of different treatment groups on the survival of tumor-producing mice injected with Raji cells expressing luciferase;

figure 8 is a flow cytometric analysis of humanized CAR-T cells in treated mice.

Detailed Description

The present inventors have extensively and intensively studied and, as a result of extensive screening, developed a humanized CD19 antigen-binding single-chain antibody (scFv) for the first time. The CAR based on the novel humanized scFv and the corresponding immune cells can specifically target and recognize the human CD19 antigen, effectively kill and eliminate tumor cells expressing the human CD19 antigen, and have the advantages of lower immunogenicity, longer survival in vivo, higher safety, higher efficiency and the like. The present invention has been completed based on this finding.

Specifically, the inventor uses mouse anti-human CD19 FMC63 antibody as the basis, to humanize light chain framework region VL FR1, VL FR2, VL FR3, VL FR4 and heavy chain framework region VH FR1, VH FR2, VH FR3 and VH FR4, and to retain antibody fragments (CDR regions) directly contacted with antigen as much as possible, modifies the framework region of the murine antibody scFv with highly homologous human framework regions, and screens single chain antibody which can maintain specificity and affinity and reduce immunogenicity and toxic and side effects through affinity remodeling. The experimental results show that when the humanized scFv polypeptide is applied to human, the humanized scFv polypeptide has the characteristics of high affinity and low immunogenicity to human CD19 antigen.

Term(s) for

As used herein, the terms "scFv of the invention", "single chain antibody of the invention" or "humanized CD19 antigen-binding single chain antibody of the invention" are used interchangeably and refer to a single chain antibody as described in the first aspect of the invention.

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, comprising any of the anti-CD 19 antibodies of the invention and compositions thereof. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

"sequence identity" as referred to herein means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between a sequence described in the present invention and a sequence with which it is identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

Antibodies

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Light chains are classified as either kappa or lambda depending on the constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof. The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus. The 3 CDR regions of the light chain refer to VL-CDR1, VL-CDR2 and VL-CDR 3; the 3 CDR regions of the heavy chain are referred to as VH-CDR1, VH-CDR2 and VH-CDR 3.

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. The humanized antibody can overcome the heterogenous reaction induced by the chimeric antibody carrying a great deal of murine protein components.

The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., CD 19). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include:

(i) fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains;

(ii)F(ab')₂a fragment comprising a bivalent fragment of two Fab fragments connected by a disulfide bridge on the chain compare region;

(iii) an Fd fragment consisting of the VH and CH1 domains;

(iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

Fv antibodies (scFv) contain the antibody heavy chain variable region, the light chain variable region, but no constant regions, and have a minimal antibody fragment with a complete 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 term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interest, NIH Publication 91-3242).

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which refers to a polypeptide having at most 5, preferably at most 4, more preferably at most 2, and most preferably at most 2 amino acids replaced by amino acids with similar or similar properties, compared with the amino acid sequence of the antibody of the present invention, and having substantially unchanged binding activity.

anti-CD 19 humanized antibodies

The invention provides an anti-CD 19 humanized antibody. Specifically, the present invention provides a highly specific and high affinity humanized antibody against CD19 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.

Specifically, the humanized CD19 antigen-binding single-chain antibody provided by the invention is based on an FMC63 antibody of a mouse anti-human CD19 antigen, and is used for humanizing a light chain framework region VL FR1, VL FR2, VL FR3, VL FR4 and a heavy chain framework region VH FR1, VH FR2, VH FR3 and VH FR 4;

the amino acid sequence of humanized VL FR1 is shown as SEQ ID No. 1;

the amino acid sequence of humanized VL FR2 is shown in SEQ ID No. 2;

the amino acid sequence of humanized VL FR3 is shown in SEQ ID No. 3;

the amino acid sequence of humanized VL FR4 is shown in SEQ ID No. 4;

the amino acid sequence of humanized VH FR1 is shown as SEQ ID No. 5;

the amino acid sequence of humanized VH FR2 is shown as SEQ ID No. 6;

the amino acid sequence of humanized VH FR3 is shown as SEQ ID No. 7;

the amino acid sequence of humanized VH FR4 is shown in SEQ ID No. 8.

The FMC63 antibody of the murine anti-human CD19 antigen is an antibody reported in the prior art. The amino acid sequence of the FMC63 antibody of the mouse anti-human CD19 antigen is shown as SEQ ID No. 25. The single chain antibody comprises a light chain, a connecting region and a heavy chain in sequence.

The light chain in the engineered single chain antibody comprises 3 light chain complementarity determining regions (VL CDRs) and 4 humanized light chain framework regions (VL FRs); the heavy chain comprises 3 heavy chain complementarity determining region VH CDRs and 4 humanized heavy chain framework region VH FRs; the sequence of the segments in the light chain is humanized VL FR1-VL CDR 1-humanized VL FR2-VL CDR 2-humanized VLFR3-VL CDR 3-humanized VL FR 4.

The amino acid sequence of the VL CDR1 is preferably shown as SEQ ID No. 9.

The amino acid sequence of the VL CDR2 is preferably shown in SEQ ID No. 10.

The amino acid sequence of the VL CDR3 is preferably shown in SEQ ID No. 11.

The sequence of the segments in the heavy chain is humanized VH FR1-VH CDR 1-humanized VH FR2-VH CDR 2-humanized VH FR3-VH CDR 3-humanized VH FR 4.

The amino acid sequence of the VH CDR1 is preferably shown as SEQ ID No. 12.

The amino acid sequence of the VH CDR2 is preferably shown as SEQ ID No. 13.

The amino acid sequence of the VH CDR3 is preferably shown as SEQ ID No. 14.

Preferably, the amino acid and nucleotide sequences of each FR and each VL CDR of a VL are as shown in SEQ ID Nos. 15 and 17.

Variable region	FR or CDR	Position in SEQ ID No. 15	Position in SEQ ID No. 17
				VL	FR1	1-23	1-69
VL	FR2	35-49	103-147
				VL	FR3	56-88	166-264
VL	FR4	97-107	289-321
				VL	CDR1	24-34	70-102
VL	CDR2	50-55	148-165
				VL	CDR3	89-96	265-288

Preferably, the amino acid and nucleotide sequences of each FR and each VH CDR of a VH are as shown in SEQ ID Nos 16 and 18.

Variable region	FR or CDR	Position in SEQ ID No. 16	Position in SEQ ID No. 18
				VH	FR1	1-25	1-75
VH	FR2	36-49	106-147
				VH	FR3	65-96	193-288
VH	FR4	110-120	328-360
				VH	CDR1	26-35	76-105
VH	CDR2	50-64	148-192
				VH	CDR3	97-109	289-327

The amino acid sequence of the humanized CD19 antigen-binding single-chain antibody is preferably shown as SEQ ID No. 19.

Preferably, the nucleotide sequence of the humanized CD19 antigen-binding single-chain antibody is shown as SEQ ID No. 20.

The invention provides a humanized anti-human CD19 antigen chimeric antigen receptor, which sequentially comprises a leader peptide, the humanized CD19 antigen binding single-chain antibody, a hinge region, a transmembrane region and an intracellular region.

The leader peptide is preferably a CD8leader chimeric receptor signal peptide;

the hinge region is preferably a CD28hinge chimeric receptor hinge;

the transmembrane region is preferably CD28 TM;

the intracellular domain preferably comprises, in order, 41-BB and CD3 zeta;

the nucleotide sequence of the humanized anti-human CD19 antigen chimeric antigen receptor is shown in SEQ ID No. 22. The nucleotide sequence of the humanized chimeric antigen receptor of the human CD19 antigen can be prepared by adopting a conventional artificial synthesis method.

CAR

The invention also provides a CAR that targets CD 19. Preferably, the structure of the CAR of the invention is represented by formula I below:

L1-scFv19-H1-TM1-C1-CD3ζ (I)

in the formula,

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

l1 is an optional signal peptide sequence;

scFv19 is a humanized scFv of the present invention targeting CD19 (as an antigen binding domain);

h1 is an optional hinge region;

TM1 is a transmembrane domain;

c1 is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ.

In another preferred embodiment, said L1 is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, said H1 is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, TM1 is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, C1 is a costimulatory signaling molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

CAR-immune cells and methods of making same

The invention provides an immune cell expressing a CAR construct of the invention. The immune cells are not particularly limited in the present invention, and representative immune cells include (but are not limited to): t cells or natural killer cells.

In the present invention, there is also provided a method for preparing a CAR-immune cell, comprising:

(A) providing an immune cell to be modified; and

(B) introducing a CAR expression cassette into the immune cell to be engineered, wherein the CAR expression cassette expresses a CAR construct of the invention, thereby obtaining an engineered immune cell.

Preferably, the method comprises the steps of: a coding sequence for a synthetic humanized Chimeric Antigen Receptor (CAR) against human CD19 antigen; and (3) transfecting the obtained CAR coding sequence to a virus preparation cell through a vector system, packaging into a CAR-containing virus, and transducing the CAR sequence to an immune cell through the virus or directly transducing the CAR sequence to the immune cell through methods such as electrotransformation.

In the present invention, the method of transfection is not particularly limited, and a transfection method well known in the art may be used.

Preferably, in the present invention, the preparation method further comprises: and (3) carrying out function and effectiveness detection on the obtained engineered immune cells.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof as described above or an ADC thereof or a corresponding CAR-immune cell (such as a CAR-T cell, or a CAR-NK cell), and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition of the invention can be directly used for binding CD19 protein molecules, and thus can be used for preventing and treating CD19 related diseases. In addition, other therapeutic agents may also be used simultaneously.

The humanized CD19 antigen binding single chain antibody or CAR or the immune cell can be used for treating human CD19 positive tumor diseases and CD19 positive immune diseases.

In the present invention, CD19 positive neoplastic diseases include (but are not limited to): acute lymphoblastic leukemia positive for CD19, acute myeloid leukemia positive for CD19, chronic lymphocytic leukemia positive for CD19, or acute lymphoma positive for CD 19.

The main advantages of the invention include:

(a) the invention provides a humanized anti-human CD19 antigen Chimeric Antigen Receptor (CAR), which is formed by connecting a leader peptide, a single-chain antibody for recognizing human CD19 antigen, a hinge region, a transmembrane region and an intracellular region in sequence to form a humanized anti-human CD19 antigen chimeric antigen receptor sequence. The humanized CAR provided by the invention has the characteristics of higher affinity and specificity and small immunogenicity with CD19 antigen, overcomes various defects of animal-derived CAR, ensures that immune cells prepared from the humanized CAR have long-term curative effect, and avoids disease repetition and drug safety.

(b) The invention provides a coding sequence of a Chimeric Antigen Receptor (CAR) containing a humanized anti-human CD19 antigen in an immune cell, which has a treatment effect on human CD19 positive tumor diseases, and due to humanized modification, compared with a product which is not subjected to humanized modification, the immune cell product has lower immunogenicity, can survive in vivo for a longer time, and has the characteristics of higher safety and higher efficiency. In vivo experiments and in vivo experiments show that: compared with the original mouse-derived anti-CD 19CAR-T, the humanized anti-CD 19CAR-T can kill the target cell Hela-CD19 with CD19 positive more effectively, has higher cytokine release level, and inhibits the growth of Raji tumor cells with CD19 positive in mice more effectively.

(c) The humanized CD19 antibody of the invention has an affinity comparable to that of murine antibodies.

In conclusion, the humanized anti-CD 19CAR-T can kill target cells more safely and effectively.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

Humanized design of scFv against human CD19 antigen

The FMC63 antibody of mouse anti-human CD19 antigen is used as a parent antibody, and a framework region is subjected to humanized modification on the basis of an amino acid sequence of an scFv chain of the parent antibody to design a humanized antibody of the anti-human CD19 antigen.

Firstly, determining a core sequence of FMC63 through molecular docking simulation, and determining the amino acid sequences of the CDR regions of the variable regions of the heavy chain and the light chain of the scFv sequence, namely CDR1, CDR2 and CDR3 of the light chain VL, and CDR1, CDR2 and CDR3 of the heavy chain VH; then, in NCBI/lgBLAST software, the sequence analysis and alignment are carried out on the human sequence in the database and the scFv sequence of FMC63, and the framework region sequence corresponding to the human antibody variable region sequence with higher homology is selected as a human template. The framework sequence of FMC63 was replaced by a modification of the framework sequence other than the CDR regions of the selected human template, i.e., the FR sequences of the framework regions L FR1, L FR2, LFR3, L FR4, H FR1, H FR2, H FR3 and H FR4 were replaced from murine to human. Combining the human FR sequences and the CDR regions to obtain a plurality of scFv sequences, and screening out sequences with higher affinity through molecular docking simulation.

Compared with the original murine FMC63scFv sequence, the humanized PMC288scFv sequence has the following differences:

1)L FR1

the original amino acid sequence: DIQMTQTTSSLSASLGDRVTISC (SEQ ID NO:23, 1-23)

Humanized sequence: DIQMTQSPSSLSASVGDRVTITC (SEQ ID No:1)

2)L FR2

The original amino acid sequence: WYQQKPDGTVKLLIY (SEQ ID NO:23, positions 35-49)

Humanized sequence: WYQQKPGKAPKLLIY (SEQ ID No:2)

3)L FR3

The original amino acid sequence: SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC (SEQ ID NO:23, 56-88 th)

Humanized sequence: SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID No:3)

4)L FR4

The original amino acid sequence: TFGGGTKLEI (position 97-107 of SEQ ID NO: 23)

Humanized sequence: TFGGGTKVEI (SEQ ID No:4)

5)H FR1

The original amino acid sequence: EVKLQESGPGLVAPSQSLSVTCTVS (SEQ ID NO:24, 1-25 th position)

Humanized sequence: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID No:5)

6)H FR2

The original amino acid sequence: WIRQPPRKGLEWLG (SEQ ID NO:24, positions 36-49)

Humanized sequence: WVRQAPGKGLEWVS (SEQ ID No:6)

7)H FR3

The original amino acid sequence: SRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA (SEQ ID NO:24, 65-96 th position)

Humanized sequence: SRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID No:7)

8)H FR4

The original amino acid sequence: WGQGTSVTVSS (SEQ ID NO:24 at positions 110-120)

Humanized sequence: WGQGTLVTVSS (SEQ ID No:8)

9) The differences in the entire scFv sequence are as follows (CDR regions underlined):

the amino acid sequence of murine scFv is as follows:

wherein, the amino acid sequence of the light chain is as follows:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK(VL，SEQ ID No:23)；

the amino acid sequence of the heavy chain is as follows:

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(VH，SEQ ID No:24)。

the amino acid sequence of the humanized scFV after modification is shown in SEQ ID No. 19, wherein the underlined is a linker peptide sequence (linker).

Wherein the amino acid sequence of the light chain is as follows:

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGGGTKVEIK(VL，SEQ ID No:15)；

wherein the amino acid sequence of the heavy chain is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGVSLPDYGVSWVRQAPGKGLEWVSVIWGSETTYYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(VH，SEQ ID No:16)。

example 2

The nucleotide sequence of the Chimeric Antigen Receptor (CAR) of the human CD19 is artificially synthesized, the sequence is shown as SEQ ID No:21, and EcoRI and XbaI enzyme cutting sites are added at two ends:

and (2) carrying out double enzyme digestion reaction on the lentiviral vector and the synthesized DNA sequence by using restriction enzymes EcoRI and XbaI respectively, and carrying out ligation reaction on the digested lentiviral vector and the DNA fragment by using T4DNA ligase (purchased from TaKaRa) after the reaction is finished to obtain the lentiviral vector for expressing the chimeric antigen receptor. The method for preparing lentiviral vector plasmids specifically expressing Chimeric Antigen Receptor (CAR) was performed according to the instructions of QIAGEN endotoxin-free plasmid dainti kit.

The amino acid sequence of the Chimeric Antigen Receptor (CAR) is shown as SEQ ID No. 21.

Wherein,

the leader peptide of CD8 is shown in SEQ ID No. 21 at positions 1-21:

MALPVTALLL PLALLLHAAR P

the hinge region of CD8 is the 266-310 position in SEQ ID No. 21:

TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACD

the CD8 transmembrane region is shown as the position 311-334 in SEQ ID No. 21:

IYIWAPLAGT CGVLLLSLVI TLYC

the 41-BB co-stimulatory factor region is the 335-376 th position in SEQ ID No. 21:

KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL

the CD3zeta region is the 377-488 th site in SEQ ID No. 21:

example 3

Preparation and purification of viruses

Lentivirus packaging was performed using cell factory expanded cultured HEK293FT (ATCC) cells. A mixture of 29mLOptiMEM, 2016. mu.L of packaging plasmid, and 504. mu.L of lentiviral vector plasmid DNA (1ug/uL) was mixed with a mixture of 28.5mL of OptiMEM and 3024. mu.L of transfection reagent to prepare a transfection solution, which was transferred to 293FT cell factory and treated with 5% CO at 37 ℃ to prepare a mixture₂The culture was carried out overnight in an incubator. The following day virus collection medium was changed (1L DMEM containing 20 mL)Fetal bovine serum and 60 μ L of 0.1M sodium butyrate). Collecting culture solution of cell factory, i.e. virus mixed solution, at 48h and 72h after transfection respectively, centrifuging at 3000rpm (2100g) for 10min to clarify virus-containing culture medium, filtering with 0.45 μm filter, digesting with nuclease (37 deg.C water bath for 6h), ultrafiltering and concentrating with 500KDa hollow fiber filter, finally, ultrafiltering at 4 deg.C for 16h to purify virus solution, resuspending with appropriate amount of PBS, and storing at-80 deg.C.

Example 4

CAR-T preparation method

Mixing fresh peripheral blood with PBS buffer solution according to the volume ratio of 1:1, sliding 25mL of blood/PBs mixed solution along a side tube of a centrifuge tube, adding the mixed solution above 15mL of Ficoll separating medium, centrifuging at 400g for 30min, and sucking the PBMC layer to a new centrifuge tube by using a pipette. PBMC cells were washed with PBS buffer, diluted in the appropriate ratio and counted, and 5000 ten thousand PBMC cells aspirated were mixed with 1.2mL of magnetic beads containing 5000 ten thousand CD3/CD28Dynabeads, mixed and transferred to a T175 flask for overnight culture to activate T cells. Thawing the virus solution on ice the next day, and mixing 5 × 10⁶TUs and 250. mu.L of a 1mg/ml DEAE-dextran solution were added to a T175 flask and on the third day, 5X 10⁶TUs into flasks transduce T cells. And on the fifth day, removing the magnetic beads by using a magnetic frame, transferring the cell suspension into a G-Rex container, and putting the G-Rex container into an incubator for continuous culture. The cells were sampled and the corresponding cell detection was completed.

Example 5

Flow cytometry detection of CAR Positive Rate

mu.L of each cell suspension was pipetted from the flask into a flow tube, the cells were washed once with PBS, and the cells were suspended with 100. mu.L of FACS buffer, and 2. mu.L of goat serum was pipetted into the cell suspension prepared in example 4 and ice-cooled for 5 min. 1 μ L of biotinylated goat anti-human Fab'2 antibody was pipetted into one of the tube cell suspensions and 1 μ L of biotinylated goat IgG was pipetted into the other tube cell suspension, and the two tubes were separately labeled for 30min in ice bath. Add 3ml of pre-chilled FACS buffer, centrifuge at 300g for 5min to remove supernatant, and leave 80. mu.L of liquid. Mu.l PE-conjugated streptavidin antibody, 4. mu.l FITC conjugated anti-CD 4 antibody, 2. mu.l APC conjugated anti-CD 8 antibody and 5. mu.l of 7-AAD solution were pipetted into each flow tube and ice-cooled for 30 min. Adding 3mL of precooled buffer, centrifuging 300g for 5min to remove supernatant, reserving 80 mu L of liquid, adding 200 mu L of buffer to mix the cells uniformly, and detecting by an up-flow meter. CAR cell ratios were analyzed using flow software gating.

Cells were analyzed by flow cytometry for antibodies specific for the Fab'2 portion of human IgG. Humanized CD19CAR-T cells, control T cells and mock CAR-T cells were stained with biotinylated antibody against the Fab'2 portion of human IgG. Stained cells were detected with PE-conjugated streptavidin and counted in the figure. The antibody binds to a human antibody and a humanized scFv. More than 80% of the T cells bound to the anti-Fab' 2 antibody and were therefore CAR-T positive cells. In contrast, T cells not transduced with virus and T cells transduced with mock CAR-encoding virus showed negative staining for Fab'2 antibody (figure 2).

Example 6

Method for detecting lethality

CAR-T cell lethality was measured by Real-time cellular Assay (RTCA) using HeLa cells stably expressing CD 19.

HeLa-CD19 target cells were cultured overnight, and then effector cells [ murine CD19CAR-T cells, humanized CD19CAR-T cells prepared in example 4, mock CAR-T cells or control T cells (empty vector CAR-T, prepared as CAR-T, but without CAR sequence in the plasmid and viral vectors used) ]: HeLa-CD19 target cells were cultured in a number ratio of 10: mixed culture at a ratio of 1. Real-time cell analysis was performed at the ACEA workstation with impedance of HeLa-CD19 monolayer on the Y-axis and time of effector cell addition on the X-axis. The cytotoxicity calculation formula at the end of the measurement (46 h after the addition of the effector cells) is shown as a formula a;

(ii) cytotoxicity ═ [ (X-Y)/X ]. 100% of formula a

Where X is the normalized impedance of the target cell without effector cells and Y is the normalized impedance of the target cell effective for effector cells. There were very significant differences between humanized CD19CAR-T cells and CD19CAR-T cells and mock CAR-T cells and control T cell treated groups (. p < 0.0001).

Non-humanized FMC63CD19 CAR-T cells served as positive controls. Humanized CD19CAR-T cells were as cytotoxic as conventional CD19CAR-T cells and were significantly more cytotoxic than control T cells and mock CAR-T cells (fig. 3A and 3B).

Example 7

Detection of factor Release

Humanized CD19CAR-T cells were evaluated for IFN- γ secretion during RTCA assay.

The medium was centrifuged to remove the cells, and IFN-. gamma.secretion was measured using an ELISA kit for human IFN-. gamma.according to the kit instructions.

The results are shown in FIG. 4. As can be seen from figure 4, the humanized CD19CAR-T cells and CD19CAR-T cells produced high levels of INF- γ release on HeLa-CD19 cells, whereas the mock CAR-T cells or control T cells did not. There were significant differences between humanized CD19CAR-T cells and CD19CAR-T cells and mock CAR-T cells and control T cell treated groups (. p < 0.015). High cytokine release levels indicate good efficacy.

Example 8

Effect of animal experiments

Humanized CD19CAR-T cells were tested in a xenograft tumor model, NSG immunodeficient mice injected with Raji leukemia cells expressing luciferase, following the specific procedure:

1. 20 NSG immunodeficient mice were prepared and randomly grouped on day 0 (D0) into PBS-injected (i.e., PBS), mock CAR-T cell-injected (i.e., mock CAR-T), CD19CAR-T cell-injected (i.e., CD19 CAR-T) and humanized CD19CAR-T injected (i.e., huCD19 CAR-T), 5 mice per group, each individually labeled.

2. Injections were performed at D0 for each mouse. All mice were injected with Raji leukemia cells (100ul, 5X 10) expressing luciferase marker⁶One); in addition, PBS (100uL) was injected into PBS-injected mice, and simulated CAR-T cells (100uL, 5X 10) were injected into simulated CAR-T cell-injected mice⁶One), CD19CAR-T cell injection group was injected with CD19CAR-T cells (100uL, 5X 10)⁶One), huCD19CAR-T cell injection group was injected with huCD19CAR-T cells (100uL, 5X 10)⁶One).

3. Monitoring Raji leukemia cells in mice: bioimaging photographs were taken of each mouse at D7, D14, D21, respectively, to observe the distribution and survival of Raji leukemia cells in the mice.

4. The body weight of each mouse was measured: to monitor the survival of the groups of mice, the body weight of each mouse was measured at D0, D7, D14, D21, D28, D35, D42, D49, D56, D63, D70, D77, D84, respectively.

And (4) counting the survival rate: from D0 to D84, the mortality of each mouse was recorded and the survival rate of each group of mice was counted.

The results are shown in FIG. 5. Fig. 5 is a bioimaging of mice injected with Raji cells expressing luciferase. As can be seen in FIG. 5, the mice were injected with PBS, mock CAR-T cells, CD19CAR-T cells, or humanized CD19CAR-T cells on day 1, and the PBS-treated mice died before day 21. As can be seen from figure 5, the humanized CD19CAR-T cells almost completely blocked Raji tumor growth in mice.

The humanized CD19CAR-T cell injected group mice showed little weight change, indicating that the injection of CAR-T cells did not affect the daily life and feeding of the mice (fig. 6).

Fig. 7 is the survival rate of mice injected with Raji cells expressing luciferase. Mice were injected with PBS on day 0, either mock CAR-T cells, CD19CAR-T cells or humanized CD19CAR-T cells. For huCD19CAR-T cells and mock CAR-T cell treated groups, p ═ 0.0072(Mantel-Cox test). As can be seen from figure 7, the humanized CD19CAR-T cells prolonged the survival of the mice; in fact, 3 of 5 mice treated with humanized CD19CAR-T cells survived 7 weeks after a single treatment.

Figure 8 is a flow cytometric analysis of humanized CAR-T cells in treated mice. Peripheral blood leukocytes of CAR-T cell treated mice were stained with antibodies against human CD3 and human Fab'2 or FMC 63. The left panel is the percentage of leukocytes expressing human CD 3. The right panel is the percentage of human CD3+ leukocytes expressing FMC63(CD19 CAR-T cells) or human Fab'2 sequences (huCD19 CAR-T cells). On study day 7, the treated mice were analyzed by flow cytometry for the frequency of human T cells and CAR-T cells in peripheral blood leukocytes. T cells were detected with anti-CD 3 antibody, CD19CAR-T cells were detected with antibody specific for FMC63CD19 CAR, humanized CD19CAR-T cells were detected with anti-human Fab'2 antibody. Approximately 3% of the leukocytes in huCD19CAR-T cell treated mouse blood were human T cells, and 10% of these T cells were huCD19CAR-T cells (figure 8).

This result indicates that the immunogenicity of the humanized CD19CAR-T cells of the present invention is significantly reduced.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Wuhansian medical technology Limited

<120> humanized CD19 antigen-binding single-chain antibody and chimeric antigen receptor, immune cell and application thereof

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180 185 190

Trp Val Ser Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala

195 200 205

Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Tyr

210 215 220

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 22

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatattc agatgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg 120

accattacct gccgcgcgag ccaggatatt agcaaatatc tgaactggta tcagcagaaa 180

ccgggcaaag cgccgaaact gctgatttat cataccagcc gcctgcatag cggcgtgccg 240

agccgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctgcag 300

ccggaagatt ttgcgaccta ttattgccag cagggcaaca ccctgccgta tacctttggc 360

ggcggcacca aagtggaaat taaaggctcc acctctggat ccggcaagcc cggatctggc 420

gagggatcca ccaagggcga agtgcagctg gtggaaagcg gcggcggcct ggtgcagccg 480

ggcggcagcc tgcgcctgag ctgcgcggcg agcggcgtga gcctgccgga ttatggcgtg 540

agctgggtgc gccaggcgcc gggcaaaggc ctggaatggg tgagcgtgat ttggggcagc 600

gaaaccacct attataacag cgcgctgaaa agccgcttta ccattagccg cgataacagc 660

aaaaacaccc tgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat 720

tgcgcgaaac attattatta tggcggcagc tatgcgatgg attattgggg ccagggcacc 780

ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cctggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgctaa tag 1473

<210> 23

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 23

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 120

<212> PRT

<213> mouse (Mus musculus)

<400> 24

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

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

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 25

<211> 245

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

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

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

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

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

Claims

1. A humanized CD19 antigen-binding single-chain antibody, characterized in that, based on FMC63 antibody of mouse anti-human CD19 antigen, the light chain framework region VL FR1, VL FR2, VL FR3, VL FR4 and the heavy chain framework region VH FR1, VH FR2, VH FR3, VH FR4 are humanized; wherein,

the amino acid sequence of humanized VL FR1 is shown as SEQ ID No. 1;

the amino acid sequence of humanized VL FR2 is shown in SEQ ID No. 2;

the amino acid sequence of humanized VL FR3 is shown in SEQ ID No. 3;

the amino acid sequence of humanized VL FR4 is shown in SEQ ID No. 4;

the amino acid sequence of humanized VH FR1 is shown as SEQ ID No. 5;

the amino acid sequence of humanized VH FR2 is shown as SEQ ID No. 6;

the amino acid sequence of humanized VH FR3 is shown as SEQ ID No. 7; and

the amino acid sequence of humanized VH FR4 is shown in SEQ ID No. 8.

2. The humanized CD19 antigen-binding single chain antibody of claim 1, wherein the heavy chain variable region of the single chain antibody comprises three complementarity determining regions CDRs: VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region of the single chain antibody comprises the following three complementarity determining regions CDRs: VL CDR1, VL CDR2 and VL CDR3, wherein the 6 CDRs are identical to the 6 CDRs of the FMC63 antibody against murine CD19 antigen.

3. The humanized CD19 antigen-binding single chain antibody of claim 1, wherein the 6 CDRs in the single chain antibody have the following structures:

VL CDR1, shown in SEQ ID No. 9;

VL CDR2, shown in SEQ ID No: 10;

VL CDR3, shown in SEQ ID No. 11;

VH CDR1, shown as SEQ ID No: 12;

VH CDR2, shown as SEQ ID No. 13; and

VH CDR3, shown in SEQ ID No: 14.

4. The humanized CD19 antigen-binding single chain antibody of claim 1, wherein the single chain antibody comprises, in order, a light chain, a connecting region, and a heavy chain, or comprises, in order, a heavy chain, a connecting region, and a light chain;

5. The humanized CD19 antigen-binding single chain antibody of claim 4, wherein the segments of the light chain are linked in the order: humanized VL FR1-VL CDR 1-humanized VL FR2-VL CDR 2-humanized VL FR3-VL CDR 3-humanized VL FR 4;

the sequence of the connection of the segments in the heavy chain is: humanized VH FR1-VH CDR 1-humanized VH FR2-VH CDR 2-humanized VH FR3-VH CDR 3-humanized VH FR 4.

6. The humanized CD19 antigen-binding single chain antibody of claim 2, wherein the heavy chain variable region has the sequence shown in SEQ ID No. 16 and the light chain variable region has the sequence shown in SEQ ID No. 15.

7. The humanized CD19 antigen-binding single chain antibody of claim 1, wherein the amino acid sequence of the humanized CD19 antigen-binding single chain antibody is set forth in SEQ ID No. 19.

8. A humanized anti-human CD19 antigen Chimeric Antigen Receptor (CAR), comprising in sequence an optional leader peptide, the humanized CD19 antigen binding single chain antibody of any one of claims 1 to 7, a hinge region, a transmembrane region, and an intracellular region.

9. A polynucleotide encoding the humanized CD19 antigen-binding single chain antibody of claim 1 or the humanized Chimeric Antigen Receptor (CAR) of anti-human CD19 antigen of claim 8.

10. An immune cell expressing an exogenous humanized CD19 antigen-binding single chain antibody according to any one of claims 1 to 7 or a chimeric antigen receptor of humanized anti-human CD19 antigen according to claim 8.