DE112018002302B4 - Anti-human CD19 antigen chimeric antigen receptor and its application - Google Patents

Abstract

The chimeric antigen receptor (CAR) of anti-human CD19 antigen, characterized in that it consists of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen, hinge domain, transmembrane domain and intracellular signal domain, which are connected one after the other , where the amino acid sequence of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen is as in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37.

Description

Technical part

The invention pertains to the field of genetic engineering, relates to the chimeric antigen receptor of anti-human CD19 antigen and its uses, and also relates to the lentivirus vector and uses including the chimeric antigen receptor of anti-human CD19 antigen.

Background technology

When mouse antibodies are used directly to treat the human body, the heterogeneity of the mouse antibody leads to a human anti-mouse antibody response (HAMA), which results in a short half-life of the antibody that is quickly removed in the circulatory system and on Loses effectiveness. It is therefore necessary to modify the mouse monoclonal antibody in order to improve the degree of humanization of the antibody and to weaken HAMA. Antibody is one of the most important molecules in the immune system. The stability of the antibody produced in vivo by humoral immunity is the result of natural evolution. However, mouse-derived or humanized antibodies produced by cellular and genetic engineering are a type of recombinant protein that can be easily degraded in vivo during production, transport, storage and use. When used in vivo for tumor treatment, the affinity, specificity of the antibody and tolerance under various pH conditions, especially the weakly acidic environment within the tumor, affect the stability and biological activity of the antibody. It can be seen that improving the stability of the antibody against the drug is very important for the formation of the antibody. In addition, the optimization of the function of the antibody effect plays an important role in the clinical application of antibodies. However, the stability and reliability of antibodies continues to amaze the antibody industry for a long time, which is also a major bottleneck in the entire field of biological research. We need to humanize the mouse antibody and modify and improve its stability, specificity and affinity.

The chimeric antigen receptor (CAR) is an artificial receptor that simulates TCR function and includes antigen recognition domain, hinge domain, transmembrane domain, and intracellular signaling domain. When the antigen (receptor) on the surface of tumor cells combines with the antibody (ligand) of CAR, the signal is transmitted to the cell via the hinge domain and the transmembrane domain, and then the signal is converted into an activation signal by intracellular signal domain to activate the effector cell. The effector cells kill tumor cells by secreting perforin or producing cytokines. In the meantime, the effector cells expand themselves in order to further intensify the immunological effect.

CD19 is a transmembrane protein (differentiation cluster 19 protein, CD19) on the surface of B cells that is closely related to the activation, signal transduction and growth regulation of B cells. It is a functional receptor molecule on the B-lymphocyte surface. When the B cell antigen receptor (BCR) recognizes the antigen, it constructs a double antigen binding model of the B cell, is involved in the transport of Ca2 + in the B cell and regulates the activation and proliferation of the B cell. CD19 as an important marker can be used for the diagnosis and prognosis of leukemia, lymphoma and diseases of the immune system.

Although there are many clinical application antibodies, they are far from meeting the requirements of clinical application, and there are still many technical problems: In view of the degree of humanization, the optimized combination and the specificity and stability of CAR, and the better ability the use of non-human monoclonal antibodies (e.g. mouse monoclonal antibodies) to remove tumor cells can be technically deficient. On the one hand, mouse antibodies cannot normally mediate complement-dependent dissolution or dissolve human target cells through antibody-dependent cytotoxicity or Fc receptor-mediated phagocytosis. On the other hand, the human host recognizes the non-human monoclonal antibody as the immune response induced by the HAMA (Human Anti-Mouse Antibody) response of the exogenous protein. It is therefore necessary to further study and optimize the CD19 antibody.

Currently, the CAR technique has been studied in many hematomas and solid tumors, particularly hematological tumors. However, it remains to be explored how the scFv sequence can be selected with high specificity and stability and the optimal CAR combination can be formed.

Content of the invention

In view of the above, the present invention aims to provide a chimeric antigen receptor of anti-human CD19 antigen. The chimeric antigen receptor of anti-human CD19 antigen of the present invention can be more stably expressed on T lymphocytes, and has high specificity and better ability to remove tumor cells.

• Der chimäre Antigenrezeptor (CAR) von Anti-Human-CD19-Antigen besteht aus dem Einzelketten-Antikörper-Fragment (scFv) zur Erkennung des Human-CD19-Antigens, Gelenkdomäne, Transmembrandomäne und intrazellulärer Signaldomäne, die nacheinander verbunden sind. Die Aminosäuresequenz von einem Einzelketten-Antikörper-Fragment (scFv) zur Erkennung des Human-CD19-Antigens, wird durch SEQ ID NR.1 oder SEQ ID NR. 2 oder SEQ ID NR. 3 oder SEQ ID NR. 4 oder SEQ ID NR. 35 oder SEQ ID NR. 36 oder SEQ ID NR. 37 dargestellt.

To achieve the above purpose, the technical solution of the present invention is as follows:

• The chimeric antigen receptor (CAR) of anti-human CD19 antigen consists of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen, hinge domain, transmembrane domain and intracellular signal domain which are connected in sequence. The amino acid sequence of a single chain antibody fragment (scFv) for recognition of the human CD19 antigen is represented by SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 35 or SEQ ID NO. 36 or SEQ ID NO. 37 shown.

The CAR of the present invention must overcome two technical barriers, one is to find a more stable and effective humanized monoclonal antibody (scFv) to recognize the CD19 antigen, and the other is to obtain the best combination mode of CAR.

The amino acid sequence of the scFv for recognizing the human CD19 antigen of the present invention is obtained by humanized modification. Why is the modification necessary? Since the CAR used to recognize the CD19 antigen cannot be stably expressed on T lymphocytes, in particular T lymphocytes from patients, the expression of CAR on the surface of T cells decreased significantly with the increase in the cultivation time after transfection . The more humanized scFv can effectively reduce the immunogenicity of CAR and improve the sustainability and safety of CAR-T in vivo. The scFv for recognizing the human CD19 antigen of the present invention is modified from the mouse anti-human CD19 monoclonal antibody FMC63. The amino acid sequence of the CDR domain of the light and heavy chain variable domains of FMC63, a monoclonal antibody strain of anti-human CD19 antigen, is shown in Tables 1, 2, 3 and 4, 5, 6. Amino acid sequence 1 QDISKY 2 HTS 3 QQGNTLPYT 4th GVSLPDYG 5 IWGSETT 6 AKHYYYGGSYAMDY

The method of humanization modification is as follows: the skeletal sequence other than the key residue was modified with reference to the skeletal domain sequence of the human antibody by humanization, and then the humanized antibody was mutated at random, and the amino acid sequence of the humanized single chain antibody (scFv) of anti Human CD19 antigen such as SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37 are preferably obtained. The monoclonal antibody consists of a light chain variable domain, a heavy chain variable domain and a constant domain of IgG Fc. The nucleic acid sequence of the humanized single chain antibody (scFv) of anti-human CD19 antigen of SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37 is shown in SEQ ID No. 51, SEQ ID No. 52 and SEQ ID No. 53 are shown.

Preferably, the amino acid sequence of the variable domain of the heavy chain of the humanized single chain antibody (scFv) of anti-human CD19 antigen is shown in SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40. The amino acid sequence of the light chain variable domain is shown in SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43.

As another preferred solution of the present invention, the antibody heavy chain variable domain comprises at least one, two or three modified, but not more than 30, 20 or 10 modified amino acid sequences in the heavy chain variable domain amino acid sequence, which is shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, or sequences with 95-99% identity with that in SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40 amino acid sequence shown.

The light chain variable domain of the antibody comprises at least one, two or three modified, but not more than 30, 20 or 10 modified amino acid sequences in the amino acid sequence of the light chain variable domain shown in SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43, or sequences with 95-99% identity with the amino acid sequence shown in SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43.

Furthermore, the amino acid sequence of the hinge domain is shown in SEQ ID NO: 5 or SEQ ID NO: 44.

Furthermore, the transmembrane domain is the amino acid sequence shown in CD8 ™ or CD28 ™ or SEQ ID NO: 59. The amino acid sequence of CD8 ™ is as shown in SEQ ID NO: 6 and the amino acid sequence of CD28 ™ is as shown in SEQ ID NO: 7.

Furthermore, the intracellular signal domain is CD28 and / or CD137 and / or CD3. The amino acid sequence of CD28 is as shown in SEQ ID NO: 8, the amino acid sequence of CD137 is as shown in SEQ ID NO: 9, and the amino acid sequence of CD3 is as shown in SEQ ID NO: 10.

The intracellular signal domain is preferably CD28 and CD3.

The intracellular signals are preferably CD137 and CD3.

Furthermore, the amino acid sequence of CAR is in SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 or SEQ ID No. 45 or SEQ ID No. 46 or SEQ ID No. 47 are shown.

1. 1) Die Gensequenz von CAR zur Synthese von Anti-Human-CD19-Antigen umfasst Propeptid, verschiedene Einzelketten-Antikörper-Fragment (scFv) von Anti-Human-CD19-Antigen, Gelenkdomäne, Transmembrandomäne und intrazelluläre Signaldomäne. Die Propeptid-Nukleinsäuresequenz ist in SEQ ID Nr. 31 gezeigt.
2. 2) Verbinden Sie die Gensequenz des DNA-Fragments mit der Restriktionsendonuklease- oder Gibson-Klonierungsmethode, um den CAR zu erhalten, der den Lentivirusvektor exprimiert.

The second object of the present invention is to provide a manufacturing method for the lentivirus vector of CAR, which comprises the following steps:

1. 1) The gene sequence of CAR for the synthesis of anti-human CD19 antigen includes propeptide, various single chain antibody fragments (scFv) of anti-human CD19 antigen, hinge domain, transmembrane domain and intracellular signal domain. The propeptide nucleic acid sequence is shown in SEQ ID NO: 31.
2. 2) Join the gene sequence of the DNA fragment using the restriction endonuclease or Gibson cloning method to obtain the CAR that expresses the lentivirus vector.

Furthermore, the nucleotide sequence of CAR after step 1) is as in SEQ ID No. 23 or SEQ ID No. 24 or SEQ ID No. 25 or SEQ ID No. 26 or SEQ ID No. 27 or SEQ ID No. 28 or SEQ ID No. 29 or SEQ ID No. 30 or SEQ ID No. 48 or SEQ ID No. 49 or SEQ ID No. 50.

Furthermore, after step 2), the lentivirus vector is packaged and cleaned.

The third object of the present invention is to provide a lentivirus vector obtained by the production method.

The lentivirus vector is obtained within the scope of the process, such a lentivirus vector having a high positive expression rate in infected cells, a high infection efficiency in primary immune cells (e.g. T lymphocytes) of patients who are difficult to infect. In addition, the expression cells of CAR transduced with the lentivirus vector can stably express the CAR during the culture process, which will not cause a decrease in the positive expression rate of CAR over time. Thus, T cells infected with the lentivirus vector have the function of killing target cells.

This invention also aims to provide an above-mentioned CAR or the use of the above lentivirus vector in the production of CAR immune cells expressing human CD19 target antigen.

Furthermore, the immune cells are T lymphocytes, NK cells, macrophages or DC cells.

The method for introducing the vector into the immune cell according to the present invention may be the gene gun method, the electric transfer method and the virus transfer method. As a preferred method, the method of introducing the vector into the immune cell is the virus delivery method.

The invention also aims to provide a T cell infected with the lentivirus vector.

The object of the invention is also to provide an above-mentioned CAR or the use of the above-mentioned lentivirus vector or the above-mentioned T-cell in the production of malignant tumor medicaments for B-cells.

Furthermore, the cells or tissues of the B-cell malignant tumor may express CD19.

CD19 mutations are associated with severe immune deficiency syndrome and B-cell lymphoma. Specific B-cell lymphoma tumors that accurately express CD19 antigen include, but are not limited to, B-cell acute lymphoid leukemia (B-ALL), B-cell promyelocytic leukemia, blastic plasmacytic dendritic cell neoplasm (BPDC), diffuse large B-cell lymphoma, follicular lymphoma, small- or large-cell follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma undriticytoid celiac disease and other hematological tumors.

B-cell malignancies also include acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL), B-cell flodgkin lymphoma (b-hl) and non-Hodgkin lymphoma (B. -NHL).

The invention also aims to provide the use of the single chain antibody (scFv) amino acid sequence to recognize human CD19 antigen in the manufacture of medicaments for the treatment of B-cell malignant tumors.

In addition, the cells of the malignant B-cell tumor can express CD19.

It is also an object of the invention to provide a method of treating a disease associated with the expression of CD19 by cells or tissues, including administering to mammals an effective amount of cells expressing the CAR.

Also, the CD19-related diseases expressed from the cells or tissues are B-cell-related diseases and B-cell malignant tumors.

Furthermore, the cells expressing CAR molecules are administered in combination with other active substances that improve the effectiveness of the cells expressing CAR molecules or improve the side effects.

The side effects are related to the cells used that express CAR molecules.

Further, the cells that express CAR molecules are administered in combination with other drugs that target or act on CD19 molecules. For cells expressing CAR molecules, they can be administered in combination with other known active ingredients and therapeutic drugs. "Combined administration" herein means that two or more different treatments are given to individual patients in the course of their illness, for example after the individual patient has been diagnosed with an illness and before the illness has been cured or eliminated or treatment is discontinued for other reasons , the two or more treatments are delivered to the individual patient. In some embodiments, treatment continues at the beginning of the second treatment delivery, thereby overlapping the administration. This is sometimes referred to as "concurrent treatment delivery" or "parallel treatment delivery". In other embodiments, another treatment delivery begins after a treatment delivery has ended. In some embodiments, in either case, the treatment is more effective because of the combined administration. For example, the second treatment is more effective than the administration of the second treatment without the first treatment, for example the same effect can be observed with the second treatment with less administration, or the second treatment reduces symptoms to a greater extent, or a similar situation can be observed with the first treatment. In some embodiments, the decrease is others Parameters associated with the delivery of treatment leading to symptoms or disease state are greater than those observed when one treatment is delivered without another. The therapeutic effect is partially added, fully added or greater than the sum of the two. The delivery can take place in such a way that the effect of the first treatment applied can still be recorded at the time of the second treatment.

The cell expressing the CAR molecule and at least one other therapeutic agent can be administered simultaneously, in the same composition or in separate compositions, or sequentially.

In some embodiments, the expressed CAR molecule can be co-expressed in the same cell with other target CAR molecules for the treatment of malignant tumors, such as e.g. the co-expressed CAR molecule and the co-expressed CAR gene-modified cell of the target CD22 molecule are used to treat malignant tumors. Or the cell that expresses the CAR molecule and the CAR expression cell that targets other targets, such as: B. CAR-T cells are combined to treat malignant tumors.

In some embodiments, the expressed CAR molecule can be modified. In particular, a "switch" molecule at the 3 'or 5' end of the expressed CAR molecule can be designed to regulate the expression of the CAR molecule. In particular, a tag gene expression tag protein at the 3 'or 5' end of the expressed CAR molecule can be designed to track CAR expression cells or to regulate CAR expression.

In other aspects, the cell that expresses the CAR molecule can be used in combination with the following therapies in the treatment concept: surgery, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents, such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation. The cell expressing the CAR molecule and the activator targeting extracellular matrix proteins such as tenscin, such as anti-tenonin antibodies, 211At-labeled anti-tenonin antibodies, can also be combined in the treatment concept. In some embodiments, the cells expressing CAR molecules and immunomodulators such as interferon α, interferon β, TGF-β2 peptide inhibitors, or poly-ICLC can be combined in one therapeutic concept.

It is also an object of the invention to provide a medicament for treating mammalian patients with diseases related to the expression of CD19 in cells or tissues. The drug includes cells expressing the CAR molecule and pharmaceutically acceptable vectors or adjuvants.

The so-called “vector” refers to a cloning vector or an expression vector, including, but not limited to, one or more plasmids (expression plasmids, cloning vectors, small rings, micro-vectors, double microchromosomes), retroviruses and lentivirus vector constructs.

In addition, the invention also aims to provide an application of the amino acid sequence of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen in the production and detection of a vector of B cell malignant tumor cells capable of expressing CD19 . The amino acid sequence of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen is shown in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4. The modified polypeptide segment has more stable and specific properties than before.

The invention also aims at an application of amino acid sequences contained in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37 are shown in the preparation of CAR skeleton.

In general, the scFV-CAR in the present invention, after being expressed in the immune cells, can not only maintain the positive rate of CAR in the process of cell culture of the patient, but also improve the ability of CAR-T to multiply and kill tumors . It has no toxic or side effects against the protonegative cells and can be used for the targeted treatment of tumors.

The term “human CD19” refers to human self-cell surface differentiation antigen 19 (differentiation cluster 19 protein, CD19), which can be detected and expressed in B-cell tumors. The amino acid sequence of human CD19 can be seen in UniProt / SwissProt with the ID of P15391. The nucleotide sequence coding for human CD19 can be seen with the ID of NM_001178098. Human CD19 is expressed in most B cell tumors such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

The term “humanized monoclonal antibody” according to the present invention is an artificially modified mouse monoclonal antibody. The mouse monoclonal antibody is modified and re-expressed using gene cloning and recombinant DNA technology. Most of its amino acid sequence is replaced with a human sequence which essentially retains the affinity and specificity of the parent mouse monoclonal antibody, reduces its heterogeneity and is advantageous for the application of the antibody to humans.

In the invention, the term “single chain antibody variable domain fragment” (scFv) refers to an antibody fragment that is composed of an amino acid sequence of the antibody VL domain and an amino acid sequence of the VH domain that is formed by a connector polypeptide and has the ability to bind antigens

In the invention, the term "chimeric antigen receptor" (CAR) is an artificially modified receptor that can anchor specific molecules (such as antibodies) for recognizing tumor antigens on immune cells (such as T cells), whereby immune cells recognize tumor antigens or virus antigens and infected tumor cells or virus Can kill cells.

In the invention, the term “co-stimulatory molecules” refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134 / 0X40, CD137 / 4-1BB, CD40, etc., which the second signal from immune cells by binding to their Activate ligands, improve the proliferation capacity of immune cells and the secretion function of cytokines and extend the survival time of activated immune cells.

In the present invention, the term "monoreceptor tyrosine base activation motifs" (1 TAM) refers to the amino acid sequence based on tyrosine (Y) and the cytoplasmic domains of receptors related to immune cell activation (such as BCR / 1gα / 1gβ, TCR / CD3, FcαR, and FcRγ, etc.) which are two tyrosine residues separated by approximately 13 other amino acid residues (YXX [L / V] X7-11YXX [L / V]). Tyrosine is the phosphorylation site of protein kinase. After phosphorylation, it can combine with signal molecules that are downstream of the signal transmission path, which leads to cell activation.

1. 1) Der Anti-Human-CD19-Antigen zum CAR-Erkennen gemäß der vorliegenden Erfindung kann in Immunzellen (T-Zellen, NK-Zellen usw.) stabiler exprimiert werden, kann CD19-Antigen-positive Tumorzellen effektiv abtöten, hat eine bessere Fähigkeit zum Entfernen von Tumorzellen, kann nicht nur die stabile Expression von CAR-Molekülen nach Infektion von primären Immunzellen (z. B. T-Lymphozyten) von Patienten, die schwer zu infizieren sind, aufrechterhalten, sondern auch die Fähigkeit von CAR-T zur Proliferation und Tötung von Tumoren kann gestärkt werden, und es hat keine toxischen und Nebenwirkungen gegen die protonegativen Zellen und kann zur gezielten Behandlung von Tumoren eingesetzt werden.
2. 2) Der modifizierte CAR gemäß der vorliegenden Erfindung weist einen hohen Humanisierungsgrad auf, kann die Immunogenität von CAR wirksam verringern und die Nachhaltigkeit und Sicherheit von CAR-T in vivo verbessern.
3. 3) Der CAR gemäß der vorliegenden Erfindung kann auf T-Lymphozyten, insbesondere T-Lymphozyten von Patienten, stabil exprimieren und kann zur adoptiven Zelltherapie für Tumore bei der Herstellung von Arzneimitteln zur Behandlung von Tumoren des hämatologischen Systems verwendet werden.

The invention has the following advantageous effects:

1. 1) The anti-human CD19 antigen for CAR recognition according to the present invention can be expressed more stably in immune cells (T cells, NK cells, etc.), can effectively kill CD19 antigen positive tumor cells, has better ability for removing tumor cells, can not only maintain stable expression of CAR molecules after infection of primary immune cells (e.g. T lymphocytes) of difficult to infect patients, but also the ability of CAR-T to proliferate and killing of tumors can be strengthened and it has no toxic and side effects against the protonegative cells and can be used for targeted treatment of tumors.
2. 2) The modified CAR according to the present invention has a high degree of humanization, can effectively reduce the immunogenicity of CAR, and improve the sustainability and safety of CAR-T in vivo.
3. 3) The CAR according to the present invention can stably express on T lymphocytes, in particular T lymphocytes of patients, and can be used for adoptive cell therapy for tumors in the production of medicaments for the treatment of tumors of the hematological system.

Figure list

• 1 is a structural simulation of the variable domain of the humanized monoclonal antibody of anti-human CD 19 antigen.
• 2 Figure 13 is a structural diagram of CAR of target CD19 and for the design and identification of the lentivirus expression vector.
• 3 Figure 10 shows the cell phenotype test results of CAR-T expressing target CD19 after 12 days of cell culture.
• 4th Figure 11 shows the killing effect of CAR-T expressing target CD19 on CD19 positive and negative tumor cells.
• 5 Figure 11 shows the proliferation results of CAR-T expressing target CD19 after stimulation and activation by CD 19 positive cells.
• 6 Figure 11 shows the treatment outcome of a CD19-positive transplanted tumor of CAR-T from target CD19, in the mouse blood-transplanted tumor model.
• 7th Figure 13 is a schematic diagram for the purification of humanized monoclonal antibody from anti-human CD19 antigen.
• 8th shows affinity test diagram of a humanized monoclonal antibody.
• 9 Figure 10 shows the half-life diagram of a humanized monoclonal antibody.
• 10 Figure 13 is the structural diagram of CAR of Target CD19.
• 11 Figure 10 shows the cell phenotype test results of CAR-T, expressing target CD19 after 12 days of cell culture.
• 12 Figure 11 shows the killing effect of CAR-T expressing target CD19 on CD19 positive and negative tumor cells.
• 13 Figure 8 shows the cytokine release from CAR-T expressing target CD19 after stimulation and activation by CD19 positive cells.
• 14th Figure 11 shows the treatment outcome of a CD19-positive transplanted tumor of CAR-T from target CD19, in the mouse blood-transplanted tumor model.
• 15th is an in vivo treatment experiment in mice of CAR-T of target CD19 with various stimulation signals on human Raji-Luc tumor cells.

Specific embodiments

The preferred embodiments of the present invention are described in detail below with reference to the accompanying figures. The experimental procedure without specifying the specific conditions in the preferred embodiment are generally applicable to conventional conditions as described in Experimental Guidelines for Molecular Cloning (Third Edition, J. Sambrook et al.), Or to those recommended by manufacturers will. The embodiments serve to better explain the content of the invention, but are not limited to the embodiments. Therefore, it is still within the scope of the present invention for those skilled in the art to make non-essential improvement and adaptation of the embodiment according to the above invention.

1. (1) Die Aminosäuresequenz der CDR-Domäne der variablen Domänen der leichten und schweren Kette von FMC63, einem monoklonalen Antikörperstamm von Maus-Anti-Human-CD19-Antigen, ist in SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 und SEQ ID Nr. 41, SEQ ID Nr. 42 und SEQ ID Nr. 43. gezeigt. 1 ist eine Struktursimulation der variablen Domäne des humanisierten monoklonalen Antikörpers von Anti-Human-CD19-Antigen. Nach Sequenzanalyse und Vergleich in der IMGT/BLAST-Datenbank wurde die humane Antikörpersequenz mit der höchsten Homologie als modifizierte Vorlage ausgewählt.
2. (2) Der Antragsteller wendet zwei Arten der Modifikation an:
   • a. Nach der molekularen Docking-Simulation sind die Aminosäurereste, die mit dem Andocken von Antigen-Antikörpern am engsten verwandt sind, in Tabelle 1 aufgeführt. Die andere Skelettsequenz als der Schlüsselrest wurde durch Humanisierung unter Bezugnahme auf Skelettregionsequenz des menschlichen Antikörpers modifiziert, und dann wurde der humanisierte Antikörper zufällig mutiert, und dann wird die Affinität durch die Methoden des Antigen-Antiktirper-Affinitäts-Screenings und der molekularen Docking-Simulation bewertet und vorhergesagt. Die Aminosäuresequenz des humanisierten Einzelketten-Antikörpers (scFv) von Anti-Human-CD19-Antigen wird vorzugsweise erhalten und ist wie in SEQ ID Nr. 35, SEQ ID Nr. 36 und SEQ ID Nr. 37 gezeigt.

Tabelle 1 Schlüsselaminosäurereste Schlüsselaminosäurereste V DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH L SGVPSRFSGGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK V EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE H TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSS b. 15 scFv-Sequenzen wurden durch zufällige Mutation nach Ersetzen der Skelettdomäne des menschlichen Antikörpers erhalten. Die optimale Sequenz wurde durch Antikörperreinigung und Stabilitätsmerkmale gescreent.Embodiment Design of the humanized monoclonal antibody of anti-human CD19 antigen.

1. (1) The amino acid sequence of the CDR domain of the light and heavy chain variable domains of FMC63, a monoclonal antibody strain of mouse anti-human CD19 antigen, is shown in SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43. 1 is a structural simulation of the variable domain of the humanized monoclonal antibody of anti-human CD19 antigen. After sequence analysis and comparison in the IMGT / BLAST database, the human antibody sequence with the highest homology was selected as the modified template.
2. (2) The applicant applies two types of modification:
   • a. After the molecular docking simulation, the amino acid residues that are most closely related to the docking of antigen antibodies are listed in Table 1. The skeletal sequence other than the key residue was modified by humanization with reference to the skeletal region sequence of the human antibody, and then the humanized antibody was mutated at random, and then the affinity is evaluated by the methods of antigen-anti-affinity screening and molecular docking simulation and predicted. The amino acid sequence of the humanized single chain antibody (scFv) of anti-human CD19 antigen is preferably obtained and is as shown in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

Table 1 Key Amino Acid Residues Key amino acid residues V DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH L. SGVPSRFSGGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK V EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE H TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSS b. 15 scFv sequences were obtained by random mutation after replacing the skeletal domain of the human antibody. The optimal sequence was screened by antibody purification and stability features.

The results of the random mutation of the monoclonal antibody are as shown in Table 2.

The following embodiments screen the scFv sequences obtained by the two methods, so that the following embodiments are described separately. Table 2 humanized1 DIQMTQSPSSLSASVGDRVTSGCRASQDISKYLNWYQQKPGKAPRLLJYHTSRLHSGVP SRFSGSGSGTOYTILTISSLQPEDFATYYCCIOGNTLPYTFIGGGTRLEIKGSTSGSGKPOSGE GSTKGQVQLQESGPGLVKPSQTLSLTCTVSGVTLPDYGVSWIRQPPGKALEWLGVIWG SETTYYNSALKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWG QGSSVTVSS humanized2 DIQMTQSPSSLSASVGDRVTIGCRASQDISKYLNWYQQKPGKAPRLUYHTSRLHSGVP SRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGE GSTKGQVQLQESGPGLVKPSQTLSLTCTVSGVTLPDYGVSWIRQPPGKALEWLGVIWGI ETTYYNSALKTRLISKDNSKNQVVLTMTNMNDPVDTATYYCAKHYYYGGSYAMDYWG QGSSVTVSS humanized3 DIQMTQSPSSLSASVGDRVTIPVRSDWDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTLSLTCTVSGVTLPDYGVSWIRQPPGKALEWLGVIWGIE TTYYNSAIKTRLTISKDNSKNQYVITMTNMGPVOTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized4 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTTPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTLSITCTVSGVSLPDYGVSWIRQPPGKALEWLGVIWGSE TTYYNSALKTRLTlSKONSKNQVVLTMTNMOPVOTATYYCAKHYYVGGSYAMOYWGQ GSSVTVSS humanized5 DIQMTQSPSSLSASVGDRVTTTCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTlSSLQPEDFAIYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTiSITCTVSGtVSLPDYGVSWIRCLPPGKALEWIGVIWGSE TTYYNTSLKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized6 DIOMTCLSPSSLSASVGDRVTIGCRASQDISKYLNWYQQKPGKAPRLUYHTSRLHSGVP SRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGE GSTKGQVqLQESGPGLVKPSQTLSLTCTVSSGVSLPDYGVSWIRQPPRKALEWLGVIWGS ETTYYTTSLKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized7 DIQMTQSPSSLSASVGDRVTIGCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVP SRFSGSGSGTCN ^- RLTISSLQPEDFATYYCQCLGNTLPYTFGGGTRLEIKGSTSGSGKPGSGE GSTKGQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPRKALEWLGVIWGS ETTYYTTSLKTRLTlSKONSKNQVVLTMTNMDGVOTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized8 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKALEWLGVIWGSETTYY NTSLKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGSSV TVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGPRVTITCRASQDISKYLNWYQCI KPGICAPRLUVHTSRLHSGVPSRFSGSGSGTDVTLTISSLQPEDFATYYCQQGNTLPYTFG GGTRLEIK humanized9 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTRIEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSOTLSLTCTVSGVSLPDYGVSWIROPPGKALEWLGVIWGSE TTYYSTSLKTRLTISKDNSKNNQVVILTMTNMDPVOTATYYCAKHYYYGGSYAMDYWGQG SSVTVSS humanized10 DIQMTQSPSSLSASVGORVTITCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTOYTLTISSLQPEOFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESPGLVKPSQTLSLTCTVSGVSLPOYGVSWIRQPPRKALEWLGVIWGSE TTYYQTSUKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized11 DIQMTQSPSSLSASVGORVTITCRASQDISKYLNWYQCIKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEOFATYFCQCIGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPGKALEWLGVIWGS ETTYYNSSLKTRLTISKONSKNQWLTMTNLDPVDTATYYCAKHYYYGGSYAMDYWGQ GSSVTVSS humanized12 DIQMTQSPSSLSASVGORVTIGCRASQDSKYLTWYOOKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEOFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTLSLTCTVSGVTLPOYGVSWIRQPPGKALEWLGVIWGIE TTYYNSSLKTRLTISKONSKIMQWLTMTIMLDPVDTATYYCAKHYYYGGSYAMDYWGQG SSVTVSS humanized13 QVQLQESGPGLVKPSQTLSLTCTVSGVTLPDYGVSWIRQPPGKALEWLGVIWGIETTYY NSSLKTRLTISKDNSKNQVVLTMTNLDPVDTATYVCAKHYVYGGSYAMDVWGOGSSVT VSSGSTSGSGKPGSGEGSTKGDICLMTCXSPSSLSASVGDRVTIGCRASQDISKYLTWYQQ KPGKAPRLUYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFG GGTRLEIK humanized14 DIQMTQSPSSLSASVGORVTITCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEOFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGSGEG STKGQVQLQESGPGLVKPSQTISLTCTVSGVSLPDYGVSWIRQPPGICALEWLNVIWGSE TSYYSTSLKTRLTISKDNSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQG SSVTVSS humanized15 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKALEWLNVIWGSETSYY STSLKTRLTISKONSKNCIVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGCIGSSVT VSSGGGGSGGGGSGGGGSDKQNATQSPSSLSASVGDRVTITCRASCIDISKVLNWYQQK PGKAPRLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATVYCQQGNTLPYTFGG GTRLEIK

Part I describes a mode of motivation for concept a

Embodiment 1: Production of a Lentivirus Expressing Human CD19 Antigen (CD19) Targeting CAR

Construction of a CAR Gene Sequence Targeting Human CD19 Antigen

The CAR sequences, which sequentially contain the propeptide (also known as the signal peptide), scFv of anti-human CD19 antigen, hFc hinge domain, transmembrane domain, and intracellular signal segment are synthesized. The structure is in shown. The nucleotide sequence of the propeptide is shown in SEQ ID NO: 5 and the amino acid sequence is shown in SEQ ID NO: 6. The amino acid sequences of hCD19-1, hCD19-2, hCD19-3, the humanized single chain antibody (scFv) of anti-human CD19 antigen are as in SEQ ID No. 35, SEQ ID No. 36 and SEQ ID No. 37 and the nucleotide sequences are as shown in SEQ ID No. 51, SEQ ID No. 52 and SEQ ID No. 53. The nucleotide sequence of the hFc hinge domain is shown in SEQ ID NO: 54 and the amino acid sequence is shown in SEQ ID NO: 44. The nucleotide sequence of the transmembrane domain is shown in SEQ ID NO: 55 and the amino acid sequence is shown in SEQ ID NO: 59. The nucleotide sequence of the intracellular signal segment is shown in SEQ ID No. 56 or SEQ ID No. 57.

The final synthesized CAR nucleotide sequence of humanized anti-CD19 is shown in SEQ ID No. 48, SEQ ID No. 49, and SEQ ID No. 50. The unhumanized mouse anti-control antibody is designated mCD19 and the CAR nucleotide sequence of scFv is shown in SEQ ID NO: 58.

Then the PCR amplification was carried out with the CAR sequence as a template. Samples were added to the reaction system according to the instruction manual of KOD FX NEO DNA Polymerase (purchased from Toyobo Company). The PCR reaction conditions were 5 minutes before denaturation at 95 ° C. Denaturation at 95 ° C. for 10 s, annealing at 55 ° C. for 15 s, extension at 68 ° C. for 30 s, there are a total of 30 cycles. After the amplification products had been identified, DNA fragments were recycled using the PCR Fragment Recovery kit (Promega). See the instruction manual for the specific methods. The CAR was recovered through disposal and DNA fragments were sent to biotechnology company Nanjing Genscript for sequencing.

Construction of the lentivirus vector pI-CAG-CD19-8h-28TM-28Z expressing CAR

The cloned gene sequence encoding CAR is digested by restriction endonuclease and the restriction endonuclease reaction is carried out according to the instruction manual. After disposal, the products of the restriction endonuclease are linked to the synthesized CAR sequence fragment in order to obtain the CAR expression vector from pI-CAG-CD19-8h-28TM-28Z which is loaded with various scFvs. According to the different loaded scFv, the CAR expression vectors are referred to as mCD19, hCD19-1, hCD19-2, hCD19-3 lentivirus vectors.

The structure is in shown. Please transfer the lentivirus vector to E. coli TOP10, select the monoclonal virus and carry out the culture for 12 hours and extract the plasmid with the plasmid extraction kit (Invitrogen). Refer to the instruction manual for specific methods. The extracted plasmids were identified by restriction endonuclease HindIII electrophoresis and the results are in 2 shown.

Embodiment 2. Production of CAR-modified T cells with CD19 antigen

Packaging of lentivirus

In this embodiment, the calcium phosphate method is used to package lentivirus. See Instructions for Experiments on Molecular Cloning (3rd Edition, J. Sambrook, et al.).

Purification of the lentivirus

The virus supernatant was collected, centrifuged and filtered and transferred to a new centrifuge tube. According to the amount of virus supernatant, 50% PEG6000 (w / v) and 4 ml of NaCl were added, and then use medicinal salt water to make the final concentration of PEG6000 8.5%, the final concentration of NaCl 0.3 m, and let cool in the refrigerator at 4 ° C. The sample was centrifuged for 30 minutes at 4 ° C and 5000 rpm and the supernatant was discarded. The Virus was resuspended in DMEM culture medium containing 10% FBS at 200 µL and packaged in a 1.5 ml EP tube and stored at -80 ° C for readiness.

Determination of the lentivirus titer

293T cells were infected with the virus. After 72 hours of infection, the cells were centrifuged at 1000 rpm for 5 minutes to collect the cells. The genome was extracted with the genome extraction kit of the QIAamp DNA Blood Mini Kit purchased from Qiagen (product no. 511004). Follow the kit's instruction manual. qRT-PCR was used to determine the virus titer. The data were analyzed with the analysis software and the virus titer calculated according to the standard curve. The results were presented in TU / mL.

T cells infected with lentivirus

Isolation of monocytes from human peripheral blood

60 ml of peripheral blood was withdrawn through the blood withdrawal tube with anticoagulant, and then 30 ml each was divided into 50 ml centrifuge tubes, and 7.5 ml of hydroxyethyl starch was added for dilution. Natural settlement at room temperature (18-25 ° C). The supernatant plasma was collected and centrifuged for 15 minutes. Then the precipitate is resuspended with normal saline, added to the lymphocyte separation solution according to the volume ratio of 1: 1 and the second white lymphocyte layer is removed after the gradient centrifugation. Wash the cells twice with normal saline, and then resuspend them with normal saline to obtain monocytes from human peripheral blood.

T lymphocytes infected with lentivirus vector

The RPMI 1640 complete culture medium with 10% FBS was used to cultivate the newly prepared peripheral blood lymphocytes (PBMC) of the mononuclear cells, and the anti-CD3 monoclonal antibody was added for activation on the first day. The first three days were infected with lentivirus. Mcd19, hcd19-1, hcdl9-2, and hcdl9-3 lentivirus vectors were added to each. Uninfected peripheral blood lymphocytes (PBMC) were used as a blank control. After 24 hours, the culture medium was replaced with RPMI 1640 whole culture medium containing 500 IU / mL recombinant human IL-2 and the culture was continued for 10 to 20 days.

Embodiment 3. Testing of CAR Expression of Targeted Human CD19 Antigen

The expression of CAR in T cells was checked by flow cytometry. The infected T cells which were cultured for 14 days were collected. The cells were resuspended with PBS solution containing 1% FBS (v / v). The cell density was adjusted to 1 × 10 6 cells / ml. The collected cells were divided into 6 tubes, of which there were 100µL in each tube. 20 μ LFITC L-labeled mouse monoclonal anti-human CD3 antibody (Biolegend), PerCP-labeled mouse anti-human CD8 monoclonal antibody (Biolegend), Alexa Fluor 647 labeled protein L (Thermofisher) added to each tube. It was incubated for 30 minutes at 4 ° C., washed twice with PBS solution and detected by upward cytometry. The results are in 3 shown. The FL1 channel is CD3 positive and the L-positive protein of the FL4 channel indicates that the T cell CAR positive rate is greater than 70%. The FL3 channel is the fraction of CD8-positive cells in CD3-positive cells and protein L-positive cells. The above results indicated that the mCD19, hCD19-1, hCD19-2, hCD19-3 lentivirus vectors were successfully transfected into T cells. The positive rate of CAR expression was over 70%, which could be used for further identification experiments. 40% of the successfully transfected T cells can be used for further identification experiments.

Embodiment 4. Verification of anti-tumor activity of T lymphocytes of chimeric antigen receptor expressing target CD19.

The CD19 positive Raji cells stably expressing firefly luciferase (Raji LUC) and the K562 cells stably expressing firefly luciferase and human CD19 antigen (referred to as K562-hCD19-1uc) were used as target cells , while the CD19-negative K562 cells stably expressing firefly luciferase (designated K562-1uc) were used as control cells, and the effector cells were CAR T cells infected with mCD19-, hCD19-1-, hCD19-2, hCD19-3 lentivirus vectors infected T cells and uninfected PBMC cells were used as controls. According to the target effect ratios of 10: 1, 5: 1 and 1: 1, the effector cells were placed with three holes in each group. The killing effect was demonstrated using the standard method provided with the Steady Glo Luciferase Assay System Kit (Promega Cat. # E2520). The kill rate was calculated using the following formula: •

Cell kill rate = (1-fluorescence intensity of the target cell co-culture pore of the effector cell / fluorescence intensity of the individual target cell pores) x100%

The kill results of CAR-T cells with different effect target ratios are shown in FIG. 4A / B / C, and the IFN factor release results from CAR-T cells after 24 hours are shown in FIG 4D shown. The results showed that the T lymphocytes from CAR expressing CD19 antigen had a significant killing effect on CD19 positive tumor cells, but a weak killing effect on CD19 negative cells that was not significantly different from the PBMC group. ELISA experiment showed that CAR-T cells release IFN-γ, which indicates that the stimulation signal from CAR-T cells is functioning normally. At the same time, by comparing the killing effect of different CAR-T cells, it was found that the killing effect of CAR-T on CD19 positive cells in the hCD19-3 lentivirus transfection group was not significantly different from that of the mCD19 lentivirus transfection group prior to motivation , but its killing effect on CD19 negative cells was significantly reduced, showing that the CAR improved specific killing performance after humanization of scFv.

Embodiment 5. Detection of the ability for long-term proliferation and cytokine secretion after CAR-T stimulation for the expression of target CD19

Proof of long-term proliferation ability after CAR-T stimulation for the expression of target CD19

The CAR T cells infected with virus vector were stimulated and activated in vitro and cultured for a long time. The proportion of CAR-T cells with double positive CD3 and Protein-L was regularly detected by flow cytometry. Then the percentage of CAR-T cells is multiplied by the total number of cells so that the number of CAR-T cells can be calculated and the proliferation curve can be drawn to test whether CAR-T cells have the ability to exhibit long-term proliferation after stimulation and to evaluate their clinical therapeutic potential. CD19 positive Raji cells and CD19 transfected K562 cells (referred to as K562-hCD19) were used as target cells to co-culture with CAR-T to stimulate activation of CAR-T cells and CD19 -negative K562 cells were used as control cells. CAR T cells and target cells were co-cultured at a target effect ratio of 5: 1, and cytokine release was detected by ELISA. The number of CAR-T cells was counted on the 3rd and 7th day after the stimulation and the positive ratio of CD4 and CD8 of CD3 and protein-L-double positive CAR-T cells and the ratio of CD45RA + / RO - / CD62L +, CD45RA- / RO + / CD62L + and CD45RA + / - / CD62L- cells in CD4- and CD8-positive cells were detected by flow cytometry. If the number of cells increased on day 7, the target cells were sown for the second stimulation. If the number of cells did not increase after n-times stimulation, the proliferation curve of CAR-T cells was statistically analyzed during the stimulation of the target cells, as in FIG 5A shown. The results showed that the proliferation ability of stimulated CAR T cells of the hCD19-2 group and the hCD19-3 group did not differ significantly from that of the mCD19 group. This indicated that these two chimeric receptor T cells with human scFv retained approximately the same proliferation ability after CAR-T stimulation as that of mCD19.

Evidence of the ability to release cytokines upon stimulation of CAR-T to express target CD19

The target cells of the 24-well plate, 5 × 10 4 cells / well. CAR-T cells and target cells were co-cultured at a target effect ratio of 5: 1. After 24 hours, the supernatant from each well was collected by centrifugation. After 48 hours of stimulation, fresh culture medium was added in a ratio of 1: 1. If the number of cells increased on day 7, the target cells were seeded for the second stimulation and the supernatant was collected 24 hours after the stimulation and activation. When the number of cells no longer increased after stimulation n times, the cell culture supernatant was collected for the last time. It was found that the number of CAR T cells no longer increased after stimulation and activation three times. The cell supernatant was collected 24 hours after each stimulation or activation. After centrifugation, the amount of IFN-γ, IL-2 and TNF-α cytokine released was detected by ELISA. The results are shown in Figure 5B / C / D. The results showed that the hCD19-3 lentivirus infection group had better IFN-γ, IL-2 and TNF-α cytokine releasing ability after stimulation three times, showing that the factor releasing performance after long-term stimulation was better than that of mCD19 and better had clinical application prospects than that of mCD19-CAR T cells.

Embodiment 6. Validation of the anti-tumor activity of CAR-T expressing target CD19 in animal model

In order to verify the anti-tumor effect of CAR-T for the expression of target CD19 in an animal model, a mouse model of a transplanted tumor of the human CD19-positive tumor cell line was created.

The mice used for the in vivo verification are NOD.Cg-PrkdcscidII2rgtm1Sug / JicCrl, which are referred to as NOG mice for short. It was bred by Mamoru Ito of the Japanese Institute for Experimental Animals (CIEA). It is a strain of mice with severe immunodeficiency that was established by hybridizing NOD / SCID mice and IL-2 receptor knockout mice of the γ chain (IL2r γ KO). This is the most common mouse strain worldwide in connection with the CAR-T in vivo tumorigenesis experiment. The mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology GmbH and bred and tested in an SPF environment in accordance with the relevant national laws and regulations for laboratory animals. The tumor target cell used for in vivo verification is Raji (referred to as Raji-luc), a CD19 positive cell line with stable expression of firefly luciferase, which is used for in vitro verification of prophase. The effector cells for the therapeutic injection were CAR-T cells infected with the lentivirus vectors mCD19, hCD19-1, hCD19-2, and hCD19-3, while PBMC cells were not infected.

The images were taken with the IVIS imaging system from PerkinElmer to show tumor growth. The imaging results and the mouse survival curve are in 6 shown. The results show that CAR T cells transfected with hCD19-3 lentivirus show better anti-tumor activity than other groups. All 8 mice in this group survived after 30 days.

Based on the above results, compared with the CAR-T of mouse anti-human CD19 that was not modified by humanization, the CAR-T of anti-human CD19 antigen obtained using the humanization modification concept of the invention was, a better killing specificity on in vitro cell experiment and better therapeutic effect in vivo animal experiment and clinical therapeutic value in the treatment of recurrent acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (CLL), B-cell non - Hodgkin's lymphoma (B-NHL) and other CD19-positive tumors of the haematological system in children and adults.

Part II B Type of modification concept

Embodiment 1. Purification of a humanized monoclonal antibody from target human CD 19 antigen

In order to construct a stable cell line which stably expresses a humanized antibody, the supernatant of the cell was filtered through a 0.45 μm membrane and the filtrate was obtained. It was diluted with an equal volume of equilibrium buffer. The pH was measured. Refer to the GE product manual for information on the individual steps involved in cleaning with AKTAPrime. After the ultrafiltration and concentration, the 0.22 µm needle filter was used for sterilization and filtration. It is then repackaged separately, marked and placed in the refrigerator at -80 ° C for preservation. Sampling and testing. SDS and Western blot were used for testing. For specific steps, see the GE Antibody Purification Guide. The test results are in 7th shown. Four monoclonal antibodies were selected in order to obtain high purity antibodies, namely scFv humanized 4 amino acid sequence SEQ ID No. 1, scFv humanized 5 amino acid sequence SEQ ID No. 2, scFv humanized 9 amino acid sequence SEQ ID No. 3 and scFv humanized 11 amino acid sequence SEQ ID No. 4.

Embodiment 2. Testing the Specificity of a Humanized Monoclonal Antibody Targeting Human CD19 Antigen

After the monoclonal antibody scFv was labeled with its His-tag, it was cloned into a plasmid vector and transfected into HEK293 cells. The supernatant of the cells was filtered through a 0.45 μm membrane and the filtrate was obtained. It was diluted with an equal volume of equilibrium buffer. The pH was measured. After the ultrafiltration and concentration, the 0.22 µm needle filter was used for sterilization and filtration. It is then packed separately and stored at -80 ° C for preservation put in the fridge. Take various humanized antibodies and CD19 positive cells Raji and CD19 negative cells K562, incubate for 30 minutes, wash off excess antibodies, add FITC-His antibody and stain for 30 minutes, wash then remove the unlabeled antibody and perform flow cytometry. The test results are shown in Table 3, four monoclonal antibodies can identify positive Raji cells and cannot identify negative K562 cells, namely, scFv-humanized 4-amino acid sequence ID No. 1. The positive cell detection rate was 90.82%, the negative cell detection rate was 0.7%. ScFv humanized 5 amino acid sequence SEQ ID No. 2, positive cell detection rate was 84.04%, negative cell detection rate was 0.5%. ScFv humanized 9 amino acid sequence SEQ ID No. 3, positive cell detection rate was 87.75%, negative cell detection rate was 0.4%. ScFv humanized 11 amino acid sequence SEQ ID No. 4, positive cell detection rate was 80.60%, negative cell detection rate was 0.19%. The control group consisted of commercial antibodies, the positive cell detection rate was 91.47%, the negative cell detection rate was 0.88%. Table 3 Specificity test of the antibody% grouping Raji K562 grouping Raji K562 Humanized 1 10.00 0.90 Humanized 8 6.14 0.20 Humanized 2 0.87 0.60 Humanized 9 87.75 0.40 Humanized 3 3.71 6.80 Humanized 10 19.81 8.2 Humanized 4 90.82 0.70 Humanized 11 80.60 0.19 Humanized 5 84.04 0.50 Humanized 12 9.65 0.80 Humanized 6 4.50 0.90 Humanized 13 1.00 0.40 Humanized 7 9.87 1.23 Humanized 14 0.98 0.33 Humanized 15 0.87 0.12 Commercial antibody 91.47 0.88

Embodiment 3: Determination of the stability of the humanized monoclonal antibody of target CD19

Detection of antibody affinity at room temperature

After the selected monoclonal antibody scFv had been labeled with its His-tag, it was cloned into a plasmid vector and transfected into HEK293 cells. scFV-His was purified. scFV-His and the negative control were diluted with PBS for 6 gradients. Raji cells with positive CD19 expression were each incubated. The positive rate of RajiCD19 was checked by flow cytometry under the gradient of the flow detection concentration, and the flow positive rate and MFI statistic were performed and the affinity of various scFvs for CD19 was analyzed. Three independent replicates were run in the experiment and the results are in 8th and Table 4 shown. The larger the Kd (nM), the smaller the affinity. Table 4 Humanized 4 Humanized 5 Humanized 9 Humanized 11 affinity 1.993 g / mL 6.216 g / mL 4.757 g / mL 18.496ug / mL WM (Kda) 27.29 27.32 27.31 27.30 Kd (nM) 73.0304 227.5256 174.1853 677.2794

Antibody half-life test:

The CD19-expressing tumor cells were divided into four groups, to each of which four kinds of scFvs were added. Each group was set with five gradient concentrations, each incubated for 1 hour at 4 ° C. The unconjugated antibody was removed by centrifugation. The antibody was dissociated at 37 ° C and the activity of the antibody was detected 15 minutes, 30 minutes, 45 minutes and 60 minutes after the dissociation. The results are in 9 and Table 5 shown, where the dissociation rate reflects the half-life of the antibody and the smaller the absolute value of the dissociation rate, the longer the half-life of the antibody. Table 5 Humanized 4 Humanized 5 Humanized 9 Humanized 11 Dissociation rate -4.138 ± -2.744 ± -0.593 ± ± 0.976 ± (MFI) 0.2746 0.2476 0.0809 0.2076

Embodiment 4: Production of a CAR Lentivirus Expressing Target Human CD19 Antigen

Construction of a CAR Gene Sequence Targeting Human CD19 Antigen

The CAR sequences, which sequentially contain the propeptide (also known as the signal peptide), scFv of anti-CD19 antigen, hlgG-Fc hinge domain, transmembrane domain and intracellular signal segment are synthesized. The structure is in shown. The nucleotide sequence of the propeptide is shown in SEQ ID NO: 31. The nucleotide sequence of humanized ScFv of anti-human CD19 antigen is shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 22. The amino acid sequence of the hIgG-Fc hinge domain is as shown in SEQ ID No. 5. The amino acid sequence of CD8 ™ or CD28 ™ in the transmembrane domain is as shown in SEQ ID No. 6 and SEQ ID No. 7. The amino acid sequences of intracellular signal segments CD28, CD137 and CD3 are as shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10. The final synthesized CAR nucleotide sequence of humanized anti-CD19 is shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30 and the amino acid sequence is shown in SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 are shown. The non-humanized mouse anti-control antibody was named mCD19.

PCR amplification, the reaction system is operated according to the operating instructions from KOD FX NEO DNA Polymerase (purchased from Toyobo). The DNA fragments were then recycled using the recovery kit (Promega). See the instruction manual for the specific methods. The CAR was recovered through disposal and DNA fragments were sent to biotechnology company Nanjing Genscript for sequencing.

Construction of the Lentivirus Vector Expressing CAR

According to the different loading with scFv, CAR expression vectors are each used as lentivirus vectors PCAR M19, PCAR M20, PCAR 011, PCAR 012, PCAR 013, PCAR 014, PCAR 015, PCAR 016, PCAR 017, PCAR 018, PCAR 019, PCAR 020, PCAR 021, PCAR 022, PCAR 023, PCAR 024, PCAR 025, PCAR 026. Of these, PCAR M19 and PCAR M20 were mouse control.

The enzyme cutting reaction was carried out according to the instruction manual. The enzyme cut products were separated by agarose gel electrophoresis. The DNA fragments were recycled using the agarose gel DNA fragment recovery kit. Then, the target fragments and vector fragments were linked by T4 ligase (purchased from Promega Company) to obtain lentivirus vectors expressing CAR. The structure is in shown. The combination of 4 CAR structures with four humanized antibodies. There are 16 combinations in total. The plasmid extraction kit (Invitrogen) extracts plasmids. Please refer to the instruction manual for specific methods.

Embodiment 5. Production of CAR-T of CD19 antigen

• (1) Packaging of the lentivirus: same as Embodiment 2 of Part I.
• (2) Purification of lentivirus: same as Embodiment 2 of Part I.
• (3) Determination of the lentivirus titer: same as in embodiment 2 of part I.
• (4) T cells infected with lentivirus
• 1) Isolation of human peripheral blood monocytes: same as Embodiment 2 of Part I.
• 2) T lymphocytes infected with lentivirus vector

The RPMI 1640 complete culture medium with 10% FBS was used to cultivate the newly prepared peripheral blood lymphocytes (PBMC) of the mononuclear cells. After activation of the anti-CD3 monoclonal antibody, he was infected with lentivirus. Lentivirus vectors were added to each and uninfected peripheral blood lymphocytes (PBMC) were used as a blank control. After 24 hours, the culture medium was replaced with RPMT 1640 whole culture medium containing 500 IU / mL recombinant human IL-2 and the culture was continued for 10 to 20 days. The acquired chimeric antigen receptor T cells were named as follows: PCAR M19, PCAR M20, PCAR 011, PCAR 012, PCAR 013, PCAR 014, PCAR 015, PCAR 016, PCAR 017, PCAR 018, PCAR 019, PCAR 020, PCAR 021, PCAR 022, PCAR 023, PCAR 024, PCAR 025, PCAR 026, PCAR 003.

Testing of CAR expression of the target human CD19 antigen

The infected T cells were collected and cultured for 10 days. The cell density was adjusted to 1 × 10 6 cells / ml. The collected cells were repackaged separately. The positive rate of Protein-L was demonstrated by flow cytometry. The results showed that different CD19-CAR combinations showed a positive rate of T lymphocytes on the 4th and 10th day of culture. Results of CAR positive rate of T cells infected with various viruses are as shown in Table 3. Among them, PCAR 019, PCAR 020, PCAR 021, PCAR 022, PCAR 023, PCAR 024, PCAR 025 and PCAR 026 have higher infection efficiency. Furthermore, PCAR M19, PCAR M20 was used as a control to carry out the positive rate test of T cells that were infected with PCAR 019-, PCAR 020-, PCAR 021-, PCAR 022-, PCAR 023-, PCAR 024-, PCAR 025-, PCAR 026 virus that had been cultured for 4 and 10 days. The results of the positive rate test are in 11 and Table 6 shown. The infection efficiency of T cells PCAR021, PCAR022 and PCAR025 is good and the probability of stable self-activation with a positive rate of CAR is low with the cultivation time. Table 6 CART% positive rate statistics grouping 4 days 10 days grouping 4 days 10 days Control T 0.00 0.00 Control T 0.00 0.00 PCAR M19 24.87 34.60 PCAR M20 54.75 78.4 PCAR 019 53.71 66.80 PCAR 023 39.81 48.2 PCAR 020 65.82 79.70 PCAR 024 20.60 25.90 PCAR 021 64.04 66.50 PCAR 025 69.65 60.80 PCAR 022 54.50 59.90 PCAR 026 61.00 75.40

Embodiment 6. Verification of anti-tumor activity of T lymphocytes expressing target CD19-CAR

The specific steps are the same as in Embodiment 4 of Part 1. The target ratio for the killing effect is 1: 1.

The kill results are in 12A / B and Table 7 shown. The results show that the T lymphocytes from CAR with the optimized combination of humanized monoclonal antibody and scFv have a significant killing effect on CD19-positive tumor cells. Table 7 CAR kill rate% grouping Raji-Luc K562-Luc grouping Raji-Luc K562-Luc Control T 10.26 21.53 Control T 6.68 3.96 PCAR 003 29.61 73.39 PCAR M20 50.76 79.24 PCAR 019 35.44 71.46 PCAR 023 49.42 78.94 PCAR 020 38.13 73.68 PCAR 024 46.13 81.67 PCAR 021 31.33 70.33 PCAR 025 44.25 79.64 PCAR 022 35.08 72.46 PCAR 026 50.27 81.82

Embodiment 7. Testing of CAR-T Cytokine Secretion Ability to Express CD19

The cytokine IFN-γ was detected using the Elisa and kit from BD. Article no. of the kit: 555142, production lot number: 6266958, for specific steps see the instruction manual of the kit

Determination: Set the blind hole to zero, measure the absorbance (OD value) of each hole one after the other at a wavelength of 450 nm and carry out the determination within 15 minutes after adding the separating solution.

The results are in 13 and Table 8, which shows secretion of IFN-γ 24 hours after target cells were killed by CAR-T cells with an effective target ratio of 1: 1. The CAR-T co-stimulatory signal of the optimized combination of humanized monoclonal antibody and scFv can function normally and excrete IFN-γ.

By comparing the killing effects of different CAR-T cells, it was found that the killing activities of the CAR-T cells PCAR 019, PCAR 020, PCAR 021, PCAR 022, PCAR 023, PCAR 024, PCAR 025 and PCAR 026 of the selected combination of monoclonal antibody scFv was higher than that of the combination of unselected 8 CAR T cells, and the killing effect on CD19 positive cells was significant. Table 8 Detection of cytokines (pg / ml) grouping Raji-Luc K562-Luc grouping Raji-Luc K562-Luc Control T 164.8 84.06 Control T 307.6 283.6 PCAR 003 18490 27500 PCAR M20 7015 8458 PCAR 019 20490 28120 PCAR 023 11580 10620 PCAR 020 20030 29470 PCAR 024 10730 11930 PCAR 021 16940 27930 PCAR 025 10660 12910 PCAR 022 15200 28010 PCAR 026 13190 15490

Embodiment 8. Validation of the anti-tumor activity of CAR-T expressing target CD19 in animal model

The construction of the transplanted tumor model in mice was the same as Embodiment 6 in the first part. The effector cells for therapeutic injection were CAR T cells infected with the lentivirus vectors PCAR 019, PCAR 020, PCAR 021, PCAR 022, PCAR 023, PCAR 024, PCAR 025 and PCAR 026.

The control cells were CAR-T cells PCAR M19 and PCAR M20 of the normal saline group, uninfected PBMC cells and an unmodified scFv combination of FMC63 antibodies. In the first part, the CAR modified with monoclonal antibody was named as PCAR 003 as a control of the killing effect in vivo.

Three days after tumor development, CAR-T cells were injected intravenously with 5E05 cells / rat. After the injection of CAR-T cells, pictures were taken every 7 days with the IVS imaging system from PerkinElmer to show the tumor growth. The survival of mice was observed and recorded every day. The results are shown in Table 9 and Table 10. PCAR 021, PCAR 022, PCAR 023, PCAR 025 and PCAR 026 showed a better therapeutic effect compared to the mouse control PCAR M19, PCAR M20 and CARPCAR003 of the antibody combination before the modification. The smaller the fluorescence value, the livelier the mice and the better the therapeutic effect. Table 9 average value 2d 9d 16d 23d 30d 37d 44d grouping to survive 2 9 16 23 30th 37 44 Saline 0/6 3.27E + 06 1.23E + 09 1.32E + 10 Control T 0/7 2.78E + 06 9.51E + 08 8.15E + 09 PCAR M19 0/7 7.74E + 05 1.84E + 08 5.04E + 09 5.03E + 09 PCAR 003 1/7 3.14E + 06 1.59E + 07 2.72E + 08 3.36E + 09 4.77E + 09 4.56E + 08 PCAR 019 1/7 2.87E + 06 1.14E + 07 1.95E + 08 1.03E + 09 5.97E + 09 3.94E + 09 PCAR 020 1/7 2.84E + 06 3.19E + 06 1.86E + 07 3.05E + 08 6.67E + 08 2.04E + 09 PCAR 021 4/7 3.12E + 06 2.48E + 06 8.32E + 06 2.01E + 07 1.56E + 09 6.64E + 08 2.05E + 09 PCAR 022 2/5 3.40E + 06 4.88E + 06 3.67E + 07 2.91E + 07 2.12E + 09 2.20E + 09 3.40E + 09 Table 10 average value 7d 2ID 35d 49d 63d 77d grouping to survive 7th 21st 35 49 63 77 Saline 0/7 1.30E + 06 1.49E + 08 4.98E + 09 2.08E + 08 PCAR M20 1/7 1.29E + 06 8.15E + 08 4.28E + 09 I.5IE + 10 5.13E + 08 1.42E + 09 PCAR 023 5/7 9.19E + 05 6.66E + 06 5.35E + 07 2.24E + 08 9.13E + 08 3.98E + 09 PCAR 024 1/7 1.44E + 06 1.42E + 08 3.77E + 09 7.85E + 09 1.37E + 10 4.70E + 07 PCAR 025 6/7 7.97E + 05 2.70E + 06 7.21E + 07 3.39E + 09 1.28E + 10 2.94E + 09 PCAR 026 3/6 1.35E + 06 1.52E + 08 2.07E + 09 6.01E + 08 3.35E + 09 6.47E + 09

The imaging results and mouse survival curves are in the 14th and 15th shown. The results show that PCAR 021, PCAR 022, PCAR 023 and PCAR 025 have a better anti-tumor effect than other groups. All experimental mice survived after 30 days.

Based on the above results, compared with the CAR-T of mouse anti-human CD19 which was not modified by humanization, the CAR-T of anti-human CD19 antigen obtained by the humanization modification concept of the invention , a better killing specificity in vitro cell experiment and shows a better therapeutic effect in vivo animal experiment. In particular, the amino acid sequences of PCAR 021, PCAR 022, PCAR 023, PCAR 025 and PCAR 026, the scFv humanized 4-amino acid sequence SEQ ID No. 1, scFv humanized 9-amino acid sequence SEQ ID No. 3 and scFv humanized obtained after the second optimization 11 amino acid sequence SEQ ID No. 4 are shown in SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 17 and SEQ ID No. 18, respectively, have a good therapeutic effect in animals. Our PCAR series CAR has clinical utility in the treatment of recurrent B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and B-cell non-Hodgkin lymphoma (B-NHL), and others CD19-positive tumors of the hematological system in children and adults.

Finally, the above embodiments are only used to explain the technical solution of the invention, not as a limitation. Although the invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solution of the invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solution of the invention which is included in the scope to fall within the claims of the invention.

Claims (24)

The chimeric antigen receptor (CAR) of anti-human CD19 antigen, characterized in that it consists of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen, hinge domain, transmembrane domain and intracellular signal domain, which are connected one after the other , where the amino acid sequence of the single chain antibody fragment (scFv) for recognition of the human CD19 antigen is as in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37. CAR after Claim 1 , characterized in that the amino acid sequence of the hinge domain is as shown in SEQ ID NO: 5 or SEQ ID NO: 44. CAR after Claim 1 , characterized in that the transmembrane domain is the amino acid sequence shown in CD8TM or CD28TM or SEQ ID NO: 59, wherein the amino acid sequence of CD8TM is as shown in SEQ ID NO: 6 and the amino acid sequence of CD28TM is as shown in SEQ ID NO. 7 shown. CAR after Claim 1 , characterized in that the intracellular signal domain is CD28 and / or CD137 and / or CD3, where the amino acid sequence of CD28 is as shown in SEQ ID NO: 8, the amino acid sequence of CD137 is as shown in SEQ ID NO: 9 and the amino acid sequence of CD3 is as shown in SEQ ID NO: 10. CAR according to one of the Claims 1 to 4th , characterized in that the amino acid sequence of CAR as in SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 or SEQ ID No. 45 or SEQ ID No. 46 or SEQ ID No. 47 is shown. Manufacturing method of the lentivirus vector of CAR according to one of the Claims 1 to 4th , characterized in that it comprises the following steps: 1) The gene sequence of CAR for the synthesis of anti-human CD19 antigen comprises propeptide, various single chain antibody fragments (scFv) of anti-human CD19 antigen, hinge domain, Transmembrane domain and intracellular signal domain, where the propeptide nucleic acid sequence is as shown in SEQ ID NO: 31. 2) Join the gene sequence of the DNA fragment using the restriction endonuclease or Gibson cloning method to obtain the CAR that expresses the lentivirus vector. Manufacturing process according to Claim 6 , characterized in that: Step 1) the nucleotide sequence of CAR as in SEQ ID No. 23 or SEQ ID No. 24 or SEQ ID No. 25 or SEQ ID No. 26, or SEQ ID No. 27 or SEQ ID No. 28 or SEQ ID No. 29 or SEQ ID No. 30 or SEQ ID No. 48 or SEQ ID No. 49 or SEQ ID No. 50 is shown. Manufacturing process according to Claim 6 , characterized in that after step 2) the lentivirus vector is packaged and cleaned. Lentivirus vector obtained from the manufacturing process according to Claim 8 . Use of a CAR after the Claims 1 to 5 or a lentivirus vector Claim 8 for the production of CAR immune cells that express human CD19 antigen. Use after Claim 10 , characterized in that the immune cells are T lymphocytes, NK cells, macrophages or DC cells. T cell after Claim 9 infected with the lentivirus vector. Use of a CAR after the Claims 1 to 5 or a lentivirus vector according to the Claims 8 or a T-cell after the Claims 12 for the manufacture of drugs for a malignant B-cell tumor. Use after Claim 13 , characterized in that the cells or tissues of the malignant B-cell tumor can express CD19. Use after Claim 13 , characterized in that the malignant B-cell tumor is acute lymphoblastic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), B-cell Hodgkin lymphoma (b-hl) and non-Hodgkin lymphoma (B-NHL) includes. The use of the amino acid sequence of the single chain antibody fragment (scFv) to recognize the human CD19 antigen Claim 1 in the manufacture of medicines for the treatment of malignant B-cell tumors. Use after Claim 16 , characterized in that the cells of the malignant B-cell tumor can express CD 19. Cells that CAR after one of the Claims 1 to 5 for use in the treatment of a disease associated with the expression of CD19 in cells or tissues in a mammal. Cells for use after Claim 18 , characterized in that the cell or tissue expresses CD19-related diseases as B-cell-related diseases and B-cell malignant tumors. Cells for use after Claim 18 , characterized in that the cells that express CAR molecules are administered in combination with other active ingredients that improve the effectiveness of the CAR molecules-expressing cells or improve the side effects. Cells for use after Claim 18 , characterized in that the cells expressing CAR molecules are administered in combination with other agents that target or act on CD19 molecules. Medicinal product for the treatment of mammalian patients with diseases which are related to the expression of CD19 in cells or tissue, characterized in that the medicinal product comprises cells according to one of Claims 1 to 5 expressing CAR molecule and pharmaceutically acceptable vectors or adjuvants. The use of the amino acid sequence of the single chain antibody fragment (scFv) to recognize the human CD19 antigen Claim 1 in the production and accurate detection of a vector of B-cell malignant tumor cells capable of expressing CD19. The use of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37 in the preparation the antigen recognition domain in the CAR skeleton.

Images