CN113307871A - 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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CN113307871A
CN113307871A CN202010125005.2A CN202010125005A CN113307871A CN 113307871 A CN113307871 A CN 113307871A CN 202010125005 A CN202010125005 A CN 202010125005A CN 113307871 A CN113307871 A CN 113307871A
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CN113307871B (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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    • C07K16/2803—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. Specifically, the invention provides a CD19 antibody, wherein the antibody targets a CD19 molecule with a first exon deleted, and can be used for treating and detecting recurrent and drug-resistant tumors. The invention also provides CAR-T cells containing the specificity of the novel CD19, 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, more specifically to novel
Preparation of anti-CD 19 antibodies and CD19-CAR-T cells and uses thereof.
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 lymphocytic leukemia and some non-acute lymphoblastic leukemias ALL highly express CD 19. Expression of CD19 on plasma cells further suggests that it can be expressed on different B cell tumors such as multiple myeloma, plasmacytoma, farfuge. CD19 is therefore considered a target for a variety of hematologic tumors.
In the existing CD19 antibody and CD19-CAR-T cell therapy for leukemia or lymphoma, the target antigen is often completely or partially deleted due to the loss of the CD19 antigen fragment, resulting in the relapse of leukemia after the patient is treated. Furthermore, in the current CD19-CAR-T cell therapy, in the case of CAR structure being misintroduced into tumor cells, it can lead to the expression of anti-CD 19 CARs on tumor cells, by virtue of scFV in the CAR structural component binding to the tumor's own CD19 molecule, thereby masking the tumor's CD19 molecule, such that the tumor cells are no longer recognized by scFV of the same structure on CD19-CAR-T cells, leading to the failure of the final therapy that the tumor cells cannot be killed.
Therefore, there is an urgent need in the art to develop a novel anti-CD 19 antibody that can recognize a different epitope of CD19, avoiding off-target, and a CD19-CAR-T cell comprising the same.
Disclosure of Invention
The invention aims to provide a CD19 antibody and a CD19-CAR-T cell aiming at recurrent tumors and preparation and application thereof.
In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining regions CDRs:
CDR1 shown in SEQ ID NO. 1,
CDR2 shown in SEQ ID NO. 2, and
CDR3 shown in SEQ ID NO. 3;
or,
CDR1 shown in SEQ ID NO. 9,
CDR2 shown in SEQ ID NO. 10, and
a CDR3 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 by at least one amino acid and can retain the binding affinity of CD 19.
In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 7.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 15.
In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region according to the first aspect of the invention.
In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.
In a third aspect of the invention, there is provided a light chain variable region of an antibody, comprising the following three complementarity determining regions CDRs:
or,
CDR 1' shown in SEQ ID NO. 4,
CDR 2' as shown in SEQ ID No. 5, and
CDR 3' as shown in SEQ ID No. 6;
or,
CDR 1' shown in SEQ ID NO. 12,
CDR 2' as shown in SEQ ID NO. 13, and
14 CDR 3' as shown in SEQ ID No.;
wherein any one of the amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity of CD 19.
In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 8.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 16.
In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region according to the third aspect of the invention.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.
In a fifth aspect of the invention, there is provided an antibody having:
(1) a heavy chain variable region according to the first aspect of the invention; and/or
(2) A light chain variable region according to the third aspect of the invention;
alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, 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 is not more than 40%, preferably 20%, more preferably 10% of the total number of amino acids in the original amino acid sequence.
In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 7, preferably 1 to 3, and more preferably 1.
In another preferred embodiment, the at least one amino acid sequence that is 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 derived sequence has an affinity KD for CD19 of 0.01nM to 1nM, preferably 0.02nM to 0.1 nM.
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 is targeted to a first exon deleted CD19 molecule (not binding to the 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 facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention there is provided a CAR construct comprising a scFV fragment of the antigen binding region of a monoclonal antibody which is a binding region which specifically binds to CD19, said scFV having 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 cell is 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 cell is from a human or non-human mammal (e.g., a mouse).
In another preferred embodiment, the immune cell is from a primate (preferably a human).
In another preferred embodiment, the immune cell is selected from the group consisting of:
(i) a chimeric antigen receptor T cell (CAR-T cell);
(ii) chimeric antigen receptor NK cells (CAR-NK cells); or
(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, (γ δ) T cells, monocytes, or macrophages.
In another preferred embodiment, the immune cell is a CD19-CAR-T cell
In a ninth aspect of the 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, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or 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 folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, 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 include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indobenzodiazepines (indoxazepines) and benzodiazepines (oxyphenoxazepines)), 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, maytansinoid, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthrax toxin dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a chain, modeccin a chain, alpha-sarcina, gelonin, mitogelonin (mitogellin), restrictocin (rettstricon), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.
In another preferred embodiment, the conjugated 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 includes:
(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 a combination thereof.
In another preferred embodiment, the immunoconjugate is an antibody drug conjugate.
In another preferred embodiment, the antibody drug conjugate is represented by the following formula:
Figure BDA0002394143980000061
wherein:
ab is an antibody against CD19,
LU is a joint;
d is a drug;
and subscript p is a value selected from 1 to 10, preferably 1 to 8.
In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a 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 present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the immune cell of the eighth aspect of the present invention, the antibody drug conjugate of the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; (b) preparing a medicament specifically targeting tumor cells expressing CD 19; and/or (c) for preparing a chimeric antigen receptor-modified immune cell.
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 tumor cells; and/or
(3) Detecting tumor cells expressing CD19 protein.
In another preferred embodiment, the detection reagent, detection plate or kit is used for diagnosing a tumor expressing CD 19.
In another preferred embodiment, the medicament is used for treating or preventing CD19 expressing tumors, tumor migration, or tumor resistance.
In another preferred embodiment, the tumor comprises solid tumor and blood 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 blood-borne tumor.
In another preferred embodiment, said 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, kidney cancer, salivary cancer, thymic epithelial cancer, thyroid cancer, ovarian cancer, prostate cancer, rectal cancer, brain 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: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to 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 preparation.
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) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or
(2) A recombinant protein according to the sixth aspect of the invention;
(3) a CAR construct according to 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 SEQ ID No.: 19, respectively.
In another preferred embodiment, the nucleotide sequence encoding the light chain variable region is as set forth in SEQ ID No.: 18 or 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, bacteriophages, 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 according to the thirteenth aspect of the invention or having a polynucleotide according to the twelfth aspect of the invention integrated into its genome.
In a fifteenth aspect of the invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of CD19 in a sample, said method comprising the steps of:
(1) contacting said sample in vitro with an antibody according to the fifth aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD19 in the sample.
In a sixteenth aspect of the present invention, there is provided a detection board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an 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 secondary antibody against the antibody of the fifth aspect of the invention;
alternatively, the kit comprises a detection 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 a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;
(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a nineteenth aspect of the present invention, there is provided a method of treating a tumor expressing CD19, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.
In another preferred example, the method further comprises: administering to a subject in need thereof an additional agent or treatment for combination therapy.
In another preferred embodiment, the other drug or treatment comprises: anti-tumor immunotherapy drugs, tumor targeting drugs, tumor chemotherapy drugs and tumor radiotherapy.
In another preferred embodiment, the anti-tumor immunotherapy medicament comprises PD-1 and PD-L1 monoclonal antibodies.
In a twentieth aspect of the present invention, there is provided a method of producing a chimeric antibody, comprising the steps of:
after cloning the nucleotide sequence 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 into an expression vector containing the nucleotide sequence of the human antibody constant region, 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 CDR region in 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 is implanted into a nucleotide sequence template containing an FR region of a human antibody, and then cloned into an expression vector containing a constant region of a human antibody, followed by expressing the humanized antibody by transfecting animal cells.
In a twenty-second aspect of the 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 according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.
In a twenty-third aspect of the invention, there is provided a method of inhibiting tumor growth 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 said antibody, or a CAR-T cell expressing said antibody.
In another preferred embodiment, the drugs can be administered alone or in combination including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, radiation therapy.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows that after 3 immunizations of mice, antibodies against CD19(hCD19-D-mFc) with a deletion of the first exon were generated in the serum.
FIG. 2 shows that HG27 antibody, HG28 antibody and anti-full length CD19 antibody all bound 293FT/hCD19-F cells.
FIG. 3 shows that HG27 antibody and HG28 antibody are able to bind to CD19-DhFc fusion protein (first exon deleted CD19 fused to human Fc fragment), whereas CD19 antibody (HIB19) is unable to bind to CD19-DhFc fusion protein.
Fig. 4 shows that HG27 and HG28 antibodies do not compete for binding to human CD19 (HIB19) (on fig. 4), and bind to different epitopes. Whereas HG27 and HG28 compete with each other for binding to the same epitope (fig. 4 below).
FIG. 5 shows that CAR-T cells were incubated with 3 target cells at different target ratios, indicating that G27 CAR-T and HG28 CAR-T cells have killing ability with equal potency to existing CAR-T cells derived from a clone of CD19 antibody (CD 19-CAR-T).
Detailed Description
The present inventors have extensively and intensively studied and, for the first time, unexpectedly found a novel anti-CD 19 antibody and a CD19-CAR comprising the same. Compared with the existing antibodies, the antibody of the invention recognizes a different epitope of CD19 and targets a CD19 molecule with a deletion of a first exon. According to the activity test result, the antibody of the invention can be combined with CD19 antigen with high specificity and KD of 9.32 x 10-2nM、2.09×10-2nM has obvious antitumor activity, and may be used in treating and detecting recurrent and medicine resistant tumor.
Term(s) for
As used herein, the term "conjugate" refers to a soluble receptor or fragment or analog thereof, or an antibody or fragment or analog thereof, capable of binding to a target.
As used herein, the terms "CD 19 conjugate", "CD 19 antibody", "anti-CD 19 antibody", "antibody of the invention" have the same meaning and refer to an antibody or fragment thereof or analog thereof that specifically recognizes CD19 and binds to CD 19.
As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of a 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 a therapeutic agent, either internally or externally, comprising any of the CD19 conjugates of the invention and compositions thereof to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.
Antibodies
As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or different classes called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3, and IgG 4. Light chains are classified as kappa or lambda chains by differences in the constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which 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) is composed of 3 CDR regions and 4 FR regions, which are sequentially arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), designated LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain, the hypervariable region of the Heavy Chain (HCDR), are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDR2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1). The four FR regions in the native heavy and light chain variable regions are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The term "antigen-binding fragment," as used herein, refers to a Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen-binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell transfected with a vector by splicing a V region gene of a murine antibody to a C region gene of a human antibody into a chimeric gene. Not only retains the high specificity and affinity of the parent mouse antibody, but also ensures that the humanized Fc segment can effectively mediate the biological effect function.
As used herein, the term "humanized antibody", is a variable region engineered version of a murine antibody of the invention, having CDR regions derived from (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; that is, the CDR sequence of the mouse antibody is grafted to the framework sequences of different types of human germline antibodies. Because the 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 an expression vector.
In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 or 1 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
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 cell-mediated reactions in which nonspecific cytotoxic cells expressing Fc γ R, upon recognition of bound antibody on target cells, cause lysis of the target cells. In various aspects, enhancing ADCC effector function may mean enhanced potency or enhanced efficacy. "potency" as used in the experimental context means the concentration of antibody (half maximal effective concentration) when observing a particular therapeutic efficacy EC 50. "efficacy" as used in the experimental context means the maximum possible effector function of the antibody at saturation levels.
The term "ADCP" or "antibody-dependent cell-mediated phagocytosis" as used herein includes cell-mediated reactions in which non-specific cytotoxic cells expressing Fc γ R recognize bound antibodies on target cells and cause phagocytosis of the target cells.
The term "CDC" or "complement dependent cytotoxicity" as used herein includes reactions in which one or more complement protein components recognize bound antibodies on target cells, which then results in lysis of the target cells.
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 Fc γ R 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: comprising an extracellular domain capable of binding an antigen, a transmembrane domain and a polypeptide which allows cytoplasmic signals to reach the domain (i.e. an intracellular signal domain), an intracellular signal domain refers to a protein which transmits information into cells via a defined signaling pathway by producing second messengers to modulate the activity of the cells, or a protein which functions as an effector by responding to such messengers, comprises a primary signal domain and may also include a functional signaling domain derived from a stimulatory molecule as defined below (i.e. a co-stimulatory signal domain). The intracellular signal domain produces a signal that can promote immune effector functions of the cells of the CAR (e.g., CAR T cells), examples of which include cytolytic and helper activities, including cytokine secretion, e.g., in the CART cells.
The term "primary signaling domain" modulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signaling domain is initiated by, for example, binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.). The primary signaling domain that functions in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or signaling motifs of ITAMs. Examples of primary signal domains comprising ITAMs that are particularly useful in the present invention include, but are not limited to, sequences derived from TCR ξ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d, in particular inventive CARs, the intracellular signaling domain comprises an intracellular signaling sequence in any one or more of the inventive CARs, such as the primary signal domain of CD3 ξ.
The term "co-stimulatory signaling domain" refers to a "co-stimulatory molecule," an cognate binding partner on a T cell, that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules or their ligands that are not 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, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), 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 containing a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the 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-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.
The term "CD 3 ζ" is defined herein as the protein provided by GenBan accession number BAG36664.1, or equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc. The "CD 3 zeta domain" is defined as an amino acid residue from the cytoplasmic domain of the zeta chain sufficient to functionally convey the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 164 of GenBan accession No. BAG36664.1, functional orthologs thereof-equivalent residues from non-human species such as mouse, rodent, monkey, ape and the like.
The term "CD 19" herein refers to a member of the TNFR superfamily having the amino acid sequence of GenBank acc.no. aaa62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.; "CD 19 co-stimulatory domain" is defined as the amino acid sequence 214-255 of GenBank ACC.No. AAA62478.2, or equivalent residues from non-categorical species such as mouse, rodent, monkey, ape, etc. In one aspect, the "CD 19 co-stimulatory domain" is SEQ ID NO: 35, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
anti-CD 19 antibodies
The term "CD 19" includes, but is not limited to, variants, isoforms, and species homologs of human CD 19. In certain instances, the humanized antibodies of the invention may cross-react with CD19 of species other than human. In some cases, the antibody may be completely specific for one or more human CD19 proteins and may exhibit species or other types of non-human cross-reactivity. The complete amino acid sequence of exemplary human CD19 has SwissPort accession number P15391. CD19 is also known as B cell surface antigen B4, B cell antigen CD19, CD19 antigen or Leu-12. Human CD19 is known by Entrez Gene as Gene ID: 930, referred to by HGNC as HGNC: 1633. CD19 may be encoded by a gene called CD 19. The use of "human CD 19" herein encompasses all known or yet to be discovered allelic and polymorphic forms of human CD 19.
In the present invention, the CD19 molecule used as an immunizing 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 the epitope corresponding to the first exon of the CD19 molecule.
In the existing CD19 antibody and CD19-CAR-T cell therapy for leukemia or lymphoma, the target antigen is often completely or partially deleted due to the loss of the CD19 antigen fragment, resulting in the relapse of leukemia after the patient is treated. In response to these relapse mechanisms, the present invention provides novel CD19 antibodies, designed to target a novel epitope on CD19 molecules, not only to the original CD19 molecule, but also to existing CD19 antibodies and CD19 molecules that have been depleted of partial antigen fragments following CD19-CAR-T therapy, and thus the CAR-T cell therapy of the present invention can be used to treat both the original ALL/lymphoma patients and the patients who have relapsed after the aforementioned existing therapy;
in the existing CD19-CAR-T cell therapy, in the case of CAR structure being misintroduced into tumor cells, it can result in anti-CD 19 CAR being expressed on tumor cells, by virtue of scFV in CAR structural components binding to the tumor's own CD19 molecule, thereby masking the tumor's CD19 molecule, such that the tumor cells are no longer recognized by scFV of the same structure on CD19-CAR-T cells, resulting in failure of the final therapy in which the tumor cells cannot be killed. The scFV of the antibody of the invention can recognize different epitopes of CD19, the antibody from which it is derived or the scFV of CAR-T cells can recognize the masked tumor cells, and can be used to treat patients who have failed current CAR-T cell therapy for the reasons described above.
Meanwhile, the anti-CD 19 antibody or its scFv of the present invention can be used to detect tumor cells in the patients who failed the above-mentioned treatment or who recur after the treatment due to antigen deletion or modulation, which cannot be recognized and detected by the existing CD19 antibody itself.
The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, preferably a humanized antibody, and more preferably a fully humanized antibody.
The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.
Among them, 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 the CD19 molecule with the first exon deleted, i.e., do not bind to the 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 an antibody or fragment thereof that binds human CD 19. The invention thus provides an antibody or fragment thereof having a variable region of a heavy and/or light chain 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 region 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. Herein with respect to the identity of the amino acid sequence or the percentage of amino acid residues in the sequence that are identical to the humanized antibody or fragment thereof that binds human CD 19. Thus, sequence identity can be determined by standard methods commonly used to compare the similarity in amino acid positions of two polypeptides. The two polypeptides are aligned for a best match 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 open and gap penalties, and a scoring matrix such as PAM250 (standard scoring matrix; see Dayhoff et al, in Atlas of Protein Sequence and Structure, Vol. 5, supplement III (1978)) may be used in conjunction with the computer program. For example, percent identity can be calculated as: the total number of identical matches is multiplied by 100 and divided by the total length of the longer sequence in the matching span and the number of empty bits poured into the longer sequence in order to align the two sequences.
The invention also provides a humanized antibody fragment that binds human CD19, said fragment being selected from the group consisting of Fab, Fab '-SH, Fd, dAb, F (ab') 2, scFv, bispecific single chain Fv dimers, diabodies, triabodies, and scFv genetically fused to the same or a different antibody. 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 of the invention, vectors, and host cells comprising the nucleic acids or vectors. The nucleic acid may be located in an intact cell, in a cell lysate, or in a 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 of antibodies or encoding VH and VL segments. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary depending on 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 is known in the art, a variety of expression vectors are commercially or otherwise available. Antibodies can be expressed using the present invention.
In a preferred embodiment, the nucleic acid encoding the heavy chain variable region is as set forth in SEQ ID No.: 17 or SEQ ID No.: 19 is shown in the figure; and/or, the nucleic acid encoding the light chain variable region is as set forth in SEQ ID No.: 18 or 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 SEQ ID No.: 17 and the nucleic acid encoding the light chain variable region is shown as the sequence table SEQ ID No.: 18 is shown in the figure; the nucleic acid encoding the heavy chain variable region is shown in a sequence table SEQ ID No.: 19 and the nucleic acid encoding the light chain variable region is shown as the sequence table SEQ ID No.: shown at 20.
Specifically, the numbering of the above nucleotide sequences is shown in Table 2:
TABLE 2CD19 antibody Gene sequence numbering
Clone number Heavy chain protein variable region Light chain protein variable regions
HG27 17 18
HG28 19 20
The method for preparing the nucleic acid is a preparation method conventional in the art, and preferably, comprises the following steps: obtaining the nucleic acid molecule coding the protein by gene cloning technology, or obtaining the nucleic acid molecule coding the protein by artificial complete sequence synthesis method.
Those skilled in the art know that the base sequence of the amino acid sequence encoding the above protein may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The homologue of the polynucleotide of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the protein sequence within a range in which the activity of the antibody is maintained.
Preparation of antibodies
Any method suitable for producing monoclonal antibodies may be used to produce the anti-CD 19 antibodies of the invention. For example, an animal may be immunized with a linked or naturally occurring CD19 homodimer or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
In the invention, a fusion protein of a human CD19 membrane-derived protein with a deleted first exon and a mouse-derived Fc fragment is used as an immunizing antigen to generate a CD19 specific antibody, and the antibody is screened for biological activity. An exemplary method for producing an anti-CD 19 antibody of the invention is described in example 1.
Fully humanized antibodies 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 sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.
Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
An exemplary method of humanizing 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, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell is any of various host cells conventionally used in the art, provided that the above recombinant expression vector is stably self-replicating 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 in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.
Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
Chimeric antigen receptor T cells comprising anti-CD 19 antibody
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., 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 surface of the cell. In some embodiments, a cell (e.g., a T cell) is 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 example, 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 equivalent affinity binding capacity 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 response, 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 scFv from which it was derived.
In another preferred embodiment, the CAR of the invention combines the antigen binding domain of a particular antibody and an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, the CD3 ξ chain, the 4-1BB and CD28 signaling modules, and combinations thereof. In another preferred embodiment, the invention provides a cell (e.g., 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 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 example, the CD19-CAR comprises at least one intracellular signaling domain selected from the group consisting of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 ξ signaling domain, and any combination thereof. In another preferred example, the CD19-CAR comprises at least one intracellular signaling domain from one or more co-stimulatory molecules other than CD137(4-1BB) or CD 28.
In another preferred embodiment, the CAR of the invention increases the IFN β expression element while combining the antigen binding domain of a particular antibody and an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, the CD3 ξ chain, the 4-1BB and CD28 signaling modules, and combinations thereof. In another preferred embodiment, the invention provides a cell (e.g., 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.
Applications 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 prevention and/or treatment of a CD 19-related disease. The CD 19-associated disease includes cancer, an autoimmune disease, a viral infection, a graft-versus-host disease, an inflammatory disease, an immunological 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 gland 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 an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
The pharmaceutical composition of the invention can be directly used for binding CD19 protein molecules, and thus can be used for preventing and treating CD19 related diseases. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 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 generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Detection use and kit
The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The invention also provides a method for detecting cells over expressing CD19 protein, which comprises the following steps: and (3) contacting the protein with a sample to be detected in vitro, and detecting the combination of the protein and the sample to be detected.
The expression "overexpression" is conventional in the art and refers to the overexpression of the CD19 protein in the RNA or protein of the sample to be tested (due to increased transcription, altered post-transcriptional processing, translation, post-translational processing and protein degradation), as well as to the local overexpression and increased functional activity (e.g.in the case of increased enzymatic hydrolysis of the substrate) due to altered protein transport patterns (increased nuclear localization).
The means of detection of such binding is conventional in the art, and is preferably FACS detection.
The present invention provides a composition for detecting cells overexpressing CD19 protein, comprising the above protein as an active ingredient. Preferably, it further comprises a compound consisting of a functional fragment of the above-mentioned protein as an active ingredient.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The main advantages of the invention include:
(a) the CD19 antibody can target the CD19 molecule with partial antigen fragment deletion after the existing CD19 antibody and CD19-CAR-T cell treatment, and can be used for treating recurrent leukemia.
(b) The scFV can identify tumor cells which are partially shielded from CD19 molecules after the prior CD19-CAR-T cell treatment, and can be used for treating patients who fail the CAR-T cell treatment due to the reasons.
(c) The CD19 antibody or its scFV of the present invention can also be used to detect tumor cells in patients who have failed the above-mentioned treatment or who have relapsed after the treatment due to antigen deletion or modulation.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
EXAMPLE 1 preparation of anti-CD 19 antibody
(one) immunization of mice and serum titer determination
Female, 8-week-old Balb/c mice were selected, and a fusion protein (hCD19-D-mFc) of human-derived CD19 extracellular protein and murine Fc fragment expressed by E.coli and deleted in the first exon was used as an immunizing antigen. When the vaccine is used for the first time, 100 mu g of hCD19-D-mFc fusion protein is emulsified with an equal amount of CFA adjuvant, and the vaccine is used for 3 times in a subcutaneous injection manner at intervals of 1 week. Peripheral blood was taken 2 weeks after immunization to test serum titer. Incubating the serum and leukemia cells CA46 or Raji cells expressing human CD19 at 4 ℃ for 30min, washing twice with PBS, adding a secondary antibody, incubating at 4 ℃ for 30min, washing twice with PBS, and detecting the serum titer by using a flow cytometer.
As shown in FIG. 1, 3 times of immunization resulted in the production of antibodies against hCD19-D-mFc in the serum of mice.
(II) screening of antibody-producing hybridoma cells
The spleen of the immunized mouse was isolated and fused with SP2/0 cells using PEG 1500. The fused cells were suspended in 20% FCS IMDM medium and plated in 96-well plates, HAT was added for selection, and the supernatants were screened by ELISA and FACS to give two positive clones, HG27 antibody and HG28 antibody.
The screened anti-CD 19 antibodies (HG27 antibody and HG28 antibody) were incubated with 293FT cells expressing human CD19 extracellular full-length protein (hCD19-F) and binding activity was detected by flow cytometry.
The results are shown in FIG. 2, and HG27, HG28 and anti-CD 19 antibodies all bound 293FT/hCD19-F cells.
(III) ELISA for detecting the binding of antibody to CD19 protein
ELISA experiments were performed using the fusion protein of first exon-deleted CD19 and human Fc fragment (hCD19-D-hFc) as substrates to identify whether the selected antibodies specifically bind to the first exon-deleted CD19 protein.
Coating the fusion protein (hCD19-D-hFc) on an ELISA half-well plate, standing overnight at 4 ℃, washing, blocking for 1h by 10% FCS PBS, adding HG27 or HG28 antibody or CD19 antibody (HIB19, Biolegend) with different concentrations, washing, adding anti-mIgG-HRP for acting for 1h, adding TMB substrate for developing for 15min, adding a stopping solution, and detecting by an ELISA method.
As shown in FIG. 3, HG27 and HG28 were able to bind to CD19-D-hFc protein, whereas CD19 antibody (HIB19, Biolegend) was unable to bind to CD19-D-hFc protein.
(IV) competitive binding assay for antibody epitopes (epitopes)
Antibody binding/competition assays were performed using the known CD19 positive leukemia cell CA46 as the target cell.
Human lymphoma cells (CA46) were used as target cells, incubated with 10ug of excess HG27 and HG28 antibodies at 4 ℃ for 30min, washed with 1% calf serum in PBS, added with 0.1ul of anti-human CD19 antibodies (anti-hCD19-FITC, eBioscience) or 0.1ug of streptavidin (streptavidin) labeled homemade antibodies HG27-biotin and HG28-biotin, incubated at 4 ℃ for 20min, and then analyzed by flow cytometry.
The results are shown in fig. 4, where there was no competitive binding between the two cloned antibodies HG27 and HG28 and the human CD19 antibody (eBioscience, HIB19) (above fig. 4), HG27 and HG28 compete with each other for binding to the same epitope (below fig. 4).
Example 2 first exon deleted CD 19-specific chimeric antigen receptor modified T cell preparation
Construction of first exon deleted CD19 chimeric antigen receptor lentivirus expression vector
Extracting RNA from hybridoma cells (HG27 and HG28), synthesizing DNA chain by reverse transcription, connecting to T vector, selecting clone, sequencing, and determining amino acid sequence and CDR region of antibody. CD19 plasmids (HG27 and HG28 plasmids) with deleted first exon are mixed with pRSV-Rev, pMDLg/pRRE and pCMV-VSVG auxiliary plasmids according to a certain proportion respectively, and 293FT cells are co-transfected. 48h after transfection, cell culture supernatants containing HG27 and HG28 lentivirus were collected and centrifuged at 3000rpm for 5min at 4 ℃. The supernatant was filtered through a 0.22uml filter and frozen at-80 ℃ for use.
(II) preparation of first exon deleted CD19 chimeric antigen receptor T lymphocytes
50ml of fresh peripheral blood of the volunteers were taken and Peripheral Blood Mononuclear Cells (PBMC) were isolated by lymphocyte separation medium, density gradient centrifugation method. The cells were labeled with magnetic beads using the Pan T cell isolation Kit (whirlpool), and the purified T lymphocytes were isolated. And (4) activating and proliferating the T lymphocytes by using the purified T cells and CD3/CD28 magnetic beads.
Activated T lymphocytes were collected and resuspended in RPMI1640 medium. Infection with HG27 and HG28 lentiviruses, respectively, at 1X 106Activated T lymphocytesThe cell suspension was added to a 6-well plate and incubated overnight at 37 ℃ in a 5% CO2 incubator. The next day, the medium was centrifuged again and replaced with fresh medium, and the culture was continued for another 2 days with addition of fresh medium. At day 8 of culture, CAR-T cells (HG27 CAR-T, HG28 CAR-T) were harvested by centrifugation and resuspended in an appropriate freezing medium and frozen in liquid nitrogen for use.
The results show that HG27 CAR-T cells and HG28 CAR-T cells were successfully prepared.
Example 3 comparison of killing Capacity of CAR-T cells to kill acute lymphoblastic leukemia cells
Collecting the prepared chimeric antigen receptor T cells (HG27 CAR-T, HG28 CAR-T) and target cells (chronic myelogenous leukemia cell K562, leukemia cell line Nalm6, and human Burkitt's lymphoma cell CA46), counting, and adjusting CAR-T cell density to 1 × 106And/ml, co-culturing according to the effective target ratio of 2:1, 10:1 and 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 the total cells was measured, the target cells were labeled with an antibody (CD19-Fc), and the cell killing effect was compared by flow cytometry, and the results are shown in FIG. 5.
The results are shown in FIG. 5, HG27 CAR-T or HG28 CAR-T cells have killing ability with equal efficacy to existing CAR-T cells (CD19-CAR-T) derived from clone of CD19 antibody.
The clone number of the existing CD19 antibody is FMC63(B19) (FITC), and CD19-CAR-T is self-made.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Fuzhou Tuo Xintiancheng Biotech Co., Ltd
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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 Ala Cys Ala Gly Ala Cys
165 170 175
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 (10)

1. An antibody heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):
CDR1 shown in SEQ ID NO. 1,
CDR2 shown in SEQ ID NO. 2, and
CDR3 shown in SEQ ID NO. 3;
or,
CDR1 shown in SEQ ID NO. 9,
CDR2 shown in SEQ ID NO. 10, and
a CDR3 shown in SEQ ID No. 11;
wherein any one of the amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one amino acid and capable of retaining the AXL binding affinity.
2. An antibody heavy chain having the heavy chain variable region of claim 1.
3. An antibody light chain variable region comprising the following three Complementarity Determining Regions (CDRs):
CDR 1' shown in SEQ ID NO. 4,
CDR 2' as shown in SEQ ID No. 5, and
CDR 3' as shown in SEQ ID No. 6;
or,
CDR 1' shown in SEQ ID NO. 12,
CDR 2' as shown in SEQ ID NO. 13, and
14 CDR 3' as shown in SEQ ID No.;
wherein any one of the amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one amino acid and capable of retaining the AXL binding affinity.
4. A light chain of an antibody, wherein said light chain has the variable region of the light chain of claim 3.
5. An antibody, wherein said antibody has:
(1) the heavy chain variable region of claim 1; and/or
(2) The light chain variable region of claim 3;
alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
6. A recombinant protein, said recombinant protein having:
(i) 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; and
(ii) optionally a tag sequence to facilitate 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 AXL and wherein the scFV has a heavy chain variable region according to claim 1 and a light chain variable region according to claim 3.
8. A recombinant immune cell expressing an exogenous CAR construct of claim 7.
9. An antibody drug conjugate, 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, a cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof;
preferably, the antibody moiety is coupled to the coupling moiety via a chemical bond or a linker,
more preferably, the linker is selected from the group consisting of: 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid imidinyl succinate (MCC), Maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), and disubstituted maleimide linker.
10. Use of an active ingredient 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, 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 ingredients are used (a) to prepare a detection reagent, a detection plate or a kit; (b) preparing a medicament specifically targeting tumor cells expressing CD 19; and/or (c) for preparing a chimeric antigen receptor-modified immune cell.
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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