CN111166878A

CN111166878A - Preparation method and application of combination of tumor antigen-targeted antibody and iNKT (immunoglobulin KT) cell

Info

Publication number: CN111166878A
Application number: CN202010009708.9A
Authority: CN
Inventors: 徐建青; 张晓燕; 丁相卿; 廖启彬; 王婧
Original assignee: Shanghai Sinobay Bio Tech Co ltd
Current assignee: Shanghai Sinobay Bio Tech Co ltd
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2020-05-19
Anticipated expiration: 2040-01-06
Also published as: CN111166878B

Abstract

The invention provides an application of a combination of an antibody targeting a tumor antigen and an immune cell iNKT in preparation of an anti-tumor medicament. The invention also provides a preparation method of the composition. The combination of the invention adopts iNKT cells as effector cells, and combines the antibodies of the targeted tumor antigens, thereby realizing higher killing activity of the tumor target cells and further improving the anti-tumor capability.

Description

Preparation method and application of combination of tumor antigen-targeted antibody and iNKT (immunoglobulin KT) cell

Technical Field

The present invention relates to the field of immunotherapy of tumors. Specifically, the present invention relates to the use of a combination of an antibody targeting a tumor antigen and iNKT cells, a kit for immunotherapy of tumors using a combination of an antibody targeting a tumor antigen and iNKT cells, and a method for preparing the same.

Background

Tumor immunotherapy can target tumor-induced immune escape and immunosuppressive tumor microenvironment, enhance the anti-tumor immune response of organisms by activating immune cells in vivo, specifically remove tumor foci, inhibit tumor growth, and break immune tolerance. Because the tumor immunotherapy has small side effect and obvious treatment effect, the method is called as the fourth major tumor treatment technology after tumor surgery, radiotherapy and chemotherapy. In 12 months of 2013, the cancer immunotherapy is evaluated by the Science journal and is the first breakthrough of ten scientific researches around the world.

The current tumor immunotherapy is mainly to enhance the anti-tumor immune response. The method mainly comprises two types, one is passive immunotherapy, which directly attacks tumor cells by using effector cells or molecules of the immune system, and comprises antibody or derivatives thereof (such as antibody-drug conjugates) targeted therapy, adoptive immune cell therapy (such as lymphokine activated killer cells, natural killer T cells, dendritic cells and the like), and genetically engineered T cells (such as chimeric antigen receptor CAR-T, T cell receptor TCR-T and the like). The other is "active" immunotherapy, which enhances the immune system by regulating endogenous immune regulation mechanisms or immune activation mechanisms, such as using type I interferons, Toll-like receptor agonists, interferon gene agonists, and the like to enhance the antigen uptake, processing, and presentation abilities of antigen presenting cells; dendritic cell vaccine and anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibody are utilized to enhance the activation and amplification of undifferentiated naive T cells; the effect of the immune response is enhanced by ex vivo stimulation of cancer patients after expansion of tumor infiltrating T cells (TILs).

Immunopotentiation strategies do control tumor growth to some extent, but the effectiveness of inhibiting multiple types of tumors is yet to be improved. Notably, while immune activators increase the body's immune response to tumors, they also increase the risk of an immune response at supraphysiological levels. For example, CAR-T cells are loaded with an antigen binding domain and an intracellular co-stimulatory domain from the receptor, recognition of the antigen is not limited by expression of the major histocompatibility complex, and can more effectively attack tumor cells. But with consequent off-target toxicity and supraphysiological responses such as acute cytokine release syndrome. Increasing clinical trials have demonstrated that combination treatment strategies for tumors, such as immunotherapy combined with chemotherapy, radiotherapy, oncolytic virus therapy, targeted therapy, etc., offer great advantages in terms of improved therapeutic efficacy and reduced side effects compared to monotherapy.

Natural killer T cells (NKTs) are an important cell type in tumor immunotherapy strategies, among which type I NKT cells, i.e., inkts, express a constant T cell antigen receptor (TCR), can secrete anti-tumor-associated cytokines upon activation by α -GalCer glycolipid antigen stimulation, enhance Natural Killer (NK) and Cytotoxic T Lymphocyte (CTL) activity, iNKT cells can also exert important anti-tumor and anti-infection effects by affecting tumors or in Antibody-Dependent Cellular Cytotoxicity (Antibody-Dependent Cellular Cytotoxicity, ADCC), in various solid or hematological clinical trials, show good therapeutic effects for patients by subsequent transfusion of ex vivo expanded iNKT cells, the effectiveness and safety of NKT cell immunotherapy tumors have been widely recognized, japan's profound labour province has approved this approach for clinical use, iNKT cell therapy techniques can also be used in combination with other surgical, chemotherapeutic drugs, ADCC, etc., are a major challenge for the targeted therapy of tumors, especially for the treatment of tumors with low expression of tumor targeting drugs, such as the anticancer drugs d.

The target of Monoclonal Antibodies (mAbs) for tumor therapy encompasses tumor cell-associated antigens (e.g., CD19, CD20, CD22, Her2), epithelial and angiogenic receptors (e.g., EGFR, VEGF, VEGFR2), and immune checkpoint key molecules (e.g., CTLA4, PD-1, PD-L1), among others. The effect of killing tumor cells can be improved through the following mechanisms: (1) directly blocking or activating ligand-receptor signal transduction activity, and inducing apoptosis; (2) immune Cell-mediated killing of tumor cells, including Antibody-Dependent Cell Phagocytosis (ADCP) with ADCC, Complement-Dependent Cytotoxicity (CDC); (3) genetic engineering modification techniques to increase the accuracy of targeting tumors, such as single-chain variable Fragment (scFv) genetically modified T cells; (4) blocking antibodies to key molecules of immune checkpoints can enhance the killing effect of T cells on tumor cells by reducing immune tolerance. (5) The antibody targeting tumor vasculature and stroma antagonizes vascular receptors or ligands, induces ablation of vascular and stromal cells and inhibits tumor cell proliferation. The antibody has the advantages of more specific action target and less side effect. The antibody drugs currently in clinical use are accumulated by more than 30 from early Rituximab (1997), Trastuzumab Trastuzumab (1998), Dewauzumab Durvaumab (2017), Oxytuzumab Inotuzumab ozogamicin (2017), Moxetuzumab pasudotox (2018) and Cemiplimab-rwlc (2018) which are successfully developed in recent years, and show promising anti-tumor effects, but most antibodies still have difficulty in achieving long-term effective inhibition and killing of tumor cells when used alone.

AIDS (AIDS) is a disease which causes the immune function to be damaged after the human body is infected by HIV virus, and the patient is easy to develop various life-threatening opportunistic infections and malignant tumors in the later period of AIDS. Because of the immune deficiencies associated with such patients, the choice of treatment regimen and dosage is very prudent. (Zhao Peng, Zhao Hua, Tu Bo. AIDS with diffuse large B-cell lymphoma 1 case report [ J ]. Depression Leguminosae, 2016,37(10):1117-1118.)

For most tumors, such as diffuse large B-cell lymphoma positive for CD20, treatment with its targeting antibody alone may increase cure for the tumor; but due to the immunodeficiency nature of aids patients, this further increases the suppression of the patients 'humoral immunity, resulting in the development of opportunistic infections that are harmful to the patient's treatment. Therefore, the dosage of the targeted antibody drug needs to be appropriate, and this results in a decrease in the therapeutic effect.

How to solve the problems of immunogenicity of antibody drugs, such as anti-drug antibody (ADA) reaction, tolerance of long-term use of tumor targets and long-term durability of single blocking signal transduction pathways, is a challenge that cannot be avoided by tumor antibody drugs. Due to the above drawbacks of the prior art, it is necessary to develop an antitumor drug and a treatment method having an excellent therapeutic effect and low side effects on patients.

Disclosure of Invention

Therefore, the present invention aims to provide the use of a combination of an antibody targeting a tumor antigen and iNKT cells for the preparation of an antitumor drug, as well as kits for immunotherapy of tumors using a combination of an antibody targeting a tumor antigen and effector cells and methods for their preparation. The tumor is selected from the group consisting of diffuse large B-cell lymphoma, sarcoma, glioma, high-grade glioma, blastoma, osteosarcoma, plasmacytoma, histiocytoma, large bowel cancer, hematopoietic cancer, testicular cancer, ovarian cancer, squamous cell carcinoma, adenocarcinoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, and squamous cell carcinoma of the head and neck;

preferably, the tumor is a malignancy associated with AIDS; the malignant tumor complicated with AIDS is preferably selected from diffuse large B cell lymphoma complicated with AIDS, Kaposi sarcoma complicated with AIDS and skin mucosa squamous carcinoma complicated with AIDS;

preferably, the tumor is AIDS-complicated diffuse large B-cell lymphoma.

Preferably, the medicament is an injection, a topical pharmaceutical dosage form or an oral preparation.

Preferably, the medicament is a tablet, a capsule, a film agent or a granule.

Preferably, the drug is a sustained release agent or a non-sustained release agent. Preferably, wherein the antibody targeting a tumor antigen comprises an Fc sequence; more preferably, the antibody targeting the tumor antigen comprises an Fc sequence comprising the a330L/I332E mutant sequence.

in one aspect, the antibody targeting a tumor antigen is an antibody that targets PD-L1, CD47, CD52, CTLA4, RANKL, CD19, CD20, CD22, CD30, CD33, CD38, GD2, EGFR, VEGF, VEGFR, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, PD-1, PDGF-R α, SLAMF7, and/or CCR 4;

preferably, the antibody targeting a tumor antigen is an antibody targeting CD 20;

preferably, the antibody comprises a heavy chain as set forth in SEQ ID NO. 2 and a light chain as set forth in SEQ ID NO. 4.

Preferably, the coding nucleotide sequence of the antibody heavy chain is shown as SEQ ID NO. 1, and the coding nucleotide sequence of the antibody light chain is shown as SEQ ID NO. 3.

In a specific embodiment, the inventors provide a combination of an antibody targeting CD20 comprising the heavy chain shown in SEQ ID NO. 2 and the light chain shown in SEQ ID NO. 4 and iNKT cells.

The inventor finds that the therapeutic monoclonal antibody and the iNKT cell are used together, so that the iNKT cell can be better guided to the tumor cell, the ADCC (antibody-mediated isothermal amplification) effect of the antibody can be improved, and the tumor can be better controlled; in addition, the combination of the antibodies with the iNKT cells which can increase the immune function can help the patient to repair the immune system, reduce the occurrence of opportunistic infection and be beneficial to the alleviation of the state of an illness.

In another aspect, the present invention provides a kit comprising:

antibodies targeting tumor antigens and iNKT cells.

Preferably, the number of iNKT cells is 10⁷Is as follows to 10¹⁰the kit according to the present invention, wherein the antibody targeting a tumor antigen is an antibody targeting PD-L1, CD47, CD52, CTLA4, RANKL, CD19, CD20, CD22, CD30, CD33, CD38, CD147, GD2, EGFR, VEGF, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, PD-1, PDGF-R α, SLAMF7 and/or CCR 4;

preferably, the antibody comprises a heavy chain as set forth in SEQ ID NO. 2 and a light chain as set forth in SEQ ID NO. 4;

The kit according to the present invention, wherein the kit comprises an antibody targeting CD20 and iNKT cells;

wherein the antibody targeting CD20 has a heavy chain shown as SEQ ID NO. 2 and a light chain shown as SEQ ID NO. 4.

In a further aspect, the invention provides the use of the combination or the kit in the preparation of an anti-tumor medicament. In a further aspect, the invention provides a method of making the combination or kit, the method comprising the steps of:

1) preparing antibodies targeting tumor antigens, and

2) iNKT cells were expanded in vitro.

The method according to the present invention, wherein, in step 2), the iNKT cells are expanded by a method comprising the steps of:

a. specifically expanding iNKT cells;

preferably, the iNKT cells are expanded using α -galactosylceramide (α -GalCer) and stimulated to proliferate with CD1 d-expressing cells loaded with α -galactosylceramide, while cytokines IL-2 and IL-7 are added to help the iNKT cells grow;

b. further carrying out iNKT cell number amplification and guiding function directed differentiation;

preferably, the CD1d expression cells loaded with alpha-galactosylceramide stimulate iNKT cells to proliferate, IL-2, IL-7 and IL-15 are added at the same time, and IL-12 is added in the culture system 1-2 days before the culture is finished to guide the iNKT cells to differentiate directionally;

preferably, the CD1 d-expressing cell is selected from the group consisting of dendritic cells, other cells expressing CD1d, and other artificially modified DC cell-like antigen presenting cells.

In another aspect, the invention provides a method of treating a tumor, the method comprising: administering a combination or kit of the invention to a subject in need thereof; or administering a combination or kit prepared according to the methods of the invention to a subject in need thereof;

preferably, the tumor is selected from diffuse large B-cell lymphoma, sarcoma, glioma, high grade glioma, blastoma, osteosarcoma, plasmacytoma, histiocytoma, large bowel cancer, hematopoietic cancer, testicular cancer, ovarian cancer, squamous cell carcinoma, adenocarcinoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, or head and neck squamous cell carcinoma;

preferably, the tumor is an AIDS-complicated malignancy selected from AIDS-complicated diffuse large B-cell lymphoma, AIDS-complicated kaposi's sarcoma, or AIDS-complicated cutaneous mucosal squamous carcinoma;

preferably, the tumor is AIDS-complicated diffuse large B-cell lymphoma.

The antibody targeting the tumor antigen has a synergistic effect with the iNKT cell. In a preferred embodiment, an antibody targeting a tumor antigen and iNKT cells are administered to a subject in need thereof simultaneously or sequentially; for example, an antibody targeting a tumor antigen is administered first followed by iNKT cells, or an antibody targeting a tumor antigen is administered first followed by iNKT cells; more preferably, iNKT cells are administered first followed by an antibody targeting a tumor antigen. Preferably, the antibody targeting the tumor antigen and iNKT cells may be administered by subcutaneous injection, intravenous injection, or intramuscular injection. The dosage of the antibody targeting a tumor antigen to be administered is not particularly limited, and generally depends on the actual conditions such as the condition of the patient or the kind of the antibody. For example, in a preferred embodiment, the specific antibody targeting tumor cells may be administered in a dose of 5mg to 200mg per dose; more preferably, the specific antibody targeting tumor cells is administered at a dose of 10 mg/time.

Preferably, the iNKT cells are administered at a dose of 10⁷Is as follows to 10¹⁰One cell/time; more preferably, the iNKT cell is administered at a dose of 1.3 to 2.5X 10⁹One cell/time.

In a preferred embodiment, the iNKT cells may be administered in a dose of 1.3X 10⁹One cell per time to 2.5X 10⁹One cell/time. The number of times of reinfusion of iNKT cells is not particularly limited and is generally determined according to the actual situation such as the condition of the patient.

One exemplary antibody according to the invention is an anti-CD 20 antibody (11B8) comprising: the heavy chain nucleotide sequence shown in SEQ ID NO. 1, the amino acid sequence shown in SEQ ID NO. 2 corresponding to the heavy chain nucleotide sequence, the light chain nucleotide sequence shown in SEQ ID NO. 3, and the amino acid sequence shown in SEQ ID NO. 4 corresponding to the light chain nucleotide sequence.

1, heavy chain nucleotide sequence of CD20 antibody 11B8

ATGGAATTAGGCCTCTCTTGGGTGTTCCTCGTGGCTATTCTCAAGGGAGTGCAGTGCGAGGTGCAGCTGGTGCAGTCTGGAGGCGGGCTCGTGCATCCTGGCGGCTCCCTGAGACTGTCTTGCACCGGAAGCGGGTTCACCTTCTCTTACCACGCTATGCACTGGGTGCGCCAGGCTCCTGGCAAGGGACTGGAGTGGGTGAGCATTATCGGAACCGGCGGCGTGACATACTACGCTGACTCTGTGAAGGGCAGATTCACAATTAGCCGCGACAACGTGAAGAACTCCCTGTACCTCCAGATGAACAGCCTCAGAGCCGAGGACATGGCTGTGTACTACTGCGCTAGAGACTACTACGGCGCCGGATCTTTCTACGACGGCCTGTACGGTATGGACGTGTGGGGCCAGGGCACAACAGTGACCGTGTCTAGC

2, heavy chain amino acid sequence of antibody 11B8 of CD20 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MELGLSWVFLVAILKGVQCEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSI IGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAGSFYDGLYGMDVWGQGTTVTVSS

3, light chain nucleotide sequence of CD20 antibody 11B8

ATGGAGGCTCCCGCTCAGCTGCTGTTCCTGCTCCTGCTGTGGCTGCCTGACACAACTGGAGAGATCGTGCTGACCCAGTCTCCCGCTACACTGTCTCTGAGCCCTGGCGAGCGCGCCACCCTGTCTTGCAGGGCCTCTCAGTCCGTTTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGACAGGCCCCAAGACTCCTCATATATGACGCTTCTAACCGCGCCACCGGCATCCCAGCTAGGTTCAGCGGGTCCGGATCTGGAACCGACTTCACACTCACAATTTCTAGCCTCGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGGTCCGACTGGCCACTCACATTCGGCGGCGGGACAAAGGTGGAGATTAAG

4, light chain amino acid sequence of antibody 11B8 of CD20 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIK

Compared with the prior art, the invention has the following advantages:

the inventor finds that the iNKT cell is used as an effector cell and is combined with a tumor antigen specific high-killing antibody for treatment, so that the tumor target cell killing activity is higher, and the anti-tumor capacity is further improved. Specifically, the inventors found that, in the treatment of tumors, if the immune effector cell iNKT is used in combination with a therapeutic monoclonal antibody targeting tumor cells, the iNKT cell has both strong CD1d targeting tumor killing effect and immune regulation effect, and the iNKT cell is accurately targeted to a tumor local lesion by a tumor-specific targeting antibody, and the ADCC effect of the antibody is used to more effectively improve the specificity of tumor killing. The combination of the antibodies with the iNKT cells which can increase the immune function can help the patients to repair the immune system, reduce the occurrence of opportunistic infection and be beneficial to the alleviation of the illness state. Compared with single therapy, the combination can greatly reduce the dosage of immune cells and antibodies and the frequency of reinfusion, relieve the pain of tumor patients and reduce the treatment cost.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 shows a flow chart for a patient using a conventional treatment method and a treatment method according to the invention. Wherein the approximate treatment for the patient is as follows:

the patient finds that the abdomen is wrapped up in 6 months in 2018, the abdominal abdomen is free from red swelling and pain and is accompanied with fever, the puncture pathology shows diffuse large B cell lymphoma, and the HIV virus is positive; the patient is then treated against the HIV virus, first and second line chemotherapy, and local radiation therapy. Chemotherapy and local radiotherapy reduce tumor burden, slightly reduce tumor, but rebound after stopping the treatment. The patient was treated with iNKT cells in combination with CD20 antibody on 18 12 months in 2018 and 3 courses of immunotherapy by 22 months 1 in 2019.

Figure 2 shows the MRI image results for a lesion in the abdomen of a patient. The figure shows that after the patient has undergone local radiotherapy of the tumor, the tumor shrinks 5 days before the first course of cell reinfusion, but rebounds after the drug withdrawal. After three courses of iNKT cells in combination with CD20 antibody, tumor size was significantly reduced and no rebound occurred.

FIG. 3 is a graph of the major and minor diameters of a lesion tumor in the abdomen of a patient;

FIG. 4 is a volume map of a lesion tumor in the abdomen of a patient.

Detailed Description

The present invention is described in detail below with reference to specific clinical trials.

The CD20 antibody was produced and prepared by GMP (good manufacturing practice) workshop of Shanghai offshore biotechnology, Inc.;

iNKT cells were obtained from autologous PBMC cells of the patient by in vitro expansion culture. And, the patient voluntarily joined the study and signed an informed consent.

EXAMPLE 1 clinical course of treatment, results and conclusions of the combinations of the invention

1. Clinical course of treatment

1.1 clinical diagnosis of patients

The patient finds that the abdomen is wrapped up, and the abdominal abdomen is not red, swollen and painful and is accompanied with fever; the confirmed diagnosis of puncture pathology is as follows: diffuse large B-cell lymphoma and Human Immunodeficiency Virus (HIV) positive (figure 1).

1.2 routine treatment protocol for patients

After the patient's condition is confirmed, the patient is treated with tenofovir/emtricitabine + dorsifvir for AIDS (AIDS). One month later, i.e., 2018, 7, 5, R-EPOCH (rituximab, doxorubicin, vincristine, and etoposide, wherein three chemotherapeutics, doxorubicin, vincristine, and etoposide, are administered together continuously for 96 hours without interruption (one chemotherapeutic is administered one bag a day for four bags) and finally vincristine (no flow rate control) is administered orally, prednisone, 18 tablets per day) for the treatment of diffuse large B-cell lymphoma. After one month, the medicine is replaced by R-ICE (Rituximab 375 mg/m)²Intravenous drip, day 1; ifosfamide 1200 mg/m²Intravenous drip for 2-6 days; carboplatin AUC ═ 5, intravenous drip, day 2; etoposide 100mg/m²Intravenous drip, on day 2-4. ) Chemotherapy, the tumor mass is slightly reduced; however, bone marrow suppression, cytomegalovirus retinitis (CMVR) and other symptoms are caused by R-ICE chemotherapy, and the tumor is not obviously resolved. After one month of R-ICE chemotherapy, the patient carries out local radiotherapy on the tumor, and the radiotherapy relieves the tumorThe tumor mass slightly decreased with burden, but the tumor rebounded after radiation therapy ceased (fig. 1).

1.3 treatment regimens Using the combination of iNKT cells and CD20 antibodies of the invention

One month after the end of the radiotherapy, the patients were treated with iNKT cells in combination with CD20 antibody, the first course was carried out 18 days 12 and 2018 (by reinfusion in the form of intravenous injection), and reinfusion was carried out once again 21 days 12 and 2018, with iNKT cell numbers of 2 × 10⁹，1.8×10⁹The dose of the CD20 antibody is 10 mg; after 3 weeks, a second course of treatment is given, with reinfusion in 2019, 1/8 days, 2019

Re-transfusing once again at 11 days 1 month in year, the number of the re-transfused iNKT cells is 1.3 multiplied by 10⁹，1.5×10⁹The dose of the CD20 antibody is still 10 mg; after 2 weeks, a third course of treatment was given, with reinfusion occurring 22 days 1 month in 2019 and once again 25 days 1 month in 2019, with reinfusion iNKT cell numbers of 2 × 10⁹，2.5×10⁹The dose of CD20 antibody was also 10mg (as shown in Table 1).

TABLE 1 application rates of the combinations of the invention

1.4 detection of tumors in patients

Detecting the proportion of effective iNKT cells by flow cytometry;

tumor size was detected by abdominal Magnetic Resonance Imaging (MRI).

2 results

2.1 changes in tumor size

After the patient is treated by chemotherapy, the tumor volume is large (the major diameter is 13 cm, and the minor diameter is 7.5 cm), the tumor does not have a significant reduction trend, and symptoms such as bone marrow suppression, CMVR and the like appear. One month after myelosuppression, CMVR, etc. had occurred, local radiotherapy of the tumor was performed, i.e. 5 days before the first course of cell reinfusion, the tumor tended to shrink (12 cm long diameter and 5.2 cm short diameter), but the tumor began to rebound after cessation of radiotherapy (fig. 2-4).

Since conventional therapy failed to control tumor growth, patients began treatment with iNKT cells in combination with CD20 antibody. In the first course of treatment, 7 days after cell reinfusion is finished, the tumor begins to shrink, and the short diameter of the tumor is reduced to 7.4 cm; iNKT cell therapy with CD20 antibody was continued for a third treatment period, 3 days after the end of which tumor continued to shrink, with the long diameter decreasing to 9.1 cm, the short diameter decreasing to 4.5 cm, and no further tumor rebound (fig. 2-4). The patient's tumor volume was reduced from the initial 750 cubic centimeters to 150 cubic centimeters (shown in FIGS. 2-4) compared to the pre-treatment (2018.12.13) image results.

2.2 Primary concomitant response and treatment after treatment with iNKT cells in combination with CD20 antibody

After treatment of patients with iNKT cells in combination with CD20 antibody, symptoms of fever due to chills and tremors were observed. After the treatment, the treatment starts to appear about 3 hours, the heat peak reaches 40 ℃, the physical cooling and the oral ibuprofen defervesce, and then the patient can recover to be normal. Meanwhile, the phenomenon of leucocyte quantity reduction begins to appear 2 days after the iNKT cell and CD20 antibody treatment, and the leucocyte treatment can be adopted to restore to the normal leucocyte level according to symptoms.

3 conclusion

The patient currently received no other treatment for tumors except iNKT cells in combination with CD20 antibody therapy and received a fourth course of cell reinfusion on 3, 15 days 2019.

Compared with the result of the image before treatment (2018.12.13), the longest diameter of the tumor of the patient is shortened by 25%, and the volume of the tumor is reduced by 44%. No rebound occurs until now (2 months total) after the last cell reinfusion of the third course.

Currently, rituximab is clinically used for treating diffuse large B cell lymphoma positive to CD20, and the dosage of the antibody is about 600mg for patients with weight of 60Kg and height of about 1.7 m. The composition of the iNKT cell and the antibody can greatly reduce the dosage of the antibody, and the dosage of the antibody is only 10mg each time, so that the drug resistance of an organism can be greatly reduced, and a more prominent treatment effect is achieved.

In conclusion, the combination of the invention can obviously reduce the tumor load of patients with HIV combined diffuse large B cell lymphoma, can effectively control the rebound of tumors, and has no obvious side effect. The iNKT cells are used as effector cells and combined with tumor antigen specific antibodies for treatment, so that the tumor suppressor has a higher tumor suppression effect and can effectively improve the anti-tumor capacity.

Sequence listing

<110> Shanghai Xinwan Biotech Co., Ltd

<120> preparation method and use of combination of tumor antigen-targeting antibody and iNKT cell

<130>DIC19110105

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>432

<212>DNA

<213>Artificial Sequence

<400>1

atggaattag gcctctcttg ggtgttcctc gtggctattc tcaagggagt gcagtgcgag 60

gtgcagctgg tgcagtctgg aggcgggctc gtgcatcctg gcggctccct gagactgtct 120

tgcaccggaa gcgggttcac cttctcttac cacgctatgc actgggtgcg ccaggctcct 180

ggcaagggac tggagtgggt gagcattatc ggaaccggcg gcgtgacata ctacgctgac 240

tctgtgaagg gcagattcac aattagccgc gacaacgtga agaactccct gtacctccag 300

atgaacagcc tcagagccga ggacatggct gtgtactact gcgctagaga ctactacggc 360

gccggatctt tctacgacgg cctgtacggt atggacgtgt ggggccaggg cacaacagtg 420

accgtgtcta gc 432

<210>2

<211>144

<212>PRT

<213>Artificial Sequence

<400>2

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

1 5 10 15

Val Gly Cys Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val His

20 25 30

Pro Gly Gly Ser Leu Ala Leu Ser Cys Thr Gly Ser Gly Pro Thr Pro

35 40 45

Ser Thr His Ala Met His Thr Val Ala Gly Ala Pro Gly Leu Gly Leu

50 55 60

Gly Thr Val Ser Ile Ile Gly Thr Gly Gly Val Thr Thr Thr Ala Ala

65 70 75 80

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

85 90 95

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Met Ala Val Thr

100 105 110

Thr Cys Ala Ala Ala Thr Thr Gly Ala Gly Ser Pro Thr Ala Gly Leu

115 120 125

Thr Gly Met Ala Val Thr Gly Gly Gly Thr Thr Val Thr Val Ser Ser

130 135 140

<210>3

<211>381

<212>DNA

<213>Artificial Sequence

<400>3

atggaggctc ccgctcagct gctgttcctg ctcctgctgt ggctgcctga cacaactgga 60

gagatcgtgc tgacccagtc tcccgctaca ctgtctctga gccctggcga gcgcgccacc 120

ctgtcttgca gggcctctca gtccgtttct tcttacctcg cttggtatca gcagaagccc 180

ggacaggccc caagactcct catatatgac gcttctaacc gcgccaccgg catcccagct 240

aggttcagcg ggtccggatc tggaaccgac ttcacactca caatttctag cctcgaaccc 300

gaggacttcg ccgtgtacta ctgccagcag aggtccgact ggccactcac attcggcggc 360

gggacaaagg tggagattaa g 381

<210>4

<211>127

<212>PRT

<213>Artificial Sequence

<400>4

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn ArgAla Thr Gly Ile Pro Ala

65 70 75 80

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

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

100 105 110

Asp Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

115 120 125

Claims

1. Use of a combination of an antibody targeting a tumor antigen and iNKT cells in the preparation of an anti-tumor medicament, said tumor being selected from the group consisting of diffuse large B-cell lymphoma, sarcoma, glioma, high-grade glioma, blastoma, osteosarcoma, plasmacytoma, histiocytoma, large bowel cancer, hematopoietic cancer, testicular cancer, ovarian cancer, squamous cell carcinoma, adenocarcinoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer and squamous cell carcinoma of the head and neck;

preferably, the tumor is AIDS-complicated diffuse large B-cell lymphoma.

2. The use according to claim 1, wherein the medicament is an injection, a topical pharmaceutical dosage form, or an oral formulation;

preferably, the drug is a sustained release agent or a non-sustained release agent;

preferably, the medicament is a tablet, a capsule, a film agent or a granule.

3. The use of claim 1, wherein the antibody targeting a tumor antigen comprises an Fc sequence; preferably, the antibody targeting the tumor antigen comprises an Fc sequence comprising the a330L/I332E mutant sequence.

4. the use according to any one of claims 1 to 3, wherein the antibody targeting a tumor antigen is an antibody targeting PD-L1, CD47, CD52, CTLA4, RANKL, CD19, CD20, CD22, CD30, CD33, CD38, CD147, GD2, EGFR, VEGF, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, PD-1, PDGF-R α, SLAMF7 and/or CCR 4;

5. The use of any one of claims 1 to 4, wherein the antibody is a CD 20-targeting antibody comprising a heavy chain set forth in SEQ ID NO 2 and a light chain set forth in SEQ ID NO 4.

6. A kit comprising an antibody targeting a tumor antigen and iNKT cells for use according to claim 1.

7. the kit of claim 6, wherein the antibody targeting a tumor antigen is an antibody targeting PD-L1, CD47, CD52, CTLA4, RANKL, CD19, CD20, CD22, CD30, CD33, CD38, CD147, GD2, EGFR, VEGF, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, PD-1, PDGF-Ra, SLAMF7 and/or CCR 4;

8. The kit of claim 6 or 7, wherein the kit comprises an antibody targeting CD20 and iNKT cells;

9. A method of preparing a combination for use according to any one of claims 1 to 5 or a kit according to any one of claims 6 to 8, the method comprising the steps of:

1) preparing antibodies targeting tumor antigens, and

2) iNKT cells expanded in vitro.

10. The method according to claim 9, wherein, in step 2), the iNKT cells are expanded by a method comprising the steps of:

a. specifically expanding iNKT cells;

preferably, the first and second electrodes are formed of a metal,

the CD1d expression cell loaded with alpha-galactosylceramide stimulates the iNKT cell to proliferate, meanwhile, IL-2, IL-7 and IL-15 are added, 1-2 days before the culture is finished, IL-12 is added into the culture system to guide the iNKT cell to differentiate directionally;