CN109879966B - Humanized design and expression verification based on murine CD19 antibody - Google Patents

Abstract

The invention provides an anti-CD 19 humanized monoclonal antibody and a preparation thereof. In particular, the invention provides a novel anti-CD 19 humanized antibody. The antibody of the invention can be combined with CD19 antigen with high specificity, and has higher affinity and bioactivity, low immunogenicity, stable structure and good pharmacy. The humanized antibody has good stability in the antibody preparation, and is used for preparing a medicament for preventing or treating CD19 related diseases.

Description

Humanized design and expression verification based on murine CD19 antibody

Technical Field

The invention relates to the field of medicines, in particular to an anti-CD 19 humanized monoclonal antibody and a preparation thereof.

Background

The humanized antibody is characterized in that a murine component is further reduced on the basis of a chimeric antibody, a murine anti-CDR region is reserved, other parts of the humanized antibody are partially replaced by a human antibody, the humanized antibody is superior to the murine antibody in disease treatment because the reduction of the murine component in the antibody can reduce immunological rejection of the body, and the humanized antibody has the other advantages that the half life period in vivo is long, the half life period of the murine antibody is less than one day, and the humanized antibody can reach days and sometimes even longer.

Clinical treatment of murine mabs is limited because they elicit human anti-mouse antibody responses (HAMA) in clinical treatment. The antibody humanization technology can greatly reduce the immunogenicity of the murine monoclonal antibody.

Thus, in view of the role and function of CD19 in various types of related diseases, there remains a need in the art to develop anti-CD 19 humanized antibodies suitable for the treatment of patients.

Disclosure of Invention

The invention aims to carry out humanized design and expression on a murine CD19 antibody (FMC63 clone), obtain a humanized antibody with the affinity of one order of magnitude with the murine antibody, and reduce the possible immunogenicity of the murine antibody in a human body to the maximum extent.

The invention also aims to provide a high-affinity high-bioactivity CD19 humanized antibody and application thereof.

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

(1a) a light chain variable region with a sequence shown as SEQ ID NO. 3;

(1b) a light chain variable region derived from the sequence shown in SEQ ID No.3 and having the function of (1a) the light chain variable region, which is formed by substituting, deleting, modifying and/or adding 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 in the amino acid sequence shown in SEQ ID No. 3;

(2a) a light chain variable region with a sequence shown as SEQ ID NO. 5; or

(2b) A light chain variable region derived from the sequence shown in SEQ ID NO.5 and having the function of the light chain variable region of (2a) formed by substituting, deleting, modifying and/or adding 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 in the amino acid sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the light chain variable region has an amino acid mutation corresponding to the sequence shown in SEQ ID No.1 selected from the group consisting of: threonine (T) at position 7, leucine (L) at position 15, serine (S) at position 22, tyrosine (Y) at position 71, serine (S) at position 72, phenylalanine (F) at position 87, leucine (L) at position 104, or a combination thereof.

In another preferred embodiment, the mutation of the light chain variable region corresponding to the sequence shown in SEQ ID No.1 is selected from the group consisting of: T7S, L15V, S22T, Y71F, S72T, F87Y, L104V, or a combination thereof.

In another preferred embodiment, the light chain variable region has an amino acid mutation corresponding to the sequence shown in SEQ ID No.1 selected from the group consisting of: aspartic acid (D) at position 1, methionine (M) at position 4, threonine (T) at position 7, serine (S) at position 9, serine (S) at position 10, aspartic acid (D) at position 17, isoleucine (I) at position 21, lysine (K) at position 45, valine (V) at position 58, tyrosine (Y) at position 71, serine (S) at position 72, phenylalanine (F) at position 87, or a combination thereof.

In another preferred embodiment, the mutation of the light chain variable region corresponding to the sequence shown in SEQ ID No.1 is selected from the group consisting of: D1E, M4L, T7S, S9A, S10T, D17E, I21L, K45R, V58I, Y71F, S72T, F87Y, or a combination thereof.

In another preferred embodiment, the light chain variable region has the sequence shown in SEQ ID NO.3 or 5.

In a second aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the first 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 third aspect of the present invention, there is provided a heavy chain variable region of an antibody, wherein the heavy chain variable region is selected from the group consisting of:

(1c) a heavy chain variable region with a sequence shown as SEQ ID NO. 4;

(1d) a heavy chain variable region derived from the sequence shown in SEQ ID No.4 and having (1c) the function of the heavy chain variable region, which is formed by substituting, deleting, modifying and/or adding 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 in the amino acid sequence shown in SEQ ID No. 4;

(2c) a heavy chain variable region with a sequence shown as SEQ ID NO. 6; or

(2d) A heavy chain variable region derived from the sequence shown in SEQ ID NO.6 and having the function of the heavy chain variable region of (2c) by substituting, deleting, modifying and/or adding 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 in the amino acid sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the heavy chain variable region has an amino acid mutation corresponding to the sequence shown in SEQ ID NO.2 selected from the group consisting of: serine (S) at position 17, valine (V) at position 20, leucine (L) at position 48, leucine (L) at position 67, methionine (M) at position 82, leucine (L) at position 85, isoleucine (I) at position 92, or a combination thereof.

In another preferred embodiment, the mutation of the heavy chain variable region corresponding to the sequence shown in SEQ ID NO.2 is selected from the group consisting of: S17T, V20L, L48I, L67V, M82L, L85V, I92V, or a combination thereof.

In another preferred embodiment, the heavy chain variable region has an amino acid mutation corresponding to the sequence shown in SEQ ID NO.2 selected from the group consisting of: glycine (G) at position 10, serine (S) at position 15, serine (S) at position 17, serine (S) at position 19, valine (V) at position 20, glycine (G) at position 44, glycine (G) at position 49, leucine (L) at position 85, serine (S) at position 115, or a combination thereof.

In another preferred embodiment, the mutation of the heavy chain variable region corresponding to the sequence shown in SEQ ID NO.2 is selected from the group consisting of: G10A, S15T, S17T, S19T, V20L, G44A, G49A, L85M, S115T, or a combination thereof.

In another preferred embodiment, the heavy chain variable region sequence is as shown in SEQ ID NO.4 or 6.

In a fourth aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the third aspect of the invention.

In another preferred embodiment, the heavy chain of said 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 fifth aspect of the invention, there is provided an antibody having:

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

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

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

In another preferred embodiment, the antibody has a light chain variable region as shown in SEQ ID No.3 or 5; and/or a heavy chain variable region as set forth in SEQ ID No.4 or 6.

In another preferred embodiment, the antibody has a mutation selected from the group consisting of:

(a1) in another preferred embodiment, the light chain variable region has an amino acid mutation corresponding to the sequence shown in SEQ ID No.1 selected from the group consisting of: threonine (T) at position 7, leucine (L) at position 15, serine (S) at position 22, tyrosine (Y) at position 71, serine (S) at position 72, phenylalanine (F) at position 87, leucine (L) at position 104, or a combination thereof; and/or

(b1) The heavy chain variable region has amino acid mutation corresponding to the sequence shown in SEQ ID NO.2 and selected from the following group: serine (S) at position 17, valine (V) at position 20, leucine (L) at position 48, leucine (L) at position 67, methionine (M) at position 82, leucine (L) at position 85, isoleucine (I) at position 92, or a combination thereof.

In another preferred embodiment, the antibody has a mutation selected from the group consisting of:

(a2) the light chain variable region has amino acid mutation corresponding to the sequence shown in SEQ ID NO.1 and selected from the following group: aspartic acid (D) at position 1, methionine (M) at position 4, threonine (T) at position 7, serine (S) at position 9, serine (S) at position 10, aspartic acid (D) at position 17, isoleucine (I) at position 21, lysine (K) at position 45, valine (V) at position 58, tyrosine (Y) at position 71, serine (S) at position 72, phenylalanine (F) at position 87, or a combination thereof; and/or

(b2) The heavy chain variable region has amino acid mutation corresponding to the sequence shown in SEQ ID NO.2 and selected from the following group: glycine (G) at position 10, serine (S) at position 15, serine (S) at position 17, serine (S) at position 19, valine (V) at position 20, glycine (G) at position 44, glycine (G) at position 49, leucine (L) at position 85, serine (S) at position 115, or a combination thereof.

In another preferred embodiment, the light chain variable region sequence of the antibody is shown as SEQ ID No.3 or 5; and/or the heavy chain variable region sequence of the antibody is shown as SEQ ID NO.4 or 6.

In another preferred embodiment, the light chain variable region sequence of the antibody is shown as SEQ ID NO. 3; and the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 4.

In another preferred embodiment, the antibody has a light chain variable region having the sequence shown in SEQ ID No. 5; and the antibody has a heavy chain variable region with a sequence shown as SEQ ID NO. 6.

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

In another preferred embodiment, the antibody specifically binds to CD 19.

In another preferred embodiment, the antibody has an affinity for human CD19 with a KD value (M) of 2.0E-10 to 1.0E-8.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

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

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

In another preferred embodiment, the antibody is in the form of a drug conjugate.

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

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

(ii) optionally a tag sequence to facilitate 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 a seventh aspect of the invention, there is provided an antibody preparation comprising:

(a) an antibody according to the fifth aspect of the invention; and

(b) a vector, said vector comprising: a buffer, sterile water, and optionally a surfactant.

In another preferred embodiment, in said formulation, said antibody is present at a concentration of 5-100 mg/mL; preferably 10-70mg/mL, more preferably 20-60 mg/mL.

In another preferred embodiment, the buffer is selected from the group consisting of: a citric acid buffer system, a histidine buffer system, or a combination thereof.

In another preferred embodiment, the buffer is a histidine buffer system.

In another preferred embodiment, the histidine buffer system is present in the formulation in a concentration of 0.5-20mM, preferably 1-10 mM.

In another preferred embodiment, the citric acid buffer system comprises histidine and histidine hydrochloride, preferably 1-10mM histidine and 1-10mM histidine hydrochloride, based on the total weight of the antibody formulation.

In another preferred embodiment, the buffer is a citric acid buffer system.

In another preferred embodiment, the concentration of the citric acid buffer system in the formulation is 5-100mM, preferably 10-50 mM.

In another preferred embodiment, the citric acid buffer system comprises sodium citrate and sodium chloride, preferably 10-50mM sodium citrate and 50-200mM sodium chloride, based on the total weight of the antibody formulation.

In another preferred embodiment, the surfactant is selected from the group consisting of: tween 80, tween 20, or a combination thereof.

In another preferred embodiment, the surfactant is present in the formulation in an amount of 0.005 to 0.2 wt.%, based on the total weight of the antibody formulation.

In another preferred embodiment, the surfactant is tween 80, and the concentration is 0.02 to 0.1 wt%.

In another preferred embodiment, the formulation has a pH in the range of 5.0 to 7.5, preferably 5.5 to 7.

In another preferred embodiment, the formulation is an injectable formulation.

In another preferred embodiment, the formulation comprises: an antibody according to the fifth aspect of the invention, a histidine buffer system, a surfactant, and the balance sterile water for injection.

In another preferred embodiment, the formulation comprises:

20-60mg/mL of the antibody of the fifth aspect of the invention;

histidine buffer system 1-10 mM;

tween 800.02-0.1 wt%;

and sterile water for injection as the balance, and the pH of the formulation is preferably in the range of 5.5 to 6.5.

In an eighth aspect of the invention, there is provided a kit comprising an antibody preparation according to the seventh aspect of the invention, and a container for holding the antibody preparation.

In a ninth aspect of the invention there is provided a CAR construct wherein the scFv of the antigen binding region is a binding region which specifically binds to CD19 and has a light chain variable region according to the first aspect of the invention and a heavy chain variable region according to the third aspect of the invention.

In a tenth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the ninth aspect of the invention.

In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

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

(a) an antibody moiety selected from the group consisting 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, or a combination thereof; and

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

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

In a twelfth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: the variable region of a light chain according to the first aspect of the invention, the light chain according to the second aspect of the invention, the variable region of a heavy chain according to the third aspect of the invention, the heavy chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the immune cell according to the tenth aspect of the invention, the antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof, wherein the active ingredient is for use in the administration of a pharmaceutical composition comprising the active ingredient

(a) Preparing a detection reagent or a kit;

(b) preparing a medicament or preparation for preventing and/or treating CD19 related diseases; and/or

(c) Preparing a medicament or a preparation for preventing and/or treating cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

In another preferred embodiment, the tumor is a tumor highly expressing CD 19.

In another preferred embodiment, the medicament or preparation is used for preparing a medicament or preparation for preventing and/or treating diseases related to CD19 (positive expression).

In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).

In another preferred embodiment, the detection reagent or the kit is used for diagnosing CD19 related diseases.

In another preferred embodiment, the detection reagent or kit is used for detecting CD19 protein in a sample.

In another preferred embodiment, the detection reagent is a detection sheet.

In a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: a light chain variable region according to the first aspect of the invention, a light chain according to the second aspect of the invention, a heavy chain variable region according to the third aspect of the invention, a heavy chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the tenth aspect of the invention, an antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

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

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition is used for treating tumors.

In another preferred embodiment, the tumor is a tumor highly expressing CD 19.

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

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

(2) A recombinant protein according to the sixth aspect of the invention;

(3) a CAR construct according to the ninth aspect of the invention.

In a fifteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the fourteenth aspect of the invention.

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

In a sixteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fifteenth aspect of the invention or having integrated into its genome a polynucleotide according to the fourteenth aspect of the invention.

In a seventeenth aspect of the invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of CD19 protein in a sample, the method comprising the steps of:

(1) contacting said sample in vitro with an antibody according to the fifth aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD19 protein in the sample.

In an eighteenth aspect of the present invention, there is provided a detection board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the eleventh aspect of the invention.

In a nineteenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody directed against the antibody according to the fifth aspect of the invention;

alternatively, the kit comprises a detection plate according to the eighteenth aspect of the invention.

In a twentieth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the sixteenth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

In a twenty-first aspect of the present invention, there is provided a method of treating a disease associated with abnormal expression or function of CD19, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

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 shows a diagram of a vector construction for single chain antibody expression.

FIG. 2 shows the results of single chain antibody flow cytometry.

FIG. 3 shows an electrophoretogram of expression purification of the full antibody.

FIG. 4 shows the sequence alignment before and after antibody humanization, where (A) is clone T1L1H4 and (B) is clone T4L2H 2.

FIG. 5 shows the results of the coupling buffer selection.

FIG. 6 shows the results of the final coupling amount of the ligand.

FIG. 7 shows the kinetic constant measurements of different mAbs with CD19, where (A) and (B) represent chimeric antibodies (chimeric); (C) and (D) represents T1L1H 4; (E) and (F) represents T4L2H 2.

Detailed Description

The present inventors have unexpectedly obtained an anti-CD 19 humanized antibody having excellent affinity and good structural stability through extensive and intensive studies and extensive screening. Specifically, the humanized antibody template has similarity and human use frequency in selecting the humanized framework region, the framework regions of the humanized antibody template T1-T4 (such as two groups of sequences T1H4, T1L1, T4H2 and T4L 2) are selected for humanization, and the obtained humanized antibody is subjected to mutation screening. The humanized antibody, especially the mutated humanized antibody, has affinity similar to that of the chimeric antibody, and the solubility and endogenous fluorescence of the humanized antibody are preliminarily studied to verify that the humanized antibody has preliminary druggability, so that the humanized antibody can be further developed into humanized monoclonal antibody medicine for target treatment in the future. And the humanized antibody is subjected to primary druggability research, and the preparation formula of the anti-CD 19 humanized antibody is screened and determined. Specifically, the invention carries out humanized design and modification on a murine CD19 antibody (FMC63 clone), expresses a corresponding humanized antibody, and finally obtains 2 humanized antibody sequences (T1L1H4 and T4L2H2) with consistent affinity with the murine antibody through flow verification and affinity verification. The present invention has been completed based on this finding.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j. diol. chem,243, p3558 (1968).

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. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of an anti-CD 19 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological 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. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

"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%.

CD19

The CD19 molecule is a potential target for treating B-line tumor, CD19 expresses CD19 molecule on the surface of most B-cell malignant tumor cells, while cells expressing CD19 molecule on the surface of normal cells are limited to B cells and follicular dendritic cells, and more importantly, the surface of pluripotent hematopoietic stem cells does not express CD19 molecule.

Murine FMC63 variable region sequence information

FMC63 mouse monoclonal antibody light chain variable region (SEQ ID NO.1)

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

FMC63 mouse Single antibody heavy chain variable region (SEQ ID NO.2)

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

Humanized T1L1H4 variable region sequence information

Humanized monoclonal antibody T1L1H4 light chain variable region (SEQ ID NO.3)

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKVEI

Humanized monoclonal antibody T1L1H4 heavy chain variable region (SEQ ID NO.4)

QVQLQESGPGLVAPSETLSLTCTVSGVSLPDYGVSWIRQPPRKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

Humanized T4L2H2 variable region sequence information

Variable region of humanized monoclonal antibody T4L2H2 light chain (SEQ ID NO.5)

EIVLTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPDRFSGSGSGTDFTLTISRLEPEDIAVYYCQQGNTLPYTFGQGTKLEI

Humanized monoclonal antibody T4L2H2 heavy chain variable region (SEQ ID NO.6)

QVTLRESGPALVKPTQTLTLTCTFSGVSLPDYGVSWIRQPPGKALEWLAVIWGSETTYYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGTTVTVSS

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. The same class of Ig can be divided into different subclasses according to the difference of amino acid composition of the heavy chain region and the number and position of the disulfide bonds of the heavy chain, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda depending on the constant region. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The antibody light chain of the invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

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 LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

The term "murine antibody" is used in the present invention as a monoclonal antibody against CD19 prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with the CD19 antigen and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine CD19 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody.

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. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity.

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 contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest 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 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 term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the CD19 molecule). Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-7M, e.g. less than about 1O^-8M、1O^-9M or lO^-10M or less affinity (KD) binding.

The term "competes for binding" refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of CD19 and binds to the antigen as a monoclonal antibody of the invention. An antibody that binds to the same epitope as a monoclonal antibody of the invention refers to an antibody that recognizes and binds to the amino acid sequence of CD19 recognized by a monoclonal antibody of the invention.

The term "KD" or "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the invention are present in an amount less than about 10^-7M, e.g. less than about 10^-8M、10^-9M or l0^-10M or less dissociation equilibrium constant (KD) binds to CD 19.

As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.

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, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

anti-CD 19 humanized antibodies

The present invention provides an anti-CD 19 humanized antibody (hereinafter referred to as CD19 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.

In 1986, Jones et al firstly transplanted the heavy chain CDR of the murine monoclonal antibody to the framework region of the heavy chain of the human antibody, and then assembled with the light chain of the murine monoclonal antibody into a complete antibody and kept the affinity similar to that of the original murine monoclonal antibody, thereby providing a thought for the development of antibody humanization technology. Queen et al succeeded in constructing a humanized antibody against CD25 in 1989 by a CDR grafting method in which a human antibody Eu framework region was humanized and amino acids of a murine antibody were retained at partial sites of the framework region to maintain affinity. In 1992 Presta et al reported a successful humanization method by CDR grafting using human antibody subgroup consensus (consensus sequence) as a template. Pedersen et al, 1994, reported humanization of antibodies using surface remodeling (resurfacing). Hsiao et al, 1994, reported humanization methods for CDR grafting with human antibody Germine sequence framework regions. Jespers et al succeeded in constructing a humanization method by a method using a phage library (Shuffling library) in 1994.

The choice of human framework regions in antibody humanization is generally two, one is a known mature antibody and one is a human Germline sequence. Known mature antibody framework regions often contain somatic mutation sites that may confer potential immunogenicity. Compared with a mature antibody, the human Germline sequence framework region is theoretically lower in immunogenicity, more flexible in structure and strong in plasticity, and can easily accept different CDR regions. The human antibody Germline gene has certain bias in the use frequency of a human body, and the antibody obtained by selecting the Germline framework region with high use frequency for humanization has the advantages of low immunogenicity, high expression quantity, stable structure and the like, so that the Germline sequence with the highest similarity with a murine antibody is not selected during humanization, the similarity and the use frequency of the human body are considered, and the framework regions of IGKV3-20 x 01 and IGHV1-69 x 01 are selected for humanization through a large number of experimental screens. The invention selects the human antibody Germline framework region for CDR transplantation, so that the constructed humanized antibody has more stable structure, high expression quantity, low immunogenicity and higher druggability.

Specifically, as described in the first to fifth aspects of the present invention:

FMC63 humanized antibody light chain variable region

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKVEI(SEQ ID NO.:3)

FMC63 humanized antibody heavy chain variable region

QVQLQESGPGLVAPSETLSLTCTVSGVSLPDYGVSWIRQPPRKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO.:4)

FMC63 humanized antibody light chain variable region

EIVLTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPDRFSGSGSGTDFTLTISRLEPEDIAVYYCQQGNTLPYTFGQGTKLEI(SEQ ID NO.5)

FMC63 humanized antibody heavy chain variable region

QVTLRESGPALVKPTQTLTLTCTFSGVSLPDYGVSWIRQPPGKALEWLAVIWGSETTYYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGTTVTVSS(SEQ ID NO.6)

In another preferred embodiment, the constant region of the humanized antibody is identical to the constant region of the anti-CD 19 murine monoclonal antibody FMC 63.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')₂Or other known antibody derivatives in the art, and any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.

The antibodies of the invention may be humanized antibodies, CDR grafted and/or modified antibodies targeting CD 19.

In the above-mentioned aspect of the present invention, the number of amino acids to be 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%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

The invention successfully carries out humanization transformation on the CD19 mouse monoclonal antibody, the humanized antibody reaches the affinity similar to that of a chimeric antibody, preliminary study on the solubility and endogenous fluorescence of the humanized antibody proves that the humanization has preliminary druggability, and the humanized monoclonal antibody can be further developed into a humanized monoclonal antibody medicament for targeted therapy in the future.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies may be used to produce the CD19 antibodies of the invention. For example, an animal may be immunized with a linked or naturally occurring CD19 protein or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of CD19 may be used as an immunogen (antigen) for the production of non-human antibodies specific for CD19, which antibodies are screened for biological activity. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculovirus vectors, plasmids and non-viral vectors.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

An exemplary method of humanizing a CD19 antibody of the invention is described in example 1.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Antibody formulations

The antibody has different stability in different preparation buffers, and is represented by the change of charge heterogeneity, degradation, polymerization and the like of antibody molecules, and the change of the quality properties is related to the physicochemical properties of the antibody, so that the preparation buffers suitable for the antibody need to be screened according to the physicochemical properties of different antibodies in the development process of antibody drugs. The currently commonly used antibody preparation buffer systems include phosphate buffer, citric acid buffer, histidine buffer, and the like, and according to the antibody properties, saline ions with different concentrations or excipients such as sorbitol, trehalose, sucrose, and the like, and a proper amount of surfactants such as tween 20 or tween 80 and the like are added to maintain the stability of the antibody.

The antibody preparation of the invention is as described in the seventh aspect of the invention.

The antibody drug combination preparation can effectively inhibit side reactions such as aggregation precipitation, hydrolysis, oxidation, deamidation and the like of the humanized antibody, and can effectively improve the stability of the product under the conditions of pressurization (high temperature, strong light irradiation, freeze thawing and the like), acceleration and long-term refrigeration.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, 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 pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Detection use and kit

The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention

(1) The present invention has developed a humanized CD19 antibody.

(2) The invention obtains a humanized CD19 antibody sequence.

(3) The invention can reduce the immunological rejection of the murine CD19 antibody in human body.

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.

Experiments in which specific conditions are not specified in the examples or test examples of the present invention are usually performed under conventional conditions or under conditions recommended by the manufacturers of raw materials/goods; reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1

Firstly, experimental steps

1. Humanization design

Humanized design is carried out according to amino acid sequence information (FMC63 clone) of CD19 antibody heavy chain and light chain, the CDR region sequence of an original antibody is kept unchanged, according to the result of germline alignment and the result of antibody result simulation, four different humanized antibody templates T1-T4 are respectively selected for the heavy chain and the light chain, and the framework region after the humanization is subjected to back mutation (back mutation), and candidate humanized antibody sequences are designed. Specific sequence information is shown in tables 3 and 4.

2. Humanized antibody gene synthesis and expression vector construction

The humanized single-chain antibody fragments designed above are respectively subjected to gene synthesis, and used as templates, overlap PCR is carried out, a VL- (G4S)3-VH single-chain antibody, namely, a heavy chain and a light chain of a T1-T4 template are respectively combined pairwise, and the combined single-chain antibody is subcloned into a single-chain antibody expression vector pFUSE-hIgG1-Fc2 by using an enzyme cutting site EcoRI-BglII (the vector schematic diagram is shown in figure 1, and the Fc sequence is human IgG1 Fc). After the vector was verified to be free of errors by sequencing, endotoxin-free plasmids were prepared using a Qiagen plasmid macrodrawer.

3. Humanized antibody expression and purification

3.1 the LVTransm transfection reagent and the single chain antibody expression vector are taken out from the refrigerator, thawed at room temperature, blown up and down by a pipette gun and mixed completely. Remove PBS or HBSS buffer and warm to room temperature. And (3) adding 130 mu g of pFUSE-hIgG1-Fc2 into one hole of a 6-hole plate by taking 2mL of PBS, blowing and beating the mixture up and down by a pipette gun, fully and uniformly mixing, adding 400 mu L of LVTransm, immediately blowing and beating the mixture up and down by a pipette, uniformly mixing, and standing the mixture at room temperature for 10 minutes.

3.2 the DNA/LVTransm complex was added to 50mL of 293F-SVP16 cells, and mixed well by gentle shaking. The cells were incubated at 37 ℃ with 5% CO₂After culturing in an incubator at 130RPM for 6-8 hours, 50mL of fresh CD-CHO medium is added, and the cells are returned to the incubator for further culture.

3.3 after continuous culture for 7 days, the culture supernatant was collected by centrifugation, filtered through a 0.45 μm filter, and the filtrate was transferred to a sterile centrifuge tube to purify the single-chain antibody using a Protein A column.

4. Flow assay for binding of humanized antibodies to target proteins

4.1 recovering K562 null cells and K562-CD19 recombinant cell strains from liquid nitrogen, and using 1640 and 10% FBS complete medium to adjust the cell state to the logarithmic growth phase.

4.2, dividing the two cells into a plurality of parts respectively, wherein the number of each part of cells is 1 x 10^6, using 1mL of PBS to resuspend the cells, respectively adding purified humanized single-chain antibodies, and each single-chain antibody needs to be incubated with blank K562 and K562-CD19, fully mixing the mixture evenly, and then incubating the mixture at room temperature for half an hour.

4.3, 800Xg room temperature centrifugation for 5 minutes, remove the antibody containing supernatant, use PBS to wash cells 3 times;

4.4, adding 2uL of Anti-human IgG (Anti-human IgG) marked by PE, fully and uniformly mixing, and incubating for 30min at room temperature in a dark place;

4.5, 800Xg room temperature centrifugation for 5 minutes, remove the secondary antibody containing supernatant, use PBS to wash cells 3 times;

4.6, using 500uL PBS heavy suspension cells, flow analysis.

Second, experimental results

1. Sequencing results of Single chain antibody expression vectors

All constructed single chain antibody expression vectors were Sanger sequenced and all antibody expression vectors were completely correct except for the occurrence of a point mutation in the humanized framework region FR1 from clone T1L2H 1.

2. Single chain antibody flow cytometry assay

As a result: according to the flow detection result, 2 humanized carriers (T1L1H4 and T4L2H2) with higher flow detection affinity are selected from different humanized antibody templates to construct a full antibody carrier, express and purify the full antibody and use for affinity detection. The results are shown in FIG. 2.

3. Expression purification of full antibody

The results of the expression purification of the full antibody are characterized in FIG. 3.

In FIG. 3, lane 1 represents the electrophoretic protein band of the chimeric antibody, lane 2 represents the electrophoretic protein band of the humanized T1L1H4 antibody, and lane 3 represents the electrophoretic protein band of the humanized T4L2H2 antibody.

4. Alignment of sequences before and after antibody humanization

The results of alignment before and after antibody humanization are shown in FIG. 4.

In FIG. 4, Panel A refers to the alignment of humanized T1L1H4 with the heavy and light chain variable regions of murine FMC 63; panel B shows the alignment of humanized T4L2H2 with the heavy and light chain variable regions of murine FMC 63.

5. Biacore affinity assay

5.1 ligand coupling Pre-enrichment experiment

The experimental conditions are as follows:

ligand: CD 19;

test pH: 10mM Acetate 5.5/5.0/4.5/4.0;

flow rate: 10 mul/min;

buffer solution: HBS-EP + buffer;

the results of the selection are shown in FIG. 5, and Acetate pH 5.0 was selected as the coupling buffer.

5.2 ligand coupling experiments

The experimental conditions are as follows:

ligand: CD 19;

coupling buffer: 10mM Acetate 5.0;

target coupling amount: 1000 RU;

the results are shown in FIG. 6, with a final coupling amount of 1035.8 RU.

5.3 binding assays for HM 1711091-chimera, HM1711091-T1L1H4, HM1711091-T4L2H2 and CD19

The experimental conditions are as follows:

flow rate: 30 mul/min;

binding time: 180 s;

dissociation time: 180 s;

regeneration reagent: glycine 1.5,30 s;

1) chimerism (chimera) 3.125/6.25/12.5/25/50/100ug/ml

The results are shown in FIGS. 7A and 7B.

Kinetic constants for HM 1711091-chimera and CD19 are as follows:

binding rate constant ka ═ 1.725X 10⁵M-1s-1；

Dissociation rate constant kd 0.004416 s-1;

dissociation equilibrium constant KD is 2.560X 10^-8M。

2)T1L1H4:3.125/6.25/12.5/25/50/100ug/ml

The results are shown in FIGS. 7C and 7D.

Kinetic constants for HM1711091-T1L1H4 and CD19 are as follows:

binding rate constant ka ═ 2.779X 10⁵M-1s-1；

Dissociation rate constant kd 0.004406 s-1;

dissociation equilibrium constant KD is 1.586 x 10^-8M。

3)T4L2H2:3.125/6.25/12.5/25/50/100ug/ml

The results are shown in FIGS. 7E and 7F.

Kinetic constants for HM1711091-T4L2H2 and CD19 are as follows:

binding rate constant ka of 9.855X 10⁴M-1s-1；

Dissociation rate constant kd 0.002799 s-1;

dissociation equilibrium constant KD is 2.840X 10^-8M。

The results of the binding constant experiments for the three antibodies to CD19 are shown in table 2.

TABLE 2 binding constants of the three antibodies to CD19

The results show that: according to the results of the affinity assays (fig. 7 and table 2), the dissociation equilibrium constants KD of the humanized antibodies T1L1H4, T4L2H2 and the Chimeric (Chimeric) were in the same order of magnitude, indicating that the humanized antibodies and the Chimeric antibodies had the same affinity.

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.

TABLE 3 humanized candidate antibody amino acid sequence information

TABLE 4 nucleic acid coding sequence information for humanized candidate antibodies

Sequence listing

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 6

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr 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 Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 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 Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

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

1 5 10 15

Thr Leu Ser Leu 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 Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 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 Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 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 Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 11

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro 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 Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 12

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 14

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro 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 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 15

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro 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 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 16

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 16

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro 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 Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 17

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr 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 Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr 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 Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 20

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 21

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 21

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 22

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 23

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 106

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 25

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 26

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 27

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat ccggcagcct 120

cctagaaaag gcctggaatg gatcggcgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 28

<211> 371

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

caggttcagc tgcaagagtc tggacctgga ctggtggccc ctagcgaaac actgagcctg 60

acctgtacag ttagcggcgt gtccctgcct gactatggcg tgtcctggat cagacagcct 120

cctcggaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

agctggaaat c 371

<210> 29

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccttca ccataagcag cctgcagcct 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 30

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccttta caatcagcag cctgcagcaa 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 31

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagcct 120

ggcggcgctc ccaagctgct gatctaccac acaagcagac tgcacagcgg cgtgcccagc 180

agattttctg gctctggcag cggcaccgac ttcaccttta caatcagcag cctgcagcaa 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 32

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcac 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccctggt caccatctcc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 33

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcac 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 34

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaaaccc 120

ggcaaggtgc ccaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 35

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaagaccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 36

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaagaccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga caatcagcag cctgcagcaa 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 37

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 38

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 39

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgaaca acatggaccc cgtggacacc gccacctact actgtgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 40

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggacttcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 41

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 42

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatcagcag gctggaacaa 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 43

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

caagtgaccc tgagagagtc tggccctgct ctggtcaagc ccacacagac cctgacactg 60

acctgcacct tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggacacca gcaagaacca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 44

<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

caagtgaccc tgagagagtc tggccctgct ctggtcaagc ccacacagac cctgacactg 60

acctgcacct tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggacacca gcaagaacca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 45

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggacttcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggccag 300

ggcaccaagc tggaaatc 318

<210> 46

<211> 318

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggccag 300

ggcaccaagc tggaaatc 318

Claims (19)

1. An anti-CD 19 humanized antibody, wherein the antibody has a light chain variable region and a heavy chain variable region, and the sequence of the light chain variable region is shown as SEQ ID NO.3, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 4;

or the sequence of the light chain variable region is shown as SEQ ID NO.5, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 6.

2. A recombinant protein, said recombinant protein having:

(i) the antibody of claim 1; and

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

3. An antibody preparation, comprising:

(a) the antibody of claim 1; and

(b) a vector, said vector comprising: buffer, sterile water.

4. The antibody formulation of claim 3, wherein said carrier further comprises a surfactant.

5. An antibody preparation according to claim 3, wherein said preparation comprises:

the antibody of claim 1 20-60 mg/mL;

histidine buffer system 1-10 mM;

tween 800.02-0.1 wt%;

and sterile water for injection as the balance, and the pH of the preparation is in the range of 5.5 to 6.5.

6. A kit comprising the antibody preparation of claim 3 and a container holding said antibody preparation.

7. A CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to CD19 and the scFv has a light chain variable region as set forth in SEQ ID No.3 and a heavy chain variable region as set forth in SEQ ID No.4, or the scFv has a light chain variable region as set forth in SEQ ID No.5 and a heavy chain variable region as set forth in SEQ ID No. 6.

8. A recombinant immune cell expressing an exogenous CAR construct of claim 7.

9. An antibody drug conjugate, comprising:

(a) an antibody portion which is the antibody of claim 1; and

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

10. The antibody drug conjugate of claim 9, wherein the detectable label comprises a radionuclide.

11. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 2, the immune cell of claim 8, or the antibody drug conjugate of claim 9, or a combination thereof, the active ingredient being for use

(a) Preparing a detection reagent or a kit;

(b) preparing a medicament or preparation for preventing and/or treating CD19 related diseases; and/or

(c) Preparing a medicament or a preparation for preventing and/or treating cancer or tumor.

12. A pharmaceutical composition, comprising:

(i) an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 2, the immune cell of claim 8, or the antibody drug conjugate of claim 9, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

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

(1) the antibody of claim 1;

(2) the recombinant protein of claim 2; and

(3) the CAR construct of claim 7.

14. A vector comprising the polynucleotide of claim 13.

15. A genetically engineered host cell comprising the vector or genome of claim 14 having the polynucleotide of claim 13 integrated therein.

16. A method for detecting CD19 protein in a sample in vitro, the method comprising the steps of:

(1) contacting the sample with the antibody of claim 1 in vitro;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of CD19 protein in the sample, and wherein the method is a non-diagnostic method.

17. A test board, said test board comprising: a substrate and a test strip comprising the antibody of claim 1 or the antibody drug conjugate of claim 9.

18. A kit, comprising:

(1) a first container comprising the antibody of claim 1; and/or

(2) A second container comprising a secondary antibody against the antibody of claim 1;

alternatively, the kit comprises an assay plate according to claim 17.

19. A method of producing a recombinant polypeptide, said method comprising:

(a) culturing the host cell of claim 15 under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the antibody of claim 1 or the recombinant protein of claim 2.

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