CN111166878B

CN111166878B - Preparation method and application of combination of antibody targeting tumor antigen and iNKT cell

Info

Publication number: CN111166878B
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: 2024-01-02
Anticipated expiration: 2040-01-06
Also published as: CN111166878A

Abstract

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

Description

Preparation method and application of combination of antibody targeting tumor antigen and iNKT cell

Technical Field

The present invention relates to the field of immunotherapy of tumors. In particular, 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 methods for their preparation.

Background

Tumor immunotherapy can target tumor-induced immune escape and immunosuppressive tumor microenvironment, and can specifically remove tumor focus, inhibit tumor growth and break immune tolerance by activating immune cells in vivo to enhance organism anti-tumor immune response. The fourth largest tumor treatment technique after tumor surgery, radiotherapy and chemotherapy is called because of small side effect and obvious treatment effect of tumor immunotherapy. In 12 months 2013, tumor immunotherapy was the first breakthrough by ten scientific studies worldwide by Science journal of rating.

Current tumor immunotherapy is mainly to enhance anti-tumor immune responses. Two main classes are "passive" immunotherapy, in which tumor cells are directly challenged with effector cells or molecules of the immune system, including antibody or its derivatives (e.g., antibody-drug conjugates) targeted therapies, adoptive immune cell therapies (e.g., lymphokine-activated killer cells, natural killer T cells, dendritic cells, etc.), genetically engineered T cells (e.g., chimeric antigen receptor CAR-T, T cell receptor TCR-T, etc.). The other category is "active" immunotherapy, in which the immune system is enhanced by modulating endogenous immune regulation or activation mechanisms, for example, by enhancing antigen uptake, processing and presentation by antigen presenting cells using type I interferons, toll-like receptor agonists and interferon gene agonists, etc.; utilizing dendritic cell vaccines and anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibodies and the like to enhance activation and expansion of undifferentiated naive T cells; the effects of the immune response are enhanced by reinfusion of cancer patients after ex vivo stimulation of expanded tumor infiltrating T cells (tumor infiltrating lymphoeytes, TILs).

The immunopotentiation strategy does control tumor growth to some extent, but the effectiveness of inhibiting multiple types of tumors remains to be enhanced. Notably, immune activators increase the risk of an immune response at a supraphysiological level while increasing the immune response of the body to a tumor. For example, CAR-T cells load the antigen binding domain and the intracellular co-stimulatory domain from the receptor, and recognition of the antigen is not limited by expression of the major histocompatibility complex, and can more effectively attack tumor cells. But with the consequent consequences of a response at an supraphysiological level such as off-target toxicity and acute cytokine release syndrome. The increasing clinical trials have demonstrated that combined treatment strategies for tumors, such as immunotherapy in combination with chemotherapy, radiation therapy, oncolytic virus therapy, targeted therapies, etc., have great advantages over monotherapy in terms of improved therapeutic effectiveness and reduced side effects.

Natural killer T cells (Natural killer T cell, NKT) are an important cell type in tumor immunotherapy strategies. Wherein, the type I NKT cells, i.e. inKT express constant T cell antigen receptor (TCR), can secrete anti-tumor related cytokines after the stimulation and activation of alpha-GalCer glycolipid antigens, and improve the activities of natural killer cells (NK) and Cytotoxic T Lymphocytes (CTL). iNKT cells can also exert important anti-tumor and anti-infective effects by affecting tumor microenvironment or in Antibody-dependent cellular cytotoxicity (ADCC). In clinical trials of various solid tumors or hematological malignancies, adoptive reinfusion of in vitro expanded iNKT cells to patients showed good therapeutic efficacy. The effectiveness and safety of NKT cell immunotherapy tumors has been widely accepted. The method has been approved in clinical use by the Japanese Ministry of labor. In addition, iNKT cell therapy techniques may be used in combination with other tumor therapy techniques, such as surgery, chemotherapy, radiation therapy, and the like. The major challenge faced by iNKT cell therapy is the low targeting, especially the very limited therapeutic effect on tumor cells that do not express CD1d molecules.

The target of tumor therapeutic monoclonal antibodies (Monoclonal Antibodies, mAbs) encompasses tumor cell associated antigens (e.g., CD19, CD20, CD22, her 2), epithelial and angiogenic receptors (e.g., EGFR, VEGF, VEGFR 2), and immune checkpoint key molecules (e.g., CTLA4, PD-1, PD-L1), and the like. The effect of killing tumor cells can be improved by the following mechanisms: (1) Directly blocking or activating ligand-receptor signal transduction activity, inducing apoptosis; (2) Immune cell mediated killing of tumor cells, including Antibody-dependent cellular phagocytosis (ADCP) and ADCC, complement-dependent cytotoxicity (CDC); (3) Genetic engineering modification techniques increase tumor targeting accuracy, such as antibody single-chain variable fragment (scFv) genetically modified T cells; (4) Blocking antibodies to immune checkpoint key molecules can enhance the killing effect of T cells on tumor cells by reducing immune tolerance. (5) Antibodies targeting tumor vasculature and stroma antagonize vascular receptors or ligands, induce vascular and stromal cell ablation and thereby inhibit proliferation of tumor cells. The antibody has the advantages of more specific acting targets and fewer side effects. The antibody drugs currently used in clinic range from early Rituximab (1997), trastuzumab (1998), to successful de-valumab (2017), oxtuzumab Inotuzumab ozogamicin (2017), moxetumomab pasudotox (2018), cemiplimab-rwlc (2018), which are developed in recent years, to 30 or more, showing a fully desirable antitumor effect, but most antibodies alone still have difficulty in achieving long-term effective inhibition and killing of tumor cells.

AIDS is a disease that causes impaired immune function after infection of the human body by HIV virus, and patients develop various life-threatening opportunistic infections and malignant tumors easily at the late stage of AIDS. Because such patients are associated with immunodeficiency, the treatment regimen is selected and the dosage is very careful. (Zhao Peng, zhao Hua, tu Bo. AIDS complicated with diffuse large B cell lymphoma 1 report [ J ]. Lesion of Legiosa medical college of academy of medicine, 2016,37 (10): 1117-1118.)

For most tumors, such as CD20 positive diffuse large B-cell lymphomas, treatment with their targeting antibodies alone can increase the cure of the tumor; however, due to the immunodeficiency characteristics of aids patients, this further increases the suppression of humoral immunity in the patient, resulting in opportunistic infections that are detrimental to patient treatment. Thus, the targeted antibody drug dose needs to be in an appropriate amount, which can lead to a decrease in therapeutic efficacy.

How to solve the problems of immunogenicity existing in antibody drugs, such as anti-drug antibody (ADA) reaction, tolerance of tumor targets in long-term use and long-acting performance of single blocking signal transduction channels, is an unavoidable challenge of tumor antibody drugs. Because of the defects in the prior art, an anti-tumor medicament and a treatment method with good treatment effect and low side effect on patients need to be developed.

Disclosure of Invention

It is therefore an object of the present invention to provide a use of a combination of an antibody targeting a tumor antigen and iNKT cells for the preparation of an anti-tumor drug, a kit for immunotherapy of tumors using a combination of an antibody targeting a tumor antigen and effector cells, and a method for their preparation. The tumor is selected from diffuse large B-cell lymphoma, sarcoma, glioma, high grade glioma, blastoma, osteosarcoma, plasmacytoma, histiocytoma, carcinoma of large intestine, hematopoietic cancer, testicular cancer, ovarian cancer, squamous cell carcinoma, adenocarcinoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, and head and neck squamous cell carcinoma;

preferably, the tumor is an AIDS-concurrent malignancy; the AIDS-concurrent malignancy is preferably selected from AIDS-concurrent diffuse large B-cell lymphoma, AIDS-concurrent kaposi's sarcoma, and AIDS-concurrent skin mucosa squamous carcinoma;

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

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

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

Preferably, the medicament 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 a tumor antigen comprises an Fc sequence comprising an a330L/I332E mutant sequence.

In one aspect, the antibody that targets a tumor antigen is an antibody that targets the following 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-Rα, SLAMF7 and/or CCR4;

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

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

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

In a specific embodiment, the present inventors provide a CD 20-targeting antibody comprising the heavy chain shown in SEQ ID NO. 2 and the light chain shown in SEQ ID NO. 4 in combination with an iNKT cell.

The inventor of the invention discovers that the therapeutic monoclonal antibody is used together with the iNKT cells, so that the iNKT cells can be better led to tumor cells, the ADCC effect of the antibody can be improved, and the tumor can be better controlled; in addition, the combined use of the iNKT cells for enhancing immune function can also help patients repair immune system, reduce occurrence of opportunistic infection and facilitate alleviation of illness.

In another aspect, the invention provides a kit comprising:

antibodies and iNKT cells targeting tumor antigens.

Preferably, the iNKT cells are 10 in number ⁷ From one to 10 ¹⁰ And each. The kit according to the invention, wherein the antibody targeting a tumor antigen is an antibody targeting the following targets: PD-L1, CD47, CD52, CTLA4, RANKL, CD19, CD20, CD22, CD30, CD33, CD38, CD147, GD2, EGFR, VEGF, PIGF, VEGFR, VEGFR, PSMA, HER2, AXL, ROR2, PD-1, PDGF-Rα, SLAMF7 and/or CCR4;

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

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

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

In a further aspect, the invention provides the use of the combination or kit in the manufacture of an anti-tumour medicament. In yet another aspect, the invention provides a method of preparing said combination or kit, said method comprising the steps of:

1) Preparing an antibody targeting a tumor antigen, 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 amplifying iNKT cells;

preferably, the iNKT cells are expanded using α -galactosylceramide (α -GalCer) and proliferation of the iNKT cells is stimulated with CD1d expressing cells loaded with α -galactosylceramide, while cytokines IL-2 and IL-7 are added to assist in the growth of the iNKT cells;

b. further carrying out the amplification of the number of the iNKT cells and the directional differentiation of the guiding function;

preferably, CD1d expressing cells loaded with alpha-galactosyl ceramide stimulate proliferation of iNKT cells, IL-2, IL-7 and IL-15 are added simultaneously, and IL-12 is added to the culture system 1-2 days before the end of the culture to induce directional differentiation of iNKT cells;

preferably, the CD1d expressing cells are 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 method 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, colorectal 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 skin mucosa squamous carcinoma;

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

The antibody targeting a tumor antigen of the present invention has a synergistic effect with the iNKT cells. 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 iNKT cell is administered first followed by an antibody targeting a tumor antigen; more preferably, iNKT cells are administered prior to administration of antibodies targeting tumor antigens. Preferably, the tumor antigen-targeting antibody and iNKT cells are administered by subcutaneous injection, intravenous injection, or intramuscular injection. The amount 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 antibody. For example, in a preferred embodiment, the specific antibody targeting tumor cells may be administered at a dose of 5mg to 200mg per time; 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 10 ⁷ From one to 10 ¹⁰ Individual cells/times; more preferably, the iNKT cells are administered at a dose of 1.3 to 2.5X10 ⁹ Individual cells/time.

In a preferred embodiment, the iNKT cells are administered at a dose of 1.3x10 ⁹ Individual cells/time to 2.5X10 ⁹ Individual cells/time. The number of times of reinfusion of iNKT cells is not particularly limited, and generally depends on the actual conditions such as the condition of the patient.

An exemplary antibody according to the invention is an anti-CD 20 antibody (11B 8) comprising: the heavy chain nucleotide sequence shown in SEQ ID NO. 1 and the amino acid sequence corresponding thereto shown in SEQ ID NO. 2, and the light chain nucleotide sequence shown in SEQ ID NO. 3 and the amino acid sequence corresponding thereto shown in SEQ ID NO. 4.

The heavy chain nucleotide sequence of SEQ ID NO. 1, CD20 antibody 11B8

ATGGAATTAGGCCTCTCTTGGGTGTTCCTCGTGGCTATTCTCAAGGGAGTGCAGTGCGAGGTGCAGCTGGTGCAGTCTGGAGGCGGGCTCGTGCATCCTGGCGGCTCCCTGAGACTGTCTTGCACCGGAAGCGGGTTCACCTTCTCTTACCACGCTATGCACTGGGTGCGCCAGGCTCCTGGCAAGGGACTGGAGTGGGTGAGCATTATCGGAACCGGCGGCGTGACATACTACGCTGACTCTGTGAAGGGCAGATTCACAATTAGCCGCGACAACGTGAAGAACTCCCTGTACCTCCAGATGAACAGCCTCAGAGCCGAGGACATGGCTGTGTACTACTGCGCTAGAGACTACTACGGCGCCGGATCTTTCTACGACGGCCTGTACGGTATGGACGTGTGGGGCCAGGGCACAACAGTGACCGTGTCTAGC

SEQ ID NO. 2, the heavy chain amino acid sequence of CD20 antibody 11B8 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MELGLSWVFLVAILKGVQCEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSI IGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAGSFYDGLYGMDVWGQGTTVTVSS

The light chain nucleotide sequence of SEQ ID NO. 3, CD20 antibody 11B8

ATGGAGGCTCCCGCTCAGCTGCTGTTCCTGCTCCTGCTGTGGCTGCCTGACACAACTGGAGAGATCGTGCTGACCCAGTCTCCCGCTACACTGTCTCTGAGCCCTGGCGAGCGCGCCACCCTGTCTTGCAGGGCCTCTCAGTCCGTTTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGACAGGCCCCAAGACTCCTCATATATGACGCTTCTAACCGCGCCACCGGCATCCCAGCTAGGTTCAGCGGGTCCGGATCTGGAACCGACTTCACACTCACAATTTCTAGCCTCGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGGTCCGACTGGCCACTCACATTCGGCGGCGGGACAAAGGTGGAGATTAAG

SEQ ID NO. 4, light chain amino acid sequence of CD20 antibody 11B8 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIK

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

the inventor of the invention discovers that the treatment is carried out by adopting the iNKT cells as effector cells and combining with the tumor antigen specific high-killing antibody, has higher tumor target cell killing activity and further improves the anti-tumor capability. 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 antibody can have both strong CD1d targeting tumor killing effect and immune regulation effect of iNKT cells, and accurately target the iNKT cells to local lesions of the tumors by the tumor specific targeting antibody, and simultaneously, the specificity of tumor killing can be more effectively improved by utilizing the ADCC effect of the antibody. The combined use of iNKT cells that increase immune function may also help the patient repair the immune system, reduce the occurrence of opportunistic infections, and facilitate remission of the condition. Compared with monotherapy, the combination can greatly reduce the dosage of immune cells and antibodies and the frequency of reinfusion, and reduce the pain of tumor patients and the treatment cost.

Drawings

Embodiments of the present 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 patient is approximately treated as follows:

the patient finds abdominal mass in 6 months in 2018, has no red swelling and pain and is accompanied by fever, and puncture pathology shows that the patient has diffuse large B cell lymphoma and HIV virus positive; the patient is then treated with anti-HIV virus, first-line and second-line chemical therapy, and local radiation therapy. Chemotherapy and local radiotherapy reduce tumor burden, so that the tumor is slightly reduced, but the tumor rebounds after stopping the medicine. The patient was treated with iNKT cells in combination with CD20 antibodies on 12 months 2018 and on 22 months 1.2019, and the patient was treated with a total of 3 courses of immunotherapy.

Fig. 2 shows MRI image results for a patient's abdominal lesion. The figure shows that the patient had contracted the tumor 5 days before cell reinfusion in the first course of treatment after local radiotherapy of the tumor, but had rebounded after withdrawal. After three courses of iNKT cells in combination with CD20 antibody, the tumor size was significantly reduced and there was no rebound.

FIG. 3 is a long and short diameter plot of a patient's abdominal lesion tumor;

fig. 4 is a volume map of a patient's abdominal focal tumor.

Detailed Description

The invention will be described in detail with reference to specific clinical trials.

CD20 antibodies are produced by GMP (good manufacturing practice) workshops of offshore biotechnology limited;

iNKT cells were obtained from PBMC cells autologous to the patient by in vitro expansion culture. And, the patient voluntarily joins the study and signs an informed consent.

EXAMPLE 1 Combined clinical treatment procedure, results and conclusion of the invention

1. Clinical treatment procedure

1.1 clinical diagnosis of patients

The patient finds abdominal mass without red swelling and pain and with fever; the diagnosis of puncture pathology is as follows: diffuse large B-cell lymphoma and Human Immunodeficiency Virus (HIV) positive (fig. 1).

1.2 conventional treatment regimen for patients

After the patient's condition is diagnosed, the patient is treated with tenofovir/emtricitabine+dortezovir for AIDS (Acquired Immune Deficiency Syndrome, AIDS). One month later, i.e., 7 months and 5 days in 2018, R-EPOCH (rituximab, doxorubicin, vincristine and etoposide) was performed, wherein three chemotherapeutics, doxorubicin, vincristine and etoposide, were administered together for continuous 96 hours without interruption (one chemotherapeutic for four bags per day) and finally 18 doses per day of vincristine (last controlled flow rate) oral prednisone) was administered for the treatment of diffuse large B-cell lymphomas. Due to poor therapeutic effect, after one month, the patient was changed to R-ICE (rituximab 375mg/m ² Intravenous drip, day 1; ifosfamide 1 200mg/m ² Intravenous drip, day 2-6; carboplatin AUC = 5, intravenous drip, day 2; etoposide 100mg/m ² Intravenous drip, day 2-4. ) Chemotherapy, tumor mass is slightly reduced; however, due to R-ICE chemotherapy, bone marrow suppression, giant cell viral retinitis (CMVR) and other symptoms are caused, and tumor regression is not obvious. After one month of R-ICE chemotherapy, the patient locally took radiation therapy to the tumor, and this radiation treatment reduced the tumor burden, slightly reduced the tumor mass, but the tumor bounced after the radiation treatment was stopped (FIG. 1).

1.3 treatment regimen Using the iNKT cells and CD20 antibody combinations of the invention

One month after the end of radiotherapy, the patient is treated with iNKT cells in combination with CD20 antibody, the first treatment course is carried out on day 12 and 18 of 2018 (reinfusion in the form of intravenous injection), and reinfusion is carried out on day 21 of 2018 and 12, and the number of reinfusion iNKT cells is 2×10 respectively ⁹ ，1.8×10 ⁹ CD20 antibody doses were 10mg each; after 3 weeks, a second course of treatment was performed, with a return infusion at 2019, 1 month and 8 days, 2019

Reinfusion was performed once again on 11 days of 1 month, and the number of reinfusion iNKT cells was 1.3x10, respectively ⁹ ，1.5×10 ⁹ In each case, the CD20 antibody dose was still 10mg; after 2 weeks, a third treatment course was performed, and the cells were returned at 22 days of 2019, 1 month and 25 days of 2019, and the number of iNKT cells was 2×10, respectively ⁹ ，2.5×10 ⁹ The CD20 antibody dose was also 10mg (as shown in table 1).

TABLE 1 application of the combinations of the invention

1.4 method for detecting tumor of patient

Detecting the proportion of available iNKT cells using flow cytometry;

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

2 results

2.1 variation in tumor size

After the chemotherapy is finished, the tumor volume of the patient is large (the long diameter is 13 cm, the short diameter is 7.5 cm), no obvious shrinkage trend exists, and symptoms such as bone marrow suppression, CMVR and the like appear. After one month of symptoms such as bone marrow suppression and CMVR, local radiotherapy of the tumor is performed, i.e., 5 days before cell reinfusion in the first course of treatment, the tumor tends to shrink (length is 12 cm and length is 5.2 cm), but the tumor begins to rebound after stopping radiotherapy (shown in FIGS. 2-4).

Since conventional therapies fail to control tumor growth, patients begin iNKT cell-in-CD 20 antibody therapy. 7 days after the cell reinfusion of the first course, the tumor begins to shrink, and the tumor short diameter is reduced to 7.4 cm; the iNKT cell-CD 20 antibody therapy was continued for a third course of treatment, and the tumor continued to shrink 3 days after the end of the third course of treatment, with a decrease in major diameter to 9.1 cm, a decrease in minor diameter to 4.5 cm, and no tumor rebound (shown in fig. 2-4). Compared to the pre-treatment (2018.12.13) imaging results, the patient tumor volume was reduced from an initial 750 cubic centimeters to 150 cubic centimeters (shown in fig. 2-4).

2.2 treatment with iNKT cells in combination with CD20 antibodies followed mainly by response and treatment measures

After treatment with iNKT cells in combination with CD20 antibodies, the patient developed symptoms of cold-tremor and fever. The heat peak reaches 40 ℃ after about 3 hours after treatment, the physical temperature is reduced, ibuprofen is orally taken for defervescence, and then the patient can recover to be normal. Meanwhile, the phenomenon of leucocyte quantity reduction starts to appear 2 days after the treatment of the iNKT cells combined with the CD20 antibody, and the normal leucocyte level can be recovered by adopting the leucocyte rising treatment according to symptoms.

Conclusion 3

The patient currently received no other treatment for the tumor than iNKT cell-associated CD20 antibody therapy and received a fourth course of cell feedback on day 3 and 15 of 2019.

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

At present, clinical rituximab is used for treating CD20 positive diffuse large B cell lymphoma, 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 cells and the antibodies can greatly reduce the dosage of the antibodies, and each dosage of the antibodies only needs 10mg, so that the drug resistance of organisms can be greatly reduced, and more outstanding treatment effects are achieved.

In a combined way, the combination can obviously reduce tumor load of patients with HIV combined diffuse large B cell lymphoma, can effectively control tumor rebound, and has no obvious side effect. The iNKT cells are taken as effector cells, and are combined with tumor antigen specific antibodies for treatment, so that the anti-tumor agent has a higher tumor inhibition effect and can effectively improve the anti-tumor capability.

Sequence listing

<110> Shanghai Xinwan biotechnology Co., ltd

Preparation method and application of <120> antibody targeting tumor antigen and iNKT cell combination

<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 Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Phe Thr Phe

35 40 45

Ser Tyr His Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Ile Ile Gly Thr Gly Gly Val Thr Tyr Tyr Ala Asp

65 70 75 80

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser

85 90 95

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Asp Tyr Tyr Gly Ala Gly Ser Phe Tyr Asp Gly Leu

115 120 125

Tyr Gly Met Asp Val Trp Gly Gln 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 Arg Ala 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 manufacture of an anti-tumor drug, wherein the tumor is an AIDS-concurrent diffuse large B-cell lymphoma;

the antibody comprises a heavy chain variable region shown as SEQ ID NO. 2 and a light chain variable region shown as SEQ ID NO. 4, and targets CD 20; and, the antibody targeting a tumor antigen comprises an Fc sequence comprising an a330L/I332E mutant sequence;

the iNKT cells are in vitro expanded iNKT cells, and the iNKT cells are expanded by a method comprising the steps of:

a. specifically amplifying iNKT cells;

amplifying iNKT cells using α -galactosyl ceramide and stimulating proliferation of iNKT cells with α -galactosyl ceramide-loaded CD1 d-expressing cells, while adding cytokines IL-2 and IL-7 to assist in iNKT cell growth;

the CD1d expression cells loaded with alpha-galactosyl ceramide stimulate the proliferation of the iNKT cells, simultaneously IL-2, IL-7 and IL-15 are added, and IL-12 is added into a culture system 1-2 days before the end of the culture to guide the directional differentiation of the iNKT cells;

the CD1d expressing cells are selected from the group consisting of dendritic cells.

2. The use of claim 1, wherein the medicament is an injection.