CN110205296B - Combination, use and preparation of antibodies with Fc mutants and effector cells - Google Patents

Abstract

The present invention provides a combination of an antibody targeting a tumor antigen with an Fc mutant and a high potency immune effector cell, wherein the Fc mutant has the a330L/I332E mutation; the effector cell is an effector cell expressing CD 16. The invention also provides application of the combination in preparing anti-tumor medicaments. The invention also provides a preparation method of the composition. The combination of the invention adopts NKT cells as effector cells, and combines with an antibody of a targeting tumor antigen with Fc mutant, thereby realizing higher killing activity of the tumor target cells and further improving the anti-tumor capability.

Description

Combination of antibody with Fc mutant and effector cell, application and preparation method

Technical Field

The present invention relates to the field of immunotherapy of tumors. In particular, the present invention relates to a combination of an antibody targeting a tumor antigen with an Fc mutant and effector cells for high-efficiency immunization, a kit for immunotherapy of tumors using the combination of an antibody targeting a tumor antigen with an Fc mutant and effector cells, and a method for preparing the same.

Background

The antibody is an immunoglobulin capable of specifically binding to an antigen, and has a molecular weight of about 150 kD. Antibodies can be digested by papain to Fab and Fc fragments. The Fab fragment has two antigen binding sites and functions to specifically bind to antigen, while the Fc fragment plays a crucial role in the effector functions of the antibody. The effector functions of most antibodies are mediated through the interaction of the Fc fragment with Fc receptors. Human Fc receptors are three Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD 16). Fc γ riii (CD16) in turn includes Fc γ riiii a and Fc γ riiii B. Fc γ rlib is found only in neutrophils, whereas Fc γ rliiia is expressed in macrophages, monocytes, Natural Killer (NK) cells and T cell subsets. Fc γ RIIIA is the only Fc receptor expressed on NK cells and can exert antibody-dependent cell-mediated cytotoxicity (ADCC). Namely, after the target cells bound by the antibody are recognized by Fc gamma RIIa, NK cells are activated to synthesize and secrete cytokines, such as IFN-gamma and granzyme, to mediate cytotoxic functions.

Natural killer T cells (NKT cells) are a heterogeneous population of specialized T lymphocytes, distinct from T lymphocytes, B lymphocytes, natural killer cells (NK cells), a novel class of T lymphocytes, belonging toclass 4 immune cells. The NKT cell has the characteristics of both NK cells and T cells, can quickly respond to the exposure of immunogen, has the accurate identification function of adaptive immune cells to the immunogen, and induces various response reactions. NKT cells are divided into two classes, including invariant NKT (invariant NKT, iNKT, type I NKT) and variant NKT (variant NKT, type II NKT); among them, iNKT expresses a constant T cell antigen receptor (TCR), a V α 14-J α 281 α chain in mice and a V α 24-J α Q chain in humans. The anti-tumor and anti-infection functions are mainly played in the body; variant NKTs possess diverse TCR repertoires and exert immune-modulatory effects mainly through secretion of various cytokines.

In recent years, great progress has been made in the research of phenotypic characteristics, distribution and development, immunological effects and relationship with diseases, tumor treatment and the like. Unlike traditional T cells, NKT cells express TCRs that are recognized by the conserved, non-polymorphic MHC class H-like molecule CD1 d. The best stimulator effector of iNKT cells is an extract from the corpus cavernosum or symbiotic microorganisms, alpha-galactosylceramide (alpha-GalCer). NKT Cells stimulate Dendritic Cells (DC) to secrete a large amount of IL-12 by activating IL-12 receptors on the surface of a membrane under the stimulation of self ligands and synthetic ligands, so that differentiation and maturation of DC-free Cells are promoted; meanwhile, NKT cells secrete a large amount of IFN-gamma, and the IFN-gamma can act on NK cells to secrete a large amount of perforin to kill target cells; IL-12 secreted by dendritic cells acts on primary CD4+ T cells, promotes the transformation of adaptive immunity to Th1 type, and enhances cellular immune response. Under the combined action of the above factors, the target cells infected by virus or intracellular parasitic bacteria and the tumor target cells can be mediated to be dissolved and destroyed. NKT can also directly kill the above target cells via the human Apoptosis-related Factor/human Apoptosis-related Factor ligand (Fas/FasL) pathway by expressing human Apoptosis-related Factor ligand (FasL). The activated NKT cells can secrete cytokines such as IL-4, IFN-gamma and the like to play an immunoregulation role. In addition, NKT cells secrete various chemotactic cytokines and the like to participate in inflammatory reactions.

NKT cells have great application prospects in resisting tumors, resisting infection, inhibiting autoimmune diseases and transplanting tolerance as novel immune regulatory cells. CD3+/CD16+ CD56+ T cells are not a typical population of NKT cells, but are a broader population of T cell subsets, also conforming to the original definition of NKT.

NKT cells are used for tumor therapy to exert a better tumor control effect, but due to the complexity of the tumor microenvironment, NKTs generally do not exert a better anti-tumor effect in tumor therapy, particularly in the treatment of solid tumors.

Disclosure of Invention

Therefore, it is an object of the present invention to provide a combination of an antibody targeting a tumor antigen with an Fc mutant and effector cells for high-efficiency immunization, and a kit for immunotherapy of tumors using the combination of an antibody targeting a tumor antigen with an Fc mutant and effector cells and a method for preparing the same.

In one aspect, the invention provides a combination of an antibody targeting a tumor antigen with an Fc mutant, wherein the Fc mutant has the a330L/I332E mutation (ali antibody), and an effector cell that is a CD 16-expressing effector cell.

The combination according to the invention, wherein the Fc mutant is derived from the Fc segment of the humanantibody subtype IgG 1;

preferably, the Fc mutant has an amino acid sequence shown as SEQ ID NO. 1.

1, namely the amino acid sequence EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK mutated in the Fc segment A330L/I332E

More preferably, the encoding nucleotide sequence of the Fc mutant has the nucleotide sequence shown as SEQ ID NO. 2.

2, Fc fragment A330L/I332E mutant nucleotide sequence

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCACTCCCCGAGGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Preferably, the antibody targeting a tumor antigen is an antibody targeting the following targets: PD-L1, CD47, CD19, CD20, CD22, CD30, CD33, CD38, GD2, EGFR, VEGF, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, SLAMF7, and/orCCR 4.

The combination according to the invention, wherein the effector cells are selected from NKT cells or NK cells.

The inventor of the invention finds that the antibody carrying the Fc segment A330L/I332E mutation can be combined with effector cells expressing CD16 to improve ADCC effect of the antibody and control tumors better.

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

antibodies targeting tumor antigens with Fc mutants; wherein the Fc mutant has the A330L/I332E mutation; and

an effector cell;

wherein the effector cell is an effector cell expressing CD 16.

The kit of the invention, wherein the Fc mutant is derived from an Fc segment of a humanantibody subtype IgG 1;

preferably, the Fc mutant has an amino acid sequence shown as SEQ ID NO. 1;

more preferably, the encoding nucleotide sequence of the Fc mutant has a nucleotide sequence shown as SEQ ID NO. 2;

preferably, the antibody targeting a tumor antigen is an antibody targeting the following targets: PD-L1, CD47, CD19, CD20, CD22, CD30, CD33, CD38, GD2, EGFR, VEGF, PIGF, VEGFR2, PSMA, HER2, AXL, ROR2, SLAMF7, and/orCCR 4.

The kit according to the present invention, wherein the effector cells are selected from NKT cells or NK cells.

The kit according to the invention, wherein the antibody is administered in an amount of 1-5mg/kg, preferably 2 mg/kg.

Preferably, the effector cells are administered in an amount of 1-9X 10⁹One effector cell per time, more preferably2X 10⁹One effector cell per time.

Preferably, the dosage form of the reagent comprises an injection dosage form, an external medicine dosage form and an oral dosage form.

Preferably, the agent can be administered by subcutaneous injection, intravenous injection, intramuscular injection.

Preferably, the agent is selected from the group consisting of tablets, capsules, films and granules.

Preferably, the dosage form of the agent includes a sustained release dosage form and a non-sustained release dosage form.

In another aspect, the invention provides the use of the combination or the kit in the preparation of an anti-tumor drug, an anti-pathogen drug or a drug with low immunity.

Preferably, the tumor may be melanoma, prostate cancer, renal cell carcinoma, neuroblastoma, pancreatic cancer, breast cancer, lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, cervical cancer, bladder cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, or a hematologic neoplastic disease.

In a further aspect, the invention provides a method of making the combination or kit, the method comprising the steps of:

1) preparing an antibody targeting a tumor antigen having an Fc mutant, wherein the Fc mutant has the a330L/I332E mutation; and

2) effector cells expressing CD16 were expanded in vitro.

According to the method of the invention, in step 2), the effector cells are NKT cells;

preferably, the NKT cells are cultured by a method comprising the steps of:

specifically amplifying type I NKT cells;

preferably, the specific stimulator alpha-galactosylceramide (alpha-GalCer) is used for expanding the type I NKT cells, and the CD1d expression cells loaded with the alpha-GalCer stimulate the proliferation of the type I NKT cells, and simultaneously the cytokines IL-2 and IL-7 are added to assist the growth of the type I NKT cells;

secondly, further performing the quantity amplification of the I-type NKT cells and guiding the function directional differentiation;

preferably, the CD1d expression cells loaded with alpha-GalCer stimulate the proliferation of type I NKT cells, simultaneously add interleukin-2 (IL-2), interleukin-7 (IL-7) and interleukin-15 (IL-15) to assist the expansion of the type I NKT cells and guide the differentiation, and add interleukin-12 (IL-12) to the culture system 1-2 days before the culture is finished to guide the directional differentiation of the type I NKT cells.

Preferably, the CD1d expressing cell is selected from a dendritic cell, other cells expressing CD1d, or other artificially modified dendritic cell-like antigen presenting cells.

In a preferred embodiment, the NKT cells are cultured by:

(1) resuspending and adjusting the concentration of Peripheral Blood Mononuclear Cells (PBMC), adding alpha-GalCer, and culturing type I NKT cells;

(2) adding the dendritic cells loaded with the alpha-GalCer into a culture system of the type I NKT cells on the 7 th day of culture, and simultaneously adding IL-2 and IL-7 to keep the concentration of the alpha-GalCer in the culture system of the type I NKT cells unchanged;

(3) culturing for 14 days, adding the dendritic cells loaded with the alpha-GalCer into the type I NKT cell culture system again, adding IL-15 at the same time, and keeping the concentration of the total alpha-GalCer, IL-2 and IL-7 of the culture system unchanged;

(4) on the 20 th day of culture, IL-12 was added to the culture system while keeping the concentrations of α -GalCer, IL-2, IL-7 and IL-15 in the system constant, and cells were collected on the 21 st day of culture.

Preferably, the dendritic cells loaded with alpha-GalCer are prepared by the following method: resuspending and adjusting PBMC concentration, adding IL-14 and GM-CSF to induce differentiation of dendritic cells at working concentrations of 500U/mL and 50ng/mL, and adding alpha-GalCer to the dendritic cell culture system on day 6 of culture for pre-incubation for 24 h.

In addition, the initial PBMC concentration used for culturing type I NKT cells was5X 10⁵/mL～3×10⁶mL, initial concentration of PBMC for inducing differentiateddendritic cells 1X 10⁶/mL～5×10⁶The working concentration of the alpha-GalCer is 50 ng/mL-500 ng/mL; the working concentration of the IL-2 is 10U/mL-100U/mL, the working concentration of the IL-7 is 20 ng/mL-200 ng/mL, and the working concentration of the IL-12 is 10 ng/mL-100 ng/mL, and the working concentration of the IL-15 is 10 ng/mL-100 ng/mL. The source of the type I NKT cells may be PBMCs, purified CD3+ T cells, or purified NKT cells; the culture medium for the type I NKT cell expansion can be X-VIVO-15 serum-free culture medium or RPMI1640 culture medium containing 10% FBS or autologous serum, and the RPMI1640 culture medium containing 10% FBS or autologous serum is used in the dendritic cell induction process.

The method according to the present invention, wherein the antibody is an antibody having the A330L/I332E mutation in the Fc region and specifically targets a tumor antigen;

one exemplary antibody according to the invention is an anti-CD 20 antibody (11B8) comprising: the heavy chain shown as SEQ ID NO. 3 and the corresponding amino acid sequence shown as SEQ ID NO. 4, and the light chain shown as SEQ ID NO. 5 and the corresponding amino acid sequence shown as SEQ ID NO. 6.

3, heavy chain nucleotide sequence of CD20 antibody 11B8

ATGGAATTAGGCCTCTCTTGGGTGTTCCTCGTGGCTATTCTCAAGGGAG TGCAGTGCGAGGTGCAGCTGGTGCAGTCTGGAGGCGGGCTCGTGCAT CCTGGCGGCTCCCTGAGACTGTCTTGCACCGGAAGCGGGTTCACCTTC TCTTACCACGCTATGCACTGGGTGCGCCAGGCTCCTGGCAAGGGACTG GAGTGGGTGAGCATTATCGGAACCGGCGGCGTGACATACTACGCTGAC TCTGTGAAGGGCAGATTCACAATTAGCCGCGACAACGTGAAGAACTCC CTGTACCTCCAGATGAACAGCCTCAGAGCCGAGGACATGGCTGTGTAC TACTGCGCTAGAGACTACTACGGCGCCGGATCTTTCTACGACGGCCTGT ACGGTATGGACGTGTGGGGCCAGGGCACAACAGTGACCGTGTCTAGC SEQ ID NO:4, the heavy chain amino acid sequence of the CD20 antibody 11B8 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MELGLSWVFLVAILKGVQCEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSIIGTGGVTYYADSVKGRFTISRDNVKNSLYL QMNSLRAEDMAVYYCARDYYGAGSFYDGLYGMDVWGQGTTVTVSSSEQ ID NO 5, light chain nucleotide sequence of CD20 antibody 11B8

ATGGAGGCTCCCGCTCAGCTGCTGTTCCTGCTCCTGCTGTGGCTGCCTG ACACAACTGGAGAGATCGTGCTGACCCAGTCTCCCGCTACACTGTCTC TGAGCCCTGGCGAGCGCGCCACCCTGTCTTGCAGGGCCTCTCAGTCCG TTTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGACAGGCCCCAA GACTCCTCATATATGACGCTTCTAACCGCGCCACCGGCATCCCAGCTAG GTTCAGCGGGTCCGGATCTGGAACCGACTTCACACTCACAATTTCTAG CCTCGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGGTCCGA CTGGCCACTCACATTCGGCGGCGGGACAAAGGTGGAGATTAAG SEQ ID NO:6, the light chain amino acid sequence of the CD20 antibody 11B8 (wherein the underlined parts are CDR1, CDR2 and CDR3, respectively)

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPE DFAVYYCQQRSDWPLTFGGGTKVEIK

Another exemplary antibody according to the invention is an anti-EGFR antibody (cetuximab) comprising the heavy chain shown in SEQ ID NO. 7 and the amino acid sequence shown in corresponding SEQ ID NO. 8, and the light chain shown in SEQ ID NO. 9 and the amino acid sequence shown in corresponding SEQ ID NO. 10.

7, i.e. the heavy chain nucleotide sequence CAGGTGCAGCTGAAGCAGAGCGGCCCAGGCCTCGTGCAGCCTAGCCA GAGCCTGTCTATTACATGCACAGTGTCCGGCTTCTCTCTGACCAACTAC GGCGTGCACTGGGTGAGACAGTCTCCTGGCAAGGGCCTGGAGTGGCT CGGCGTGATTTGGTCTGGCGGCAACACCGACTACAACACCCCTTTCAC ATCTAGGCTCAGCATTAACAAGGACAACTCTAAGTCTCAGGTGTTCTTC AAGATGAACTCCCTCCAGTCCAACGACACCGCCATTTACTACTGCGCC CGCGCCCTGACATACTACGACTACGAGTTCGCCTACTGGGGCCAGGGC ACACTCGTGACCGTGTCC of the EGFR antibody cetuximab

SEQ ID NO 8, the heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAL TYYDYEFAYWGQGTLVTVS of the EGFR antibody cetuximab

9 SEQ ID NO, i.e. the nucleotide sequence GACATCCTGCTGACACAGAGCCCCGTGATTCTCAGCGTGAGCCCAGGC GAGAGGGTGTCTTTCTCTTGCCGCGCCTCCCAGTCCATTGGCACAAAC ATCCACTGGTATCAGCAGAGGACCAACGGCTCCCCTAGACTCCTCATTA AGTACGCCTCTGAGTCTATTAGCGGCATTCCATCTAGGTTCAGCGGCTC TGGCTCCGGCACCGACTTCACCCTGTCTATCAACTCTGTGGAGTCCGA GGACATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCTACAAC CTTCGGCGCCGGCACCAAGCTAGAACTGAAG of the light chain of the EGFR antibody cetuximab

10, light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASE SISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE LK of the EGFR antibody cetuximab

The point mutation A330L/I332E of the Fc mutant means that glycine (A) is mutated to leucine (L) at the 330 amino acid position and isoleucine (I) is mutated to glutamic acid (E) at the 332 amino acid position in the Fc amino acid sequence.

In another aspect, the invention provides a method of treating a tumor or pathogen infection or immune deficiency, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a combination or kit of the invention; or administering to a subject in need thereof a therapeutically effective amount of a combination or kit prepared according to the methods of the invention.

The antibody with the Fc mutant and targeting the tumor antigen has a synergistic effect with the effector cells.

In a preferred embodiment, administering the antibody targeting a tumor antigen with an Fc mutant and the effector cell to a subject in need thereof comprises simultaneous administration or sequential administration; for example, an antibody targeting a tumor antigen with an Fc mutant is administered first followed by administration of effector cells, or an antibody targeting a tumor antigen with an Fc mutant is administered first followed by administration of effector cells; more preferably, the effector cells are administered first followed by the antibody targeting the tumor antigen with the Fc mutant.

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

the inventor finds that the NKT cell is used as an effector cell and combined with a tumor antigen specific high-killing antibody for treatment, so that the tumor target cell has higher killing activity, and the anti-tumor capacity is further improved.

Drawings

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

FIG. 1 is a schematic of the in vitro expansion of CD3+ CD56+ CD16+ NKT cells using the methods of the invention;

FIG. 2 shows the construction map of CD20 antibody 11B8 and EGFR antibody cetuximab expression vector plasmids according to the present invention; wherein FIG. 2A is a map of AbVec-hIgG1WT expression vector plasmid construction, containing an unmutated antibody Fc segment for expression of the heavy chain portion of the unmutated antibody; FIG. 2B is AbVec-hIgG1ALIE plasmid (having the A330L/I332E mutation described herein) for expressing the heavy chain portion of a mutant antibody; FIG. 2C is a map of AbVec-hIgKappapa expression vector plasmid construction for expression of the light chain portion of the antibody.

FIG. 3 shows the function of antibody binding of 11B 8-unmutated and 11B 8-mutated antibodies according to the invention; it can be seen from the figure that both the 11B 8-unmutated antibody and the 11B 8-mutated antibody bind Raji cells (lymphoma cell line).

FIG. 4 shows Raji cell killing results of NKT cells incubated with Raji cells alone in vitro in combination with either 11B 8-unmutated or 11B 8-mutated antibodies incubated with Raji cells. As can be seen from the figure, the NKT cell combined with the 11B 8-mutant antibody can obviously improve the killing effect of the Raji cell.

FIG. 5 shows the in vivo protection of NKT cells either in combination with 11B 8-unmutated or 11B 8-mutated antibodies; wherein FIG. 5A is tumor size control of NKT cells treated alone, in combination with either 11B 8-unmutated or 11B 8-mutated antibodies on Raji B lymphoma inoculated B-NDG mice; FIG. 5B is a graph of the effect of NKT cell therapy alone, NKT cells in combination with either 11B 8-unmutated or 11B 8-mutated antibody on the survival of Raji B lymphoma vaccinated B-NDG mice. As can be seen from the figure, the NKT cell combined 11B 8-unmutated antibody and the NKT cell combined 11B 8-mutated antibody can obviously improve the killing effect on Raji tumor of a B lymphoma tumor model in vivo, and the NKT cell combined 11B 8-mutated antibody group has obviously stronger killing effect.

FIG. 6 shows the function of cetuximab-unmutated antibody and cetuximab-mutated antibody binding; as can be seen from the figure, both cetuximab-unmutated antibodies and cetuximab-mutated antibodies bind to A549 cells.

FIG. 7 shows the killing results of A549 cells by in vitro NKT cells incubated with A549 cells (lung cancer cell lines) alone, and NKT cells incubated with A549 cells in combination with a cetuximab-unmutated antibody or a cetuximab-mutated antibody. As shown in the figure, the combination of NKT cells and cetuximab-mutant antibodies can obviously improve the killing effect of A549 cells.

FIG. 8 shows the in vivo protection of NKT cells either in combination with a cetuximab-unmutated antibody or a cetuximab-mutated antibody; NKT cells treated alone, combined with cetuximab-unmutated antibody or cetuximab-mutated antibody, exert tumor size control in B-NDG mice vaccinated with A549 lung carcinoma. It can be seen from the figure that the NKT cell combined cetuximab-unmutated antibody and the NKT cell combined cetuximab-mutated antibody can obviously improve the killing effect on the lung cancer tumor model A549 tumor in a mouse body, and the NKT cell combined cetuximab-mutated antibody group has obviously better killing effect.

Detailed Description

Example 1: peripheral blood PBMC isolation

1. 30-50 mL of heparin anticoagulated human peripheral blood is taken to be put into a centrifuge tube, and the peripheral blood is treated by the following steps of 1:1, diluting and uniformly mixing;

2. another 50mL centrifuge tube was filled with 15mL of lymphocyte separation medium (STEMCELL, cat # 07851) in a volume ratio of lymphocyte separation medium to blood diluent of 1: 2, slowly adding the mixed blood diluent into the upper layer of the lymphocyte separation liquid along the tube wall to ensure that the mixed blood diluent and the lymphocyte separation liquid form clear layers, and centrifuging for 30 minutes at 300 rpm;

3. after the centrifugation is finished, sucking the mononuclear cell layer into a new 50mL centrifuge tube, adding 30mL of X-VIVO-15(LONZA company, cat # 04-418Q) culture medium, washing once, centrifuging for 5 minutes at 800g, and removing supernatant;

4. adding 20mL of X-VIVO-15 culture medium, uniformly mixing by blowing and sucking, centrifuging for 10 minutes at room temperature of 200g, and removing supernatant. Add 10mL X-VIVO-15 medium to the suspension.

Example 2: induction of differentiation of dendritic cells (dendritic cells) for stimulation of proliferation of type I NKT cells

1. The PBMC concentration was adjusted to1X 10 by using a medium containing 10% Fetal Bovine Serum FBS (FBS) (Biological Industry, cat. No. 04-001-1A) RPMI-1640(Roswell Park mental Institute, RPMI) (Corning, cat. No. 10-040-CVR)⁶Individual cells/mL. Spread into 25cm²A flask with a diagonal neck and a vented cap (T25 cell culture flask, from Thermo, cat # 156340) at 37 deg.C and 5% CO₂The cells were incubated for 1 hour.

2. The flask was removed and the supernatant and non-adherent cells removed. The cell surface was washed 2 times with 10% FBS-containing RPMI1640 medium, then 5mL of 10% FBS-containing RPMI1640 medium was added, and the cytokines GM-CSF (R & D, cat # 215-GM-500) and IL-4(R & D, cat # 204-IL-050) were added at working concentrations of 500U/mL and 50ng/mL, respectively.

3. On the fourth day of culture, the culture system was supplemented with 3mL of a medium containing the above-mentioned working concentrations of GM-CSF and IL-4.

4. On the sixth day of culture, α -GalCer (Sigma, cat # 158021-47-7) was added to the medium system to a working concentration of 100 ng/mL.

5. On the seventh day of culture, cells were collected.

Example 3: in vitro expansion of CD3+ CD56+ CD16+ NKT cells

1. PBMC concentration was adjusted to3X 10 with X-VIVO-15 cell culture medium⁶cells/mL, add α -GalCer to a working concentration of 100ng/mL, spread toIn a 6-well plate.

2. On day 3 of culture, medium system was replenished and α -GalCer was added to working concentration.

3. On day 7 of culture, the α -GalCer-loaded dendritic cells prepared in example 2 (about 1X 10)⁵) Adding into NKT cell culture system, and simultaneously supplementing stimulating factors to working concentration: alpha-GalCer 100ng/mL, IL-2 (R)&Company D, cat # 202-IL-500)100U/mL and IL-7 (R)&Company D, cat # 207-IL-025)20 ng/mL. One PBMC was revived and used to induce differentiated dendritic cells to re-stimulate NTK cells in the same manner as in example 2.

4. Onday 10 of culture, fluid was replenished and α -GalCer, IL-2 and IL-7 were added at the same concentrations as above.

5. On day 14 of culture, the dendritic cells loaded with α -GalCer were again added to the NKT cell culture system, and simultaneously supplemented with the stimulation factors α -GalCer, IL-2 and IL-7 at the same concentrations as above. And IL-15(R & D, cat. No. 247-ILB-025) was added to the culture system at a concentration of 20 ng/mL.

6. On day 17 of culture, fluid was replenished and α -GalCer, IL-2, IL-7 and IL-15 were added at the same concentrations as described above.

7. Onday 20 of culture, fluid was replenished and α -GalCer, IL-2, IL-7 and IL-15 were added at the same concentrations as above. In addition, IL-12(R & D, cat 419-ML-500) was added to a working concentration of 20 ng/mL.

8. On day 21 of culture, cells were harvested, 100ul of cell product was taken, and the following fluorescent antibodies were added: anti-CD3-PB (BD Pharmingen, cat # SP34-2), anti-CD56-PE-Cy7(BD Pharmingen, cat # NCAM16.2), anti-CD16-FITC (BD Pharmingen, cat # 3G8) were incubated at 4 ℃ for 30 minutes, and the ratio of the target cell population in the product was measured by flow-assay.

As shown in FIG. 1, the amplification products were dominated by the population of CD3+ CD56+ NKT cells, with a population of NKT cells that were CD3+ CD56+ CD16+ (FIG. 1).

Example 4: construction of mutant Fc segment AbVec-hIgG1ALIE expression plasmid

Firstly, an AbVec-hIgG1WT carrier is used as a template for PCR reaction, and an Fc segment is artificially synthesized. The specific sequences of the two primers (Fc-ALIE-F and Fc-ALIE-R) used for PCR were as follows:

SEQ ID NO:11Fc-ALIE-F：

CCAACAAAGCCCTCCCACTCCCCGAGGAGAAAACCATCTC；

SEQ ID NO:12Fc-ALIE-R：

GAGATGGTTTTCTCCTCGGGGAGTGGGAGGGCTTTGTTGG

the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; carrying out program denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 30 seconds, extending at 72 ℃ for 1 minute, and reacting for 30 cycles; a further 10 minutes of extension at 72 ℃ was carried out, terminating at 25 ℃.

The endonuclease used herein was purchased from Thermo Scientific, unless otherwise specified, and the same shall apply hereinafter.

PCR product verification and clone construction: and 9 mu L of reaction product is taken out, 1 mu L of endonuclease DpnI is added, the enzyme digestion reaction is carried out for 2 hours, the methylated template plasmid is removed, the product is transformed into Escherichia coli E.coli TOP10, and the product grows on a culture plate containing ampicillin overnight. On day 2, randomly selecting a single colony, sequencing, and successfully cloning the mutant Fc segment AbVec-hIgG1ALIE expression vector plasmid after verifying that the whole sequence is correct.

Example 5: construction of expression plasmid for CD20 antibody 11B8

DNA sequences shown in SEQ ID NO. 3 and SEQ ID NO. 5 were artificially synthesized, and PCR amplification was performed using the synthesized DNA sequences as templates and the following primers.

Primers for amplifying the heavy chain of the antibody were:

SEQ ID NO:13 11B8-VH-F：

GCAACCGGTATGGAATTAGGCCTCTCTTGG

SEQ ID NO:14 11B8-VH-R：

TGGTCGACCGGCTAGACACGGTCACTGTTGT

primers for amplifying the light chain of the antibody were:

SEQ ID NO:15 11B8-VK-F：

GCAACCGGTATGGAGGCTCCCGCTCAGC

1611B 8-VK-R: ACCGTACGCTTAATCTCCACCTTTGTC PCR reaction sequence: pre-denaturation at 94 ℃ for 5 min; carrying out program denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 30 seconds, extending at 72 ℃ for 50 seconds, and reacting for 30 cycles; a further 10 minutes of extension at 72 ℃ was carried out, terminating at 25 ℃.

Recovery and cloning construction of PCR products: after the amplification, the target gene was separated in 2% agarose gel, the gel was cut and recovered, the PCR fragment was recovered using a Sanprep column DNA gel recovery kit (Promega corporation, cat # A9282), and the antibody heavy chain gene recovery product was cleaved with both the AbVec-hIgG1WT (FIG. 2A) and AbVec-hIgG1ALIE (FIG. 2B) vectors using the enzymes AgeI and SalI. Similarly, both the recovered antibody light chain gene product and the AbVec-hIgKappa (FIG. 2C) vector were double digested with the restriction enzymes AgeI and BsiWI. After the cleavage, the fragment and the vector were ligated with T4DNA ligase overnight at 4 ℃ and the ligation product was transformed into E.coli TOP10 (Deuterobio, Cat. No. CH5001C) on day 2 and grown overnight on ampicillin-containing plates. On day 3, single colonies were randomly picked for PCR identification, and positive clones were selected for double enzyme digestion identification. And after sequencing is carried out to verify that all the sequences are correct, the heavy chain gene and the light chain gene of the antibody 11B8 are successfully cloned.

Cell supernatants from 293T transfected with AbVec-hIgG1-WT-11B8-VH plasmid and AbVec-hIgKappa-11B8-VK plasmid, and cell supernatants from 293T transfected with AbVec-hIgG1-ALIE-11B8-VH plasmid and AbVec-hIgKappa-11B8-VK plasmid were pooled. The antibody was purified by Protein G Sepharose column (GE, cat. No. 28-9031-34), and the purified antibody was dissolved in PBS buffer. Both the purified 11B-unmutated and 11B 8-mutated antibodies were able to bind to Raji cells (fig. 3).

Example 6: NKT cells with 11B 8-unmutated antibody/11B 8-mutated antibody in vitro killing of lymphoma cells (Raji cells)

1. Target cells (T) (1X 10) were double-stained with PKH-26(sigma-Aldrich, cat # MINI26) (2. mu.M) and CFSE (molecular probes, cat # C1157) (5. mu.M)⁶A cell).

2. Resuspend cells in 1mL complete medium, add 1. mu.L CFSE, incubate at 37 ℃ for 10min, and stop staining with 1mL of pre-cooled complete medium.

3. Target cells were washed with PBS buffer, centrifuged at 400g for 5 min, the supernatant discarded, and cells resuspended in 100. mu.L ofsolvent C. Another 100. mu.L of the solvent C was added to 0.4. mu.L of PKH-26 and mixed well. After the 2 tubes of liquid were mixed well and reacted at room temperature for 2 minutes, 200. mu.L of FBS was added and incubated at room temperature for 1 minute to terminate the staining. 400g, after 10min centrifugation, the supernatant was discarded.

4. Resuspending target cells in RPMI1640 medium at a cell density of1X 10⁵mL, at 50. mu.L of cell suspension perwell 1X 10⁵The cells were plated in a 96-well round bottom plate, 50. mu.L of diluted 11B 8-unmutated or 11B-mutated antibody was added to the plate, the final concentration of antibody was 10. mu.g/mL, and the plate was incubated at 37 ℃ for 1h, and each sample was plated in duplicate.

5. mu.L of NKT cells were added at a ratio of 1:1 effector/target (E: T) and then the reaction was continued at 37 ℃ for 4 hours.

6. Finally, the cells were washed twice with PBS and analyzed by flow cytometry (BD Pharminge, model forttesa). 5000 non-gated events were collected, wherein the ratio of PKH-26high positive/CFSE negative cell population represents ADCC killing activity of the sample.

The results are shown in fig. 4, and compared with the addition of NKT cells alone or the combination of NKT cells with 11B8-WT antibody, NKT cells with 11B8-ali can significantly improve killing of Raji cells in vitro.

Example 7: in vivo back infusion of CD3+ CD56+ CD16+ NKT cells and 11B8-WT/11B8-ALIE antibody for treating Raji B lymphoma

1. To pair

(B-NSGTM) mice were implantedsubcutaneously 5X 10⁵The tumor length and tumor length of the mice were recorded daily in Raji cells (100. mu.L), and the tumor volume was calculated by the following formula.

According to the ethical regulations of animal experiments, when the tumor diameter of a mouse exceeds 2cm in any direction, the mouse is euthanized, and the experimental mouse is marked as dead (about 10 days of tumor formation is expected).

2. Tumor volume calculation formula: tumor volume (mm)³) Long diameter x wide diameter²)/2。

3. 10 days after inoculation of mice with tumor Raji cells, tumorigenic mice were randomly divided into four groups (6 mice each) of untreated control group, NKT cell-treated group, NKT cell +11B 8-unmutated antibody-treated group, and NKT cell +11B 8-mutated antibody-treated group, respectively. The administration mode is orbital venous return infusion. The drug was administered by reinfusion once more on each ofday 3 and 10 after the administration.

A: untreated control group: the same volume of physiological saline;

b: NKT cell treatment group: 5X 10⁶A plurality of NKT cells;

c: NKT cells +11B 8-unmutated antibody treatment group:5X 10⁶2mg/kg 11B 8-unmutated antibody was added to individual NKT cells;

d: NKT cell +11B 8-mutant antibody treatment group:5X 10⁶2mg/kg 11B 8-unmutated antibody was added to individual NKT cells.

4. Tumor growth curve monitoring: after cell reinfusion, tumor size was monitored daily using a vernier caliper for 39 days. Measuring the long diameter and the wide diameter of the tumor body by using a vernier caliper, and calculating the tumor volume; survival of mice bearing tumors for 50 days.

The results are shown in FIG. 5, where the NKT cell-treated group controls Raji B lymphoma in B-NDG mice to some extent compared to the untreated control group, but tumor volumes of more than 2000mm were observed in both groups on day 39³(ii) a The NKT cell +11B 8-unmutated antibody and the NKT cell +11B 8-mutated antibody treatment groups can obviously control the growth of Raji B lymphoma of a B-NDG mouse and can control the tumor volume of the mouse to be 1000mm³The following (fig. 5A); the NKT cell +11B 8-mutant antibody treatment group was able to significantly improve the survival rate of mice vaccinated with Raji B lymphoma B-NDG (fig. 5B).

Example 8: construction of EGFR antibody cetuximab expression plasmid

Artificially synthesizing DNA sequences shown in SEQ ID NO. 9 and SEQ ID NO. 11, and amplifying the heavy chain and the light chain of the EGFR antibody cetuximab by adopting PCR by taking the synthesized DNA as a template.

The heavy chain amplification primers of the EGFR antibody cetuximab include:

SEQ ID NO:17CETUXIMAB-VH-F：

GCAACCGGTCAGGTGCAGCTGAAGCAGAG

SEQ ID NO:18CETUXIMAB-VH-R：

TGGTCGACCGGGACACGGTCACGAGTGTGC

the light chain amplification primers for EGFR antibody CETUXIMAB include:

SEQ ID NO:19CETUXIMAB-VK-F：

GCAACCGGTGACATCCTGCTGACACAGAG

SEQ ID NO:20CETUXIMAB-VK-R：

ACCGTACGCTTCAGTTCTAGCTTGGTGC

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; carrying out program denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 30 seconds, extending at 72 ℃ for 50 seconds, and reacting for 30 cycles; a further 10min extension at 72 ℃ was carried out, ending at 25 ℃.

Recovering and cloning the PCR reaction product: after amplification is finished, separating a target gene from 2% agarose gel, cutting and recovering gel, performing PCR fragment recovery by using a Sanprep column type DNA gel recovery kit, performing double enzyme digestion on an antibody heavy chain gene recovery product, AbVec-hIgG1WT (figure 2A) and AbVec-hIgG1ALIE (figure 2B) vectors by using restriction enzymes AgeI and SalI, similarly, performing double enzyme digestion on an antibody light chain gene recovery product and AbVec-hIgKappa (figure 2C) vectors by using restriction enzymes AgeI and BsiWI, connecting the fragments and the vectors by using T4DNA ligase at 4 ℃ overnight, converting the connection product to Escherichia coli E.coli TOP10 on day 2, and growing on a culture plate containing ampicillin overnight. On day 3, single colonies were randomly picked for PCR identification, and positive clones were selected for double enzyme digestion identification. And after sequencing is carried out to verify that all the sequences are correct, the heavy chain gene and the light chain gene of the antibody 11B8 are successfully cloned.

Two sets of plasmid-transfected 293T cell supernatants were collected separately (AbVec-hIgG 1-WT-cetuximab-VH and AbVec-hIgKappa-cetuximab-VK plasmids, and AbVec-hIgG 1-ALIE-cetuximab-VH and AbVec-hIgKappa-cetuximab-VK plasmids). The antibody was purified by Protein G agarose column, and the purified antibody was dissolved in PBS buffer. Both the purified cetuximab-unmutated antibody and cetuximab-mutated antibody were able to bind to a549 cells (fig. 6).

Example 9: CD3+ CD56+ CD16+ NKT cells and cetuximab-unmutated antibody/cetuximab-mutated antibody in vitro kill A549 cells

An A549 cell in vitro killing experiment performed according to the kit instruction (Dojindo, K17);pipette 100. mu.L of resuspended A549 cells into a 96-well plate at1X 10 cells per well⁴， 37℃，5％CO₂The culture was carried out overnight.

2. The next day, antibody was added as shown in the table, 5% CO at 37 deg.C₂After 1 hour of incubation, the ratio of effective target was 5: 1 addition of NKT cells, continued at 37 ℃ with 5% CO₂The culture was carried out for 4 hours.

3. After 20. mu.L lysis buffer was added to the high control wells, 20. mu.L medium was added to the low control and background wells at 37 ℃ with 5% CO₂Incubate for 30 minutes.

4.Pipette 100 μ L of supernatant from each well into a new 96 well plate.

5. After 50. mu.L of a developing solution was added to each well, the reaction was carried out for 5 minutes in the dark at room temperature.

6. Finally, 25. mu.L of stop buffer was added to each well, and the absorbance at 490nm was immediately measured by a microplate reader.

The results are shown in fig. 7, which shows that NKT cells in combination with cetuximab-mutant antibodies significantly improve killing of a549 cells in vitro compared to NKT cells alone or NKT cells in combination with cetuximab-unmutant antibodies.

Example 10: in vivo reinfusion of NKT cells with cetuximab-unmutated antibody/cetuximab-mutated antibody for treating A549 lung cancer

1. To pair

(B-NSGTM) mice were implanted subcutaneously1X 10⁶A549 cells (100. mu.L) of (1), and then the tumor length and the tumor length of the mice were recorded every day.

According to the ethical regulations of animal experiments, when the tumor diameter of a mouse exceeds 2cm in any direction, the mouse is euthanized, and the experimental mouse is marked as dead (about 10 days of tumor formation is expected).

2. Tumor volume calculation formula: tumor volume (mm)³) Long diameter x wide diameter²)/2

3. 10 days after the mice were inoculated with tumors, the tumorigenic mice were randomly divided into four groups (6 mice each), which were an untreated control group, an NKT cell-treated group, an NKT cell + cetuximab-unmutated antibody-treated group, and an NKT cell + cetuximab-mutated antibody-type treated group, respectively. The administration mode is orbital venous return infusion. The drug was administered by reinfusion once more on each ofday 3 and 10 after the administration.

A: untreated control group: the same volume of physiological saline;

b: NKT cell treatment group: 5X 10⁶A plurality of NKT cells;

c: NKT cells + cetuximab-unmutated antibody treatment group: 5X 10⁶(ii) NKT cells plus 2mg/kg cetuximab-unmutated antibody;

d: NKT cells + cetuximab-mutant antibody treatment group:5X 10⁶2mg/kg cetuximab-mutant antibody was added to individual NKT cells.

4. Tumor growth curve monitoring: after cell reinfusion, tumor size was monitored daily using a vernier caliper for 63 days. The long diameter and the wide diameter of the tumor body are measured by a vernier caliper, and the tumor volume is calculated.

The results are shown in FIG. 8, and the NKT cell-treated group and the NKT cell + cetuximab-unmutated antibody group only weakly controlled the growth of the A549 lung cancer tumor in the B-NDG mouse, and the tumor size was 500mm onday 50³The above; the NKT cell + cetuximab-mutant antibody treatment group can obviously control the growth of the A549 tumor of the B-NDG mice, and the tumor growth of the mice in the group is controlled to be 200mm³The following.

Sequence listing

<110> Shanghai Xinwan Biotech Co., Ltd

<120> combination, use and preparation of antibody having Fc mutant and effector cell

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 232

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 2

<211> 699

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cactccccga ggagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaatga 699

<210> 3

<211> 432

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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> 4

<211> 144

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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> 5

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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> 6

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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

<210> 7

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caggtgcagc tgaagcagag cggcccaggc ctcgtgcagc ctagccagag cctgtctatt 60

acatgcacag tgtccggctt ctctctgacc aactacggcg tgcactgggt gagacagtct 120

cctggcaagg gcctggagtg gctcggcgtg atttggtctg gcggcaacac cgactacaac 180

acccctttca catctaggct cagcattaac aaggacaact ctaagtctca ggtgttcttc 240

aagatgaact ccctccagtc caacgacacc gccatttact actgcgcccg cgccctgaca 300

tactacgact acgagttcgc ctactggggc cagggcacac tcgtgaccgt gtcc 354

<210> 8

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser

115

<210> 9

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gacatcctgc tgacacagag ccccgtgatt ctcagcgtga gcccaggcga gagggtgtct 60

ttctcttgcc gcgcctccca gtccattggc acaaacatcc actggtatca gcagaggacc 120

aacggctccc ctagactcct cattaagtac gcctctgagt ctattagcgg cattccatct 180

aggttcagcg gctctggctc cggcaccgac ttcaccctgt ctatcaactc tgtggagtcc 240

gaggacatcg ccgactacta ctgccagcag aacaacaact ggcctacaac cttcggcgcc 300

ggcaccaagc tagaactgaa g 321

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccaacaaagc cctcccactc cccgaggaga aaaccatctc 40

<210> 12

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagatggttt tctcctcggg gagtgggagg gctttgttgg 40

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcaaccggta tggaattagg cctctcttgg 30

<210> 14

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tggtcgaccg gctagacacg gtcactgttg t 31

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcaaccggta tggaggctcc cgctcagc 28

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

accgtacgct taatctccac ctttgtc 27

<210> 17

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcaaccggtc aggtgcagct gaagcagag 29

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tggtcgaccg ggacacggtc acgagtgtgc 30

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gcaaccggtg acatcctgct gacacagag 29

<210> 20

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

accgtacgct tcagttctag cttggtgc 28

Claims (6)

1.A combination of an antibody targeting a tumor antigen with an Fc mutant and an effector cell, wherein the effector cell is an in vitro induced expansion of NKT cells; the Fc mutant has an amino acid sequence shown as SEQ ID NO. 1, and the antibody targeting a tumor antigen with the Fc mutant is an anti-EGFR antibody, which comprises a heavy chain shown as SEQ ID NO. 8 and a light chain shown as SEQ ID NO. 10.

2. The combination of claim 1, wherein the nucleotide sequence encoding the Fc mutant has the nucleotide sequence shown in SEQ ID NO. 2.

3. A kit, comprising:

antibodies and effector cells targeting tumor antigens with Fc mutants;

wherein the effector cells are NKT cells which are induced to expand in vitro;

the Fc mutant has an amino acid sequence shown as SEQ ID NO. 1; and

the antibody with the Fc mutant and targeting the tumor antigen is an anti-EGFR antibody, which comprises a heavy chain shown as SEQ ID NO. 8 and a light chain shown as SEQ ID NO. 10.

4. The kit of claim 3, wherein the nucleotide sequence encoding the Fc mutant has the nucleotide sequence shown in SEQ ID NO. 2.

5. Use of a combination according to claim 1 or2 or a kit according to claim 3 or 4 in the manufacture of a medicament for the treatment of lung cancer.

6. A method of making the combination of claim 1 or2 or the kit of claim 3 or 4, the method comprising the steps of:

1) preparing an anti-EGFR antibody having an Fc mutant, wherein the Fc mutant has an amino acid sequence shown as SEQ ID NO. 1, the anti-EGFR antibody comprising a heavy chain shown as SEQ ID NO. 8 and a light chain shown as SEQ ID NO. 10; and

2) expanding effector cells expressing CD16 in vitro;

wherein the effector cells are NKT cells, and the NKT cells are cultured by a method comprising the following steps:

specifically amplifying I-type NKT cells;

amplifying the type I NKT cells by using alpha-galactosylceramide (alpha-GalCer), and stimulating the proliferation of the type I NKT cells by using CD1d expression cells loaded with the alpha-galactosylceramide, and simultaneously adding cytokines IL-2 and IL-7 to assist the growth of the type I NKT cells;

secondly, further performing the quantity amplification of the I-type NKT cells and guiding the function directional differentiation;

wherein, CD1d expression cells loaded with alpha-galactosylceramide stimulate the proliferation of the type I NKT cells, IL-2, IL-7 and IL-15 are added at the same time, and IL-12 is added in a culture system 1-2 days before the culture is finished to guide the directional differentiation of the type I NKT cells;

wherein the CD1d expressing cell is selected from a dendritic cell.

Images