Pharmaceutical Combination and Use Thereof

Abstract

The present disclosure relates to a novel pharmaceutical combination and a use thereof. The pharmaceutical combination can be used to treat cancer, such as colon cancer.

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Claims

1. A pharmaceutical combination comprising a substance A and a substance B; wherein the substance A is Compound F, a crystal form thereof, a pharmaceutically acceptable salt thereof or a solvate thereof; ##STR00005## the substance B is an antibody M comprising a heavy chain CDR amino acid sequence selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, or an antigen binding fragment thereof.

2. The pharmaceutical combination as defined in claim 1, wherein the antibody M comprises: (a) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 1, a HCDR2 amino acid sequence of SEQ ID NO: 3, and a HCDR3 amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 2, a LCDR2 amino acid sequence of SEQ ID NO: 4, and a LCDR3 amino acid sequence of SEQ ID NO: 6; (b) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 7, a HCDR2 amino acid sequence of SEQ ID NO: 9, and a HCDR3 amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 8, a LCDR2 amino acid sequence of SEQ ID NO: 10, and a LCDR3 amino acid sequence of SEQ ID NO: 12; (c) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 13, a HCDR2 amino acid sequence of SEQ ID NO: 15, and a HCDR3 amino acid sequence of SEQ ID NO: 17; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 14, a LCDR2 amino acid sequence of SEQ ID NO: 16, and a LCDR3 amino acid sequence of SEQ ID NO: 18; or (d) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 19, a HCDR2 amino acid sequence of SEQ ID NO: 21, and a HCDR3 amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 8, a LCDR2 amino acid sequence of SEQ ID NO: 10, and a LCDR3 amino acid sequence of SEQ ID NO: 12; optionally, the antibody M comprises a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 7, a HCDR2 amino acid sequence of SEQ ID NO: 9, and a HCDR3 amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 8, a LCDR2 amino acid sequence of SEQ ID NO: 10, and a LCDR3 amino acid sequence of SEQ ID NO: 12.

3. The pharmaceutical combination as defined in claim 1 or 2, wherein the antibody M comprises: (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 22; (b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 25; (c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 26; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 27; or (d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 25; optionally, the antibody M comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 25.

4. The pharmaceutical combination as defined in any one of claims 1-3, wherein the antibody M comprises a heavy chain constant region of human IgG4, and a light chain constant region of human λ or κ light chain; and/or, the substance B is the antibody M; and/or, the substance A is the mesylate of Compound F; and/or, the pharmaceutical combination further comprises a pharmaceutical excipient.

5. The pharmaceutical combination as defined in any one of claims 1-4, wherein the pharmaceutical combination is in the form of a fixed combination or a non-fixed combination, optionally, in the form of a non-fixed combination.

6. A pharmaceutical composition comprising a substance A and a substance B; wherein the substance A and the substance B are as defined in any one of claims 1-4.

7. A kit comprising: a first container comprising a first pharmaceutical composition comprising a substance A; and a second container comprising a second pharmaceutical composition comprising a substance B; wherein the substance A and the substance B are as defined in any one of claims 1-4.

8. A method for treating a cancer comprising administering the pharmaceutical combination as defined in any one of claims 1-5 or the pharmaceutical composition as defined in claim 6.

9. The method as defined in claim 8, wherein the cancer is non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas or myelomas; optionally, the cancer is colon cancer; and/or, the substance A and the substance B are administrated simultaneously or separately, optionally, administrated separately; and/or, the substance A is administrated orally; and/or, the substance B is administrated by injection.

10. A pharmaceutical combination comprising a substance A and a substance C; wherein the substance A is Compound F, a crystal form thereof, a pharmaceutically acceptable salt thereof or a solvate thereof; ##STR00006## the substance C is an antibody N comprising a CDR amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or an antigen binding fragment thereof.

11. The pharmaceutical combination as defined in claim 10, wherein the antibody N comprises: (a) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 41; and (b) a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45 and 46, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50 and 51.

12. The pharmaceutical combination as defined in claim 10 or 11, wherein the antibody N comprises: (a) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising a LCDR1 amino acid sequence of SEQ ID NO: 42, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 42, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (c) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 43, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (d) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 44, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (e) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 45, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (f) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 44, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (g) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 45, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49; (h) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 45, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 50; (i) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 46, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 51; or (j) a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 46, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 48; optionally, the antibody N comprises a heavy chain variable region comprising a HCDR1 amino acid sequence of SEQ ID NO: 38, a HCDR2 amino acid sequence of SEQ ID NO: 39, and a HCDR3 amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a LCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 42, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 49.

13. The pharmaceutical combination as defined in any one of claims 10-12, wherein the antibody N comprises: (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31; (b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31; (c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 32; (d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 33; (e) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 34; (f) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 33; (g) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 34; (h) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 35; (i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 36; or (j) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 37; optionally, the antibody N comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31.

14. The pharmaceutical combination as defined in any one of claims 10-13, wherein the antibody N further comprises a heavy chain constant region of human IgG4, and a light chain constant region of human λ or κ light chain; and/or, the substance C is the antibody N; and/or, the substance A is the mesylate of Compound F; and/or, the pharmaceutical combination further comprises a pharmaceutical excipient.

15. The pharmaceutical combination as defined in any one of claims 10-14, wherein the pharmaceutical combination is in the form of a fixed combination or a non-fixed combination, optionally, in the form of a non-fixed combination.

16. A pharmaceutical composition comprising a substance A and a substance C; wherein the substance A and the substance C are as defined in any one of claims 10-14.

17. A kit comprising: a first container comprising a first pharmaceutical composition comprising a substance A; and a second container comprising a second pharmaceutical composition comprising a substance C; wherein the substance A and the substance C are as defined in any one of claims 10-14.

18. A method for treating a cancer comprising administering the pharmaceutical combination as defined in any one of claims 10-15 or the pharmaceutical composition as defined in claim 16.

19. The method as defined in claim 18, wherein the cancer is non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas or myelomas; optionally, the cancer is colon cancer; and/or, the substance A and the substance C are administrated simultaneously or separately, optionally, administrated separately; and/or, the substance A is administrated orally; and/or, the substance C is administrated by injection.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0202] The following examples further illustrate the invention, but the invention is not limited thereto.

Embodiment 1: Pharmacodynamic Study of Antibody 1.14.4 Monotherapy or in Combination with Lenvatinib Mesylate on B-hPD-1 Humanized Mouse MC38-hPD-L1 Colon Cancer Animal Model

[0203] 1. Objective of the Study

[0204] The objective of the study was to evaluate the therapeutic efficacy of antibody 1.14.4 monotherapy or in combination with Lenvatinib mesylate in the treatment of subcutaneous MC38-hPD-L1 transplanted tumor.

[0205] 2. Reagents and Equipment

TABLE-US-00005 TABLE 5 Reagents and equipment Reagents or Storage Physical equipment Supplier Art. No condition Lot. No form hIgG4 CROWN C0004-4 — AB170091 5.1 mg/mL BIO- solution in SCIENCE physio- INC logical TAICANG saline Lenvatinib Eisai Co., — — 164H0501 powder mesylate Ltd. DMEM Cellgro 10-013-  4° C. — — CVR FBS Excell FND500- −20° C. — — 120820 Vernier Hexagon 00534220 — — — caliper Metrology Physio- — — — — — logical saline

[0206] 3. Test Sample

[0207] Antibody 1.14.4 was prepared according to WO2017020858A1 (Examples 1-3).

[0208] 4. Formulation

[0209] 4.1. Formulation of hIgG4

[0210] 5.1 mg/mL solution of hIgG4 in physiological saline was diluted with physiological saline to a concentration of 0.3 mg/mL for use. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to room temperature before use.

[0211] 4.2. Formulation of Different Concentration of Lenvatinib Mesylate

[0212] Lenvatinib mesylate powder was dissolved in 3 mmol/L HCl solution to prepare 3 mg/mL stock solution of Lenvatinib mesylate. The stock solution was stored at 4° C. when not in use.

[0213] 1 mg/mL Lenvatinib mesylate solution was prepared by diluting 2.24 mL of 3 mg/mL stock solution with 4.48 mL of 3 mmol/L HCl solution. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to room temperature before use.

[0214] 0.3 mg/mL Lenvatinib mesylate solution was prepared by diluting 1.92 mL of 1 mg/mL Lenvatinib mesylate solution with 4.48 mL of 3 mmol/L HCl solution. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to room temperature before use.

[0215] 0.1 mg/mL Lenvatinib mesylate solution was prepared by diluting 1.6 mL of 0.3 mg/mL Lenvatinib mesylate solution with 3.2 mL of 3 mmol/L HCl solution. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to room temperature before use.

[0216] 4.3. Formulation of Antibody 1.14.4

[0217] Antibody 1.14.4 was diluted with physiological saline to prepare 0.3 mg/mL antibody 1.14.4 solution for use. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to room temperature before use.

[0218] 5. Animals

[0219] Species: Mus musculus;

[0220] Strain: C57BL/6;

[0221] Name: B-hPD-1 mice;

[0222] Sex: male;

[0223] Body weight: 17-21 g

[0224] Age: 7 weeks;

[0225] Number of animals: 85 (56 plus 29 spare) mice;

[0226] Animal supplier: Biocytogen Jiangsu Co., Ltd;

[0227] License number: SCXK (Jiangsu) 2016-0004;

[0228] Animal Certificate No.: 201727553.

[0229] 6. Animal Housing and Care

[0230] Animals were housed in the SPF animal laboratory of Experiment Animal Center in Biocytogen Beijing Co., Ltd using individually ventilated cages (IVC). Animals were acclimated to the environment for three days prior to initiate the study.

[0231] Temperature: 20-26° C.;

[0232] Humidity: 40%-70%;

[0233] Light: 12 hours on and 12 hours off;

[0234] Cage: Cage was made of PPSU with a volume of 320 mm×200 mm×135 mm. Corn cobs were used for animal bedding, which were sterilized by autoclave before use. Beddings were changed once a week. Each cage was identified by a cage card marking the number of animals, gender, strain, receiving date, group number, and starting date of the experiment.

[0235] Diet and water: Reverse osmosis (RO) water was autoclaved before use. Animals had free access to SPF mouse feed and sterile drinking water.

[0236] Animal identification: Each mice was identified by ear tag.

[0237] 7. Experimental Procedure

[0238] 7.1. Cell Culture

[0239] Mouse colon cancer cells MC38 were purchased from Shun Ran (Shanghai) Biotechnology Co., Ltd., and the cells were cultured in a 37° C., 5% CO.sub.2 incubator supplemented with Dulbecco's Modified Eagle's medium containing 10% inactivated fetal bovine serum. MC38 cells were genetically modified to overexpress human PD-L1 and knock out murine PD-L1, the modified cells were named as MC38-hPD-L1 cells.

[0240] 7.2. Tumor Inoculation, Group and Dosage Regimen

[0241] MC38-hPD-L1 tumor cells were resuspended in 1×PBS to a concentration of 5×10.sup.6 cells/mL. Each of the B-hPD-1 humanized mice was subcutaneously inoculated at the right lateral thorax region with 0.1 mL of the cell suspension, and a total of 85 mice were inoculated. On the 7th day after inoculation, when the average tumor volume reached about 103 mm.sup.3, the mice with moderate tumor volume were selected and randomly divided into 8 groups according to tumor volume, with 7 mice in each group, and the treatment was initiated on the same day. The dosage regimen was shown in Table 6.

TABLE-US-00006 TABLE 6 Dosage regimen Treatment Group Dose.sup.a, frequency.sup.b, route.sup.c Animals No. G1 hIgG4, 3 mg/kg, Q2D, i.p. 7 G2 Antibody 1.14.4, 3 mg/kg, Q2D, i.p. 7 G3 Lenvatinib mesylate, 1 mg/kg, QD, p.o. 7 G4 Lenvatinib mesylate, 1 mg/kg, QD, p.o. + 7 Antibody 1.14.4, 3 mg/kg, Q2D, i.p. Note: .sup.aDosing volume was 10 μL/g based on the body weight of the animal; .sup.bQD refers to once a day; Q2D refers to once every two days; .sup.cp.o. refers to orally; i.p. refers to intraperitoneal injection. The administration interval of the two drugs in the combination group was 0.5-1 h.

[0242] 7.3. Dose Adjustment or Suspension

[0243] The animals were daily checked at the time of routine monitoring. If one or more of the following conditions occur, the administration should be suspended until the animal returns to be normal.

[0244] 1) The animal's body weight loss is more than 20% of the initial body weight at the start of treatment (when the body weight loss is within 10% of the initial body weight at the start of treatment, the administration is continued).

[0245] 2) After administration, the body temperature of the animal decreases and cannot be recovered.

[0246] 3) After administration, the mobility of the animal is sluggish or abnormal.

[0247] During the experiment, no mice were subjected to suspension of administration due to weight loss.

[0248] 7.4. Termination of the Experiment

[0249] The experiment was terminated one day after the final administration of Lenvatinib mesylate group.

[0250] 7.5. Humane Endpoints for Experimental Animals

[0251] During the course of the experiment, animals should be euthanized if one or more of the following conditions occur.

[0252] 1) When the tumor volume of the animal exceeds 3000 mm.sup.3, or the average tumor volume of the whole group exceeds 2000 mm.sup.3.

[0253] 2) Tumor becomes anabrotic, necrotic or infected, and has not been recovered within one week.

[0254] 3) The animal suffers from sluggish mobility, or paralysis.

[0255] 4) The animal's body weight loss is more than 20% of the initial body weight at the start of treatment.

[0256] During the experiment, No. 52136 and No. 52158 mice in the G1 group were euthanized in advance because the tumor volume exceeded 3000 mm.sup.3. The body weight and tumor volume data of the two mice on the day of euthanasia were followed to the termination of the experiment.

[0257] 7.6. Euthanasia

[0258] Animals were euthanized with excess CO.sub.2 at the termination of the experiment or humane endpoints.

[0259] 7.7. Experimental Indicators

[0260] 7.7.1. Tumor Volume

[0261] Tumor volume was measured three times a week in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=0.5 a×b.sup.2 where a and b were the long and short diameters of the tumor, respectively.

[0262] 7.7.2. Body Weight

[0263] Animals were weighed 2-3 times a week before tumor inoculation, during animal grouping and the treatment, and before the euthanasia.

[0264] 7.7.3. General Clinical Observations

[0265] At the time of routine monitoring, the animals were checked daily for any effects of tumor growth, ulceration, mental state and behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect.

[0266] 7.7.4. Tumor Weight and Photographing

[0267] At the termination of the experiment, the animals were photographed to record the tumor bearing state after euthanasia, and the tumors of the mice were weighed and photographed.

[0268] 7.8. Evaluation Index of Drug Efficacy

[0269] 7.8.1. Tumor Growth Inhibition (TGI.sub.TV):

TGI.sub.TV(%)=[1−(T.sub.i−T.sub.0)/(V.sub.i−V.sub.0)]×100%.

[0270] T.sub.i: tumor volume of the treatment group at day i following treatment; T.sub.0: tumor volume of the treatment group at day 0 following treatment; V.sub.i: tumor volume of the control group at day i following treatment; V.sub.0: tumor volume of the control group at day 0 following treatment.

[0271] 7.8.2. Tumor Weight Inhibition (TGI.sub.TW):

[0272] At the termination of the experiment, the animals were euthanized, the tumors were separated and weighed, and the difference of tumor weight between each group was calculated. Tumor weight inhibition (TGI.sub.TW) was calculated by the formula:

[0273] TGI.sub.TW (%)=(W.sub.C−W.sub.T)/W.sub.T×100%; where We means tumor weight of control group, W.sub.T means tumor weight of treatment group.

[0274] 7.9. Statistical Analysis

[0275] The original data measured and observed must be recorded. The original data was analyzed and the results were expressed by mean and standard error (Mean±SEM). The data of body weight, tumor volume and tumor weight was statistically analyzed using independent-samples t-test. All data were analyzed using SPSS, P<0.05 is considered to be statistically significant. The results of the analysis were considered both statistical significance and biological significance.

[0276] 8. Results

[0277] 8.1. Body Weight Changes

[0278] All the experimental animals were in a good state of mobility, diet, etc. During the treatment period, the body weight of the mice had a certain degree increase. There was no significant difference in body weight between the different treatment groups and hIgG4 control group (P>0.05). No. 52158 mouse in G1 group was euthanized on the 14th day after the start of treatment because the tumor volume exceeded 3000 mm.sup.3. The weight data of this mouse on the day of euthanasia was followed to the termination of the experiment and included in the statistical analysis. The body weight changes of all animals were shown in Table 7.

TABLE-US-00007 TABLE 7 Body weight changes Animal Days after the start of treatment/weight (g) Group No. 0 3 6 8 10 12 14 16 19 20 G1 52138 19.7 19.2 19.6 19.6 20.2 20.8 22.3 22.4 21.2 22.1 52115 19.7 19.0 20.0 19.9 20.7 19.3 21.6 22.6 21.6 21.6 52175 19.7 18.9 19.7 20.3 20.7 18.9 20.3 20.5 22.0 22.6 52105 20.0 19.6 20.2 20.1 20.5 20.9 22.0 21.7 21.2 21.9 52136 20.3 19.0 20.1 19.7 20.5 20.2 22.6 23.3 23.3 23.3 52142 21.6 21.0 22.5 22.3 23.0 23.7 24.7 25.2 24.3 22.5 52158 20.2 19.4 19.8 21.0 21.6 22.8 24.1 24.1 24.1 24.1 Mean 20.2 19.4 20.3 20.4 21.0 20.9 22.5 22.8 22.5 22.6 SD 0.7 0.7 1.0 1.0 1.0 1.8 1.5 1.5 1.3 0.9 SEM 0.3 0.3 0.4 0.4 0.4 0.7 0.6 0.6 0.5 0.3 CV 3.4% 3.8% 5.0% 4.7% 4.6% 8.4% 6.6% 6.8% 6.0% 3.8% G2 52118 19.5 19.9 20.3 20.5 20.1 21.3 22.0 22.2 21.8 22.4 52149 19.2 19.4 20.2 20.3 20.0 21.1 22.0 21.7 22.4 23.3 52182 18.6 19.5 19.9 20.2 20.5 21.3 22.6 23.0 21.9 23.0 52107 20.0 19.3 20.3 20.2 20.8 20.6 22.2 22.4 21.7 22.6 52110 21.1 21.0 21.7 21.9 22.0 22.9 23.5 22.9 23.1 24.4 52147 20.2 20.2 21.3 20.8 21.1 21.4 22.3 22.6 22.7 23.2 52150 21.3 20.6 21.7 21.3 22.1 21.8 23.3 23.4 22.8 23.7 Mean 20.0 20.0 20.8 20.7 20.9 21.5 22.6 22.6 22.3 23.2 SD 1.0 0.6 0.8 0.6 0.8 0.7 0.6 0.6 0.6 0.7 SEM 0.4 0.2 0.3 0.2 0.3 0.3 0.2 0.2 0.2 0.3 CV 4.9% 3.2% 3.7% 3.1% 4.0% 3.4% 2.7% 2.5% 2.5% 2.9% G3 52104 18.6 18.8 19.4 19.5 20.2 20.1 20.9 21.6 21.2 21.5 52116 19.5 19.2 20.1 20.6 21.1 21.1 22.9 22.8 23.0 23.2 52169 19.4 18.9 19.8 20.4 20.1 21.1 22.0 22.6 23.0 23.2 52123 20.0 19.7 20.6 20.7 21.5 21.4 22.2 22.5 21.9 22.6 52143 20.2 19.3 20.3 20.3 20.4 20.7 22.0 21.9 21.4 22.1 52153 20.8 20.3 21.2 21.0 21.7 22.2 23.0 23.0 22.6 23.2 52156 20.8 20.2 20.8 21.0 21.4 21.6 23.0 22.8 20.3 22.9 Mean 19.9 19.5 20.3 20.5 20.9 21.2 22.3 22.5 21.9 22.7 SD 0.8 0.6 0.6 0.5 0.7 0.7 0.8 0.5 1.0 0.7 SEM 0.3 0.2 0.2 0.2 0.3 0.3 0.3 0.2 0.4 0.2 CV 4.0% 3.1% 3.0% 2.5% 3.2% 3.2% 3.4% 2.3% 4.6% 2.9% G4 52119 19.3 19.1 19.5 20.0 21.1 20.6 21.9 21.8 21.0 21.7 52101 19.9 19.8 20.7 20.3 20.9 20.3 21.8 21.9 21.2 21.9 52155 20.3 19.5 20.3 20.3 21.0 20.5 21.9 21.8 21.4 22.0 52130 20.3 19.4 20.5 20.9 21.8 21.6 22.7 23.2 22.9 23.2 52135 20.5 19.4 20.3 20.1 20.5 20.2 21.7 21.7 21.2 21.7 52176 20.7 20.2 20.8 21.4 22.8 22.8 23.9 24.1 23.6 23.9 52181 21.4 21.3 21.4 21.9 21.9 22.2 23.1 23.2 23.3 23.4 Mean 20.3 19.8 20.5 20.7 21.4 21.2 22.4 22.5 22.1 22.5 SD 0.7 0.7 0.6 0.7 0.8 1.0 0.8 1.0 1.1 0.9 SEM 0.2 0.3 0.2 0.3 0.3 0.4 0.3 0.4 0.4 0.3 CV 3.2% 3.7% 2.8% 3.5% 3.7% 4.9% 3.7% 4.3% 5.1% 4.1%

[0279] The body weight on the 16th day after the start of treatment was statistically analyzed using independent-samples t-test, and the result was shown in Table 8.

TABLE-US-00008 TABLE 8 Body weight on the 16th day Weight (g)/Mean ± SEM Group Treatment Animals Day 0 .sup.a Day 16 .sup.a P.sup.b Changes G1 hIgG4 3 mg/kg 7 20.2 ± 0.3 22.8 ± 0.6 — +2.6 G2 Antibody 1.14.4 3 mg/kg 7 20.0 ± 0.4 22.6 ± 0.2 0.720 +2.6 G3 Lenvatinib mesylate 1 mg/kg 7 19.9 ± 0.3 22.5 ± 0.2 0.565 +2.6 G4 Lenvatinib mesylate 1 mg/kg + 7 20.3 ± 0.2 22.5 ± 0.4 0.671 +2.2 Antibody 1.14.4 3 mg/kg Note: .sup.a Day 0 is the day of the start of treatment (i.e., the day of animal grouping), Day 16 is the 16th day after the start of treatment; .sup.bp value vs control group.

[0280] 8.2. Tumor Volume Results

[0281] Tumor growth of all animals was closely monitored during the whole period of the experiment. The tumor volume of all animals was measured three times a week and the results were recorded. The tumor volume data was shown in Table 9. No. 52158 mouse in G group was euthanized on the 14th day after the start of treatment because the tumor volume exceeded 3000 mm.sup.3. The tumor growth data of this mouse on the day of euthanasia was followed to the termination of the experiment and included in the statistical analysis.

TABLE-US-00009 TABLE 9 Tumor volume Animal Days after the start of treatment/tumor volume (mm.sup.3) Group No. 0 3 6 8 10 12 14 16 19 20 G1 52138 98.6 275.2 473.6 659.1 869.1 924.8 930.3 1265.3 1406.0 1607.2 52115 108.8 306.8 836.2 1229.0 1128.4 1297.1 1584.6 1882.1 1737.5 1859.5 52175 95.6 222.6 453.6 672.1 835.5 1033.7 1417.4 1606.0 1902.4 2305.1 52105 82.9 286.1 602.2 712.2 999.8 1262.4 1260.6 1551.3 2173.4 3175.2 52136 112.9 474.6 947.1 1198.3 1575.3 2001.3 2514.7 3080.0 3080.0 3080.0 52142 121.5 378.7 979.2 868.6 1125.2 1659.1 1316.0 1783.5 1757.9 1951.0 52158 98.9 527.6 1138.6 1934.9 2415.4 2484.3 3618.4 3618.4 3618.4 3618.4 Mean 103 353 776 1039 1278 1523 1806 2112 2239 2514 SD 13 112 268 462 558 561 939 881 807 773 SEM 5 42 101 175 211 212 355 333 305 292 CV 12.4% 31.8% 34.6% 44.4% 43.6% 36.8% 52.0% 41.7% 36.0% 30.8% G2 52118 118.2 177.1 288.1 323.2 335.8 296.5 253.7 287.9 306.0 399.4 52149 86.2 134.6 308.7 433.2 457.8 693.6 710.0 994.6 1173.0 1267.0 52182 110.7 305.3 739.6 887.1 1046.6 1557.3 1756.4 2427.2 3093.0 3146.2 52107 75.5 176.8 294.8 405.3 499.7 887.5 1053.1 1245.4 1976.9 1777.2 52110 107.4 279.0 361.3 387.6 507.7 810.9 1049.4 980.7 1194.0 982.9 52147 99.2 210.5 318.8 391.6 638.6 898.6 1010.9 1192.0 2068.6 2071.5 52150 121.1 272.7 617.4 726.5 765.6 1036.5 1245.6 1572.2 1642.7 1783.3 Mean 103 222 418 508 607 883 1011 1243 1636 1632 SD 17 64 183 212 237 379 462 653 876 876 SEM 6 24 69 80 90 143 174 247 331 331 CV 16.4% 28.8% 43.7% 41.8% 39.0% 43.0% 45.6% 52.5% 53.5% 53.7% TGI — 0.522 0.531 0.567 0.571 0.451 0.466 0.433 0.282 0.365 G3 52104 87.7 195.2 432.7 620.5 964.3 1093.4 1170.2 1341.3 1427.3 1488.5 52116 102.8 340.1 579.3 886.4 1269.5 1248.3 1678.0 2139.3 2792.6 2915.3 52169 114.6 329.8 592.7 718.2 974.5 1425.3 1754.2 2366.9 2701.0 2973.9 52123 136.5 249.5 538.7 604.1 630.4 613.4 614.3 892.3 994.4 936.1 52143 72.8 232.6 370.4 520.3 481.7 538.7 430.5 437.1 503.7 582.0 52153 96.2 264.5 456.2 498.9 777.4 807.9 1120.2 1179.2 2127.4 2258.9 52156 108.1 269.1 468.5 605.0 612.2 869.2 829.2 971.9 821.4 925.6 Mean 103 269 491 636 816 942 1085 1333 1624 1726 SD 20 52 82 132 271 328 504 692 923 990 SEM 8 19 31 50 103 124 190 2.61 349 374 CV 19.8% 19.2% 16.6% 20.7% 33.3% 34.8% 46.4% 51.9% 56.8% 57.4% TGI — 0.337 0.423 0.43 0.393 0.4 0.423 0.388 0.288 0.327 G4 52119 88.4 167.6 283.1 274.9 326.0 426.2 424.3 549.2 877.4 750.7 52101 109.0 182.9 412.8 460.7 475.7 540.2 569.9 627.3 698.5 756.6 52155 99.7 345.3 722.7 683.5 927.3 821.5 835.8 736.1 1012.4 1107.5 52130 121.0 304.0 634.0 622.3 836.1 967.5 1045.8 1137.2 1643.5 1890.7 52135 82.7 187.5 238.4 254.8 358.0 360.3 394.0 417.5 508.1 555.8 52176 119.5 318.6 603.9 660.1 909.4 1169.1 1095.3 1323.2 1450.1 1473.1 52181 97.8 262.3 350.3 328.7 492.5 549.6 537.9 562.6 718.9 696.6 Mean 103.0 253.0 464.0 469.0 618.0 691.0 700.0 765.0 987.0 1033.0 SD 15 73 189 187 264 301 291 336 417 488 SEM 6 28 71 71 100 114 110 127 158 185 CV 14.3% 28.9% 40.8% 39.8% 42.7% 43.6% 41.5% 44.0% 42.2% 47.3% TGI — 0.401 0.464 0.608 0.562 0.586 0.649 0.671 0.586 0.614

[0282] On the 16th day after the start of treatment, the tumor volume on the 16th day after the start of treatment was statistically analyzed using independent-samples t-test, and the result was shown in Table 10. It can be seen that the combination of Lenvatinib mesylate (1 mg/kg) and antibody 1.14.4 (3 mg/kg) exhibits a more significant antitumor activity than the monotherapy group.

TABLE-US-00010 TABLE 10 Tumor volume on the 16th day Tumor volume (mm.sup.3)/Mean ± SEM Group Treatment Animals Day 0 .sup.a Day 16 .sup.a TGI.sub.TV (%) P.sup.b G1 hIgG4 3 mg/kg 7 103 ± 5 2112 ± 333 — — G2 antibody 1.14.4 3 mg/kg 7 103 ± 6 1243 ± 247 43.3 0.058 G3 Lenvatinib mesylate 1 mg/kg 7 103 ± 8 1333 ± 261 38.8 0.090 G4 Lenvatinib mesylate 1 mg/kg + 7 103 ± 6  765 ± 127 67.1 0.006 antibody 1.14.4 3 mg/kg Note: .sup.a Day 0 is the day of the start of treatment (i.e., the day of animal grouping), Day 16 is the 16th day after the start of treatment; .sup.bp value vs control group (G1).

[0283] 8.3. Synergy Analysis

[0284] Synergy score was calculated using the following formula described in Clarke R. Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models[J]. Breast Cancer Research & Treatment, 1997, 46(2-3):255-278, which is incorporated by reference in its entirety:

Synergy score=((A/C)×(B/C))/(AB/C);

[0285] where A is RTV value of drug A; B is RTV value of drug B; C is RTV value of vehicle control; AB is RTV value of combination treatment with A and B respectively. [0286] Synergy score>1 represents synergistic; [0287] Synergy score=1 represents additive; [0288] Synergy score<1 represents antagonistic.

[0289] Relative tumor volume (RTV) was calculated using the following formula:

RTV=V.sub.t/V.sub.0

[0290] where V.sub.0 and V.sub.t are the average tumor volume at the start of treatment (day 0) and the average tumor volume on a certain time point (day t).

[0291] The results of average tumor volume on the 20th day and synergy score were shown in Table 11.

TABLE-US-00011 TABLE 11 Synergy analysis Tumor volume Synergy (mm.sup.3)/Mean ± SEM score Group Treatment Day 0 Day 20 @ D20 G1 hIgG4 3 mg/kg 103 ± 5 2514 ± 292 — G2 Antibody 1.14.4 3 mg/kg 103 ± 6 1632 ± 331 — G3 Lenvatinib mesylate 103 ± 8 1726 ± 374 — 1 mg/kg G4 Lenvatinib mesylate 103 ± 6 1033 ± 185 1.08 1 mg/kg + Antibody 1.14.4 3 mg/kg

[0292] Synergistic effect between Lenvatinib mesylate and antibody 1.14.4 was demonstrated as synergy score was larger than 1 in the combination group G4.

[0293] 8.4. Tumor Weight Results

[0294] In this experiment, all animals were euthanized on the 20th day after the start of treatment, and tumors were separated, weighed and photographed. The individual tumor weight data was shown in Table 12.

TABLE-US-00012 TABLE 12 Tumor weight Animal Tumor weight Group No. (g) G1 52138 1.021 52115 1.168 52175 1.583 52105 1.353 52136 2.074 52142 1.509 52158 2.365 Mean 1.582 SD 0.483 SEM 0.183 CV 30.5% G2 52118 0.329 52149 0.882 52182 2.554 52107 1.823 52110 1.200 52147 1.337 52150 1.093 Mean 1.333 SD 0.741 SEM 0.280 CV 55.6% TGI.sub.TW 15.8% G3 52104 1.039 52116 2.048 52169 2.485 52123 0.652 52143 0.408 52153 1.362 52156 0.715 Mean 1.244 SD 0.772 SEM 0.292 CV 62.0% TGI.sub.TW 21.3% G4 52119 0.818 52101 0.641 52155 0.716 52130 1.545 52135 0.457 52176 1.198 52181 0.536 Mean 0.844 SD 0.391 SEM 0.148 CV 46.4% TGI.sub.TW 46.6%

[0295] The tumor weight was statistically analyzed using independent-samples t-test, and the result was shown in Table 13. It can be seen that the combination of Lenvatinib mesylate (1 mg/kg) and antibody 1.14.4 (3 mg/kg) exhibits a more significant antitumor activity than the monotherapy group.

TABLE-US-00013 TABLE 13 Tumor weight statistical analysis Tumor weight (g) TGI.sub.TV Group Treatment Animals Mean ± SEM (%) P.sup.a G1 hIgG4 3 mg/kg 7 1.582 ± 0.183 — — G2 antibody 1.14.4 7 1.333 ± 0.280 15.8 0.470 3 mg/kg G3 Lenvatinib mesylate 7 1.244 ± 0.292 21.3 0.346 1 mg/kg G4 Lenvatinib mesylate 7 0.844 ± 0.148 65.8 0.009 1 mg/kg + antibody 1.14.4 3 mg/kg Note: .sup.ap value vs control group (G1).

Embodiment 2: Pharmacodynamic Study of Antibody 2E5 Monotherapy or in Combination with Lenvatinib Mesylate on B-hPD-1 Humanized Mouse MC38-hPD-L1 Colon Cancer Animal Model

[0296] 1. Objective of the Study

[0297] The objective of the study was to evaluate the therapeutic efficacy of antibody 2E5 as a single agent or in combination with Lenvatinib mesylate in the treatment of subcutaneous MC38-hPD-L1 transplanted tumor.

[0298] 2. Reagents and Equipment

TABLE-US-00014 TABLE 14 Reagents and equipment Reagents or Storage Physical equipment Supplier Art. No. condition Lot. No. form DMEM Cellgro 10-013-CVR 4° C. — — FBS HyClone SH30406.02 −80° C.  — — PBS Gibco 20012-027 4° C. — — Vernier Hexagon 00534220 — — — caliper Metrology 0.9% — — — — — Sodium chloride injection hIgG4 CROWN C0004-4 — AB170091 5.1 mg/mL BIO- solution in SCIENCE physio- INC logical TAICANG saline Lenvatinib Eisai Co., — — 164H0501 powder mesylate Ltd.

[0299] 3. Test Sample

[0300] Antibody 2E5 was prepared according to WO2018053709A1 (Examples 1-3).

[0301] 4. Formulation

[0302] 4.1. Formulation of hIgG4

[0303] 5.1 mg/mL solution of hIgG4 in physiological saline was diluted with 0.9% sodium chloride injection to a concentration of 0.2 mg/mL for use. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to warm to room temperature before use.

[0304] 4.2. Formulation of Different Concentration of Lenvatinib Mesylate

[0305] Lenvatinib mesylate powder was dissolved in 3 mmol/L HCl solution to prepare 3 mg/mL stock solution of Lenvatinib mesylate. The stock solution was stored at 4° C. when not in use.

[0306] 1 mg/mL Lenvatinib mesylate solution was prepared by diluting 0.12 mL of 3 mg/mL stock solution with 0.24 mL of 3 mmol/L HCl solution. 0.36 mL of 1 mg/mL Lenvatinib mesylate solution was further diluted with 3.24 mL of 3 mmol/L HCl solution to prepare 0.1 mg/mL Lenvatinib mesylate solution for use. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to warm to room temperature before use.

[0307] 4.3. Formulation of Antibody 2E5

[0308] Antibody 2E5 was diluted with 0.9% sodium chloride injection to prepare 0.1 mg/mL and 0.2 mg/mL antibody 2E5 solution for use. The prepared solution was stored at 2-8° C. when not in use, and allowed to stand to warm to room temperature before use.

[0309] 5. Animals

[0310] Species: Mus musculus;

[0311] Strain: C57BL/6;

[0312] Name: B-hPD-1 humanized mice;

[0313] Sex: male;

[0314] Body weight: 20-28 g

[0315] Age: 7 weeks;

[0316] Number of animals: 100 (60 plus 40 spare) mice;

[0317] Animal supplier: Biocytogen Jiangsu Co., Ltd;

[0318] License number: SCXK (Jiangsu) 2016-0004;

[0319] Animal Certificate No.: 201803133.

[0320] 6. Animal Housing and Care

[0321] Animals were housed in the SPF animal laboratory of Experiment Animal Center in Biocytogen Beijing Co., Ltd using individually ventilated cages (IVC). Animals were acclimated to the environment for 3-7 days prior to initiate the study.

[0322] Temperature: 20-26° C.;

[0323] Humidity: 40%-70%;

[0324] Light: 12 hours on and 12 hours off;

[0325] Cage: Cage was made of PEI with a volume of 320 mm×200 mm×135 mm. Corn cobs were used for animal bedding, which were sterilized by autoclave before use. Beddings were changed once a week. Each cage was identified by a cage card marking the number of animals, gender, strain, receiving date, group number, and starting date of the experiment.

[0326] Diet and water: Reverse osmosis (RO) water was autoclaved before use. Animals had free access to SPF mouse feed and sterile drinking water.

[0327] Animal identification: Each mice was identified by ear tag.

[0328] 7. Experimental Procedure

[0329] 7.1. Cell Culture

[0330] Mouse colon cancer cells MC38 were purchased from Shun Ran (Shanghai) Biotechnology Co., Ltd., and the cells were cultured in a 37° C., 5% CO.sub.2 incubator supplemented with Dulbecco's Modified Eagle's medium containing 10% inactivated fetal bovine serum. MC38 cells were genetically modified to overexpress human PD-L1 and knock out murine PD-L1, the modified cells were named as MC38-hPD-L1 cells.

[0331] 7.2. Tumor Inoculation, Group and Dosage Regimen

[0332] MC38-hPD-L1 tumor cells were resuspended in 1×PBS to a concentration of 5×10.sup.5 cells per 0.1 mL. Each of the B-hPD-1 humanized mice was subcutaneously inoculated at the right lateral thorax region with 0.1 mL of the cell suspension. When the average tumor volume reached about 77 mm.sup.3, the mice with moderate tumor volume were selected and randomly divided into 6 groups according to tumor volume, with 10 mice in each group, and the treatment was initiated on the same day. The dosage regimen was shown in Table 15.

TABLE-US-00015 TABLE 15 Dosage regimen Treatment.sup.a Animals Group Dose.sup.b, route Frequency.sup.c No. G1 hIgG4, 2 mg/kg, i.p. 0, 4, 8, 11, 13, 16; 10 a total of 6 times G2 Antibody 2E5, 0, 4, 8, 11, 13, 16; 10 1 mg/kg, i.p. a total of 6 times G3 Antibody 2E5, 0, 4, 8, 11, 13, 16; 10 2 mg/kg, i.p. a total of 6 times G4 Lenvatinib mesylate, QD; a total of 18 times 10 1 mg/kg, p.o. G5 Lenvatinib mesylate, Lenvatinib mesylate: 10 1 mg/kg, p.o. + QD; a total of 18 times Antibody 2E5, Antibody 2E5: 0, 4, 8, 11, 1 mg/kg, i.p. 13, 16; a total of 6 times G6 Lenvatinib mesylate, Lenvatinib mesylate: 10 1 mg/kg, p.o. + QD; a total of 18 times Antibody 2E5, Antibody 2E5: 0, 4, 8, 11, 2 mg/kg, i.p. 13, 16; a total of 6 times Note: .sup.aThe administration interval of the two drugs in the combination group (i.e., G5 and G6) was 0.5-1 h; .sup.bDosing volume was 10 μL/g based on the body weight of the animal; .sup.cp.o. means orally; i.p. means intraperitoneal injection; QD means once a day; 0, 4, 8, 11, 13, 16 means days after animal grouping (i.e., the day of the start of treatment).

[0333] 7.3. Dose Adjustment or Suspension

[0334] The animals were daily checked at the time of routine monitoring. If one or more of the following conditions occur, the administration should be suspended until the animal returns to be normal:

[0335] 1) The animal's body weight loss is more than 20% of the initial body weight at the start of treatment (when the body weight loss is within 10% of the initial body weight at the start of treatment, the administration is continued).

[0336] 2) After administration, the body temperature of the animal decreases and cannot be recovered.

[0337] 3) After administration, the mobility of the animal is sluggish or abnormal.

[0338] During the experiment, no mice were subjected to suspension of administration due to the above reasons.

[0339] 7.4. Termination of the Experiment

[0340] The experiment was terminated 2 hours after the final administration of Lenvatinib mesylate on the 17.sup.th day after the start of treatment.

[0341] 7.5. Humane Endpoints for Experimental Animals

[0342] During the course of the experiment, animals should be euthanized if one or more of the following conditions occur:

[0343] 1) When the tumor volume of the animal exceeds 3000 mm.sup.3, or the average tumor volume of the whole group exceeds 2000 mm.sup.3.

[0344] 2) Tumor becomes anabrotic, necrotic or infected, and has not been recovered within one week.

[0345] 3) The animal suffers from sluggish mobility, or paralysis.

[0346] 4) The animal's body weight loss is more than 20% of the initial body weight at the start of treatment.

[0347] During the experiment, No. 55191 mouse in the G1 group was euthanized in advance because the tumor volume exceeded 3000 mm.sup.3 on the 16.sup.th day after the start of treatment. No mice was euthanized in advance due to other clinical symptoms.

[0348] 7.6. Euthanasia

[0349] Animals were euthanized with excess CO.sub.2 at the termination of the experiment or humane endpoints.

[0350] 7.7. Experimental Indicators

[0351] 7.7.1. Tumor Volume:

[0352] Tumor volume was measured 2-3 times a week in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=0.5 a×b.sup.2 where a and b were the long and short diameters of the tumor, respectively.

[0353] 7.7.2. Body Weight

[0354] Animals were weighed 2-3 times a week before tumor inoculation, during animal grouping and the treatment, and before the euthanasia.

[0355] 7.7.3. General Clinical Observations

[0356] At the time of routine monitoring, the animals were checked daily for any effects of tumor growth, ulceration, mental state and behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect.

[0357] 7.7.4. Tumor Weight and Photographing

[0358] At the termination of the experiment, the animals were photographed to record the tumor bearing state after euthanasia, and the tumors of the mice were weighed and photographed.

[0359] 7.8. Evaluation Index of Drug Efficacy

[0360] 7.8.1. Tumor Growth Inhibition (TGI.sub.TV):

TGI.sub.TV(%)=[1−(T.sub.i−T.sub.0)/(V.sub.i−V.sub.0)]×100%.

[0361] T.sub.i: tumor volume of the treatment group at day i following treatment; T.sub.0: tumor volume of the treatment group at day 0 following treatment; V.sub.i: tumor volume of the control group at day i following treatment; V.sub.0: tumor volume of the control group at day 0 following treatment.

[0362] 7.8.2. Tumor Weight Inhibition (TGI.sub.TW):

[0363] At the termination of the experiment, the animals were euthanized, the tumors were separated and weighed, and the difference of tumor weight between each group was calculated. Tumor weight inhibition (TGI.sub.TW) was calculated by the formula:

[0364] TGI.sub.TW (%)=(W.sub.C−W.sub.T)/W.sub.T×100%; where We means tumor weight of control group, W.sub.T means tumor weight of treatment group.

[0365] 7.9. Statistical Analysis

[0366] The original data measured and observed must be recorded. The original data was analyzed and the results were expressed by mean and standard error (Mean±SEM). The data of body weight, tumor volume and tumor weight was statistically analyzed using independent-samples t-test. All data were analyzed using SPSS, P<0.05 is considered to be statistically significant.

[0367] 8. Results

[0368] 8.1. Body Weight Changes

[0369] All the experimental animals were in a good state of mobility, diet, etc. During the treatment period, the body weight of the mice had a certain degree increase. No. 55191 mouse in the G1 group was euthanized in advance because the tumor volume exceeded 3000 mm.sup.3 on the 16.sup.th day after the start of treatment. The weight data of this mouse on the day of euthanasia was followed to the termination of the experiment and included in the statistical analysis. The body weight changes of all animals were shown in Table 16.

TABLE-US-00016 TABLE 16 Body weight change Animal Days after the start of treatment/body weight (g) Group No. 0 4 8 10 12 15 17 G1 55106 24.4 23.9 25.7 25.6 27.8 28.0 28.3 55115 24.8 24.9 26.5 26.3 28.2 28.8 30.2 55136 23.4 23.8 25.7 25.2 26.6 27.6 27.9 55151 27.2 27.0 28.4 28.3 29.8 30.9 31.7 55156 24.7 23.5 25.8 25.8 27.6 28.0 30.2 55157 26.4 26.5 28.4 28.2 29.7 29.8 31.1 55164 24.5 24.4 26.3 25.8 27.0 27.8 28.0 55188 26.0 25.7 27.2 26.6 27.9 29.1 29.7 55191 23.9 24.1 26.0 25.6 26.7 27.3 27.3 55194 23.9 23.6 24.7 24.7 25.6 25.9 26.0 Mean 24.9 24.7 26.5 26.2 27.7 28.3 29.0 SD 1.2 1.3 1.2 1.2 1.3 1.4 1.8 SEM 0.4 0.4 0.4 0.4 0.4 0.4 0.6 CV  4.9% 5.1% 4.5% 4.6% 4.8% 4.9% 6.2% G2 55122 27.3 27.2 28.6 27.5 28.8 30.0 29.6 55131 27.6 27.7 29.5 29.0 30.0 31.2 31.8 55147 25.4 21.8 26.9 26.7 27.9 28.7 29.2 55150 23.8 23.3 25.0 24.3 25.5 26.9 27.6 55161 22.1 25.0 23.2 22.9 23.2 23.8 24.2 55168 24.5 25.2 26.7 26.7 27.5 28.9 29.5 55170 21.9 23.6 24.3 24.5 26.0 26.4 26.9 55178 23.9 25.1 27.0 26.4 27.8 28.4 28.2 55184 24.9 25.2 27.5 26.7 28.0 29.1 29.5 55190 27.3 27.0 28.8 27.4 28.9 29.4 29.8 Mean 24.9 25.1 26.8 26.2 27.4 28.3 28.6 SD 2.1 1.9 2.0 1.8 2.0 2.1 2.1 SEM 0.7 0.6 0.6 0.6 0.6 0.7 0.7 CV  8.3% 7.4% 7.6% 6.9% 7.2% 7.4% 7.2% G3 55108 21.9 21.4 22.7 21.8 22.5 23.9 24.2 55125 23.5 22.9 25.0 24.7 25.2 26.5 27.1 55135 28.2 27.9 28.7 27.7 28.5 30.5 30.8 55146 23.3 23.7 24.7 23.8 24.4 25.8 26.0 55149 24.1 24.0 26.1 25.1 25.8 27.8 27.8 55167 25.9 26.6 28.1 26.4 27.1 28.9 30.0 55171 26.3 27.3 28.4 26.9 26.0 26.7 27.6 55173 26.2 25.9 28.2 26.8 27.6 28.6 29.3 55182 23.7 23.2 24.8 23.4 24.4 25.6 25.5 55186 26.6 26.3 27.5 27.6 28.1 29.5 30.0 Mean 25.0 24.9 26.4 25.4 26.0 27.4 27.8 SD 1.9 2.2 2.0 2.0 1.9 2.0 2.2 SEM 0.6 0.7 0.6 0.6 0.6 0.6 0.7 CV  7.8% 8.7% 7.8% 7.8% 7.3% 7.4% 7.8% G4 55103 25.1 25.8 26.5 26.2 27.2 28.0 28.7 55104 28.0 27.7 29.5 29.7 29.7 30.6 31.8 55123 26.0 26.4 27.5 27.0 27.5 28.2 29.5 55139 25.5 25.2 25.9 25.3 26.3 27.3 27.9 55155 27.4 27.0 27.3 27.2 27.3 28.0 29.2 55163 22.7 22.6 23.8 23.7 23.9 25.5 26.1 55165 19.6 19.6 20.9 20.2 20.6 21.9 22.3 55175 23.0 23.1 25.5 24.5 25.3 26.8 27.0 55187 25.2 25.3 26.7 26.1 26.7 27.4 28.0 55198 26.0 25.8 26.4 26.8 27.5 29.2 29.7 Mean 24.9 24.9 26.0 25.7 26.2 27.3 28.0 SD 2.5 2.4 2.3 2.5 2.5 2.3 2.6 SEM 0.8 0.8 0.7 0.8 0.8 0.7 0.8 CV 10.0% 9.8% 8.9% 9.8% 9.5% 8.5% 9.1% G5 55105 23.9 23.8 24.5 24.0 23.8 24.8 24.2 55112 27.3 26.8 27.9 27.1 27.0 28.6 29.0 55120 28.5 28.5 29.2 28.2 29.3 30.0 29.6 55140 23.8 23.9 24.9 24.3 24.2 25.4 25.0 55143 26.3 25.7 26.5 25.7 26.0 26.7 26.7 55154 21.9 21.8 22.3 22.9 23.2 24.6 25.5 55162 23.3 24.2 25.0 25.4 25.7 26.2 27.4 55180 24.9 25.3 25.0 25.4 24.9 26.5 27.2 55192 24.3 23.8 24.1 23.5 24.0 24.2 24.5 55193 25.2 25.2 26.7 26.5 26.9 27.8 28.2 Mean 24.9 24.9 25.6 25.3 25.5 26.5 26.7 SD 2.0 1.9 2.0 1.7 1.9 1.9 1.9 SEM 0.6 0.6 0.6 0.5 0.6 0.6 0.6 CV  7.9% 7.5% 7.8% 6.6% 7.3% 7.1% 7.1% G6 55101 25.0 24.7 25.3 24.9 24.7 25.1 25.8 55109 22.6 22.8 23.8 23.4 22.4 24.2 24.4 55113 23.3 24.0 24.2 24.0 23.9 24.8 24.8 55121 23.6 23.5 25.0 24.1 24.1 24.6 25.2 55127 25.2 25.6 26.0 25.7 25.3 26.6 27.0 55159 27.2 26.9 28.0 27.5 27.8 29.3 29.4 55160 25.6 25.7 27.2 26.7 27.0 27.8 28.2 55185 27.4 27.6 28.2 28.0 28.6 29.9 29.9 55189 23.0 23.1 23.3 23.5 23.2 23.6 24.2 55199 26.7 25.5 26.5 26.2 26.7 27.7 28.3 Mean 25.0 24.9 25.8 25.4 25.4 26.4 26.7 SD 1.8 1.6 1.7 1.7 2.1 2.2 2.1 SEM 0.6 0.5 0.5 0.5 0.7 0.7 0.7 CV  7.1% 6.4% 6.7% 6.6% 8.2% 8.4% 8.0%

[0370] The body weight on the 17th day after the start of treatment was shown in Table 17.

TABLE-US-00017 TABLE 17 Body weight on the 17th day Weight (g)/Mean ± SEM Group Treatment Day 0 .sup.a Day 17 .sup.a Changes G1 hIgG4 (2 mg/kg) 24.9 ± 0.4 29.0 ± 0.6 +4.1 G2 Antibody 2E5 (1 mg/kg) 24.9 ± 0.7 28.6 ± 0.7 +3.7 G3 Antibody 2E5 (2 mg/kg) 25.0 ± 0.6 27.8 ± 0.7 +2.8 G4 Lenvatinib mesylate 24.9 ± 0.8 28.0 ± 0.8 +3.1 (1 mg/kg) G5 Lenvatinib mesylate 24.9 ± 0.6 26.7 ± 0.6 +1.8 (1 mg/kg) + Antibody 2E5 (1 mg/kg) G6 Lenvatinib mesylate 25.0 ± 0.6 26.7 ± 0.7 +1.7 (1 mg/kg) + Antibody 2E5 (2 mg/kg) Note: .sup.a Day 0 is the day of the start of treatment (i.e., the day of animal grouping). Day 17 is the 17th day after the start of treatment.

[0371] 8.2. Tumor Volume Results

[0372] Tumor growth of all animals was closely monitored during the whole period of the experiment. No. 55191 mouse in the G1 group was euthanized in advance since the tumor volume exceeded 3000 mm.sup.3 on the 16th day after the start of treatment. The weight data of this mouse on the day of euthanasia was followed to the termination of the experiment and included in the statistical analysis. The tumor weight changes of all animals were shown in Table 18.

TABLE-US-00018 TABLE 18 Tumor volume Animal Days after the start of treatment/tumor volume (mm.sup.3) Group No. 0 4 8 10 12 15 17 G1 55106 76.5 224.2 891.7 1227.7 1601.3 1749.5 1706.5 55115 76.1 208.8 489.8 817.4 956.0 2314.2 2792.2 55136 70.8 221.7 690.8 998.5 1512.8 1919.3 2520.8 55151 73.3 203.5 676.1 1085.3 1574.2 2049.0 3017.9 55156 87.4 287.7 1037.1 1061.1 1360.2 1657.0 1820.8 55157 79.3 289.7 1010.8 1027.8 1435.5 1912.4 3026.8 55164 78.3 272.3 1139.6 1423.1 2509.8 2814.6 3998.8 55188 80.5 223.6 1154.3 1683.4 1901.4 2538.0 3484.4 55191 80.1 376.9 1297.1 1453.9 2725.9 3185.3 3185.3 55194 69.4 133.6 350.5 345.5 574.2 681.8 838.1 Mean 77 244 874 1112 1615 2082 2639 SD 5 66 311 373 645 693 941 SEM 2 21 98 118 204 219 298 CV 6.8% 26.9% 35.5% 33.5% 39.9% 33.3% 35.7% G2 55122 74.2 133.5 274.6 279.4 573.8 689.0 982.2 55131 75.7 184.2 216.7 248.7 257.1 354.1 239.0 55147 67.5 269.2 448.7 608.9 924.5 1149.7 1379.7 55150 72.0 353.0 457.3 771.4 1312.1 1392.4 2172.7 55161 77.2 183.0 233.1 234.7 381.6 591.7 565.9 55168 80.6 140.7 511.8 717.8 1304.6 2234.1 2361.1 55170 79.2 195.8 474.9 481.8 962.7 1105.7 1330.5 55178 78.5 162.2 390.1 387.9 405.2 358.4 245.2 55184 84.6 246.0 709.7 888.0 927.0 1634.1 2060.5 55190 81.5 145.7 226.8 294.3 348.8 572.4 610.6 Mean 77 201 394 491 740 1008 1195 SD 5 69 159 240 397 613 797 SEM 2 22 50 76 126 194 252 CV 6.5% 34.4% 40.2% 48.9% 53.7% 60.9% 66.7% TGI 25.6% 60.2% 60.0% 56.9% 53.6% 56.4% G3 55108 70.6 147.7 226.8 288.3 653.9 741.2 1534.9 55125 86.2 223.8 409.0 408.0 510.6 725.4 1149.8 55135 72.3 413.3 613.1 836.3 1169.7 1205.5 1579.3 55146 75.5 162.8 283.2 309.2 372.2 480.5 755.1 55149 82.1 149.4 326.5 426.0 430.6 430.9 541.9 55167 79.1 194.5 358.3 398.3 625.7 897.0 1359.9 55171 76.6 263.1 948.9 1029.5 1663.8 2226.4 2487.6 55173 80.1 199.2 430.6 526.7 1230.2 1474.0 1726.9 55182 70.5 216.9 206.3 262.3 560.3 463.8 318.9 55186 78.2 272.8 481.6 608.9 1092.6 1137.9 1568.4 Mean 77 224 428 509 831 978 1302 SD 5 79 220 251 429 560 638 SEM 2 25 69 79 136 177 202 CV 6.6% 35.3% 51.3% 49.3% 51.6% 57.2% 49.0% TGI — 11.9% 55.9% 58.2% 51.0% 55.1% 52.2% G4 55103 76.4 200.5 372.9 334.9 510.8 658.2 571.3 55104 81.2 215.4 423.5 520.1 748.6 1225.3 1076.5 55123 75.7 185.2 420.4 309.3 291.0 266.3 220.1 55139 70.8 205.0 514.5 514.5 750.2 870.2 1285.2 55155 75.3 220.4 383.5 484.3 820.6 887.5 1080.5 55163 78.9 278.3 295.3 419.7 685.6 1155.8 1671.7 55165 79.6 181.0 332.3 451.7 606.8 733.1 792.0 55175 67.5 186.3 373.0 472.9 247.7 273.7 344.4 55187 78.8 312.5 484.6 500.3 475.1 565.7 556.7 55198 86.8 267.4 470.9 516.8 706.4 1040.9 1312.4 Mean 77 225 407 452 584 768 891 SD 5 45 69 76 198 335 470 SEM 2 14 22 24 63 106 149 CV 7.0% 20.1% 17.0% 16.7% 33.9% 43.6% 52.7% TGI — 11.3% 58.6% 63.7% 67.0% 65.6% 68.2% G5 55105 82.0 238.5 145.1 128.4 193.2 198.9 148.0 55112 82.6 200.4 354.8 363.8 658.8 595.2 696.3 55120 82.8 205.9 242.3 160.9 165.2 149.9 246.5 55140 79.6 178.6 224.7 145.0 239.9 198.9 159.9 55143 75.1 177.8 250.6 319.7 398.3 406.5 465.4 55154 69.7 185.6 311.0 311.1 596.9 670.9 872.3 55162 72.6 212.4 296.0 269.2 491.7 558.3 637.6 55180 77.1 216.2 252.8 202.6 328.3 312.1 308.1 55192 79.2 184.9 307.9 128.2 257.5 328.4 386.0 55193 71.1 95.8 95.3 141.8 78.5 158.2 215.3 Mean 77 190 248 217 341 358 414 SD 5 38 79 90 192 193 249 SEM 2 12 25 29 61 61 79 CV 6.3% 20.1% 31.7% 41.7% 56.3% 53.8% 60.2% TGI 32.7% 78.5% 86.5% 82.9% 86.0% 86.9% G6 55101 81.2 356.9 265.5 205.2 179.2 225.1 189.4 55109 76.4 244.8 209.1 199.1 277.8 292.9 368.1 55113 78.8 151.6 171.2 205.8 237.7 242.8 290.4 55121 76.2 224.3 148.5 288.4 342.6 294.7 236.5 55127 72.2 251.4 310.9 178.7 346.6 470.5 379.5 55159 69.4 144.5 208.0 147.0 237.0 276.8 230.2 55160 79.8 246.6 363.8 313.5 850.6 1362.8 1484.6 55185 73.6 139.9 127.3 155.7 215.3 271.0 197.3 55189 85.6 97.7 125.5 134.5 181.4 159.9 168.8 55199 78.5 202.5 256.4 226.6 391.0 446.8 624.8 Mean 77 206 219 205 326 404 417 SD 5 75 80 58 198 350 399 SEM 1 24 25 18 63 111 126 CV 6.1% 36.6% 36.6% 28.4% 60.8% 86.5% 95.7% TGI — 22.9% 82.2% 87.6% 83.8% 83.7% 86.7%

[0373] The tumor volume on the 17th day after the start of treatment was statistically analyzed using independent-samples t-test, and the result was shown in Table 19. There was a significant difference in the tumor volume between each treatment group and control group (P<0.05). There was a significant difference in the tumor volume between the combination group and the monotherapy group (P<0.05). There was no significant difference in the tumor volume between G2 and G3 (P>0.05).

TABLE-US-00019 TABLE 19 Synergy analysis Tumor volume (mm.sup.3)/Mean ± SEM Synergy score Group Treatment Day 0 .sup.a Day 17 .sup.a TGI.sub.TV (%) P.sup.b @ D17 G1 hIgG4 (2 mg/kg) 77 ± 2 2639 ± 298 — — G2 Antibody 2E5 (1 mg/kg) 77 ± 2 1195 ± 252 56.4 0.002 G3 Antibody 2E5 (2 mg/kg) 77 ± 2 1302 ± 202 52.2 0.002 G4 Lenvatinib mesylate (1 mg/kg) 11 ± 2  891 ± 149 68.2 <0.001 G5 Lenvatinib mesylate (1 mg/kg) + 77 ± 2 414 ± 79 86.9 <0.001 1.18 antibody 2E5 (1 mg/kg) G6 Lenvatinib mesylate (1 mg/kg) + 77 ± 1  417 ± 126 86.7 <0.001 1.15 Antibody 2E5 (2 mg/kg) Note: .sup.a Day 0 is the day of the start of treatment, Day 17 is the 17th day after the start of treatment; .sup.bp value vs control group (G1); Difference between groups: G2 versus G3, p = 0.743; G5 versus G6, p = 0.982; G2 versus G5, p = 0.013; G4 versus G5, p = 0.013; G3 versus G6, p = 0.002; G4 versus G6, p = 0.026.

[0374] Synergistic effect between Lenvatinib mesylate and antibody 2E5 was demonstrated as synergy score was larger than 1 in the combination groups G5 and G6.

[0375] 8.3. Tumor Weight Results

[0376] In this experiment, all animals were euthanized on the 17th day after the start of treatment, and tumors were separated, weighed and photographed. The individual tumor weight data was shown in Table 20.

TABLE-US-00020 TABLE 20 Tumor weight Group Animal No. Tumor weight (g) G1 55106 1.228 55115 2.179 55136 1.873 55151 2.523 55156 1.199 55157 2.062 55164 3.519 55188 2.413 55191 3.376 55194 0.485 Mean 2.086 SD 0.952 SEM 0.301 CV 0.456 G2 55122 0.949 55131 0.097 55147 1.273 55150 1.466 55161 0.419 55168 1.747 55170 0.914 55178 0.275 55184 1.549 55190 0.397 Mean 0.909 SD 0.589 SEM 0.186 CV 0.648 TGI.sub.TW 56.4% G3 55108 0.970 55125 0.809 55135 1.323 55146 0.579 55149 0.480 55167 0.969 55171 2.501 55173 1.475 55182 0.520 55186 1.175 Mean 1.080 SD 0.602 SEM 0.190 CV 0.558 TGI.sub.TW 48.2% G4 55103 0.685 55104 0.913 55123 0.259 55139 0.862 55155 0.739 55163 1.368 55165 0.745 55175 0.499 55187 0.465 55198 1.137 Mean 0.767 SD 0.326 SEM 0.103 CV 0.425 TGI.sub.TW 63.2% G5 55105 0.133 55112 0.762 55120 0.172 55140 0.177 55143 0.634 55154 0.851 55162 0.669 55180 0.508 55192 0.563 55193 0.162 Mean 0.463 SD 0.277 SEM 0.088 CV 0.598 TGI.sub.TW 77.8% G6 55101 0.164 55109 0.345 55113 0.250 55121 0.226 55127 0.409 55159 0.325 55160 0.993 55185 0.194 55189 0.381 55199 0.456 Meam 0.374 SD 0.238 SEM 0.075 CV 0.635 TGI.sub.TW  82.1% |

[0377] The tumor weight was statistically analyzed using independent-samples t-test, and the result was shown in Table 21. There was a significant difference in the tumor weight between the combination group and monotherapy group (p<0.05).

TABLE-US-00021 TABLE 21 tumor weight analysis Tumor weight (g) Group Treatment Mean ± SEM TGI.sub.TV (%) P.sup.a G1 IgG4 (2 mg/kg) 2.086 ± 0.301 — — G2 Antibody 2E5 (1 mg/kg) 0.909 ± 0.186 56.4 0.004 G3 Antibody 2E5 (2 mg/kg) 1.080 ± 0.190 48.2 0.011 G4 Lenvatinib mesylate 0.767 ± 0.103 63.2 0.002 (1 mg/kg) G5 Lenvatinib mesylate 0.463 ± 0.088 77.8 <0.001 (1 mg/kg) + Antibody 2E5 (1 mg/kg) G6 Lenvatinib mesylate 0.374 ± 0.075 82.1 <0.001 (1 mg/kg) + Antibody 2E5 (2 mg/kg) Note: .sup.ap value vs control group (G1); Difference between groups: G2 versus G3, p = 0.528; G5 versus G6, p = 0.451; G2 versus G5, p = 0.0499; G4 versus G5, p = 0.037; G3 versus G6, p = 0.003; G4 versus G6, p = 0.006.

[0378] It is to be understood that the foregoing description of the preferred embodiments is intended to be purely illustrative of the principles of the disclosure, rather than exhaustive thereof, and that changes and variations will be apparent to those skilled in the art, and that the present disclosure is not intended to be limited other than expressly set forth in the following claims.