CN111558038A - Immune checkpoint inhibitor for cancer treatment - Google Patents
Immune checkpoint inhibitor for cancer treatment Download PDFInfo
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The present invention protects an immune checkpoint inhibitor for cancer therapy, and particularly provides a monoclonal antibody, which is a humanized monoclonal antibody, can specifically bind to ILDR2, can effectively inhibit ILDR2 immunization, and has a tumor progression inhibiting function.
Description
Technical Field
The present invention relates to the field of immune checkpoint inhibitors, in particular to an immune checkpoint inhibitor for cancer therapy.
Background
Mabs targeting co-inhibitory immune checkpoints, such as PD-1 and CTLA-4, have shown clinical activity in a variety of malignancies, including melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, MSI high colorectal cancer, Merkel cell carcinoma and hodgkin lymphoma, and have altered medical oncology practices.
In particular, immune checkpoint inhibitors have been successful in melanin therapy, with approved treatment regimens including anti-PD-1 (nivolumab and pembrolizumab), anti-CTLA-4 (ipilimumab) and anti-PD-1/CTLA-4 combination (nivolumab-ipilimumab).
At present, the experience of experts on the treatment of various malignant tumors by using the immune checkpoint inhibitor is more and more abundant, and more tumor diseases are brought into the scope of immunotherapy. Meanwhile, medical experts continuously explore the combined application mode of other tumor treatment modes and immunotherapy, and strive to further improve the treatment effect of malignant tumors. However, although immune checkpoint inhibitors may temporarily reactivate CTLs, enhancing tumor control, if effector memory T cells are damaged, the clinical response may subside, causing tumor recurrence after acquired resistance or withdrawal.
In order to solve the technical problems, the invention provides a brand-new immune checkpoint inhibitor for cancer treatment, which can be used for high-efficiency adjuvant treatment of various solid tumors and non-solid tumors without side effects so as to achieve the purposes of relieving pain, slowing disease course and/or curing.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides an immune checkpoint inhibitor for cancer treatment.
The invention is realized by the following technical scheme:
an immune checkpoint inhibitor for use in the treatment of cancer, which immune checkpoint inhibitor is an antibody targeting human ILDR 2.
Further, the functional fragment of the polypeptide comprises a light chain and a heavy chain; the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequence of CDR-L1 is shown as SEQ ID NO. 1; the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 3; the heavy chain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of CDR-H1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR-H2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR-H3 is shown as SEQ ID NO. 6.
Further, the antibody and the functional fragment thereof are anti-human ILDR2 chimeric antibody and/or an active fragment thereof.
Further, the antibody and the functional fragment thereof are anti-human ILDR2 humanized antibodies and/or active fragments thereof.
Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 8.
Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 15.
Further, the antibody comprises an amino acid sequence of any Fc-terminus selected from the group consisting of human antibodies IgG1, IgG2, IgG3, IgG 4.
Further, the step of producing the immune checkpoint inhibitor comprises: culturing a host cell containing the above antibody in a medium and under suitable culture conditions; recovering the produced antibody and active fragments thereof from the culture medium or from the cultured host cells and formulating with a pharmaceutically acceptable carrier into an immune checkpoint inhibitor.
Further, the immune checkpoint inhibitor may inhibit the progression of cancer including, but not limited to, leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
It is well known in the art that an antigen binding domain refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity of action, and that sequences recognizing one target molecule are generally unable to recognize other molecular sequences.
Representative antigen binding domains include: a variable region of an antibody, a structural variant of a variable region of an antibody, a binding domain of a receptor, a ligand binding domain, or an enzyme binding domain.
IgG is an abbreviation of Immunoglobulin G (IgG), which is a major antibody component of serum, and human IgG has four subtypes based on the r-chain antigenic difference in IgG molecules: IgG1, IgG2, IgG3, IgG 4.
The binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established and well-known techniques of the art.
Host cells of the invention include, but are not limited to, E.coli, phage display systems, yeast, plant cells, animal cells.
Pharmaceutically acceptable carriers refer to immune checkpoint inhibitor carriers that are conventional in the pharmaceutical art, including but not limited to diluents, excipients, water, and the like; including but not limited to adhesives such as gelatin and polyvinylpyrrolidone; humectants such as glycerol; including but not limited to absorption enhancers such as quaternary ammonium compounds; including but not limited to surfactants such as cetyl alcohol, sodium lauryl sulfate, and the like.
The monoclonal antibody, particularly RCA-2933, can be well specifically combined with ILDR2, can effectively inhibit ILDR2 immunity, and has the functions of inhibiting cancer progression of leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma and melanoma.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.
Example 1: obtaining novel ILDR2 chimeric antibodies
The mouse immunization uses a female BALB/c mouse with the age of 6 to 8 weeks as an experimental animal (purchased from Shanghai Jiake Biotechnology Co., Ltd.), 50 mu g of human ILDR2 protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) is used for the first immunization and is fully mixed with complete Freund's adjuvant to form emulsion, the abdominal cavity of the mouse is injected according to the injection amount of 0.5 ml/injection amount, the boosting immunization is carried out every 2 weeks, 25 mu g of human ILDR2 protein is used for the boosting immunization and is fully mixed with incomplete Freund's adjuvant to form emulsion, the abdominal cavity of the mouse is injected according to the injection amount of 0.5 ml/injection amount, the boosting immunization is carried out for 3 times, after one week of the last immunization, venous blood and serum of the mouse are taken, the titer of the antibody is measured by an ELISA method, and mouse cells with high titer are selected to.
Logarithmic growth myeloma cells (SP2/0) were harvested and washed 2 times with incomplete culture medium. Preparing immune spleen cell suspension, mixing myeloma cells and spleen cells at a ratio of 1: 10, washing with incomplete culture solution in a centrifuge tube for 1 time, centrifuging at 1200rpm for 8 min, removing supernatant, homogenizing cell precipitate, preheating in 40 deg.C water bath, adding preheated 40 deg.C water solution into the cell precipitateAdding 25ml of incomplete culture medium preheated to 40 ℃ within one minute after the PEG-40001 ml appears particles to stop the action of PEG-4000, standing at room temperature, adding 2ml of HAT culture medium, slightly blowing the precipitated cells to suspend, uniformly mixing, and supplementing HAT culture medium until the concentration of splenocytes in the centrifugal tube reaches 2 × 106And/ml, subpackaging the spleen cell suspension onto a 96-well plate, culturing and observing the growth condition of the hybridoma cells, and sucking out a supernatant sample for antibody detection when the cell area is grown to be more than 1/2 of the bottom area of the well.
Hybridoma culture supernatants were screened for anti-human ILDR2 antibodies. Specifically, in step one, a 96-well high-adsorption microplate was coated with ILDR2 (available from BIOSS, solebao technologies ltd., beijing) in an amount of 100 μ L per well using a carbonate buffer solution, and then washed 3 times with the buffer solution. And step two, blocking by using a buffer solution containing 1% BSA, incubating for 1 hour at 25 ℃, blocking the sample at the blocking amount of 300 mu L/hole, washing 3 times by using the buffer solution after the incubation is finished, respectively adding 100 mu L of a supernatant sample (S1-S85) and positive serum (control, CK1-5) into a hole 1-90, incubating for 1 hour at 25 ℃, and washing 5 times by using the buffer solution. Step three, adding 100 μ L of anti-mouse IgG antibody diluted in 1/10000 ratio in buffer containing 1% BSA to each well, wherein the anti-mouse IgG antibody is labeled with horseradish peroxidase, incubating for 1 hour at 25 ℃, and washing 5 times with the buffer. And step four, adding 100 mu L of colorimetric substrate 3,3',5,5' -Tetramethylbenzidine (TMB) into each well, carrying out color development at 30 ℃ for 10min, then stopping the color development reaction, reading the absorbance at 450nm on an enzyme labeling instrument, and selecting a positive clone capable of secreting the human ILDR2 binding antibody according to the strength of OD450 nm.
The clones obtained by screening and having both the antigen-binding activity and the antigen-neutralizing activity were subjected to the measurement of the antibody DNA sequence. Cellular mRNA was first extracted using the RNAprep Pure kit (Tiangen) according to the instructions. First strand cDNA was then synthesized using the QuantScript RT kit (Tiangen). And (3) using the cDNA first chain generated by reverse transcription for subsequent PCR reaction, cloning a target band obtained by PCR amplification into a pGEM-T vector, selecting a single clone for DNA sequencing, and completing the sequencing by Nanjing Kingsler Biotech Co.
Obtaining the variable region of the antibody light chain and the antibody heavy chain by PCR amplificationA variable region, which is obtained by excluding the sequence of the skeleton region to obtain the sequence of the complementarity determining region, wherein the amino acid sequence of three complementarity determining regions CDR-L1 of the light chain is shown in SEQ ID NO:1, the amino acid sequence of CDR-L2 is shown in SEQ ID NO:2 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:3, the amino acid sequence of three complementarity determining regions CDR-H1 of the heavy chain is shown in SEQ ID NO:4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO:5 and the amino acid sequence of CDR-H3 is shown in SEQ ID NO:6, the amino acid sequence of the constant region of the antibody light chain is obtained by connecting mouse IgVH 7-21, the sequence of the constant region of the heavy chain of the antibody is mouse IgVH2-09, the sequence of the constant region of the antibody is obtained by connecting the variable region of the light chain with the constant region of the light chain of the antibody, the sequence of the full length of the heavy chain is obtained by connecting the variable region of the heavy chain with the constant region of the heavy chain of the antibody, the variable region of the constant region of the heavy chain, the variable region of the heavy chain is cloned to the eukaryotic expression vector of the cell 713 ET 713-pEN 1, the cell transfection of5cells/mL. Plasmid was added to a final concentration of 42.13. mu.g/mL and linear polyethyleneimine was added to a final concentration of 60. mu.g/mL, according to the transfection volume. And (3) putting the mixture into a cell culture box at 120rpm and 37 ℃ for culturing for 1 hour, then adding a fresh culture medium into the culture solution until the final volume is 20 times of the transfection volume, continuing culturing for 5-6 days, and collecting supernatant.
Example 2: detection of anti-human ILDR2 chimeric monoclonal antibody
The kinetic constants of the Anti-human ILDR2 chimeric monoclonal antibody (Anti-ILDR 2-M) obtained in example 1 and the binding of the Anti-human ILDR2 chimeric monoclonal antibody to the antigen thereof were measured, and the binding and dissociation between the molecule coated on the biochip and the molecule to be detected was measured by the instrument optical surface plasmon resonance technique. Specifically, Anti-ILDR2-M was dissolved in sodium acetate buffer (pH 5.0) and coupled to CM chips, followed by blocking with 1M ethanolamine. Different concentrations of Anti-ILDR2-M were injected at 30. mu.L/min for 3min during the binding phase and 30. mu.L/min for 10min during the dissociation phase, and the binding and dissociation kinetic constants were calculated by analysis using Biacore3000 software. The binding kinetic constants, dissociation kinetic constants and dissociation equilibrium constants of Anti-ILDR2-M are shown in Table 1.
TABLE 1 kinetic constants for Anti-ILDR2-M binding to its antigen
In vivo neutralization activity of Anti-ILDR2-M was determined. Specifically, female BALB/c mice (purchased from Guangdong province medical laboratory animal center) of 6-8 weeks old were selected. Each group was randomized into three groups of 10, each group being administered Anti-ILDR2-M (intravenous, single dose) at 3 three dose levels of 0.5nmol/kg,5nmol/kg, 50 nmol/kg. One hour after administration, 10 μ g of ILDR2 was subcutaneously injected into each mouse, 2 hours after injection, each mouse was subjected to orbital bleeding without anticoagulation, the blood was left to clot at room temperature for about 45 minutes, serum samples were obtained by centrifugation, and the concentration of mouse CCK in serum was measured by ELISA kit (CCK) according to the instructions, and the results showed that Anti-ILDR2-M was able to inhibit the level of CCK secreted from the mouse by human ILDR2 and substantially reduce the level of mouse CCK to an unstimulated state.
Determination of the pharmacokinetic studies of Anti-ILDR2-M in rats, specifically, 6-8 week old female SD rats (purchased from Guangdong provincial animal center for medical laboratory). Three groups of 5 were randomized and 10nmol/kg anti-ILDR2-M (intravenous, single dose) was administered to each group. At 0 point, 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours and 264 hours after administration, the orbital blood collection is not anticoagulated, the blood sample is placed at room temperature for 45 minutes until the blood coagulation, the blood sample is obtained by centrifugation, and the blood sample is frozen at-80 ℃ for testing. The Anti-ILDR2-M in serum is measured by ELISA, and the test result shows that the pharmacokinetic parameters of the Anti-ILDR2-M with the dose of 10nmol/kg for a single intravenous injection are as follows: half-life t1/2 is 512 hours; the area under the curve AUClast is 52139 nM.hr; zero concentration C0 was estimated to be 496 nM; the apparent distribution volume Vd is 121 mL/Kg; the clearance rate CL is 0.193 mL/hr/kg; the average residence time MRTlast is 168 hours.
Example 3: preparation of novel humanized ILDR2 antibody
The humanized Anti-human ILDR2 antibody is prepared by referring to the preparation method of molecular Immunol, selecting a humanized template which is best matched with Anti-ILDR2-M non-CDR region from a Germine database, wherein the template of a heavy chain variable region is human IgVH4-28 a 03, the template of a light chain variable region is human IGKV1-16 a 02, transplanting a mouse antibody CDR region to the selected humanized template, replacing the CDR region of the human template to obtain a humanized antibody heavy chain variable region, and the amino acid sequence is shown as SEQ ID NO. 7, so as to obtain the humanized antibody light chain variable region which is shown as SEQ ID NO. 8. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) obtained by selecting suitable sites for back mutation by sequence alignment are shown in table 2.
TABLE 2 heavy chain variable region amino acid sequence and light chain variable region amino acid sequence
| VH | SEQ ID NO |
| ORI | SEQ ID NO:7 |
| RCA-2931 | SEQ ID NO:9 |
| RCA-2932 | SEQ ID NO:10 |
| RCA-2933 | SEQ ID NO:11 |
| RCA-2934 | SEQ ID NO:12 |
| VL | SEQ ID NO |
| ORI | SEQ ID NO:8 |
| RCA-2931 | SEQ ID NO:13 |
| RCA-2932 | SEQ ID NO:14 |
| RCA-2933 | SEQ ID NO:15 |
| RCA-2934 | SEQ ID NO:16 |
The heavy chain variable region (SEQ ID NO: 9-12) of the humanized anti-human ILDR2 monoclonal antibody was ligated to the human antibody IgG1 heavy chain constant region (SEQ ID NO:17) to obtain the corresponding heavy chain full-length sequences, respectively. The light chain variable region (SEQ ID NO: 13-16) of the humanized anti-human ILDR2 monoclonal antibody is connected with the constant region (SEQ ID NO:18) of the human antibody Kappa light chain to respectively obtain corresponding light chain full-length sequences, all the heavy chain full-length sequences and the light chain full-length sequences are combined to obtain the humanized antibody full-length sequence, and the humanized antibody full-length sequence is connected into an LRET-713 (carrier framework pEGFP-N1) carrier through enzyme digestion.
Example 4: detection of humanized ILDR2 monoclonal antibody
Detection example 3 the kinetic constants of the binding of the humanized ILDR2 monoclonal antibody (test group numbers: ORI, RCA-2931, RCA-2932, RCA-2933, RCA-2934) and its ILDR2 antigen were obtained, and the binding and dissociation between the molecules coupled and coated on the biochip and the molecules to be detected were detected by the instrument optical surface plasmon resonance technique. Specifically, the humanized ILDR2 mabs to be detected were separately dissolved in sodium acetate buffer solution (pH 5.0) and injected to couple to CM chips, followed by blocking with 1M ethanolamine. In the binding phase, different test groups of humanized ILDR2 mab were injected at 30 μ L/min for 3min, in the dissociation phase, at 30 μ L/min for 10min with PBS buffer, and the binding and dissociation kinetic constants were calculated by analysis using Biacore3000 software. The binding kinetic constants, dissociation kinetic constants and dissociation equilibrium constants of the humanized ILDR2 mab are shown in table 3.
TABLE 3 kinetic constants for binding of humanized ILDR2 mAb to its antigen
Determination of pharmacokinetic studies of humanized ILDR2 mab in rats, specifically, 6-8 week old female SD rats (purchased from central laboratory animals in Guangdong province). Randomly divided into five groups (test group 1, test group 2, test group 3, test group 4, test group 5, 10 per group), test group 1 was given 10nmol/kg RCA-2931 (intravenous, single administration); test group 2 was given 10nmol/kg RCA-2932 (intravenous, single administration); test group 3 was given 10nmol/kg RCA-2933 (intravenous, single administration); test group 4 was given 10nmol/kg RCA-2934 (intravenous, single administration); test group 5 was given 10nmol/kg Anti-ILDR2-M (intravenous, single dose). At 0 point, 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours and 264 hours after administration, the orbital blood collection is not anticoagulated, the blood sample is placed at room temperature for 45 minutes until the blood coagulation, the blood sample is obtained by centrifugation, and the blood sample is frozen at-80 ℃ for testing.
The pharmacokinetic parameters for Anti-ILDR2-M (ORI group) at a dose of 10nmol/kg for a single intravenous injection were as follows: half life t1/2The area under the drug-hour curve AUC is 512 hourslast52139nM hr, estimated zero concentration C0 of 496nM, apparent volume Vd of 121mL/Kg, clearance CL of 0.193 mL/mL-hr/kg, mean residence time MRTlastWas 168 hours.
The pharmacokinetic parameters for RCA-2931 at a dose of 10nmol/kg for a single intravenous injection were as follows: half life t1/2Area under the curve AUC at 487 hrlastHr is 46139nM, estimated zero concentration C0 is 486nM, apparent volume of distribution Vd is 119mL/Kg, clearance CL is 0.181mL/hr/Kg, average residence time MRTlastWas 176 hours.
The pharmacokinetic parameters for RCA-2932 at a dose of 10nmol/kg for a single intravenous injection were as follows: half life t1/2Area under the time curve AUC at 502 hourslastAt 51132nM. hr, estimated zero concentration C0 of 523nM, apparent volume of distribution Vd of 124mL/Kg, clearance CL of 0.197mL/hr/Kg, average residence time MRTlastWas 201 hours.
The pharmacokinetic parameters for RCA-2933 at a dose of 10nmol/kg for a single intravenous injection were as follows: half life t1/2Area under the drug time curve AUC at 462 hourslastHr is 47141nM, estimated zero concentration C0 is 456nM, apparent volume of distribution Vd is 101mL/Kg, clearance CL is 0.173mL/hr/Kg, average residence time MRTlastIt was 179 hours.
The pharmacokinetic parameters for RCA-2934 at a dose of 10nmol/kg for a single intravenous injection were as follows: half life t1/2AUC of 490 hours, area under the time-of-drug curvelastAt 48593nM. hr, estimated zero concentration C0 of 398nM, apparent volume of distribution Vd of 116mL/Kg, clearance CL of 0.177mL/hr/Kg, mean residence time MRTlastIt was 181 hours.
The results show that the humanized ILDR2 monoclonal antibody has strong binding capacity with the antigen, wherein the test groups RCA-2933 and RCA-2934 have more outstanding binding capacity with the antigen, the binding capacity of RCA-2931 and RCA-2932 is weaker than that of RCA-2933 and RCA-2934 but better than that of ORI lease, and the pharmacokinetics of the humanized ILDR2 monoclonal antibody in a rat is close to that of a chimeric antibody.
Example 5 study of the drug Effect of humanized ILDR2 monoclonal antibody
Growth inhibition of humanized monoclonal antibody (RCA-2933) against human ILDR2 on mouse tumor grafts was examined. Experimental material human 8-week-old female mice (C57BL/6 background,supplied by Beijing Baiosai map Gene Biotechnology Co., Ltd.). After the mice are adapted to the environment, 9 mice are taken, H22 tumor cells are injected into the right armpit of each mouse, the injection part is cancerated, the tumor growth of the mice is obvious, the slicing result proves that the test is successful, the model construction is completed, the mice are loaded with the tumor, the tumor growth is observed, the weight is recorded, the mice are divided into 3 groups according to the tumor volume, S1, S2 and S3 (the volume is maximum and is approximately equal to 150 mm)3) (ii) a S4, S5, S6 (volume medium, equal to 120mm or so)3) (ii) a S7, S8, S9 (volume medium, equal to 100mm or so)3) (ii) a S3, S6 and S9 are set as a vehicle control group (equal volume of physiological saline is injected), the dosage of S1, S4 and S7 is 50nmol/kg, and the dosage of S2, S5 and S8 is 20nmol/kg, and the medicines are intraperitoneally injected, 2 times a week and continuously administered for 4 weeks. From the day of administration, tumor volume was measured 1 time per week, and its major axis a and minor axis b were measured, and the tumor volume calculation formula was: tumor volume ═ a x b2) The results are shown in Table 4.
TABLE 4 tumor volume determination table
As can be seen from table 4, the anti-human ILDR2 humanized monoclonal antibody (RCA-2933) has anti-tumor activity, and significantly inhibits the growth of mouse transplanted tumor inoculated with H22 tumor cells, wherein the RCA-2931 has more significant inhibitory effect against large or medium volume tumors.
Growth inhibition of humanized monoclonal antibody (RCA-2932) against human ILDR2 on mouse tumor grafts was examined. The experimental material was selected from female mice of 8 weeks old (C57BL/6 background, supplied by Beijing Baiosai Tokyo Gene Biotechnology Co., Ltd.). After the mice are adapted to the environment, 9 mice are taken, H22 tumor cells are injected into the right armpit of each mouse, the injection part is cancerated, the tumor growth of the mice is obvious, the slicing result proves that the test is successful, the model construction is completed, the mice are loaded with the tumor, the tumor growth is observed, the weight is recorded, the mice are divided into 3 groups according to the tumor volume, A1, A2 and S3 (the volume is maximum and is approximately equal to 150 mm)3) (ii) a A4, A5, S6 (medium in volume, equal to about 120 mm)3) (ii) a A7, A8, S9 (medium in volume, equal to or about 100 mm)3) (ii) a S3, S6 and S9 are set as a vehicle control group (equal volume of physiological saline is injected), the dosage of A1, A4 and A7 is 50nmol/kg, and the dosage of A2, A5 and A8 is 20nmol/kg, and the medicines are intraperitoneally injected and are administered for 2 times a week and continuously administered for 4 weeks. From the day of administration, tumor volume was measured 1 time per week, and its major axis a and minor axis b were measured, and the tumor volume calculation formula was: tumor volume ═ a x b2) The results are shown in Table 5.
TABLE 5 tumor volume determination table
As can be seen from table 4, the anti-human ILDR2 humanized monoclonal antibody (RCA-2932) has anti-tumor activity, and significantly inhibits the growth of mouse graft tumor inoculated with H22 tumor cells, wherein the RCA-2932 has a more significant inhibitory effect against tumors of medium volume or small volume.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
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115 120
<210>8
<211>113
<212>PRT
<213>Homo sapiens
<400>8
Glu Ile Val Leu Thr Gln Ser Pro Arg Ile Thr Ala Cys Arg Asp Leu
1 5 10 15
Ile Tyr Leu Ala Ser Tyr Leu Glu Cys Gln Ala Gln Arg Val Lys His
20 25 30
Gly Arg Cys His Ala Gln Trp Tyr Gln Ala Arg Lys Thr Gln Asn Leu
35 40 45
Leu His Arg Glu Ile Trp Gln Trp Glu His Thr Ala Arg Ser Glu Ile
50 55 60
Ile His Phe Ala Asn Met Pro Lys Val Glu Ile Lys Arg Thr Tyr Thr
65 70 75 80
Asn Ala Val Cys Tyr Asn Glu Val Ser Cys Gln Gly Val Cys Ala Arg
85 90 95
Phe Ser Gly Asn Glu Leu Trp Asp Gln Ala Cys Asp Gly Ser Tyr His
100 105 110
Arg
<210>9
<211>122
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VH RCA-2931
<400>9
Gln Val Ala Trp Val Gln Ile Gln Leu Pro Lys Trp Ser Ile Val Ser
1 5 10 15
Asp Lys Phe Tyr Ser Cys Thr Asp Tyr Cys Asn Ser Asp Arg Tyr Met
20 25 30
Glu Met Trp Glu Trp Val Phe Ala Leu Glu His Gly Gln Ala Asn His
35 40 45
Phe Cys Asp Glu Phe Val Trp Gln Arg Leu Glu Trp Ile Gly Thr Ile
50 55 60
Thr Met Arg Asp Gly Arg Asn Ser Ser Asp Ala Arg Ser Arg Val Gly
65 70 75 80
Thr Lys Ala Trp Ile Ile Gly Trp Pro Val Gly Trp Cys Trp Ala Trp
85 90 95
Gly Ala Gly Ser Ser Leu Tyr Ser Asp Arg Asn Asp Pro Met Trp Ala
100 105 110
Cys Ala Arg Val Thr Val Ser Ser Phe Ser
115 120
<210>10
<211>122
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VH RCA-2932
<400>10
Gln Val Ala Trp Val Gln Ala Trp Leu Pro Lys Trp Ser Ile Val Ser
1 5 10 15
Asp Lys Phe Tyr Ser Asn Thr Asp Tyr Cys Asn Ser Asp Arg Tyr Met
20 2530
Glu Met Trp Glu Trp Val Phe Ala Leu Glu His Gly Gln Ala Asn His
35 40 45
Phe Cys Asp Glu Phe Val Trp Gln Arg Leu Glu Trp Ile Gly Thr Ile
50 55 60
Thr Met Arg Asp Gly Arg Asn Asn Ser Asp Ala Arg Ser Arg Val Gly
65 70 75 80
Thr Lys Ala Ala Ile Ser Gly Trp Pro Val Gly Trp Cys Trp Ala Trp
85 90 95
Gly Ala Gly Ser Ser Leu Arg Ser Asp Arg Asn Asp Pro Met Trp Ala
100 105 110
Cys Ala Arg Val Thr Val Ser Ser Phe Ser
115 120
<210>11
<211>122
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VH RCA-2933
<400>11
Gln Val Ala Trp Val Gln Trp Gln Leu Pro Lys Trp Ser Ile Arg Ser
1 5 10 15
Asp Lys Phe Tyr Ser Cys Thr Asp Tyr Cys Asn Ser Asp Arg Tyr Met
20 25 30
Glu Met Trp Glu Trp Val Phe Ala Leu Glu Leu Gly Gln Ala Asn His
35 40 45
Phe Cys Asp His Phe Val Trp Gln Arg Leu Glu Trp Ile Gly Thr Ile
50 55 60
Thr Met Arg Asp Gly Arg Asn Asn Ser Asp Ala Arg Ser Arg Val Gly
65 70 75 80
Thr Lys Ala Ala Ile Ile Gly Trp Pro Val Gly Trp Cys Glu Ala Trp
85 90 95
Gly Ala Gly Ser Ser Leu Arg Ser Asp Arg Asn Asp Pro Met Trp Ala
100 105 110
Cys Ala Arg Val Thr Val Ser Ser Phe Ser
115 120
<210>12
<211>122
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VH RCA-2934
<400>12
Gln Val Ala Trp Ser Gln Trp Gln Leu Pro Lys Trp Ser Ile Val Ser
1 5 10 15
AspLys Phe Tyr Ser Cys Thr Asp Tyr Cys Asn Ser Asp Arg Leu Met
20 25 30
Glu Met Trp Glu Trp Val Phe Ala Leu Glu His Gly Gln Ala Asn His
35 40 45
Phe Cys Asp Glu Phe Val Trp Gln Arg Leu Glu Trp Ile Gly Thr Ile
50 55 60
Thr Met Arg Asp Gly Arg Asn Asn Ser Met Ala Arg Ser Arg Val Gly
65 70 75 80
Thr Lys Ala Ala Ile Ile Gly Trp Pro Val Gly Trp Cys Trp Ala Trp
85 90 95
Gly Ala Gly Ser Tyr Leu Arg Ser Asp Arg Asn Asp Pro Met Trp Ala
100 105 110
Cys Ala Arg Val Thr Val Ser Ser Phe Ser
115 120
<210>13
<211>113
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VL RCA-2931
<400>13
Glu Ile Val Leu Thr Trp Ser Pro Arg Ile Thr Ala Cys Arg Asp Leu
1 510 15
Ile Tyr Leu Ala Ser Tyr Leu Glu Cys Gln Ala Gln Arg Val Lys His
20 25 30
Gly Arg Cys His Ala Gln Trp Ala Tyr Ala Arg Lys Thr Gln Asn Leu
35 40 45
Leu His Arg Glu Ile Trp Gln Trp Glu His Thr Ala Arg Ser Glu Ile
50 55 60
Ile His Phe Ala Asn Met Pro Lys Val Glu Ile Lys Arg Thr Ala Thr
65 70 75 80
Asn Ala Val Cys Tyr Asn Glu Val Ser Cys Gln Gly Val Cys Ala Arg
85 90 95
Phe Ser Gly Asn Glu Leu Trp Asp Gln Ala Cys Asp Gly Ser Tyr His
100 105 110
Arg
<210>14
<211>113
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VL RCA-2932
<400>14
Glu Ile Val Leu Thr Gln Ser Pro Arg Ile Thr Ala Cys Arg Asp Leu
1 5 10 15
Ile Tyr Leu Ala Ser Tyr Leu Glu Cys Gln Ala Gln Arg Val Lys His
20 25 30
Gly Arg Cys His Ala Gln Gln Tyr Gln Ala Arg Lys Thr Gln Asn Leu
35 40 45
Leu His Arg Glu Ile Trp Gln Trp Glu His Thr Ala Arg Ile Glu Ile
50 55 60
Ile His Phe Ala Asn Met Pro Lys Val Glu Ile Lys Arg Thr Tyr Thr
65 70 75 80
Asn Glu Val Cys Tyr Asn Glu Val Ser Cys Gln Gly Val Cys Ala Arg
85 90 95
Phe Ser Gly Asn Glu Leu Trp Asp Gln Ala Cys Asp Gly Cys Tyr His
100 105 110
Arg
<210>15
<211>113
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VL RCA-2933
<400>15
Glu Ile Val Leu Thr Gln Ser Pro Glu Ile Thr Ala Cys Arg Asp Leu
1 5 10 15
Ile Tyr Leu Ala SerTyr Leu Glu Cys Gln Ala Gln Arg Val Lys His
20 25 30
Gly Arg Cys His Ala Gln Trp Tyr Leu Ala Arg Gln Thr Gln Asn Leu
35 40 45
Leu His Arg Glu Ile Trp Gln Trp Glu His Thr Ala Arg Ser Glu Ile
50 55 60
Ile His Phe Ala Asn Met Pro Lys Val Glu Ile Lys Arg Thr Tyr Thr
65 70 75 80
Asn Ala Ile Cys Tyr Asn Glu Val Ser Cys Gln Gly Val Cys Ala Arg
85 90 95
Phe Ser Gly Asn Glu Leu Trp Asp Gln Ala Cys Asp Gly Ser Tyr His
100 105 110
Arg
<210>16
<211>113
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>UNSURE
<223>VL RCA-2934
<400>16
Glu Ile Val Leu Thr Gln Ser Ala Arg Ile Thr Ala Cys Arg Asp Leu
1 5 10 15
Ile Tyr Leu Ala Ser Tyr Leu Glu Cys Gln Ala Gln Arg Val Lys His
20 25 30
Gly Arg Cys His Ala Gln Trp Tyr Gln Ala Arg Lys Thr Gln Asn Leu
35 40 45
Leu His Arg Glu Ile Trp Gln Trp Glu Thr Thr Ala Arg Ser Glu Ile
50 55 60
Ile His Phe Ala Asn Met Pro Lys Val Glu Ile Lys Arg Thr Tyr Thr
65 70 75 80
Asn Ala Val Leu Tyr Asn Glu Val Ser Cys Gln Gly Val Cys Ala Arg
85 90 95
Phe Ser Gly Asn Glu Leu Trp Asp Gln Ala Cys Asp Gly Tyr Tyr His
100 105 110
Arg
Claims (9)
1.An immune checkpoint inhibitor for use in the treatment of cancer, wherein the immune checkpoint inhibitor is an antibody targeting human ILDR 2.
2. The immune checkpoint inhibitor of claim 1 wherein the functional polypeptide fragment comprises a light chain and a heavy chain; the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequence of CDR-L1 is shown as SEQ ID NO. 1; the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 3; the heavy chain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of CDR-H1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR-H2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR-H3 is shown as SEQ ID NO. 6.
3. The immune checkpoint inhibitor as claimed in claim 1 wherein the antibody and functional fragments thereof are anti-human ILDR2 chimeric antibodies and/or active fragments thereof.
4. The immune checkpoint inhibitor as claimed in claim 1 wherein the antibody and functional fragments thereof are anti-human ILDR2 humanized antibodies and/or active fragments thereof.
5. The immune checkpoint inhibitor as claimed in claim 1 wherein the antibody heavy chain variable region amino acid sequence is shown as SEQ ID No. 7 and the antibody light chain variable region amino acid sequence is shown as SEQ ID No. 8.
6. The immune checkpoint inhibitor as claimed in claim 1 wherein the antibody heavy chain variable region amino acid sequence is shown as SEQ ID No. 11 and the antibody light chain variable region amino acid sequence is shown as SEQ ID No. 15.
7. The immune checkpoint inhibitor of claim 1 wherein the antibody comprises an amino acid sequence at either Fc terminus selected from the group consisting of human antibodies IgG1, IgG2, IgG3, IgG 4.
8. The immune checkpoint inhibitor according to claim 1 wherein the step of producing the immune checkpoint inhibitor comprises: culturing a host cell comprising the antibody of claim 6 or claim 7 in a culture medium and under suitable culture conditions; recovering the produced antibody and active fragments thereof from the culture medium or from the cultured host cells and formulating with a pharmaceutically acceptable carrier into an immune checkpoint inhibitor.
9. The immune checkpoint inhibitor as claimed in claim 1 wherein the immune checkpoint inhibitor may inhibit the progression of cancer including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
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| CN111574626A (en) * | 2020-05-27 | 2020-08-25 | 杭州伽南企业管理有限公司 | ILDR2 antibody, and pharmaceutical composition and application thereof |
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