阅读说明：本技术 针对cd19的全人源抗体或抗体片段、嵌合抗原受体及应用 (Fully human antibody or antibody fragment aiming at CD19, chimeric antigen receptor and application ) 是由 杨光 于 2019-12-05 设计创作，主要内容包括：本发明公开了针对CD19的全人源抗体或抗体片段、嵌合抗原受体及应用。该抗体或抗体片段或编码该抗体或抗体片段的核酸能够用于制备宿主细胞、慢病毒质粒和诊断肿瘤以及靶向性抗肿瘤药物等。本发明的抗体或抗体片段能够高特异性的与人源CD19蛋白结合。(The invention discloses a fully human antibody or antibody fragment aiming at CD19, a chimeric antigen receptor and application. The antibody or antibody fragment or the nucleic acid encoding the antibody or antibody fragment can be used for preparing host cells, lentiviral plasmids, tumor diagnosis, targeting antitumor drugs and the like. The antibody or antibody fragment of the invention can be combined with human CD19 protein with high specificity.)

1. A fully human antibody or antibody fragment directed to CD19, wherein the antibody or antibody fragment comprises: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions HCDR1, HCDR2 and HCDR 3; the light chain variable region comprises three complementarity determining regions LCDR1, LCDR2 and LCDR 3;

the amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 3. 4, 5, 6, 7 and 8; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 11. 12, 13, 14, 15, 16; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 19. 20, 21, 22, 23, 24; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 27. 28, 29, 30, 31, 32; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 35. 36, 37, 38, 39, 40; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 43. 44, 45, 46, 47, 48; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 51. 52, 53, 54, 55, 56; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 59. 60, 61, 62, 63, 64.

2. The fully human antibody or antibody fragment against CD19 of claim 1, wherein the anti-CD19 binding domain of the antibody or antibody fragment is an scFv and the amino acid sequence of the anti-CD19 binding domain comprises SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, or a functional homologue thereof having 95-99% identity to such sequences.

3. The fully human antibody or antibody fragment against CD19 according to claim 2, wherein the amino acid sequence of the scFv comprises a heavy chain variable region of SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, wherein the modified amino acid number is at least one, two or three but not more than 30.

4. The fully human antibody or antibody fragment against CD19 of claim 1, wherein the anti-CD19 binding domain of the antibody or antibody fragment is an scFv whose nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, or a functional homologue thereof having 95-99% identity to any of these sequences.

5. The fully human antibody or antibody fragment against CD19 of claim 4, wherein the scFv comprises a heavy chain variable region of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, at least one, two or three but not more than 30 modified nucleic acids.

6. A chimeric antigen receptor comprising a fully human antibody or antibody fragment against CD19 according to any one of claims 1-5.

7. The chimeric antigen receptor according to claim 6, comprising, in sequential order: a signal peptide sequence, a CD19 binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signaling domain:

the signal peptide sequence comprises CSF2 RA;

the detection tag comprises C-myc; said CD19 binding domain comprises an scFv comprising said fully human antibody or antibody fragment directed against CD 19;

the hinge region and transmembrane domain comprise: hinge region Hinge TM and transmembrane structure CD8,

the functional signaling domain comprises: CD28, 4-1BB and CD3 Zeta connected in sequence.

8. A host cell comprising the chimeric antigen receptor of claim 6 or 7.

9. A lentiviral plasmid comprising a nucleotide sequence encoding the fully human antibody or antibody fragment against CD19 of any one of claims 1-5.

10. The use of the fully human antibody or antibody fragment against CD19 according to any one of claims 1-5, wherein the fully human antibody or antibody fragment against CD19 is used for preparing a diagnostic tumor and a targeted antitumor drug.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a fully human antibody or antibody fragment aiming at CD19 and application thereof.

Background

B cell malignancies are common hematological malignancies, first-line clinical therapy has a certain efficacy, but has a high recurrence rate and poor prognosis, and CD19 has received great attention as a molecular target for B cell malignancy immunotherapy. CD19 is a normal and malignant B lymphocyte specific surface protein that plays an important role in the development, proliferation and differentiation of B cells, as well as in malignant transformation. The specificity of CD19 in B lymphocyte expression and the broad spectrum of malignancy expression thus make it a potential molecular target for B lymphocyte malignancy immunotherapy.

Various immunotherapeutic strategies targeting CD19, including Fc fragment-modified antibodies, antibody-drug conjugates, bispecific antibodies, chimeric antigen receptor-modified T cells, and the like, are being developed in the laboratory and in the clinic, achieving encouraging therapeutic effects in leukemia and lymphoma, and strongly driving the development of targeted immunotherapy.

CAR-T is known as Chimeric Antigen Receptor T-cell immunotherapy (Chimeric Antigen Receptor T-Cell immunotherapy). Novel cell therapies with significant efficacy in the treatment of acute leukemias and non-hodgkin's lymphomas are considered to be one of the most promising approaches to tumor treatment. CAR-T technology has undergone a lengthy evolution process, a new type of cell therapy that has emerged for many years but has only been improved in recent years for clinical use.

The key to this new therapeutic strategy is the recognition of an artificial receptor called a Chimeric Antigen Receptor (CAR) for the target cell, and the ability of patient T cells to express this CAR after genetic modification. In human clinical trials, scientists have extracted some of the T cells from patients through a dialysis-like process and then genetically modified in the laboratory to introduce genes encoding the CAR so that the T cells can express the new receptor. These genetically modified T cells are propagated in the laboratory and subsequently perfused back into the patient. These T cells bind to the molecule on the surface of the target cell using the CAR receptor they express, and this binding triggers an internal signal generation which then activates the T cells so strongly that they rapidly destroy the target cell.

Disclosure of Invention

The invention aims to provide a fully human antibody or antibody fragment aiming at CD19, a chimeric antigen receptor and application. The antibody or antibody fragment has high targeting property, can be specifically combined with human-derived CD19 protein, and can be used for treating hematological cancers related to CD19 expression.

To achieve the above object, the present invention provides a fully human antibody or antibody fragment against CD19, the antibody or antibody fragment comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions HCDR1, HCDR2 and HCDR 3; the light chain variable region comprises three complementarity determining regions LCDR1, LCDR2 and LCDR 3;

the amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 3. 4, 5, 6, 7 and 8; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 11. 12, 13, 14, 15, 16; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 19. 20, 21, 22, 23, 24; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 27. 28, 29, 30, 31, 32; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 35. 36, 37, 38, 39, 40; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 43. 44, 45, 46, 47, 48; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 51. 52, 53, 54, 55, 56; or

The amino acid sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 59. 60, 61, 62, 63, 64.

Preferably, the anti-CD19 binding domain of the antibody or antibody fragment is an scFv and the amino acid sequence of the anti-CD19 binding domain comprises SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, or a functional homologue thereof having 95-99% identity to such sequences.

Preferably, the amino acid sequence of said scFv comprises a heavy chain variable region of SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, wherein the modified amino acid number is at least one, two or three but not more than 30.

Preferably, the anti-CD19 binding domain of the antibody or antibody fragment is an scFv, the nucleic acid sequence of which comprises the amino acid sequence of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, or a functional homologue thereof having 95-99% identity to any of these sequences.

Preferably, the scFv comprises a heavy chain variable region of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, at least one, two or three but not more than 30 modified nucleic acids.

The invention also provides a chimeric antigen receptor which contains the fully human antibody or antibody fragment aiming at the CD 19.

Preferably, the chimeric antigen receptor comprises, connected in sequence: a signal peptide sequence, a CD19 binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signaling domain:

the signal peptide sequence comprises CSF2 RA;

the detection tag comprises C-myc; said CD19 binding domain comprises an scFv comprising said fully human antibody or antibody fragment directed against CD 19;

the hinge region and transmembrane domain comprise: hinge region Hinge TM and transmembrane structure CD8,

the functional signaling domain comprises: CD28, 4-1BB and CD3 Zeta connected in sequence.

The invention also provides a host cell comprising the chimeric antigen receptor described above.

The invention also provides a lentiviral plasmid comprising a nucleotide sequence encoding a fully human antibody or antibody fragment against CD19 as described above.

The invention also provides application of the fully human antibody or antibody fragment aiming at the CD19, and the fully human antibody or antibody fragment aiming at the CD19 is used for preparing tumor diagnosis and targeting antitumor drugs.

Has the advantages that:

(1) the antibody or antibody fragment can be combined with human CD19 protein with high specificity, the chimeric antigen receptor technology (CAR-T technology) is utilized to engineer and express the human antibody fragment which is integrated in CAR and is combined with CD19 in T cells, and the obtained chimeric antigen receptor T cells can be used for treating hematological cancers related to CD19 expression.

(2) The chimeric antigen receptor of the invention can target a malignant B cell tumor disease cell surface marker molecule CD 19.

Drawings

FIG. 1 is a lentivirus sublibrary plasmid map.

FIG. 2 shows the result of green fluorescence signal of lentivirus sublibrary package.

FIG. 3 shows the result of the Jurkat fluorescence signal of lentivirus-infected host cells.

FIG. 4a shows the blank host cell jurkat as a control, and the cells expressing the green fluorescence reporter gene are obtained by cell sorting, i.e., the cell population in Q2\ Q3 in the right picture.

FIG. 4b shows that the host cell jurkat expressing the CAR gene obtained by sorting was co-incubated with the negative cell K562 and the positive cell Raji, and the cells expressing the APC fluorescence signal were obtained by cell sorting using the negative cell as a control, i.e., the right cell population in the right panel.

FIG. 5a shows that the purified antibodies 3-F11, 4-H3, 1-B11 and 3-H5 can well recognize CD19 antigen on the surface of Raji cells by using Raji cells highly expressing CD19 as a blank control and FMC63 as a positive control.

Fig. 5b shows that the purified antibodies C135, C13, C20 and C25 can all recognize CD19 antigen on the surface of Raji cells well, using Raji cells highly expressing CD19 as blank control and FMC63 as positive control.

FIG. 6 shows activation of jurkat effector host cells incubated with target cells (detection of anti-CD69-APC/anti-CD69-PE fluorescent antibody) indicating that all human antibodies C13, C20, C25, C135, 3-F11, 4-H3, 1-B11 and 3-H5 against CD19 can better recognize CD19 on Raji cell surface.

Detailed Description

Definition of terms

An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Antibodies include single chain antibodies.

The "heavy chain" consists of a heavy chain variable region (VH) and a heavy chain constant region.

The "light chain" consists of a light chain variable region (VL) and a light chain constant region.

"scFv": the single-chain antibody (single-chain antibody fragment) is formed by connecting an antibody heavy chain variable region and a light chain variable region through a short peptide (linker) of 15-20 amino acids.

The "heavy chain variable region" and "light chain variable region" can be further subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions termed "framework regions" (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4, the variable regions of the heavy and light chains contain binding domains that can interact with antigens. In the present invention, CDRL, CDR2, CDR3 of the heavy chain variable region are represented as HCDR1, HCDR2 and HCDR3, respectively; the CDRL, CDR2, CDR3 of the light chain variable region are denoted LCDR1, LCDR2 and LCDR3, respectively.

The "constant region" may mediate the binding of the immunoglobulin to host tissues or factors.

An "antigen binding domain" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD 19).

"monoclonal antibody" refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibody compositions exhibit specific binding and affinity for a particular epitope (the portion of the antigen specifically recognized by the antigen receptor).

A "chimeric antigen receptor" consists of an extracellular antigen-binding region (consisting of a light chain and a heavy chain derived from a monoclonal antibody, joined by a flexible hinge region in between to form a single chain antibody, a transmembrane region, and an intracellular signaling region.

"CAR-T technology" refers to chimeric antigen receptor T cell immunotherapy. The scFv for recognizing tumor-associated antigen and an intracellular signal domain, namely an immunoreceptor tyrosine activation motif, are subjected to in vitro gene recombination to generate recombinant plasmids, the recombinant plasmids are transfected into T cells of a patient in vitro by a transfection technology to enable the T cells of the patient to express tumor antigen receptors, and the transfected T cells are purified and amplified in a large scale and are called chimeric antigen receptor T cells.

"95-99% identity" means homology of 95% or more (optimally 98% or more).

"modification" is a variation of an amino acid or nucleotide, including deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids or nucleotides, and addition of one or several (usually less than 20, preferably less than 10, more preferably less than 5) amino acids or nucleotides at the C-terminal and/or N-terminal, without changing the function of the protein or nucleic acid.

The invention provides a fully human antibody or antibody fragment aiming at CD19, wherein the anti-CD19 binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is specifically shown in Table 1.

Amino acid sequence of scFv

The nucleic acid sequence encoding the scFv provided by the invention comprises SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, and 57, and after translation into an amino acid sequence based on codons corresponding to bases in each sequence, the amino acid sequences of the corresponding scfvs are represented by SEQ ID NOs: 2. 10, 18, 26, 34, 42, 50, 58.

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

Some of the material sources are illustrated below:

CD19 protein: human CD19 (available from Acrobiosters, cat # CD 9-H8259).

Phage antibody library of human single-chain antibodies: constructed by the firm floros pharmaceutical technology ltd.

Streptavidin magnetic beads: purchased from Invitrogen corporation.

Enzyme-linked immunosorbent assay microplate: a96-well half-well low-turbine microplate (available from Corning Corp.).

Anti-M13 HRP antibody: from Thermo Fisher, inc.

M13 helper phage: purchased from Invitrogen corporation.

SOC culture medium: purchased from Shanghai Producers.

pCGMT phage vector: available from Addgene, Inc.

XL1-blue bacterium: purchased from agilent corporation under item number 200228.

LB solid medium plate: 5g of yeast extract, 10g of peptone, 10g of sodium chloride and 10g of agar powder were dissolved in 1L of double distilled water, autoclaved at 121 ℃ and poured onto a plate.

Developing solution ABTS solution: available from Thermo Fisher corporation under the designation 002024.

Gelred nucleic acid dye: from Thermo Fisher, inc.

pFUSE expression vector: purchased from Invitrogen corporation.

Plasmid extraction kit: purchased from QIAGEN corporation.

Restriction enzymes: purchased from Takara corporation.

And (3) recombinase: purchased from Novoprotein, inc.

lipo3000 reagent: from Thermo Fisher, inc.

Polybrene reagent: from Thermo Fisher, inc.

RMPI 1640 medium: purchased from gibco.

DMEM medium: purchased from gibco.

Myc-Tag (9B11) Mouse mAb (PE Conjugate): purchased from cell signalling, inc.

293Fectin transfection reagent: purchased from Invitrogen, cat # 12347500.

293Freestyle suspension cells: from Thermo Fisher, inc.

Immunoglobulin IgG1 constant region Fc fragment: purchased from tsingse corporation, tokyo.

Protein quantification kit by BCA method: purchased from Pierce, cat # 23252.

CBS antigen fixing solution: 1.59g of Na₂CO₃And 2.93g NaHCO₃Dissolved in 1L of water and adjusted to pH 9.6.

Anti-Human Fc HRP secondary antibody: from Thermo Fisher, inc.

96-well bottom-pass plate: purchased from Corning corporation.

Biacore instrument: purchased from GE, T200.

Protein A chip: purchased from GE company.