CN113307871B - Preparation and application of novel anti-CD 19 antibody and CD19-CAR-T cell - Google Patents

Preparation and application of novel anti-CD 19 antibody and CD19-CAR-T cell Download PDF

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CN113307871B
CN113307871B CN202010125005.2A CN202010125005A CN113307871B CN 113307871 B CN113307871 B CN 113307871B CN 202010125005 A CN202010125005 A CN 202010125005A CN 113307871 B CN113307871 B CN 113307871B
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CN113307871A (en
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黄纲雄
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Fuzhou Tuoxin Tiancheng Biotechnology Co ltd
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Fuzhou Tuoxin Tiancheng Biotechnology Co ltd
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Abstract

The invention provides preparation and application of a novel anti-CD 19 antibody and a CD19-CAR-T cell. In particular, the invention provides a CD19 antibody that targets a first exon-deleted CD19 molecule, which can be used for the treatment and detection of recurrent, drug-resistant tumors. The invention also provides a novel CD19 specific CAR-T cell, and a preparation method and application thereof.

Description

Preparation and application of novel anti-CD 19 antibody and CD19-CAR-T cell
Technical Field
The present invention relates to the field of medicine, and more particularly to novel forms
Preparation of anti-CD 19 antibody and CD19-CAR-T cell and its application are provided.
Background
B cells include pre-B cells, early-developing B cells, mature B cells, and the like. Mature B cells differentiate terminally into plasma cells and malignant B cells. Most pre-B Acute Lymphoblastic Leukemia (ALL), non-hodgkin's malignant lymphoma, B-cell Chronic Lymphocytic Leukemia (CLL), pre-lymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, and some non-acute lymphoblastic leukemia highly express CD19. The expression of CD19 on plasma cells further suggests that it may be expressed on different B cell neoplasms such as multiple myeloma, plasmacytoma, fahrenheit. CD19 is therefore considered a target for a variety of hematological neoplasms.
In the current treatment of leukemia or lymphoma with CD19 antibodies and CD19-CAR-T cells, complete or partial loss of the target antigen is often due to the loss of the CD19 antigen fragment, resulting in relapse of leukemia in patients after treatment. In addition, in the existing CD19-CAR-T cell therapy, when the CAR structure is wrongly introduced into the tumor cell, the CAR expressing anti-CD 19 on the tumor cell can be caused, and the scFV in the CAR structural component binds to the CD19 molecule of the tumor itself, so as to shield the CD19 molecule of the tumor, so that the tumor cell can not be recognized by the scFV with the same structure on the CD19-CAR-T cell, and the tumor cell can not be killed, and the final therapy fails.
Thus, there is an urgent need in the art to develop novel anti-CD 19 antibodies that can recognize different epitopes of CD19, avoiding off-target, and CD19-CAR-T cells comprising the antibodies.
Disclosure of Invention
The invention aims at providing a CD19 antibody and a CD19-CAR-T cell aiming at recurrent tumor, and preparation and application thereof.
In a first aspect of the invention there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
CDR1 as shown in SEQ ID NO. 1,
CDR2 as set forth in SEQ ID No.:2, and
CDR3 as shown in SEQ ID NO. 3;
or,
CDR1 as shown in SEQ ID NO. 9,
CDR2 as set forth in SEQ ID NO. 10, and
CDR3 as shown in SEQ ID NO. 11;
wherein any one of the amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one amino acid and which is capable of retaining CD19 binding affinity.
In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 7.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 15.
In a second aspect of the invention there is provided an antibody heavy chain having a heavy chain variable region according to the first aspect of the invention.
In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.
In a third aspect of the invention, there is provided a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:
Or,
CDR1' shown in SEQ ID NO. 4,
CDR2' shown in SEQ ID No.:5, and
CDR3' shown in SEQ ID NO. 6;
or,
CDR1' shown in SEQ ID NO. 12,
CDR2' shown in SEQ ID No.:13, and
CDR3' as shown in SEQ ID NO. 14;
wherein any one of the amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one amino acid and which is capable of retaining CD19 binding affinity.
In another preferred embodiment, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 8.
In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 16.
In a fourth aspect of the invention there is provided an antibody light chain having a light chain variable region according to the third aspect of the invention.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.
In a fifth aspect of the invention, there is provided an antibody having:
(1) The heavy chain variable region of the first aspect of the invention; and/or
(2) A light chain variable region according to the third aspect of the invention;
alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
In another preferred embodiment, the CDR regions of the humanized antibody comprise 1, 2, or 3 amino acid changes.
In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit.
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 partially or fully humanized monoclonal antibody.
In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted does not exceed 40%, preferably 20%, more preferably 10% of the total number of amino acids of the initial amino acid sequence.
In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted is 1 to 7, preferably 1 to 3, more preferably 1.
In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
In another preferred embodiment, the derivative sequence with at least one amino acid added, deleted, modified and/or substituted has the function of inhibiting cell surface CD19 or recombinant CD19 protein.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the derivative sequence has an affinity KD for CD19 of 0.01nM to 1nM, preferably 0.02nM to 0.1nM.
In another preferred embodiment, the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention targets a CD19 molecule lacking the first exon (does not bind to an epitope corresponding to the first exon of CD 19).
In a sixth aspect of the present invention, there is provided a recombinant protein having:
(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention there is provided a CAR construct whose scFV fragment of the monoclonal antibody antigen binding region is a binding region that specifically binds to CD19 and which scFV has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.
In an eighth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.
In another preferred embodiment, the cell is a mammalian cell.
In another preferred embodiment, the immune cells are ex vivo.
In another preferred embodiment, the immune cells are autologous.
In another preferred embodiment, the immune cells are non-autologous.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In another preferred embodiment, the immune cells are derived from a primate (preferably a human).
In another preferred embodiment, the immune cells are selected from the group consisting of:
(i) Chimeric antigen receptor T cells (CAR-T cells);
(ii) Chimeric antigen receptor NK cells (CAR-NK cells); or (b)
(iii) Exogenous T Cell Receptor (TCR) T cells (TCR-T cells)
In another preferred embodiment, the immune cell comprises: NK cells, T cells, NKT cells, (γδ) T cells, monocytes, or macrophages.
In another preferred embodiment, the immune cells are CD19-CAR-T cells
In a ninth aspect of the present invention, there is provided an immunoconjugate comprising:
(a) An antibody moiety selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, 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, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or a toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxybenzodiazepines)), vinca alkaloids (vilos), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-chain of jezosin, α -octacocin, gelonin, mitogellin, restrictocin (retproctrocin), phenol, enomycin, curcin, crotonin, calicheamicin, saporin (Sapaonaria officinalis), glucocorticoids, or combinations thereof.
In another preferred embodiment, the coupling moiety is a detectable label.
In another preferred embodiment, the detectable label is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, nanoparticles of any form, etc.
In another preferred embodiment, the enzyme comprises a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)).
In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra223, ru-106, na24, sr89, tb-149, th-227, xe-133 Yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the immunoconjugate is an antibody drug conjugate.
In another preferred embodiment, the antibody drug conjugate is of the formula:
Figure BDA0002394143980000061
wherein:
ab is an antibody against CD19,
LU is the linker;
d is a drug;
and subscript p is a value selected from the group consisting of 1 to 10, preferably 1 to 8.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In a tenth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the immune cell of the eighth aspect of the invention, the antibody drug conjugate of the ninth aspect of the invention, or a combination thereof, wherein the active ingredient is used in (a) the preparation of a detection reagent, a detection plate, or a kit; (b) Preparing a medicament specifically targeting a tumor cell expressing CD 19; and/or (c) for preparing chimeric antigen receptor-modified immune cells.
In another preferred embodiment, the detection reagent, detection plate or kit is used for:
(1) Detecting CD19 protein in the sample; and/or
(2) Detecting endogenous CD19 protein in the tumor cells; and/or
(3) Detecting tumor cells expressing CD19 protein.
In another preferred embodiment, the assay reagent, assay plate or kit is used to diagnose a CD19 expressing tumor.
In another preferred embodiment, the agent is used to treat or prevent a CD19 expressing tumor, tumor migration, or tumor resistance.
In another preferred embodiment, the tumor comprises a solid tumor, a hematological cancer.
In another preferred embodiment, the tumor comprises a recurrent tumor, preferably a tumor that recurs after CAR-T cell therapy, such as a recurrent hematological tumor.
In another preferred embodiment, the CD19 expressing tumor is selected from the group consisting of: bladder cancer, biliary tract cancer, brain cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, neck cancer, renal cancer, salivary cancer, thymus epithelial cancer, thyroid cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma, myeloma, or a combination thereof.
In another preferred embodiment, the tumor is a drug resistant tumor.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the immune cell of the eighth aspect of the invention, the antibody drug conjugate of the ninth aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In a twelfth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) The heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention; or (b)
(2) The recombinant protein according to the sixth aspect of the present invention;
(3) The CAR construct of the seventh aspect of the invention.
In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region is as set forth in SEQ ID No.:17 or sequence listing SEQ ID No.: 19.
In another preferred embodiment, the nucleotide sequence encoding the light chain variable region is as set forth in SEQ ID No.:18 or sequence listing SEQ ID No.: shown at 20.
In a thirteenth aspect of the invention there is provided a vector comprising a polynucleotide according to the twelfth aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a fourteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the thirteenth aspect of the invention having incorporated therein a polynucleotide according to the twelfth aspect of the invention.
In a fifteenth aspect of the present invention, there is provided a method of detecting CD19 in a sample in vitro (including diagnostic or non-diagnostic), 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 whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CD19 in the sample.
In a sixteenth aspect of the present invention, there is provided a detection plate comprising: a substrate (support) and a test strip comprising an antibody according to the fifth aspect of the invention or an immunoconjugate according to the ninth aspect of the invention.
In a seventeenth 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 second antibody against the antibody of the fifth aspect of the invention;
Alternatively, the kit comprises a test plate according to the sixteenth aspect of the invention.
In an eighteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing the host cell of the fourteenth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a nineteenth aspect of the invention, there is provided a method of treating a CD19 expressing tumor, the method comprising: administering to a subject in need thereof an antibody of the fifth aspect of the invention, an antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
In another preferred embodiment, the method further comprises: the combination therapy is administered to a subject in need thereof with other drugs or therapeutic methods.
In another preferred embodiment, the other medicament or treatment method comprises: antitumor immunotherapeutic agent, tumor targeting agent, tumor chemotherapeutic agent, and tumor radiotherapy.
In another preferred embodiment, the anti-tumor immunotherapeutic agent comprises PD-1, PD-L1 mab.
In a twentieth aspect of the present invention, there is provided a method of preparing a chimeric antibody comprising the steps of:
the heavy chain variable region according to the first aspect of the present invention and/or the light chain variable region according to the third aspect of the present invention are cloned into an expression vector comprising the nucleotide sequence of the human antibody constant region, and then the human-mouse chimeric antibody is expressed by transfecting animal cells.
In a twenty-first aspect of the present invention, there is provided a method of preparing a humanized antibody comprising the steps of:
the nucleotide sequence of the heavy chain variable region according to the first aspect of the present invention and/or the CDR region of the light chain variable region according to the third aspect of the present invention is implanted into a nucleotide sequence template containing the FR region of a humanized antibody, and then cloned into an expression vector containing the constant region of a human antibody, and then the humanized antibody is expressed by transfecting animal cells.
In a twenty-second aspect of the present invention, there is provided a method of inhibiting tumor cell growth and migration comprising the steps of: administering to a subject in need thereof an antibody of the fifth aspect of the invention, an antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
In a twenty-third aspect of the invention, there is provided a method of inhibiting the growth of a tumor in a model animal comprising the steps of: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody.
In another preferred embodiment, the drugs may be administered alone or in combination, including tumor immunotherapy, tumor targeting drugs, cytotoxic drugs, radiation therapy.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows that after 3 immunizations of mice, antibodies against CD19 (hCD 19-D-mFc) with the first exon deletion have been raised in the serum.
FIG. 2 shows that HG27 antibody, HG28 antibody and anti-full length CD19 antibody can bind to 293FT/hCD19-F cells.
FIG. 3 shows that the HG27 antibody and the HG28 antibody can bind to the CD19-DhFc fusion protein (the first exon-deleted CD19 and the human Fc fragment were fused), whereas the CD19 antibody (HIB 19) could not bind to the CD19-DhFc fusion protein.
FIG. 4 shows that the HG27 antibody and the HG28 antibody do not have competitive binding to the human CD19 antibody (HIB 19) (FIG. 4), and that the epitopes bound by the two antibodies are different. While HG27 and HG28 compete with each other for binding to the same epitope (fig. 4 bottom).
Figure 5 shows that the G27 CAR-T and HG28 CAR-T cells have the same potent killing capacity as the existing CD19 antibody clone-derived CAR-T cells (CD 19-CAR-T) by incubating CAR-T cells with 3 target cells at different potency target ratios.
Detailed Description
The present inventors have studied extensively and intensively, and have unexpectedly found a novel anti-CD 19 antibody and CD19-CAR comprising the same for the first time. Compared with the existing antibodies, the antibodies of the invention recognize different epitopes of CD19 and target CD19 molecules with the deletion of the first exon. According to the results of the activity test, the antibodies of the present invention are capable of binding to the CD19 antigen with high specificityKD of 9.32X10 respectively -2 nM、2.09×10 -2 nM has remarkable anti-tumor activity, and can be used for treating and detecting recurrent and drug-resistant tumors.
Terminology
As used herein, the term "conjugate" refers to a soluble receptor or fragment or analog thereof, or an antibody or fragment or analog thereof, that is capable of binding to a target.
As used herein, the terms "CD19 conjugate," "CD19 antibody," "anti-CD 19 antibody," "antibody of the invention," and "antibodies" have the same meaning, and refer to antibodies or fragments or analogs thereof that specifically recognize CD19 and bind to CD 19.
As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic, diagnostic, 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. Treatment of a cell includes contacting a reagent with the cell, contacting a reagent with a fluid, and contacting a fluid with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, study and diagnosis; comprising contacting the CD19 conjugate with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.
As used herein, the term "treatment" refers to the administration of an internally or externally used therapeutic agent, including any CD19 conjugates of the invention, and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.
Antibodies to
As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. 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, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HCDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by 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 DNA recombination techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.
As used herein, the term "humanized antibody", a variable region engineered version of the murine antibody of the present invention, has CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 or 1 amino acids are replaced by amino acids of similar or similar nature as compared to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
Initial residues Representative substitution Preferred substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein includes a cell-mediated reaction in which nonspecific cytotoxic cells expressing fcγr recognize antibodies bound on target cells and cause lysis of the target cells. In various aspects, enhancing ADCC effector function may mean enhanced potency or enhanced efficacy. "titers" as used in the experimental setting means the concentration of antibody (half maximum effective concentration) when observing a particular therapeutic efficacy EC 50. "efficacy" as used in the experimental context means the maximum possible effector function of an antibody at saturation level.
The term "ADCP" or "antibody-dependent cell-mediated phagocytosis" as used herein includes cell-mediated reactions in which nonspecific cytotoxic cells expressing fcγr recognize antibodies bound on target cells and cause phagocytosis of the target cells.
The term "CDC" or "complement dependent cytotoxicity" as used herein includes a reaction in which one or more complement protein components recognize antibodies bound to a target cell, which then results in lysis of the target cell.
The term "effector function" as used herein includes biochemical events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include fcγr mediated effector functions such as ADCC and ADCP, and complement mediated effector functions such as CDC.
The term "chimeric antigen receptor" or "CAR" as used herein refers to: a polypeptide comprising an extracellular domain capable of binding an antigen, a transmembrane domain and a domain that allows cytoplasmic signaling (i.e., an intracellular signaling domain), which refers to a protein that transmits information into a cell via a defined signaling pathway by generating a second messenger to modulate cellular activity, or by functioning as an effector in response to such a messenger, comprises a primary signaling domain, and may also include a functional signaling domain derived from a stimulatory molecule as defined below (i.e., a co-stimulatory signaling domain). Intracellular signaling domains generate signals that can promote immune effector functions of cells of the CAR (e.g., CAR T cells), examples of immune effector functions, such as in CART cells, including cytolytic activity and helper activity, including cytokine secretion.
The term "primary signal domain" modulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signal domain is triggered by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain acting in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or a signaling motif of ITAM. Examples of primary signal domains comprising ITAM that are particularly useful in the present invention include, but are not limited to, sequences derived from tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d, in particular inventive CARs, the intracellular signaling domain in any one or more of the inventive CARs comprises an intracellular signaling sequence, such as the primary signaling domain of CD3 ζ.
The term "costimulatory signal domain" refers to a "costimulatory molecule," which is an cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules or ligands thereof that are non-antigen receptors required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18) and CD19 (CD 137).
In one aspect of the invention, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain that contains a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino acid (ND end) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to a cell membrane.
The term "cd3ζ" is defined herein as the protein provided by genbank accession No. BAG36664.1, or equivalent residues from a non-human species such as mice, rodents, monkeys, apes, etc. The "cd3ζ domain" is defined as the amino acid residue from the cytoplasmic domain of the ζ chain that is sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 164 of genbank accession No. BAG36664.1, functional orthologs thereof-equivalent residues from non-human species such as mice, rodents, monkeys, apes, and the like.
The term "CD19" herein refers to a member of the TNFR superfamily having the amino acid sequence of GenBank acc.no. aaa62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.; "CD19 co-stimulatory domain" is defined as amino acid sequence 214-255 of GenBank ACC.No. AAA62478.2, or equivalent residues from a non-taxonomic species such as mouse, rodent, monkey, ape, etc. In one aspect, the "CD19 co-stimulatory domain" is SEQ ID NO:35, or equivalent residues from a non-human species such as mice, rodents, monkeys, apes, etc.
anti-CD 19 antibodies
The term "CD19" includes, but is not limited to, variants, isoforms and species homologs of human CD 19. In some cases, the humanized antibodies of the invention can cross-react with CD19 of a species other than human. In some cases, the antibodies may be completely specific for one or more human CD19 proteins and may exhibit species or other types of non-human cross-reactivity. An exemplary complete amino acid sequence of human CD19 has SwissPort accession number P15391.CD19 is also known as the B cell surface antigen B4, the B cell antigen CD19, the CD19 antigen or Leu-12. Human CD19 is called Gene ID by Entrez Gene: 930, called HGNC by HGNC:1633.CD19 may be referred to as the gene encoding CD 19. The use of "human CD19" herein encompasses all known or yet to be discovered alleles and polymorphic forms of human CD 19.
In the present invention, the CD19 molecule used as an immune antigen is a CD19 molecule in which the first exon is deleted, and the anti-CD 19 antibody of the present invention does not bind to an epitope corresponding to the first exon of the CD19 molecule.
In the current treatment of leukemia or lymphoma with CD19 antibodies and CD19-CAR-T cells, complete or partial loss of the target antigen is often due to the loss of the CD19 antigen fragment, resulting in relapse of leukemia in patients after treatment. Aiming at the mechanism of recurrence, the invention provides a novel CD19 antibody, designs a novel epitope which targets the CD19 molecule, not only aims at the original CD19 molecule, but also targets the CD19 molecule with partial antigen fragment deletion after the existing CD19 antibody and CD19-CAR-T treatment, so that the CAR-T cell treatment of the invention can be used for treating the original ALL/lymphoma patient and also can be used for treating the patient with recurrence after the existing treatment;
In the existing CD19-CAR-T cell treatment, under the condition that the CAR structure is wrongly introduced into tumor cells, the CAR expressing anti-CD 19 on the tumor cells can be caused, and the scFv in the CAR structural component binds with the CD19 molecule of the tumor, so that the CD19 molecule of the tumor is shielded, the tumor cells can not be identified by the scFv with the same structure on the CD19-CAR-T cells, and the tumor cells can not be killed and finally the treatment fails. The scFV of the antibody of the invention can recognize different epitopes of CD19, and the antibody derived from the scFV or the scFV of the CAR-T cells can recognize the tumor cells shielded by the scFv, and can be used for treating patients with failure of the existing CAR-T cell treatment due to the reasons.
Meanwhile, the anti-CD 19 antibody or scFV thereof of the present invention can be used for detecting tumor cells of patients who fail in the above treatment or relapse after the treatment due to the deletion or modulation of antigen, which cannot be recognized and detected by the existing CD19 antibody itself.
The antibodies of the invention may be double-or single-chain antibodies and may be selected from animal-derived antibodies, chimeric antibodies, human-animal chimeric antibodies, preferably humanized antibodies, more preferably fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as IgA, igD, igE, igG and any one or more of IgM antibodies or antibodies of other subtypes.
Wherein the animal is preferably a mammal, such as a mouse.
Specifically, the present invention provides murine antibodies HG27 and HG28, antibody HG27 comprising the heavy chain variable region shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the light chain variable region shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, antibody HG28 comprising the heavy chain variable region shown in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, and the light chain variable region shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
The antibodies of the invention target a CD19 molecule lacking the first exon, i.e. do not bind to an epitope corresponding to the first exon of CD 19.
The amino acid sequences of the antibodies of the present invention are shown in Table 1
TABLE 1
Figure BDA0002394143980000151
Figure BDA0002394143980000161
Figure BDA0002394143980000171
In another aspect of the invention, the invention provides variants of antibodies or fragments thereof that bind to human CD 19. The invention thus provides antibodies or fragments thereof having a heavy and/or light chain variable region that is at least 80% identical to the variable region sequence of a heavy or light chain. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequence identity. The identity of the amino acid sequence or percentage of amino acid residues in the sequence that are identical to a humanized antibody or fragment thereof that binds human CD19 is referred to herein. Thus, sequence identity can be determined by standard methods commonly used to compare amino acid position similarity of two polypeptides. The two polypeptides are aligned for optimal matching of their respective amino acids (along the full length of one or both sequences or along a predetermined portion of one or both sequences) using a computer program such as BLAST or FASTA. The program provides default opening penalties and default gap penalties, and scoring matrices such as PAM250 (standard scoring matrix; see Dayhoff et al, in Atlas of Protein Sequence and Structure, volume 5, phase three augmentation (1978)) may be used in conjunction with the computer program. For example, percent identity may be calculated as: the total number of identical matches is multiplied by 100, divided by the total length of the longer sequences in the match span and the number of gaps that are poured into the longer sequences in order to align the two sequences.
The invention also provides humanized antibody fragments that bind human CD19 selected from the group consisting of Fab, fab '-SH, fd, dAb, F (ab') 2, scFv, bispecific single chain Fv dimer, diabodies, triabodies, and scFv genetically fused to the same or different antibodies. Preferred fragments are scFv, bispecific single chain Fv dimers and diabodies. The invention also provides full length humanized antibodies that bind to human CD 19.
Nucleic acids, vectors and host cells
The invention also provides isolated nucleic acids encoding the antibodies and fragments thereof, vectors, and host cells comprising the nucleic acids or vectors. The nucleic acid may be in whole cells, in a cell lysate, or in partially purified or substantially purified form.
The nucleic acids of the invention may be obtained using standard molecular biology techniques, for example, by standard PCR amplification or cDNA cloning techniques, to obtain cdnas encoding the light and heavy chains or encoding the VH and VL segments of an antibody. For antibodies obtained from a library of immunoglobulin genes (e.g., using phage display techniques), one or more nucleic acids encoding the antibodies may be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary with the host cell used.
For expression of the protein, the nucleic acid encoding the antibody of the invention may be incorporated into an expression vector. A variety of expression vectors are available for protein expression. Expression vectors may include self-replicating extra-chromosomal vectors, or vectors that integrate into the host genome. Expression vectors for use in the present invention include, but are not limited to, those that enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As known in the art, a variety of expression vectors are commercially or otherwise available. Antibodies can be expressed for use in the present invention.
In a preferred embodiment, the nucleic acid encoding the heavy chain variable region is as set forth in the sequence listing SEQ ID No.:17 or sequence listing SEQ ID No.: 19; and/or, the nucleic acid encoding the light chain variable region is as set forth in the sequence listing SEQ ID No.:18 or sequence listing SEQ ID No.: shown at 20.
In a more preferred embodiment, the nucleic acid encoding the heavy chain variable region is as set forth in the sequence listing SEQ ID No.:17, and the nucleic acid encoding the light chain variable region is as set forth in the sequence listing SEQ ID No.: shown at 18; nucleic acid encoding the heavy chain variable region is as set forth in the sequence listing SEQ ID No.:19 and the nucleic acid encoding the light chain variable region is as set forth in the sequence listing SEQ ID No.: shown at 20.
Specifically, the numbers of the above nucleotide sequences are shown in Table 2:
TABLE 2CD19 antibody Gene sequence numbering
Clone number Heavy chain protein variable region Light chain protein variable region
HG27 17 18
HG28 19 20
The method for preparing the nucleic acid is a method conventional in the art, and preferably comprises the steps of: the nucleic acid molecules encoding the above proteins are obtained by gene cloning techniques or by artificial total sequence synthesis.
It is known to those skilled in the art that a nucleotide sequence encoding the amino acid sequence of the above protein may be appropriately introduced into a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. Homologs of the polynucleotides of the invention may be obtained by substitution, deletion or addition of one or more bases of the gene encoding the protein sequence within a range that retains antibody activity.
Preparation of antibodies
Any suitable method for producing monoclonal antibodies may be used to produce the anti-CD 19 antibodies of the invention. For example, animals may be immunized with linked or naturally occurring CD19 homodimers or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
In the present invention, a fusion protein of a human CD19 extramembranous protein with a first exon deleted and a murine Fc fragment is used as an immune antigen to generate a CD19 specific antibody, and the biological activity of the antibody is screened. An exemplary method of producing an anti-CD 19 antibody of the invention is described in example 1.
The fully humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. In the present invention, the antibody is an IgG antibody, and an IgG4 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.
Also, any type of light chain may 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 present invention.
An exemplary method of humanizing an anti-CD 19 antibody of the invention is described in example 3.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell is a variety of host cells conventional in the art, as long as it is capable of stably self-replicating the recombinant expression vector described above and the nucleic acid carried thereby can be efficiently expressed. In particular, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
Preferred host cells include E.coli TG1 or BL21 cells (expressing single chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full length IgG antibodies)
The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained 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 cultivated in conventional medium under suitable conditions.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are 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 using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
Chimeric antigen receptor T cells containing anti-CD 19 antibodies
In another preferred embodiment, the invention provides a plurality of Chimeric Antigen Receptors (CARs) comprising an antibody or antibody fragment engineered to enhance binding to CD19 protein. In another preferred embodiment, the invention provides a cell (e.g., a T cell) engineered to express a CAR, wherein the CAR T cell ("CART") exhibits anti-tumor properties. In another preferred embodiment, the cell is transformed with a CAR, which is expressed on the cell surface. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cell can stably express the CAR.
In another preferred embodiment, the anti-CD 19 protein binding portion of the CAR is an scFv antibody fragment. In another preferred embodiment, the antibody fragment is functional, whereby it retains an equivalent affinity binding capacity as compared to the IgG antibody from which it is derived, e.g. it binds the same antigen with comparable efficacy. In another preferred embodiment, the antibody fragment is functional, whereby it provides a biochemical reaction, which may include, but is not limited to, activating an immune reaction, inhibiting initiation of signaling from its target antigen, inhibiting kinase activity, and the like. In another preferred embodiment, the anti-CD 19 antigen binding domain of the CAR is an scFv antibody fragment that is humanized relative to the murine sequence from which it is derived.
In another preferred embodiment, the CAR of the invention combines the antigen binding domain of a particular antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, CD3 ζ chains, 4-1BB, and CD28 signaling modules, and combinations thereof. In another preferred embodiment, the invention provides a cell (e.g., a T cell) engineered to express a Chimeric Antigen Receptor (CAR), wherein the CAR T cell ("CART") exhibits anti-tumor properties. In another preferred embodiment, the antigen binding domain of the CAR comprises a humanized anti-CD 19 antibody fragment comprising a scFV. Accordingly, the present invention provides, CD 19-CARs comprising a humanized anti-CD 19 binding domain engineered into T cells, and methods of using the same for adoptive immunotherapy.
In another preferred embodiment, the CD19-CAR comprises at least one intracellular signaling domain selected from the group consisting of a CD137 (4-1 BB) signaling domain, a CD28 signaling domain, a CD3 zeta signaling domain, and any combination thereof. In another preferred embodiment, the CD19-CAR comprises at least one intracellular signaling domain derived from one or more costimulatory molecules other than CD137 (4-1 BB) or CD 28.
In another preferred embodiment, the CARs of the invention combine the antigen binding domain of a particular antibody with an intracellular signaling molecule while increasing the IFN beta expression element. For example, in some aspects, intracellular signaling molecules include, but are not limited to, CD3 ζ chains, 4-1BB, and CD28 signaling modules, and combinations thereof. In another preferred embodiment, the invention provides a cell (e.g., a T cell) engineered to express a Chimeric Antigen Receptor (CAR), wherein the CAR T cell ("CART") exhibits anti-tumor properties. In another preferred embodiment, the antigen binding domain of the CAR comprises a humanized anti-CD 19 antibody fragment comprising a scFV.
Application of
The invention provides the use of an antibody of the invention, for example for the preparation of a diagnostic formulation, or for the preparation of a medicament for the prophylaxis and/or treatment of a CD 19-related disease. The CD 19-associated disease includes cancer, autoimmune disease, viral infection, graft versus host disease, inflammatory disease, immune disease, or a combination thereof. Wherein the cancer comprises solid tumor and blood cancer, and the solid tumor is selected from the following group: bladder cancer, biliary tract cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary cancer, thymic epithelial cancer, and thyroid cancer, or a combination thereof; the autoimmune diseases include: systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, or a combination thereof.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may 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 antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical compositions of the present invention can be used directly to bind CD19 protein molecules and thus can be used to prevent and treat CD19 related diseases. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the 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 formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The 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 100 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 typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture 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 comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The invention also provides a method for detecting cells over-expressing CD19 protein, comprising the following steps: the protein contacts with the sample to be detected in vitro, and the combination of the protein and the sample to be detected is detected.
The meaning of overexpression is conventional in the art and refers to overexpression of the CD19 protein in the sample to be examined (altered by transcription, post-transcriptional processing, translation, post-translational processing and protein degradation), as well as to local overexpression and increased functional activity due to altered protein transport patterns (increased nuclear localization), as in the case of increased enzymatic hydrolysis of the substrate.
The detection means for binding is conventional in the art, preferably FACS detection.
The present invention provides a composition for detecting cells overexpressing CD19 protein, which comprises the above-mentioned protein as an active ingredient. Preferably, it further comprises a compound composed of a functional fragment of the above protein as an active ingredient.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The main advantages of the invention include:
(a) The CD19 antibody can target the CD19 molecules with partial antigen fragments deleted after the existing CD19 antibody and CD19-CAR-T cell treatment, and can be used for treating recurrent leukemia.
(b) The scFV of the present invention can identify tumor cells partially shielded from CD19 molecules after existing CD19-CAR-T cell therapies, and can be used to treat patients with CAR-T cell therapy failure due to the above reasons.
(c) The CD19 antibody or scFV thereof of the present invention can also be used to detect tumor cells of patients who fail the above-mentioned treatment or who relapse after the treatment due to antigen deletion or modulation.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1 preparation of anti-CD 19 antibodies
(one) immunized mice and serum titers determination
Female, 8-week-old Balb/c mice were selected, and a fusion protein of a human CD19 extramembranous protein and a murine Fc fragment (hCD 19-D-mFc) expressed by E.coli and deleted in the first exon was used as an immunizing antigen. In the first immunization, 100 mug of hCD19-D-mFc fusion protein is emulsified with an equivalent amount of CFA adjuvant, and the immunization is performed by subcutaneous injection for 3 times at intervals of 1 week. Serum titers were detected in peripheral blood taken 2 weeks after immunization. Serum was incubated with leukemia cells CA46 or Raji cells expressing human CD19 at 4deg.C for 30min, washed twice with PBS, incubated with secondary antibody at 4deg.C for 30min, washed twice with PBS, and the serum titers were detected using flow cytometry.
The results are shown in FIG. 1, and antibodies against hCD19-D-mFc have been raised in the serum of mice after 3 immunizations.
(II) antibody-producing hybridoma cell screening
Spleen isolated cells from immunized mice were fused with SP2/0 cells using PEG 1500. The fused cells were suspended in 20% FCS IMDM culture medium and inoculated into 96-well plates, HAT was added for selection, and supernatants were screened by ELISA and FACS to obtain two positive clones, HG27 antibody and HG28 antibody.
The screened anti-CD 19 antibodies (HG 27 antibody and HG28 antibody) were incubated with 293FT cells expressing human CD19 extramembranous full length protein (hCD 19-F) and binding activity was detected by flow cytometry.
As a result, HG27, HG28 and anti-CD 19 antibodies each bound to 293FT/hCD19-F cells, as shown in FIG. 2.
(III) ELISA detection of antibody binding to CD19 protein
ELISA experiments were performed using the fusion protein of the first exon-deleted CD19 and the human Fc fragment (hCD 19-D-hFc) as a substrate to identify whether the screened antibodies specifically bind to the first exon-deleted CD19 protein.
The fusion protein (hCD 19-D-hFc) was coated on ELISA half-plates, left overnight at 4℃and blocked with 10% FCS PBS for 1h after washing, HG27 or HG28 or CD19 antibodies (HIB 19, bioleged) at different concentrations were added respectively, after washing, anti-mIgG-HRP was added for 1h, and TMB substrate was added for 15min, after addition of stop solution, ELISA was performed.
As a result, HG27 and HG28 were able to bind to CD19-D-hFc protein, whereas CD19 antibody (HIB 19, bioleged) was unable to bind to CD19-D-hFc protein, as shown in FIG. 3.
(IV) antibody epitope (epi) competitive binding experiments
Detection of antibody binding/competition was performed using leukemia cell CA46, known to be CD19 positive, as target cell.
Human lymphoma cells (CA 46) were used as target cells, incubated with 10ug excess HG27 and HG28 antibodies at 4℃for 30min, washed with PBS containing 1% calf serum, then 0.1ul of anti-human CD19 antibody (anti-hCD 19-FITC, eBioscience) or 0.1ug of streptavidin-labeled self-made antibodies HG27-biotin and HG28-biotin were added, incubated at 4℃for 20min, and analyzed by flow cytometry after washing.
As a result, as shown in FIG. 4, there was no competitive binding between the two cloned antibodies HG27 and HG28 and the human CD19 antibody (eBioscience, HIB 19) (FIG. 4, top), and HG27 and HG28 were in competition with each other to bind to the same epitope (FIG. 4, bottom).
Example 2 preparation of CD 19-specific chimeric antigen receptor modified T cells with first exon deletion
Construction of first exon deleted CD19 chimeric antigen receptor lentiviral expression vector
RNA was extracted from hybridoma cells (HG 27 and HG 28), synthesized into DNA strands by reverse transcription, ligated into T vectors, selected for cloning, and sequenced to determine the light and heavy chain amino acid sequences and CDR regions of the antibodies. The first exon-deleted CD19 plasmid (HG 27, HG28 plasmids) was mixed with pRSV-Rev, pMDLg/pRRE and pCMV-VSVG helper plasmids in a certain ratio, respectively, and 293FT cells were co-transfected. After 48h of transfection, cell culture supernatants containing HG27 and HG28 lentiviruses were collected, respectively, and centrifuged at 3000rpm at 4℃for 5min. The supernatant was filtered through a 0.22. 0.22uml filter and stored frozen at-80℃until use.
(II) preparation of first exon deleted CD19 chimeric antigen receptor T lymphocyte
50ml of fresh peripheral blood from volunteers was taken and Peripheral Blood Mononuclear Cells (PBMC) were isolated by lymphocyte separation liquid and density gradient centrifugation. Cells were labeled with magnetic beads using Pan T cell isolation Kit (meitian gentle), and T lymphocytes were isolated and purified. And (3) activating and proliferating the T lymphocytes by utilizing the CD3/CD28 magnetic beads after purifying the T cells.
Activated T lymphocytes were collected and resuspended in RPMI1640 medium. Infection with HG27 and HG28 lentiviruses, respectively, 1X 10 6 The activated T lymphocytes were plated in 6-well plates and incubated overnight at 37℃in a 5% CO2 incubator. The next day, the culture was centrifuged again and replaced with fresh medium, and fresh medium was added every 2 days, and the expansion culture was continued. On day 8 of culture, CAR-T cells (HG 27 CAR-T, HG CAR-T) were harvested by centrifugation and resuspended in appropriate cryopreservation liquid and frozen in liquid nitrogen for later use.
The results showed that HG27 CAR-T cells and HG28 CAR-T cells were successfully prepared and obtained.
EXAMPLE 3 comparison of killing Capacity of CAR-T cells to acute Leaching leukemia cells
Taking the prepared chimeric antigen receptor T cells(HG 27 CAR-T, HG CAR-T), and target cells (chronic myelogenous leukemia cell K562, leukemia cell line Nalm6, human Burkitt's lymphoma cell CA 46), counting, and adjusting CAR-T cell density to 1×10 6 Per ml, co-cultures were performed at an effective target ratio of 2:1, 10:1, 20:1, respectively. The control cells were PBMC, CD19 CAR-T cells. After 18h of co-culture, the proportion of CD19+ tumor target cells in total cells was detected, the target cells were labeled with an antibody (CD 19-Fc), and the results were compared with the cell killing effect by flow cytometry, as shown in FIG. 5.
The results are shown in FIG. 5, where HG27 or HG28 CAR-T cells have the killing ability with equivalent efficacy to the existing CD19 antibody clone-derived CAR-T cells (CD 19-CAR-T).
The clone number of the existing CD19 antibody is FMC63 (B19) (FITC), and the CD19-CAR-T is self-made.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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Ala Cys Cys Thr Gly Thr Gly Cys Ala Thr Gly Gly Cys Cys Thr Gly
180 185 190
Gly Ala Ala Thr Gly Gly Ala Thr Thr Gly Gly Ala Gly Cys Thr Ala
195 200 205
Thr Thr Gly Ala Thr Cys Cys Thr Gly Ala Ala Ala Cys Thr Gly Gly
210 215 220
Thr Gly Gly Cys Ala Cys Thr Gly Cys Cys Thr Ala Cys Ala Ala Thr
225 230 235 240
Cys Ala Gly Ala Ala Gly Thr Thr Cys Ala Ala Gly Gly Gly Cys Ala
245 250 255
Ala Gly Gly Cys Cys Ala Cys Ala Thr Thr Gly Ala Cys Thr Gly Cys
260 265 270
Ala Gly Ala Cys Ala Ala Ala Thr Cys Cys Thr Cys Cys Ala Thr Cys
275 280 285
Ala Cys Ala Gly Cys Cys Thr Ala Cys Ala Thr Gly Gly Ala Gly Cys
290 295 300
Thr Cys Cys Gly Cys Cys Thr Cys Cys Thr Gly Ala Cys Ala Thr Cys
305 310 315 320
Thr Gly Ala Gly Gly Ala Cys Thr Cys Thr Gly Cys Cys Gly Thr Cys
325 330 335
Thr Ala Thr Thr Ala Cys Thr Gly Thr Ala Cys Ala Ala Gly Ala Cys
340 345 350
Gly Ala Ala Cys Thr Gly Gly Gly Ala Cys Cys Thr Ala Cys Thr Gly
355 360 365
Gly Gly Gly Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr
370 375 380
Cys Thr Cys Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
385 390 395
<210> 18
<211> 399
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Ala Thr Gly Ala Thr Gly Ala Gly Thr Cys Cys Thr Gly Cys Cys Cys
1 5 10 15
Ala Gly Thr Thr Cys Cys Thr Gly Thr Thr Thr Cys Thr Gly Thr Thr
20 25 30
Ala Gly Thr Gly Cys Thr Cys Thr Gly Gly Ala Thr Thr Cys Gly Gly
35 40 45
Gly Ala Ala Ala Cys Cys Ala Ala Cys Gly Gly Thr Gly Ala Thr Gly
50 55 60
Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys Ala Gly Ala Cys
65 70 75 80
Thr Cys Cys Ala Cys Thr Cys Ala Cys Thr Thr Thr Gly Thr Cys Gly
85 90 95
Gly Thr Thr Ala Cys Cys Ala Thr Thr Gly Gly Ala Cys Ala Ala Cys
100 105 110
Cys Ala Gly Cys Cys Thr Cys Cys Ala Thr Cys Thr Cys Thr Thr Gly
115 120 125
Thr Ala Ala Gly Thr Cys Ala Ala Gly Thr Cys Ala Gly Ala Gly Thr
130 135 140
Cys Thr Cys Thr Thr Ala Gly Ala Ala Ala Gly Thr Gly Ala Thr Gly
145 150 155 160
Gly Ala Cys Ala Gly Ala Cys Ala Thr Ala Thr Thr Thr Gly Ala Ala
165 170 175
Thr Thr Gly Gly Thr Thr Gly Thr Thr Ala Cys Ala Gly Ala Gly Gly
180 185 190
Cys Cys Ala Gly Gly Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Ala
195 200 205
Ala Gly Cys Gly Cys Cys Thr Ala Ala Thr Cys Thr Ala Thr Cys Thr
210 215 220
Gly Gly Thr Gly Thr Cys Thr Ala Ala Ala Cys Thr Gly Gly Ala Cys
225 230 235 240
Thr Cys Thr Gly Gly Ala Gly Thr Cys Cys Cys Thr Gly Ala Cys Ala
245 250 255
Gly Gly Thr Thr Cys Gly Cys Thr Gly Gly Cys Ala Gly Thr Gly Gly
260 265 270
Ala Thr Cys Ala Gly Gly Gly Ala Cys Ala Gly Ala Thr Thr Thr Cys
275 280 285
Ala Cys Ala Cys Thr Gly Ala Ala Ala Ala Thr Cys Ala Gly Cys Ala
290 295 300
Gly Ala Gly Thr Gly Gly Ala Gly Gly Cys Thr Gly Ala Gly Gly Ala
305 310 315 320
Thr Thr Thr Gly Gly Gly Ala Gly Thr Cys Thr Ala Thr Thr Ala Thr
325 330 335
Thr Gly Thr Thr Gly Gly Cys Ala Ala Gly Gly Ala Ala Cys Ala Cys
340 345 350
Ala Thr Thr Thr Thr Cys Cys Gly Thr Gly Gly Ala Cys Gly Thr Thr
355 360 365
Cys Gly Gly Ala Gly Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala Gly
370 375 380
Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Ala Cys Gly Gly
385 390 395
<210> 19
<211> 399
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Ala Thr Gly Gly Ala Ala Thr Gly Gly Ala Gly Cys Thr Gly Gly Gly
1 5 10 15
Thr Cys Thr Thr Thr Cys Thr Cys Thr Thr Cys Cys Thr Cys Cys Thr
20 25 30
Gly Thr Cys Ala Gly Thr Ala Ala Ala Thr Gly Cys Ala Gly Gly Thr
35 40 45
Gly Thr Cys Cys Ala Ala Thr Cys Cys Cys Ala Gly Gly Thr Thr Cys
50 55 60
Ala Ala Cys Thr Gly Cys Ala Gly Cys Ala Gly Thr Cys Ala Gly Gly
65 70 75 80
Gly Ala Cys Thr Gly Ala Gly Cys Thr Gly Gly Thr Gly Ala Gly Gly
85 90 95
Cys Cys Thr Gly Gly Gly Gly Cys Thr Thr Cys Ala Gly Thr Gly Ala
100 105 110
Cys Gly Thr Thr Gly Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys
115 120 125
Thr Thr Cys Gly Gly Gly Cys Cys Ala Cys Ala Cys Ala Cys Thr Thr
130 135 140
Ala Cys Thr Gly Ala Cys Thr Ala Thr Gly Ala Ala Ala Thr Gly Cys
145 150 155 160
Ala Cys Thr Gly Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Ala Cys
165 170 175
Ala Cys Cys Thr Ala Thr Ala Cys Ala Thr Gly Gly Cys Cys Thr Gly
180 185 190
Gly Ala Ala Thr Gly Gly Ala Thr Thr Gly Gly Ala Gly Cys Thr Ala
195 200 205
Thr Thr Gly Ala Thr Cys Cys Thr Gly Ala Ala Ala Ala Thr Gly Gly
210 215 220
Thr Gly Gly Thr Gly Thr Thr Gly Cys Cys Thr Ala Cys Ala Ala Thr
225 230 235 240
Cys Ala Gly Ala Ala Gly Thr Thr Cys Ala Ala Gly Gly Gly Cys Ala
245 250 255
Ala Gly Gly Cys Cys Ala Cys Ala Cys Thr Gly Ala Cys Thr Gly Cys
260 265 270
Ala Gly Ala Cys Ala Ala Ala Thr Cys Cys Thr Cys Cys Ala Cys Thr
275 280 285
Ala Cys Ala Thr Cys Cys Thr Ala Cys Ala Thr Gly Gly Ala Ala Cys
290 295 300
Thr Cys Cys Gly Cys Ala Gly Cys Cys Thr Gly Ala Cys Ala Thr Cys
305 310 315 320
Thr Gly Ala Gly Gly Ala Cys Thr Cys Thr Gly Cys Cys Gly Thr Cys
325 330 335
Thr Ala Thr Thr Ala Cys Thr Gly Thr Ala Cys Ala Ala Gly Ala Cys
340 345 350
Gly Ala Ala Cys Thr Gly Gly Ala Ala Cys Cys Thr Ala Cys Thr Gly
355 360 365
Gly Gly Gly Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr
370 375 380
Cys Thr Cys Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
385 390 395
<210> 20
<211> 399
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Ala Thr Gly Ala Thr Gly Ala Gly Thr Cys Cys Thr Gly Cys Cys Cys
1 5 10 15
Ala Gly Thr Thr Cys Cys Thr Gly Thr Thr Thr Cys Thr Gly Thr Thr
20 25 30
Ala Gly Thr Gly Cys Thr Cys Thr Gly Gly Ala Thr Thr Cys Gly Gly
35 40 45
Gly Ala Ala Ala Ala Cys Ala Ala Cys Gly Gly Thr Gly Ala Thr Gly
50 55 60
Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys Ala Gly Ala Cys
65 70 75 80
Thr Cys Cys Ala Cys Thr Cys Ala Cys Thr Thr Thr Gly Thr Cys Gly
85 90 95
Gly Thr Thr Ala Cys Cys Ala Thr Ala Gly Gly Ala Cys Ala Gly Cys
100 105 110
Cys Ala Gly Cys Cys Thr Cys Cys Ala Thr Cys Thr Cys Thr Thr Gly
115 120 125
Cys Ala Ala Gly Thr Cys Ala Ala Gly Thr Cys Ala Gly Ala Gly Cys
130 135 140
Cys Thr Cys Thr Thr Ala Gly Ala Ala Ala Gly Thr Gly Ala Cys Gly
145 150 155 160
Gly Ala Ala Ala Gly Ala Cys Ala Thr Ala Thr Thr Thr Gly Ala Ala
165 170 175
Thr Thr Gly Gly Thr Thr Gly Thr Thr Ala Cys Ala Gly Ala Gly Gly
180 185 190
Cys Cys Ala Gly Gly Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Ala
195 200 205
Ala Gly Cys Gly Cys Cys Thr Ala Ala Thr Cys Thr Ala Thr Cys Thr
210 215 220
Gly Gly Thr Gly Thr Cys Thr Gly Ala Ala Cys Thr Gly Gly Ala Cys
225 230 235 240
Thr Cys Thr Gly Gly Ala Gly Thr Cys Cys Cys Thr Gly Ala Cys Ala
245 250 255
Gly Gly Thr Thr Cys Ala Cys Thr Gly Gly Cys Ala Gly Thr Gly Gly
260 265 270
Ala Thr Cys Ala Gly Gly Gly Ala Cys Ala Gly Ala Thr Thr Thr Cys
275 280 285
Ala Cys Ala Cys Thr Gly Ala Ala Ala Ala Thr Ala Ala Gly Cys Ala
290 295 300
Gly Ala Gly Thr Gly Gly Ala Gly Gly Cys Thr Gly Ala Gly Gly Ala
305 310 315 320
Thr Thr Thr Gly Gly Gly Ala Gly Thr Thr Thr Ala Thr Thr Ala Thr
325 330 335
Thr Gly Cys Thr Gly Gly Cys Ala Ala Gly Gly Thr Ala Cys Ala Cys
340 345 350
Ala Thr Thr Thr Thr Cys Cys Gly Thr Gly Gly Ala Cys Gly Thr Thr
355 360 365
Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala Gly
370 375 380
Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Ala Cys Gly Gly
385 390 395

Claims (20)

1. An antibody having a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region, wherein:
(1) The heavy chain variable region includes the following three complementarity determining region CDR:
CDR1 as shown in SEQ ID NO. 1,
CDR2 as set forth in SEQ ID No.:2, and
CDR3 as shown in SEQ ID NO. 3; and is also provided with
The light chain variable region includes the following three complementarity determining region CDRs:
CDR1' shown in SEQ ID NO. 4,
CDR2' shown in SEQ ID No.:5, and
CDR3' shown in SEQ ID NO. 6;
or,
(2) The heavy chain variable region includes the following three complementarity determining region CDR:
CDR1 as shown in SEQ ID NO. 9,
CDR2 as set forth in SEQ ID NO. 10, and
CDR3 as shown in SEQ ID NO. 11; and is also provided with
The light chain variable region includes the following three complementarity determining region CDRs:
CDR1' shown in SEQ ID NO. 12,
CDR2' shown in SEQ ID No.:13, and
CDR3' shown in SEQ ID NO. 14.
2. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 7.
3. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 15.
4. The antibody of claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID No. 8.
5. The antibody of claim 1, wherein the light chain variable region has the amino acid sequence set forth in SEQ ID No. 16.
6. A recombinant protein, said recombinant protein comprising:
(i) The antibody of claim 1; and
(ii) Optionally a tag sequence to assist expression and/or purification.
7. A CAR construct, wherein the scFV fragment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to CD19, and wherein the scFV has the heavy chain variable region and the light chain variable region of the antibody of claim 1.
8. A recombinant immune cell expressing the CAR construct of claim 7 exogenously.
9. An antibody drug conjugate, comprising:
(a) An antibody moiety that is the antibody of claim 1, 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 cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof.
10. The antibody drug conjugate of claim 9, wherein the antibody moiety is conjugated to the conjugate moiety via a chemical bond or a linker.
11. The antibody drug conjugate of claim 10, wherein the linker is selected from the group consisting of: 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid imidosuccinate (MCC), ma Yaxian imidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB) and disubstituted maleimide-based linkers.
12. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 6, the immune cell of claim 8, the antibody drug conjugate of claim 9, or a combination thereof, wherein the active ingredient is used to (a) prepare a detection reagent, a detection plate, or a kit; (b) Preparing a medicament specifically targeting a tumor cell expressing CD 19; and/or (c) for preparing chimeric antigen receptor-modified immune cells.
13. A pharmaceutical composition, said pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 6, the immune cell of claim 8, the antibody drug conjugate of claim 9, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
14. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) The antibody of claim 1; or (b)
(2) The recombinant protein of claim 6;
(3) The CAR construct of claim 7.
15. A vector comprising the polynucleotide of claim 14.
16. A genetically engineered host cell comprising the vector or genome of claim 15 integrated with the polynucleotide of claim 14.
17. A method for non-diagnostic in vitro detection of CD19 in a sample, said method comprising the steps of:
(1) Contacting the sample with the antibody of claim 1 in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CD19 in the sample.
18. A test plate, said test plate comprising: a substrate and a test strip comprising the antibody of claim 1 or the antibody drug conjugate of claim 9.
19. A kit, comprising:
(1) A first container containing the antibody of claim 1; and/or
(2) A second container containing the second antibody of claim 1;
alternatively, the kit contains a test plate according to claim 18.
20. A method of producing a recombinant polypeptide, the method comprising:
(a) Culturing the host cell of claim 16 under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of claim 1 or the recombinant protein of claim 6.
CN202010125005.2A 2020-02-27 2020-02-27 Preparation and application of novel anti-CD 19 antibody and CD19-CAR-T cell Active CN113307871B (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108610420A (en) * 2016-12-13 2018-10-02 科济生物医药（上海）有限公司 The humanized antibody of anti-CD19 and the immune effector cell for targeting CD19
WO2019125070A1 (en) * 2017-12-22 2019-06-27 앱클론(주) Antibody or antigen-binding fragment thereof that specifically recognizes b cell malignancies, chimeric antigen receptor comprising same, and uses thereof
WO2019129174A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Car-t cell targeting cd19 and expressing cd40 antibody at high level of stability, and use thereof
WO2019137518A1 (en) * 2018-01-15 2019-07-18 李华顺 Specific antibody targeting cd19 and preparation method therefor as well as application thereof, and car-nk cell targeting cd19 and preparation method therefor as well as application thereof
WO2019195273A1 (en) * 2018-04-02 2019-10-10 Alamab Therapeutics, Inc. Connexin 43 antibodies and use thereof
CN110396129A (en) * 2019-07-10 2019-11-01 武汉思安医疗技术有限公司 Humanization CD19 antigen binding single-chain antibody and its Chimeric antigen receptor, immunocyte and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554414B (en) * 2015-09-18 2019-04-23 上海科济制药有限公司 Anti- CD19 human antibody and the immune effector cell for targeting CD19
JOP20180042A1 (en) * 2017-04-24 2019-01-30 Kite Pharma Inc Humanized Antigen-Binding Domains and Methods of Use

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108610420A (en) * 2016-12-13 2018-10-02 科济生物医药（上海）有限公司 The humanized antibody of anti-CD19 and the immune effector cell for targeting CD19
WO2019125070A1 (en) * 2017-12-22 2019-06-27 앱클론(주) Antibody or antigen-binding fragment thereof that specifically recognizes b cell malignancies, chimeric antigen receptor comprising same, and uses thereof
WO2019129174A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Car-t cell targeting cd19 and expressing cd40 antibody at high level of stability, and use thereof
WO2019137518A1 (en) * 2018-01-15 2019-07-18 李华顺 Specific antibody targeting cd19 and preparation method therefor as well as application thereof, and car-nk cell targeting cd19 and preparation method therefor as well as application thereof
WO2019195273A1 (en) * 2018-04-02 2019-10-10 Alamab Therapeutics, Inc. Connexin 43 antibodies and use thereof
CN110396129A (en) * 2019-07-10 2019-11-01 武汉思安医疗技术有限公司 Humanization CD19 antigen binding single-chain antibody and its Chimeric antigen receptor, immunocyte and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
曹韪凡等.CD19-CAR-T和CD19-CAR-CIK细胞对表达CD19+K562细胞株杀伤作用的比较.中国优生与遗传杂志.2016,(第06期),全文. *
袁园等.靶向CD19的CAR修饰的NK细胞对B细胞淋巴瘤的杀伤.中国肿瘤生物治疗杂志.2017,(第05期),全文. *

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