BIOMARKER FOR PREDICTING THERAPEUTIC RESPONSIVENESS TO CANCER IMMUNOTHERAPEUTIC AGENT AND USE THEREOF
20230416369 ยท 2023-12-28
Assignee
Inventors
- Sook Kyung CHANG (Anyang-si, KR)
- Jeong Kook KIM (Seongnam-si, KR)
- Ji Hyun PARK (Suwon-si, KR)
- Hee Wook SHIN (Seongnam-si, KR)
- Sun Kwang SONG (Seongnam-si, KR)
Cpc classification
G01N2333/51
PHYSICS
G01N2333/65
PHYSICS
International classification
Abstract
A method for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapeutic agent, a composition for predicting therapeutic responsiveness, and a kit for predicting therapeutic responsiveness are disclosed. It is possible to distinguish patient groups for which a cancer immunotherapeutic agent is effective from patient groups for which a cancer immunotherapeutic agent is ineffective, in advance, thus greatly enhancing the efficiency of the anticancer therapy using the cancer immunotherapeutic agent.
Claims
1. A composition for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising an agent capable of measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3.
2. The composition of claim 1, wherein the one or more genes comprise OPN.
3. The composition of claim 1, wherein the one or more genes comprise OPN and POSTN.
4. The composition of claim 1, wherein the one or more genes comprise OPN, POSTN, and IGFBP-3.
5. The composition of claim 1, wherein the cancer immunotherapy inhibits binding between PD-L1 and PD-1.
6. The composition of claim 1, wherein the cancer immunotherapy is an anti-PD-L1 antibody.
7. The composition of claim 6, wherein the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 4.
8. The composition of claim 1, wherein the agent capable of measuring an expression level of mRNA is an antisense nucleotide, a primer, or a probe which complementarily binds to the gene.
9. The composition of claim 1, wherein the agent capable of measuring an expression level of protein is an antibody that specifically binds to the protein of the gene.
10. The composition of claim 1, wherein the cancer is a solid cancer.
11. The composition of claim 1, wherein the cancer is one or more selected from the group consisting of cholangiocarcinoma, colon cancer, adenocarcinoma, rectal cancer, breast cancer, synovial sarcoma, chondrosarcoma, thyroid cancer, stomach cancer, thymus cancer, cervical cancer, glioma, brain cancer, melanoma, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lymphoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, blood cancer, parathyroid cancer, and ureteral cancer.
12. A kit for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising the composition of claim 1.
13. A method for providing information for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising: measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3 in a biological sample obtained from the cancer patient; and evaluating the patient's therapeutic responsiveness to the cancer immunotherapy, based on the measured expression level of mRNA or protein.
14. The method of claim 13, wherein the one or more genes comprise OPN.
15. The method of claim 13, wherein the one or more genes comprise OPN and POSTN.
16. The method of claim 13, wherein the one or more genes comprise OPN, POSTN, and IGFBP-3.
17. The method of claim 13, wherein the cancer immunotherapy inhibits binding between PD-L1 and PD-1.
18. The method of claim 13, wherein the cancer immunotherapy is an anti-PD-L1 antibody.
19. The method of claim 13, further comprising determining whether the patient needs to receive treatment with the cancer immunotherapy or needs to continue treatment with the cancer immunotherapy, depending on the evaluation of the patient's therapeutic responsiveness to the cancer immunotherapy.
20. The method of claim 13, wherein the expression level is measured before, after, or both before and after administration of the cancer immunotherapy.
21. The method of claim 20, wherein the expression level is measured before and after administration of the cancer immunotherapy, wherein when the gene is OPN or POSTN, the patient's therapeutic responsiveness is evaluated as being low in a case where the measured expression level is significantly higher after administration of the cancer immunotherapy than before administration of the cancer immunotherapy.
22. The method of claim 20, wherein the expression level is measured before administration of the cancer immunotherapy, wherein when the gene is OPN or POSTN, the patient's therapeutic responsiveness is evaluated as being low in a case where the measured expression level is significantly higher than an expression level thereof in other control cancer patients before administration of the cancer immunotherapy.
23. The method of claim 20, wherein the expression level is measured before and after administration of the cancer immunotherapy, wherein when the gene is IGFBP-3, the patient's therapeutic responsiveness is evaluated as being low in a case where the measured expression level is significantly lower after administration of the cancer immunotherapy than before administration of the cancer immunotherapy.
24. The method of claim 20, wherein the expression level is measured before administration of the cancer immunotherapy, wherein when the gene is IGFBP-3, the patient's therapeutic responsiveness is evaluated as being low in a case where the measured expression level is significantly lower than an expression level thereof in other control cancer patients before administration of the cancer immunotherapy.
25. The method of claim 13, wherein the expression level of mRNA is measured by one or more methods selected from the group consisting of in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR), competitive reverse transcriptase polymerase chain reaction (competitive RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), RNase protection assay (RPA), microarray, and Northern blotting.
26. The method of claim 13, wherein the expression level of protein is measured by one or more methods selected from the group consisting of Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, Ouchterlony, complement fixation assay, and protein chip.
27. A method for treating cancer based on therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising: (i) administering the cancer immunotherapy to the cancer patient; and (ii) measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3 in a biological sample obtained from the cancer patient, before, after, or both before and after administration of the cancer immunotherapy.
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE FOR CARRYING OUT INVENTION
[0030] Hereinafter, the present disclosure will be described in detail.
[0031] According to an aspect of the present disclosure, there is provided a composition for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising an agent capable of measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3.
[0032] As used herein, the predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy means predicting whether the cancer patient will respond favorably or unfavorably to the cancer immunotherapy, predicting risk of resistance to the cancer immunotherapy in the cancer patient, or predicting the patient's prognosis, such as recurrence, metastasis, and survival, after such immunotherapy. The composition according to the present disclosure is capable of providing information that allows for selection of the most appropriate immunotherapy modality for a cancer patient. More specifically, the composition according to the present disclosure is capable of predicting whether a patient will respond favorably or unfavorably to a cancer immunotherapy, thereby providing information for facilitating selection of a cancer immunotherapy and increasing therapeutic responsiveness.
[0033] As used herein, the term OPN (osteopontin) refers to a secreted adhesive glycoprotein of 33 kDa, which is capable of acting in the immune system on regulation of cytokine secretion, cell migration, or the like, or a gene encoding the same. In addition, OPN has been found to be overexpressed in several solid carcinomas, and thus may be a diagnostic marker for various carcinomas. In the present disclosure, OPN may be a biomarker for predicting therapeutic responsiveness to a cancer immunotherapy. For example, the OPN gene, which may be NCBI Gene ID 6696, is a gene belonging to chromosome 4 and may be present in various species. Specifically, the OPN gene may be a polynucleotide consisting of the nucleotide sequence of NCBI Reference Sequence NM_001040058.2.
[0034] As used herein, the term POSTN (periostin) refers to a 90 kDa protein that regulates cell adhesion and interaction with the extracellular matrix, or a gene encoding the same. POSTN is a protein overexpressed in various epithelial carcinomas such as ovarian cancer and breast cancer, and may be a diagnostic marker for various carcinomas. In the present disclosure, POSTN may be a biomarker for predicting therapeutic responsiveness to a cancer immunotherapy. For example, the POSTN gene, which may be NCBI Gene ID 10631, is a gene belonging to chromosome 13 and may be present in various species. Specifically, the POSTN gene may be a polynucleotide consisting of the nucleotide sequence of NCBI Reference Sequence NM_006475.3.
[0035] As used herein, the term IGFBP-3 (insulin-like growth factor binding protein-3) refers to one of several proteins, which bind to insulin-like growth factor (IGF)-1 and -2, or a gene encoding the same. Unlike the other IGF-binding proteins, IGFBP-3 has a unique property to associate with an acid-labile subunit (ALS) after binding of either IGF-1 or IGF-2. In addition, IGFBP-3 is known to inhibit growth of breast cancer, prostate cancer, or the like, regardless of IGF, and may be a diagnostic marker for various carcinomas. In the present disclosure, IGFBP-3 may be a biomarker for predicting therapeutic responsiveness to a cancer immunotherapy. For example, the IGFBP-3 gene, which may be NCBI Gene ID: 3486, is a gene belonging to chromosome 7 and may be present in various species. Specifically, the IGFBP-3 gene may be a polynucleotide consisting of the nucleotide sequence of NCBI Reference Sequence NM_001013398.2.
[0036] In an embodiment, the one or more genes may comprise OPN.
[0037] In an embodiment, the one or more genes may comprise OPN and POSTN.
[0038] In an embodiment, the one or more genes may comprise OPN, POSTN, and IGFBP-3.
[0039] In the present disclosure, the cancer immunotherapy may be any cancer immunotherapy, such as an immune checkpoint inhibitor, which can be used for anti-cancer immunotherapy.
[0040] In an embodiment, the cancer immunotherapy may inhibit binding between PD-L1 and PD-1.
[0041] In an embodiment, the cancer immunotherapy may be an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. Preferably, the cancer immunotherapy may be an anti-PD-L1 antibody. More preferably, for details regarding the anti-PD-L1 antibody, reference may be made to those disclosed in International Publication No. WO2017/0132562. For example, the anti-PD-L1 antibody may comprise a heavy chain variable domain and a light chain variable domain, in which the heavy chain variable domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 2. In addition, the anti-PD-L1 antibody may comprise heavy chain complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10. In addition, the anti-PD-L1 antibody may comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 2. Further, the anti-PD-L1 antibody may comprise a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 4.
[0042] In an embodiment, the agent capable of measuring an expression level of mRNA may be, but is not limited to, an antisense nucleotide, a primer, or a probe which complementarily binds to mRNA of the gene.
[0043] As used herein, the term antisense nucleotide refers to a nucleic acid-based molecule that has a sequence complementary to a target gene or polynucleotide and thus can form a dimer with the target gene or polynucleotide; and the antisense nucleotide can be used to detect the target gene. The antisense nucleotide may have an appropriate length for its increased detection specificity.
[0044] As used herein, the term primer refers to a nucleic acid sequence which can form base pairs with a complementary template of a gene or polynucleotide in a sample and serve as a starting point for copying a template strand. A sequence of the primer does not necessarily have to be exactly the same as the template, and the primer only needs to have a sequence sufficiently complementary to the template so that they can hybridize with each other. The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four different nucleoside triphosphates in an appropriate buffer solution and at an appropriate temperature. PCR conditions, and lengths of sense and antisense primers may be modified based on those known in the art.
[0045] As used herein, the term probe refers to a substance capable of specifically binding to a gene or polynucleotide to be detected in a sample, in which the binding allows to specifically identify the presence of the gene or polynucleotide in the sample. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions may be modified based on those known in the art.
[0046] The antisense nucleotide, the primer, or the probe may be preferably designed using knowledge and techniques known in the art depending on a gene or polynucleotide to be detected.
[0047] In an embodiment, the agent capable of measuring an expression level of the protein may be an oligopeptide, an antibody (such as monoclonal antibody, polyclonal antibody, and chimeric antibody), a ligand, a peptide nucleic acid (PNA), or an aptamer, which specifically binds to the protein.
[0048] Preferably, the agent capable of measuring an expression level of the protein may be, but is not limited to, an antibody that specifically binds to a protein encoded by the gene.
[0049] As used herein, the term antibody has the same meaning as is known in the art and may refer to a specific protein molecule directed against an antigenic site. In the present disclosure, the antibody is not particularly limited in its form, and may include polyclonal antibodies, monoclonal antibodies, or parts thereof as long as they have an antigen-binding property, as well as all immunoglobulin antibodies. The antibody may also include special antibodies such as humanized antibodies. For example, the antibody may include a complete form having two full-length light chains and two full-length heavy chains as well as functional fragments of the antibody molecule. The functional fragment of the antibody molecule refers to a fragment that retains at least an antigen-binding function, and examples thereof may include, but are not limited to, Fab, F(ab), F(ab)2, and Fv.
[0050] In an embodiment, the cancer may be a solid cancer. The cancer may be, but is not limited to, one or more selected from the group consisting of cholangiocarcinoma, colon cancer, adenocarcinoma, rectal cancer, breast cancer, synovial sarcoma, chondrosarcoma, thyroid cancer, stomach cancer, thymus cancer, cervical cancer, glioma, brain cancer, melanoma, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lymphoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, blood cancer, parathyroid cancer, and ureteral cancer. In addition, the cancer may be colorectal cancer.
[0051] According to another aspect of the present disclosure, there is provided a kit for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising the composition. In the kit, the predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy and the cancer are as described above.
[0052] Specifically, the kit consists of one or more other component compositions, solutions, or devices, which are suitable for assay methods.
[0053] In an embodiment, the kit may be a kit for measuring an expression level of mRNA of the gene, for example, a kit containing essential elements to perform RT-PCR. For example, the kit may contain other reagents which are known in the art to be required to perform RT-PCR.
[0054] In an embodiment, the kit may be a kit for measuring an expression level of the protein, for example, an enzyme-linked immunosorbent assay (ELISA) kit. The kit may contain other reagents which are known in the art to be required for immunological detection of antibodies.
[0055] According to still yet another aspect of the present disclosure, there is provided a method for providing information for predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy, comprising measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3 in a biological sample obtained from the cancer patient; and evaluating the patient's therapeutic responsiveness to the cancer immunotherapy based on the measured expression level of mRNA or protein. In the above-described method, the measuring an expression level of mRNA of gene, the measuring an expression level of protein, and the predicting therapeutic responsiveness of a cancer patient to a cancer immunotherapy are as described above.
[0056] As used herein, the term biological sample refers to any biological sample obtained from an individual, and examples thereof may include, but are not limited to, a sample (such as blood, serum, plasma, lymph, saliva, or urine) obtained from an individual whose therapeutic responsiveness to a cancer immunotherapy is intended to be determined, for example, a cancer patient.
[0057] In an embodiment, the method may further comprise determining whether the patient needs to receive treatment with the cancer immunotherapy or needs to continue treatment with the cancer immunotherapy, depending on the evaluation of the patient's therapeutic responsiveness to the cancer immunotherapy.
[0058] In an embodiment, the method may further comprise administering the cancer immunotherapy to the patient. The administering may be performed before, after, or both before and after measuring an expression level of mRNA or protein of the one or more genes. For example, the expression level may be measured immediately before administration of the cancer immunotherapy, and 20 to 60 days, 25 to 50 days, 30 to 45 days, or 35 to 43 days after first administration of the cancer immunotherapy. For example, the therapeutic responsiveness to the cancer immunotherapy may be evaluated by obtaining a blood sample from the cancer patient, measuring an expression level of one or more genes of OPN, POSTN, and IGFBP-3, measuring an expression level of the same gene(s) again 40 days after starting treatment with the cancer immunotherapy, and comparing the expression levels before and after the treatment with the cancer immunotherapy. Here, the therapeutic responsiveness may be evaluated based on an increased or decreased expression level, the pattern of which differs depending on each gene or a protein expressed therefrom.
[0059] In addition, the evaluating the patient's therapeutic responsiveness to the cancer immunotherapy based on the measured expression level of mRNA or protein comprises comparing an expression level of the mRNA or protein measured in the cancer patient before (and/or after) administration of the cancer immunotherapy with an expression level of the mRNA or protein measured in other control cancer patients before (and/or after) administration of the cancer immunotherapy. The expression level measured in the other control cancer patients before and/or after administration of the cancer immunotherapy may be an average expression level identified in biological samples obtained from a plurality of control cancer patients, who have been identified to have therapeutic responsiveness to the cancer immunotherapy or have no therapeutic responsiveness to the cancer immunotherapy, before and/or after administration of the cancer immunotherapy.
[0060] In an embodiment, the expression level is measured before and after administration of the cancer immunotherapy, wherein when the gene is OPN or POSTN, the patient may be evaluated as having low therapeutic responsiveness in a case where the measured expression level is significantly higher after administration of the cancer immunotherapy than before administration of the cancer immunotherapy. For example, for OPN, in a case where the expression level measured after administration of the cancer immunotherapy is 1.1 to 2 times, 1.2 to 2 times, or 1.5 to 1.8 times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having low therapeutic responsiveness to the cancer immunotherapy. In addition, in a case where the expression level measured after administration of the cancer immunotherapy is 0.4 to 1 times, 0.4 to 0.9 times, or 0.5 to times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having high therapeutic responsiveness to the cancer immunotherapy. For example, for POSTN, in a case where the expression level measured after administration of the cancer immunotherapy is 1.1 to 2 times, 1.2 to 2 times, or 1.5 to 1.8 times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having low therapeutic responsiveness to the cancer immunotherapy. In addition, in a case where the expression level measured after administration of the cancer immunotherapy is 0.4 to 1 times or 0.5 to 0.8 times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having high therapeutic responsiveness to the cancer immunotherapy.
[0061] In an embodiment, the expression level is measured before or after administration of the cancer immunotherapy, wherein when the gene is OPN or POSTN, the patient may be evaluated as having low therapeutic responsiveness in a case where the measured expression level is significantly higher than the expression level measured in other control cancer patients (for example, patients evaluated as having high therapeutic responsiveness) before or after administration of the cancer immunotherapy.
[0062] In an embodiment, the expression level is measured before and after administration of the cancer immunotherapy, wherein when the gene is IGFBP-3, the patient may be evaluated as having low therapeutic responsiveness in a case where the measured expression level is significantly lower after administration of the cancer immunotherapy than before administration of the cancer immunotherapy. For example, in a case where the expression level measured after administration of the cancer immunotherapy is 0.4 to 1 times, 0.4 to 0.9 times, or 0.5 to 0.8 times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having low therapeutic responsiveness to the cancer immunotherapy. For example, in a case where the expression level measured after administration of the cancer immunotherapy is 1.1 to 2 times, 1.2 to 2 times, or 1.5 to 1.8 times the expression level measured before administration of the cancer immunotherapy, the cancer patient may be evaluated as having high therapeutic responsiveness to the cancer immunotherapy.
[0063] In an embodiment, the expression level is measured before or after administration of the cancer immunotherapy, wherein when the gene is IGFBP-3, the patient may be evaluated as having low therapeutic responsiveness in a case where the measured expression level is significantly lower than the expression level measured in other control cancer patients (for example, patients evaluated as having high therapeutic responsiveness) before or after administration of the cancer immunotherapy.
[0064] In an embodiment, the expression level of the mRNA may be measured by one or more methods selected from the group consisting of in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR), competitive reverse transcriptase polymerase chain reaction (competitive RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), RNase protection assay (RPA), microarray, and northern blotting, but the method is not limited thereto.
[0065] In an embodiment, the expression level of the protein may be measured by one or more methods selected from the group consisting of Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, Ouchterlony, complement fixation assay, and protein chip, but the method is not limited thereto.
[0066] According to still yet another aspect of the present disclosure, there is provided a method for treating cancer based on therapeutic responsiveness of a cancer patient to a cancer immunotherapy, the method comprising (i) administering the cancer immunotherapy to the cancer patient; and (ii) measuring an expression level of mRNA or protein of one or more genes selected from the group consisting of OPN, POSTN, and IGFBP-3 in a biological sample obtained from the cancer patient before, after, or both before and after administration of the cancer immunotherapy.
[0067] In an embodiment, the method may further comprise evaluating the patient's therapeutic responsiveness to the cancer immunotherapy based on the measured mRNA or protein expression level.
[0068] In an embodiment, the method may further comprise determining whether the patient needs to receive treatment with the cancer immunotherapy or needs to continue treatment with the cancer immunotherapy, depending on the evaluation of the patient's therapeutic responsiveness to the cancer immunotherapy.
[0069] Regarding the above-described method, the cancer immunotherapy, the gene, the cancer, the biological sample, the measuring an expression level of mRNA, the measuring an expression level of protein, the evaluating therapeutic responsiveness, and the like are as described above.
[0070] Hereinafter, the present disclosure will be described in detail by way of examples. However, the following examples are only for illustrating the present disclosure, and the present disclosure is not limited to the following examples.
Example 1. Materials and Methods
Example 1.1. Information on Patient Group and Collection of Sample
[0071] Patients with a metastatic solid cancer or a locally advanced solid cancer were subjected to anti-cancer immunotherapy with an anti-PD-L1 antibody (an anti-PD-L1 antibody (IMC-001) comprising a heavy chain that comprises the amino acid sequence set forth in SEQ ID NO: 3 and a light chain that comprises the amino acid sequence set forth in SEQ ID NO: 4), and blood samples were obtained from the patients.
[0072] Specifically, intravenous administration of the antibody IMC-001 was performed every 2 weeks according to a standard 3+3 dose escalation design until disease progression or occurrence of unacceptable toxicity. A total of 15 patients (8 males and 7 females, with their average age being 58 years old) were included in 5 dose escalation cohorts (2, 5, 10, 15, and 20 mg/kg).
[0073] Blood samples were collected from the patients before initiation of dosing (Day 1), on days 3 and 8 after dosing (Days 3 and 8), and on days 15 and 43 after dosing (Days 15 and 43). The blood samples were subjected to centrifugation and serum samples were collected therefrom. All samples were immediately separated, frozen, and stored at 80 C. until use. Type of cancer and BOR information for the blood samples by date of collection are as shown in Table 1.
TABLE-US-00001 TABLE 1 Blood sample Type of cancer BOR Day 1, 3, 8, 15, 43 Cholangiocarcinoma/liver metastasis SD Day 1, 3, 8, 15, 43 Gallbladder adenocarcinoma/lymph metastasis Day 1, 3, 8, 15, 43 Cholangiocarcinoma/liver metastasis Day 1, 3, 8, 15, 43 Thymus cancer/lung and bone metastasis Day 1, 3, 8, 15, 43 Rectal cancer/lymph metastasis SD.fwdarw.PR Day 1, 3, 8, 15, 43 Rectal cancer/lung and liver metastasis PD Day 1, 3, 8, 15, 43 Thymus cancer/lung and liver metastasis Day 1, 3, 8, 15, 43 Thyroid cancer/lung, spleen, and lymph NE metastasis Day 1, 3, 8, 15 Colon cancer/liver metastasis PD Day 1, 3, 8, 15 Rectal cancer/lung metastasis Day 1, 3, 8, 15 Breast cancer Day 1, 3, 8, 15 Synovial sarcoma/lung and liver metastasis Day 1, 3, 8, 15 Chondrosarcoma/lung and bone metastasis Day 1, 3, 8, 15 Stomach cancer/rectum metastasis Day 1, 3, 8, 15 Rectal cancer/lung metastasis BOR (best overall response): Best overall response recorded from initiation of dosing until disease progression/relapse; SD: stable disease; PD: progressive disease; PR: partial response; and NE: not evaluable. The total number of patients was 15, in which SD was 4, PR was 1, PD was 9, and NE was 1.
Example 1.2. Multiplexed Assay
[0074] The blood samples from the patient group were screened for each of 55 soluble factors by analyzing serum samples using Luminex's xMAP Technology, which is a quantitative multiplexed immunoassay using automated bead-based technology (see Table 2).
TABLE-US-00002 TABLE 2 Circulating Alpha-fetoprotein (AFP) tumor Fibroblast growth factor 2 (FGF-2) marker Human epididymis protein 4 (HE4) Hepatocyte growth factor (HGF) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Leptin Macrophage migration inhibitory factor (MIF) Osteopontin (OPN) Prolactin Stem cell factor (SCF) Tumor growth factor (TGF- ) Tumor necrosis factor (TNF- ) TNF-related apoptosis inducing ligand (TRAIL) Vascular endothelial growth factor (VEGF) Cancer antigen 125 (CA125) Cancer antigen 15-3 (CA15-3) Ferritin Galectin3 Insulin growth factor binding protein-3 (IGFBP-3) Myeloperoxidase (MPO) Angiogenic Angiopoietin 2 (Ang2) marker Bone morphogenetic protein 9 (BMP-9) Epidermal growth factor (EGF) Endoglin Fibroblast growth factor 1 (FGF-1) Granulocyte colony stimulating factor (G-CSF) Heparin-binding epidermal growth factor-like growth factor (HB-EGF) Placental growth factor (PLGF) Vascular endothelial growth factor C (VEGF-C) Vascular endothelial growth factor (VEGF-D) Metastatic Dickkopf 1 (DKK-1) marker Growth differentiation factor 15 (GDF-15) Periostin (POSTN) Cytokine/ Interferon (IFN-) chemokine Interleukin 1 (IL-1) Interleukin 1 (IL-1) Interleukin 1 receptor antagonist (IL-1r) Interleukin 2 (IL-2) Interleukin 4 (IL-4) Interleukin 10 (IL-10) Interleukin 12 (IL-12) Interleukin 15 (IL-15) Interleukin 17 (IL-17) C-X-C motif ligand 9 (CXCL9) C-X-C motif ligand 10 (CXCL10) Platelet-derived growth factor AB (PDGF-AB) Platelet-derived growth factor BB (PDGF-BB) Tie-2 Chemokine ligand 5 (CCL5) Monocyte chemotactic protein 1 (MCP-1) Monocyte chemotactic protein 3 (MCP-3) Chemokine ligand 22(CCL22) Macrophage inflammatory protein 1 alpha (MIP-1) Macrophage inflammatory protein 1 beta (MIP-1)
[0075] Human premixed multiplex assay kits for fabrication of plex panels were purchased from R&D Systems (Catalog nos. LXSAHM-06, 10, 18, 21). xMAP analysis of serum was performed using Luminex 100 in Koma Biotech Inc. (Seoul, Republic of Korea). Experimental data were analyzed using 5-parameter curve fitting in MasterPlex QT 2010 software.
Example 2. Results Obtained by Performing Analysis of Soluble Factors Before and After Administration of Cancer Immunotherapy
[0076] The analysis results in Example 1.2 were obtained from Koma Biotech Inc., and serum levels of the 55 factors were analyzed to perform comparison between the SD group and the PD group. Analysis of the 55 soluble factors showed that POSTN, OPN, and IGFBP-3 were factors that produced significant experimental results which allow for identification of therapeutic responsiveness to the cancer immunotherapy.
Example 2.1. POSTN and OPN Showed Low Levels Both at Baseline and after Treatment with Anti-PD-L1 Antibody in SD Group
[0077] The analysis results for POSTN and OPN are shown in
[0078] POSTN (periostin) is an adhesion molecule and OPN (osteopontin) is an intracellularly secreted glycoprotein.
[0079] The analysis results showed that the POSTN level in the PD group was higher than that in the SD group at baseline and increased significantly until day 43, whereas the POSTN level in the SD group remained low during the test period (range of average POSTN level in the SD group: 153316.0 to 158397.3 pg/mL, see
[0080] In the SD group, OPN gradually decreased until day 43, whereas in the PD group, OPN remained higher than in the SD group starting from baseline and did not change greatly over time (range of average OPN level in the SD group: 27687 to 41942.3 pg/mL, see
[0081] For the samples obtained from the patients whose BOR changed from SD to PR, the levels of POSTN and OPN gradually decreased until day 43 and were at the lowest on day 43 (see
[0082] Therefore, for a cancer patient group, in a case where the group shows relatively low baseline levels of POSTN and OPN, or a lowered level of OPN is produced in the group after treatment with the cancer immunotherapy as compared with before treatment with the cancer immunotherapy, the group may be selected as a patient group suitable for the treatment with the cancer immunotherapy.
Example 2.2. PD Group Showed Low Level of IGFBP-3 on Day 43 after Administration of Anti-PD-L1 Antibody
[0083] The IGFBP-3 level was not significantly different between the SD and PD groups at baseline. However, the IGFBP-3 level was significantly changed during the course of treatment with the anti-PD-L1 antibody (
[0084] These results suggest that in a case where a patient shows a low IGFBP3 level after administration of the anti-PD-L1 antibody, IGFBP3 may be a biomarker which indicates that the patient has no responsiveness to the antibody.
Example 3. Animal Experiments Using Colorectal Cancer Mouse Model
[0085] A request for animal experiments using a colorectal cancer mouse model was made to Biocytogen and Gempharmatech. A colorectal cancer mouse model obtained with C57BL/6 mice and MC38-hPD-L1 tumor cells was developed by Biocytogen, and a colorectal cancer mouse model obtained with BALB/c mice and CT26-hPD-L1/hCD47 tumor cells was developed by Gempharmatech.
Example 3.1. Therapeutic Responsiveness Following Administration of IMC-001 (Biomarker: OPN)
Example 3.1.1. Changes in Tumor Volume Following Administration of IMC-001 In MC38-hPD-L1 Tumor Model
[0086] In vivo efficacy of IMC-001 was evaluated for treatment of MC38-hPD-L1 tumor in C57BL/6 mice as a colorectal cancer model.
[0087] For tumor development, the C57BL/6 mice were subcutaneously injected on the right anterior flank with 510 5 MC38-hPD-L1 tumor cells suspended in 0.1 mL PBS. The tumor-bearing mice whose average tumor size reached about 100 mm 3 were randomly enrolled in the group hIgG1 20 mg/kg, the group IMC-001 3 mg/kg, and the group IMC-001 10 mg/kg (each group consisting of 8 mice). All test substances were intraperitoneally administered to the tumor-bearing mice at a frequency of 3 times a week for 3 weeks.
[0088] The mice were sacrificed on day 21. GraphPad Prism 8 software was used to plot graphs of tumor volume, and comparison between the groups was performed using two-way ANOVA with Dunnett's multiple comparison test (p-value; *<0.05, **<0.01, ***<0.001, ****<0.0001) (
Example 3.1.2. Determination of Mouse Serum OPN Concentration by ELISA
[0089] At the time of sacrifice of the mice as in Example 3.1.1, all serum samples were collected and stored at 80 C. until analysis.
[0090] Mouse serum OPN was measured by enzyme-linked immunosorbent assay (ELISA) using the mouse OPN duoset ELISA kit (DY411, R&D Systems, Minneapolis, Minnesota, United States), and the procedure was performed according to the instructions of the manufacturer of the kit.
[0091] All samples were diluted at a ratio of 1:8,000 using 1 reagent diluent concentrate 2 from Duoset Ancillary Reagent Kit 2 (DY008, R&D Systems) within the range of the standard curve. All samples were run in duplicate.
[0092] GraphPad Prism 8 software was used to plot variable data, and then correlation between two parameters (serum OPN and relative tumor volume) was identified using Pearson's correlation analysis (
Example 3.1.3. Changes in Tumor Volume Following Administration of IMC-001 in CT26-hPD-L1/hCD47 Tumor Model
[0093] In vivo efficacy of IMC-001 was evaluated for treatment of CT26-hPD-L1/hCD47 tumor in BALB/c mice as a colorectal cancer model.
[0094] For tumor development, the BALB/c mice were subcutaneously injected on the right anterior flank with 210.sup.6 CT26-hPD-L1/hCD47 tumor cells suspended in 0.1 mL PBS. The tumor-bearing mice whose average tumor size reached about 100 mm 3 were randomly enrolled in the group hIgG1 21.4 mg/kg, the group IMC-001 2.1 mg/kg, and the group IMC-001 10.7 mg/kg (each group consisting of 8 mice). All test substances were intraperitoneally administered to the tumor-bearing mice at a frequency of twice a week for the first 2 weeks, and tumor volume was measured for 2 more weeks.
[0095] The mice were sacrificed on day 28 after the first administration of IMC-001. GraphPad Prism 8 software was used to plot graphs of tumor volume, and comparison between the groups was performed using two-way ANOVA with Dunnett's multiple comparison test (p-value; *<0.05, **<0.01, ***<0.001, ****<0.0001) (
Example 3.1.4. Determination of Mouse Serum OPN Concentration by ELISA
[0096] At the time of sacrifice of the mice as in Example 3.1.3, all serum samples were collected and stored at 80 C. until analysis.
[0097] Mouse serum OPN was measured by enzyme-linked immunosorbent assay (ELISA) using the mouse OPN duoset ELISA kit (DY411, R&D Systems, Minneapolis, Minnesota, United States), and the procedure was performed according to the instructions of the manufacturer of the kit. All samples were diluted at a ratio of 1:8,000 using 1 reagent diluent concentrate 2 from Duoset Ancillary Reagent Kit 2 (DY008, R&D Systems) within the range of the standard curve. All samples were run in duplicate.
[0098] GraphPad Prism 8 software was used to plot variable data, and then correlation between two parameters (serum OPN and relative tumor volume) was identified using Pearson's correlation analysis (
Example 3.1.5. Results
[0099] In the two colorectal cancer mouse models (MC38 and CT26) used in Examples 3.1.1 to 3.1.4, tumor growth was inhibited in the groups administered with IMC-001 as compared with each control group (hIgG1) (
[0100] In addition, as shown in
[0101] From these results, since in the groups administered with IMC-001, a lower OPN level was observed as a relative tumor volume was smaller, it was identified that OPN could be used as a biomarker to predict whether an anti-PD-L1 antibody such as IMC-001 would exert a therapeutic effect.
Example 3.2. Therapeutic Responsiveness Following Administration of IMC-001 (Biomarker: IGFBP-3)
Example 3.2.1. Changes in Tumor Volume Following Administration of IMC-001 in MC38-hPD-L1 Tumor Model
[0102] In vivo efficacy of IMC-001 was evaluated for treatment of MC38-hPD-L1 tumor in C57BL/6 mice as a colorectal cancer model.
[0103] For tumor development, the C57BL/6 mice were subcutaneously injected on the right anterior flank with 510 5 MC38-hPD-L1 tumor cells suspended in 0.1 mL PBS. The tumor-bearing mice whose average tumor size reached about 100 mm 3 were randomly enrolled in the group hIgG1 20 mg/kg, the group IMC-001 3 mg/kg, and the group IMC-001 10 mg/kg (each group consisting of 8 mice). All test substances were intraperitoneally administered to the tumor-bearing mice at a frequency of 3 times a week for 3 weeks.
[0104] The mice were sacrificed on day 21. GraphPad Prism 8 software was used to plot graphs of tumor volume, and comparison between the groups was performed using two-way ANOVA with Dunned s multiple comparison test (p-value; *<0.05, **<0.01, ***<0.001, ****<0.0001) (
Example 3.2.2. Determination of Mouse Serum IGFBP-3 Concentration by ELISA
[0105] At the time of sacrifice of the mice as in Example 3.2.1, all serum samples were collected and stored at 80 C. until analysis.
[0106] Mouse serum IGFBP-3 was measured by enzyme-linked immunosorbent assay (ELISA) using the mouse IGFBP-3 duoset ELISA kit (DY775, R&D Systems, Minneapolis, Minnesota, United States), and the procedure was performed according to the instructions of the manufacturer of the kit. All samples were diluted at a ratio of 1:2,000 using 1 reagent diluent concentrate 3 from Duoset Ancillary Reagent Kit 3 (DY009, R&D Systems) within the range of the standard curve. Specifically, the detection antibody was diluted using 1 reagent diluent concentrate 3 containing 2% heat-inactivated normal goat serum (DY005, Reagent Additive 1, R&D Systems) according to the manufacturer's recommendations. All samples were run in duplicate.
[0107] GraphPad Prism 8 software was used to plot variable data. Correlation between two parameters (serum IGFBP-3 and relative tumor volume) was identified using Pearson's correlation analysis (
Example 3.2.3. Changes in Tumor Volume Following Administration of IMC-001 in CT26-hPD-L1/hCD47 Tumor Model
[0108] In vivo efficacy of IMC-001 was evaluated for treatment of CT26-hPD-L1/hCD47 tumor in BALB/c mice as a colorectal cancer model.
[0109] For tumor development, the BALB/c mice were subcutaneously injected on the right anterior flank with 210.sup.6 CT26-hPD-L1/hCD47 tumor cells suspended in 0.1 mL PBS. The tumor-bearing mice whose average tumor size reached about 100 mm 3 were randomly enrolled in the group hIgG1 21.4 mg/kg, the group IMC-001 2.1 mg/kg, and the group IMC-001 10.7 mg/kg (each group consisting of 8 mice). All test substances were intraperitoneally administered to the tumor-bearing mice at a frequency of twice a week for 4 weeks.
[0110] The mice were sacrificed on day 28 after the first administration of IMC-001. GraphPad Prism 8 software was used to plot graphs of tumor volume, and comparison between the groups was performed using two-way ANOVA with Dunnett's multiple comparison test (p-value; *<0.05, **<0.01, ***<0.001, ****<0.0001) (
Example 3.2.4. Determination of Mouse Serum IGFBP-3 Concentration by ELISA
[0111] At the time of sacrifice of the mice as in Example 3.2.3, all serum samples were collected and stored at 80 C. until analysis.
[0112] Mouse serum IGFBP-3 was measured by enzyme-linked immunosorbent assay (ELISA) using the mouse IGFBP-3 duoset ELISA kit (DY775, R&D Systems, Minneapolis, Minnesota, United States), and the procedure was performed according to the instructions of the manufacturer of the kit. All samples were diluted at a ratio of 1:2,000 using 1 reagent diluent concentrate 3 from Duoset Ancillary Reagent Kit 3 (DY009, R&D Systems) within the range of the standard curve. Specifically, the detection antibody was diluted using 1 reagent diluent concentrate 3 containing 2% heat-inactivated normal goat serum (DY005, Reagent Additive 1, R&D Systems) according to the manufacturer's recommendations. All samples were run in duplicate.
[0113] GraphPad Prism 8 software was used to plot variable data. Correlation between two parameters (serum IGFBP-3 and relative tumor volume) was identified using Pearson's correlation analysis (
Example 3.2.5. Results
[0114] In the two colorectal cancer mouse models (MC38 and CT26) used in Examples 3.2.1 to 3.2.4, tumor growth was inhibited in the groups administered with IMC-001 as compared with each control group (hIgG1) (
[0115] In addition, as shown in
[0116] From these results, since in the groups administered with IMC-001, a higher IGFBP-3 level was observed as a relative tumor volume was smaller, it was identified that IGFBP-3 could be used as a biomarker to predict whether an anti-PD-L1 antibody such as IMC-001 would exert a therapeutic effect.