Application of pharmaceutical composition containing anti-PD-1 antibody in preparation of medicine for treating advanced non-small cell lung cancer
阅读说明：本技术 一种含有抗pd-1抗体的药物组合物在制备治疗晚期非小细胞肺癌药物中的应用 (Application of pharmaceutical composition containing anti-PD-1 antibody in preparation of medicine for treating advanced non-small cell lung cancer ) 是由 蒋涛 陈洛南 任胜祥 周彩存 王平洋 于 2021-10-11 设计创作，主要内容包括：本发明提供了一种含有抗PD-1抗体的药物组合物在制备治疗晚期非小细胞肺癌药物中的应用。此外,本发明还提供了预测该药物组合物治疗疗效的标记物、试剂盒和方法。该药物组合物包括抗PD-1抗体和细胞毒类抗癌药,该药物用于治疗EGFR突变型晚期非小细胞肺癌患者,且其经EGFR小分子酪氨酸激酶抑制剂治疗的疗效是失败的。本发明针对EGFR突变型晚期非小细胞肺癌经EGFR小分子酪氨酸激酶抑制剂治疗失败以后不存在EGFR T790M基因突变的患者提供一种有效的治疗策略,并针对该治疗策略下的患者进行基因组突变和表达数值进行高效处理,准确选出能够从抗PD-1抗体和细胞毒类抗癌药联合治疗中获益的EGFR突变型晚期非小细胞肺癌患者,指导此类患者的精准化、规范化治疗。(The invention provides application of a pharmaceutical composition containing an anti-PD-1 antibody in preparation of a medicine for treating advanced non-small cell lung cancer. In addition, the invention also provides a marker, a kit and a method for predicting the treatment effect of the pharmaceutical composition. The pharmaceutical composition comprises an anti-PD-1 antibody and a cytotoxic anticancer drug, and the drug is used for treating EGFR mutant advanced non-small cell lung cancer patients, and the curative effect of the drug on EGFR small molecule tyrosine kinase inhibitor is failed. The invention provides an effective treatment strategy for patients without EGFR T790M gene mutation after EGFR mutant type advanced non-small cell lung cancer fails to be treated by an EGFR small-molecule tyrosine kinase inhibitor, and efficiently processes genome mutation and expression numerical values of the patients under the treatment strategy, accurately selects EGFR mutant type advanced non-small cell lung cancer patients who can benefit from combined treatment of anti-PD-1 antibodies and cytotoxic anti-cancer drugs, and guides the accurate and standardized treatment of the patients.)
1. Use of a pharmaceutical composition comprising an anti-PD-1 antibody for the manufacture of a medicament for the treatment of advanced non-small cell lung cancer, wherein the pharmaceutical composition comprises an anti-PD-1 antibody and a cytotoxic anti-cancer agent, wherein the medicament is for the treatment of EGFR mutant advanced non-small cell lung cancer patients who have failed therapy with an EGFR small molecule tyrosine kinase inhibitor.
2. The use of claim 1, wherein the non-small cell lung cancer patient has an EGFR-sensitive mutation and does not have a secondary EGFR T790M mutation; preferably, the non-small cell lung cancer patient has an exon 21L858R mutation or an exon 19 deletion; more preferably, said non-small cell lung cancer patient is treated with said medicament:
(1) compared with the average M1 macrophage-to-M2 macrophage ratio of a non-small cell lung cancer patient, the M1 macrophage ratio is higher than the average value, the M2 macrophage ratio is lower than the average value, and/or the M1/M2 macrophage ratio is higher than the average value; and/or
(2) There is a mutation in the DSPP and/or TP53 genes, preferably a double mutation of DSPP and TP53 or a DSPP mutation.
3. The use according to claim 1, wherein the cytotoxic anticancer drug is an antifolate metabolizing anticancer drug and/or a platinum anticancer drug; preferably, the anti-folate metabolism anticancer agent is pemetrexed; the platinum anticancer drug is carboplatin; the anti-PD-1 antibody is preferably Tereprinimab.
4. A pharmaceutical composition for treating advanced non-small cell lung cancer, comprising an anti-PD-1 antibody and a cytotoxic anticancer agent, wherein the pharmaceutical composition is used for treating EGFR mutant advanced non-small cell lung cancer patients and the therapeutic effect of the EGFR small-molecule tyrosine kinase inhibitor treatment is failed.
5. The pharmaceutical composition of claim 4, wherein the non-small cell lung cancer patient has an EGFR-sensitive mutation and does not have a secondary EGFR T790M mutation; preferably, the non-small cell lung cancer patient has an exon 21L858R mutation or an exon 19 deletion; more preferably, said non-small cell lung cancer patient is treated with said medicament:
(1) compared with the average M1 macrophage-to-M2 macrophage ratio of a non-small cell lung cancer patient, the M1 macrophage ratio is higher than the average value, the M2 macrophage ratio is lower than the average value, and/or the M1/M2 macrophage ratio is higher than the average value; and/or
(2) There is a mutation in the DSPP and/or TP53 genes, preferably a double mutation of DSPP and TP53 or a DSPP mutation.
6. The pharmaceutical composition of claim 4, wherein the cytotoxic anticancer drug is an antifolate-metabolizing anticancer drug and/or a platinum-based anticancer drug; preferably, the anti-folate metabolism anticancer agent is pemetrexed; the platinum anticancer drug is carboplatin; the anti-PD-1 antibody is preferably Tereprinimab.
7. A kit for predicting the therapeutic efficacy of a pharmaceutical composition according to any one of claims 4 to 6, comprising:
(1) reagents for detecting mutations in the DSPP and/or TP53 genes in peripheral blood or tumor tissue of an individual;
(2) reagents and/or devices for detecting the ratio of M1 macrophages to M2 macrophages; and/or
(3) Reagents and/or devices for detecting the expression levels of: RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB.
8. A method for predicting the therapeutic efficacy of a pharmaceutical composition according to any one of claims 4 to 6, comprising detecting the presence of a mutation in the DSPP and/or TP53 gene in tumor cells and/or peripheral blood of said patient, wherein the presence of a mutation in the DSPP and/or TP53 gene, preferably a double mutation of DSPP and TP53 or the presence of a mutation in DSPP indicates that said patient responds better to therapy than a patient in the absence of said mutation.
9. The method of claim 8, further comprising detecting one or more of the following indicators:
(1) detecting the semi-quantitative numerical proportion of M1 macrophage and M2 macrophage of the patient obtained by indirect calculation of gene transcriptome data; wherein a M1 macrophage ratio above the mean, an M2 macrophage ratio below the mean, and/or an M1/M2 macrophage ratio above the mean indicates that the patient responds to treatment better than patients with an M1 macrophage ratio below the mean, an M2 macrophage ratio above the mean, and/or an M1/M2 macrophage ratio below the mean, respectively;
(2) detecting the expression level of the following genes in the patient: RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB.
Technical Field
The invention relates to the technical field of medicine, in particular to application of a pharmaceutical composition containing an anti-PD-1 antibody in preparation of a medicine for treating advanced non-small cell lung cancer.
Background
From 2004, scientists successively discovered EGFR gene mutation and its small molecule Tyrosine Kinase Inhibitor (TKI) important value in the treatment of advanced NSCLC patients with non-small cell lung cancer (NSCLC), and the treatment pattern of advanced NSCLC patients with EGFR mutation is changed day by day. Median progression-free survival (mPFS) for first-line EGFR TKIs treatments, such as erlotinib, gefitinib, icotinib, affinib, osimertinib and almonetinib, for 10-19 months. However, almost all patients with advanced NSCLC with EGFR mutations develop resistance to first-line TKI treatment. After failure of first-line TKI treatment, patients with advanced NSCLC with secondary EGFR T790M resistance mutation received osimertinib treatment with an effective rate of only 50% -60% and mPFS for only 7-8 months. For those advanced NSCLC patients without secondary EGFR T790M resistance mutation, the means of subsequent treatment is very limited, the current standard treatment regimen is still chemotherapy, with mPFS only 4-5 months. Therefore, there is an urgent need for new strategies in clinical treatment to further improve the overall prognosis of these populations after failure of EGFR-TKIs.
Programmed death receptor 1(PD-1) is mainly expressed in activated T cells and B cells, has the function of inhibiting the activation of lymphocytes, and is a normal peripheral tissue tolerance mechanism for preventing and treating over-immune stress. However, the activated T cells infiltrated in the tumor microenvironment highly express PD-1 molecules, and the activated T cells highly express PD-L1 and/or PD-L2, so that the PD-1 pathway of the activated T cells in the tumor microenvironment is continuously activated, the T cell function is inhibited, and the tumor cells cannot be killed. The therapeutic PD-1 antibody can block the pathway, partially restore the function of T cells, and enable the activated T cells to continuously kill tumor cells.
Currently, immunotherapy based on PD-1 antibodies has drastically changed the treatment pattern of advanced/metastatic NSCLC without EGFR mutations. Immune Checkpoint Inhibitors (ICIs) directed against the PD-1/PD-L1 pathway, such as nivolumab, pembrolizumab and atezolizumab, have become the standard of first and second line treatment for advanced NSCLC patients. However, EGFR mutant NSCLC patients treated with the single agent PD-L1 or PD-1 antibody after EGFR TKI failure did not show substantial survival benefit compared to patients receiving standard chemotherapy. In addition, clinical trials using the EGFR TKI and the PD-L1 antibody in combination result in high incidence of interstitial pneumonia and other safety problems, and therefore, for patients after EGFR mutant advanced non-small cell lung cancer fails to be treated by the EGFR small molecule tyrosine kinase inhibitor, better treatment strategies need to be explored, and a curative effect prediction marker of a novel treatment strategy needs to be further searched, so that patients with EGFR mutant advanced non-small cell lung cancer who can benefit from the combination treatment of the anti-PD-1 antibody and the cytotoxic anticancer drug can be accurately selected, and the accurate and standardized treatment of the patients can be guided.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides application of a pharmaceutical composition containing an anti-PD-1 antibody in preparation of a medicine for treating advanced non-small cell lung cancer, and a marker, a kit and a method for predicting the curative effect of the medicine.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides an application of a pharmaceutical composition containing an anti-PD-1 antibody in preparing a medicine for treating advanced non-small cell lung cancer, wherein the pharmaceutical composition comprises the anti-PD-1 antibody and a cytotoxic anticancer drug, the medicine is used for treating EGFR mutant advanced non-small cell lung cancer patients, and the curative effect of the EGFR mutant advanced non-small cell lung cancer patients through EGFR small-molecule tyrosine kinase inhibitor is failed.
Further, the non-small cell lung cancer patients have an EGFR sensitive mutation and do not have a secondary EGFR T790M mutation.
Furthermore, the non-small cell lung cancer patient has exon 21L858R mutation or exon 19 deletion.
Further, after the non-small cell lung cancer patient is treated by the medicine:
(1) compared with the average M1 macrophage-to-M2 macrophage ratio of a non-small cell lung cancer patient, the M1 macrophage ratio is higher than the average value, the M2 macrophage ratio is lower than the average value, and/or the M1/M2 macrophage ratio is higher than the average value; and/or
(2) There is a mutation in the DSPP and/or TP53 genes, preferably a double mutation of DSPP and TP53 or a DSPP mutation.
Further, the cytotoxic anticancer drug is an antifolate-metabolizing anticancer drug and/or a platinum-based anticancer drug.
Further, the anti-folate metabolism anticancer drug is pemetrexed; the platinum anticancer drug is carboplatin.
Further, the anti-PD-1 antibody is Tereprinimab.
In a second aspect of the invention, there is provided a pharmaceutical composition for treating advanced non-small cell lung cancer, comprising an anti-PD-1 antibody and a cytotoxic anticancer agent, wherein the pharmaceutical composition is used for treating patients with EGFR mutant advanced non-small cell lung cancer and the therapeutic effect of the pharmaceutical composition on treatment with an EGFR small molecule tyrosine kinase inhibitor is failed.
Further, the non-small cell lung cancer patients have an EGFR sensitive mutation and do not have a secondary EGFR T790M mutation.
Furthermore, the non-small cell lung cancer patient has exon 21L858R mutation or exon 19 deletion.
Further, after the non-small cell lung cancer patient is treated by the pharmaceutical composition:
(1) compared with the average M1 macrophage-to-M2 macrophage ratio of a non-small cell lung cancer patient, the M1 macrophage ratio is higher than the average value, the M2 macrophage ratio is lower than the average value, and/or the M1/M2 macrophage ratio is higher than the average value; and/or
(2) There is a mutation in the DSPP and/or TP53 genes, preferably a double mutation of DSPP and TP53 or a DSPP mutation.
Further, the cytotoxic anticancer drug is an antifolate-metabolizing anticancer drug and/or a platinum-based anticancer drug.
Further, the anti-folate metabolism anticancer drug is pemetrexed; the platinum anticancer drug is carboplatin.
Further, the anti-PD-1 antibody is Tereprinimab.
A third aspect of the present invention provides a kit for predicting the therapeutic efficacy of the above pharmaceutical composition, comprising:
(1) reagents for detecting mutations in the DSPP and/or TP53 genes in peripheral blood or tumor tissue of an individual;
(2) reagents and/or devices for detecting M1 macrophages and M2 macrophages and their ratios; and/or
(3) Reagents and/or devices for detecting the expression levels of: RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB.
In a fourth aspect, the present invention provides a method for predicting the therapeutic efficacy of the above pharmaceutical composition, which comprises detecting the presence or absence of a mutation in the DSPP and/or TP53 gene in the tumor cells and/or peripheral blood of the above patient, wherein the presence of a mutation in the DSPP and/or TP53 gene, preferably a double mutation of DSPP and TP53 or the presence of a mutation in DSPP indicates that the patient responds better to the treatment than a patient in which the mutation is not present.
Further, the method may further comprise detecting one or more of the following:
(1) detecting the semi-quantitative numerical ratio of M1 macrophage and M2 macrophage of the patient obtained by indirect calculation through gene transcriptome data; wherein a M1 macrophage ratio above the mean, a M2 macrophage ratio below the mean, and/or a M1/M2 macrophage ratio above the mean indicates that the patient responds to treatment better than patients with a M1 macrophage ratio below the mean, a M2 macrophage ratio above the mean, and/or a M1/M2 macrophage ratio below the mean, respectively;
(2) detecting the expression level of the following genes in the patient: RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention provides an effective treatment strategy for patients without EGFR T790M gene mutation after EGFR mutant type advanced non-small cell lung cancer fails to be treated by an EGFR small molecule tyrosine kinase inhibitor, namely combining an anti-PD-1 antibody, an anti-folate metabolism anticancer drug and a platinum anticancer drug, efficiently treating genome mutation and expression numerical values of the patients under the treatment strategy, accurately selecting EGFR mutant type advanced non-small cell lung cancer patients who can benefit from the combined treatment of the anti-PD-1 antibody and the cytotoxic anticancer drug, and guiding the accurate and standardized treatment of the patients.
Drawings
FIG. 1 shows the therapeutic response, progression-free survival and overall survival of anti-PD-1 antibodies in combination with chemotherapy for non-small cell lung cancer in one embodiment of the invention; wherein panel a is the maximum change in tumor size from baseline assessed according to RECIST v1.1 (n-38); the length of the bar represents the maximum decrease or minimum increase of the target lesion; # denotes that the identified partial response is classified as stable disease; patients with more than 30% reduction in target lesions but with progression to new lesions or non-target lesions; panel b shows the change in tumor burden over time in baseline individuals assessed according to RECIST v1.1 (n-38); panel c shows the assessment of progression free survival of all 40 patients in the cohort according to RECIST v 1.1; panel d shows the overall survival of all 40 patients enrolled as assessed according to RECIST v 1.1;
FIG. 2 shows the results of whole exome sequencing of a patient in one embodiment of the invention; wherein panel a highlights the mutant genes color-coded by the type of mutation in the experimental sample; panel b shows the selection of the first three combinations of different basis factors, depending on the P-value of the inter-group PFS; the dashed line in the left panel shows the threshold of the filter; the numbers on the bar graph in the right panel represent the proportion of the corresponding sample in the general population; panel c shows progression free survival (log rank test) for DSPP + TP53 co-mutant patients versus wild type patients; panel d shows the immunoinfiltration of the DSPP + TP53 co-mutation with wild type in EGFR mutant samples obtained from TCGA (Wilcoxon signed rank test); panel e shows progression free survival (log rank test) for DSPP mutant versus wild type patients; panel f shows the immunoinfiltration of DSPP mutations relative to wild type in EGFR mutation samples obtained from TCGA (Wilcoxon signed rank test); PFS, progression free survival; HR, risk ratio; p < 0.05, P < 0.01, P < 0.001, P < 0.0001;
FIG. 3 shows the results of a comprehensive analysis of the sequencing of the full exome and transcriptome of a patient according to one embodiment of the invention; wherein, the graph a is a sample correlation heat map based on an expression matrix; different colors represent different correlation coefficients; panel b is a volcano plot of 8 up-and 5 down-regulated differentially expressed genes between PR (partial response) and non-PR groups, each group of genes being labeled with the names of the first 5 genes; panel c shows the immunoassay (Wilcoxon signed rank test) for the PR and non-PR groups; panel d shows the M1/M2 macrophage ratio (Wilcoxon signed rank test) for PR vs non-PR groups; panel e shows the M1/M2 macrophage ratio of DSPP mutants from TCGA (Wilcoxon signed rank test) EGFR mutant samples to wild type tumors; p < 0.05, P < 0.01, P < 0.001, P < 0.0001;
FIG. 4 shows the process and results of whole transcriptome sequencing of a patient in one embodiment of the invention; FIG. a shows a flow chart of support vector machine selection of PR classifier; graph b shows a line graph of classification accuracy, the x-axis shows the number of genes that make up the classifier, the y-axis shows the number of 1000 cycle test passes, two fold lines represent two different accuracy thresholds, the light lines need to be correctly classified five times out of six, while the dark lines need to be monopolies of rights; FIG. c is a Venn diagram showing the intersection of candidate genes selected by differential expression analysis, support vector machine and sample specific network analysis;
FIG. 5 shows differential expression analysis and protein-protein interaction network between DSPP-mutants and wild type tumors in one embodiment of the invention; wherein panel a is a volcano plot of 536 up-and 76 down-regulated differentially expressed genes from an EGFR mutant sample obtained from TCGA (P.ltoreq.0.05, Log) between DSPP mutants and wild type tumors2The Fold Change is more than or equal to 2); panel b is a GSEA enrichment plot showing down-regulation of MAPK signaling pathway (enrichment score-0.309) in DSPP mutant and wild-type tumors;panel c shows first and critical second order genes associated with DSPP on PPI background network and local network consisting of MAPK8IP2 and its first and second order linked genes on protein-protein interaction background network;
FIG. 6 shows a comparison of IFN- γ signature gene expression in our cohort and in the TCGANSCLC cohort in accordance with an embodiment of the invention; in our cohort (a) and TCGA EGFR mutation NSCLC cohort (b), the DSPP mutation is associated with increased IFN- γ signature gene expression; ns, not significant; WT, wild type.
Detailed Description
The invention provides application of a pharmaceutical composition containing an anti-PD-1 antibody in preparation of a medicine for treating advanced non-small cell lung cancer and a marker for predicting treatment effect of the medicine, and provides a system and a method for developing and predicting treatment effect prediction of malignant tumors, particularly non-small cell lung cancer patients, by using the marker in combination with anti-PD-1 antibody chemotherapy.
Term(s) for
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless otherwise defined elsewhere herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.
By "administering," "administering," and "treating" is meant introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and via in vivo electroporation.
An "adverse effect" (AE) as referred to herein is any adverse and often unintentional or undesirable sign, symptom or disease associated with the use of medical treatment. For example, adverse reactions may be associated with activation of the immune system or expansion of immune system cells in response to therapy. The medical treatment may have one or more related AEs, and each AE may have the same or different severity level.
"tumor burden" refers to the total amount of tumor mass distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as measuring its size using calipers after the tumor is removed from the subject, or while in vivo using imaging techniques such as ultrasound, bone scans, Computed Tomography (CT), or Magnetic Resonance Imaging (MRI) scans.
The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, measuring its dimensions using calipers after the tumor is removed from the subject, or while in vivo using imaging techniques such as bone scans, ultrasound, CT, or MRI scans.
The terms "subject", "individual", "object" include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject" and "patient" are used interchangeably herein.
"antibody" as used herein refers to any form of antibody that achieves the desired biological or binding activity. It is therefore used in its broadest sense, but is not limited to, monoclonal, polyclonal, multispecific, humanized full-length human, chimeric and camel-derived single domain antibodies. An "antibody" specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region comprising three constant domains CH1, CH2 and CH 3. Each light chain comprises a light chain variable region (VL) and a light chain constant region comprising a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Generally, from N-terminus to C-terminus, both light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. Amino acids are typically assigned to each domain according to the following definitions: sequences of Proteins of Immunological Interest, Kabat et al; national Institutes of Health, Bethesda, Md.; 5 th edition; NIH publication No. 91-3242 (1991): kabat (1978) adv.prot.chem.32: 1 to 75; kabat et al, (1977) j.biol.chem.252: 6609-6616; chothia et al (1987) Jmol.biol.196: 901-917 or Chothia et al, (1989) Nature 341: 878-883.
The term "antibody" includes: naturally occurring and non-naturally occurring abs; monoclonal and polyclonal Ab; chimeric and humanized abs; human or non-human Ab; ab is fully synthesized; and a single chain Ab. Non-human abs may be humanized by recombinant methods to reduce their immunogenicity in humans.
Unless specifically indicated otherwise, "antibody fragment" or "antigen-binding fragment" as used herein refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, e.g., a fragment that retains one or more CDR regions. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; nanobodies and multispecific antibodies formed from antibody fragments.
"chimeric antibody" refers to antibodies and fragments thereof as follows: wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity.
"human antibody" refers to an antibody comprising only human immunoglobulin sequences. A human antibody may contain murine carbohydrate chains if it is produced in a mouse, mouse cells, or a hybridoma derived from a mouse cell. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.
"humanized antibody" refers to antibody forms containing sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain a minimal sequence derived from a single side of a non-human immunoglobulin. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.
Herein, the term "cancer" or "malignancy" refers to a wide variety of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distal parts of the body through the lymphatic system or blood stream. Examples of cancers suitable for treatment or prevention using the methods, medicaments and kits of the invention include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of cancer include squamous cell cancer, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, nasopharyngeal cancer, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.
The term "non-small cell lung cancer" classifies non-small cell lung cancer (NSCLC) into three categories based on the appearance and other characteristics of the cancer cells: squamous Cell Carcinoma (SCC), adenocarcinoma, and Large Cell Carcinoma (LCC). SCC accounts for approximately 25-30% of all lung cancer cases. SCC is closely associated with smoking, usually in the central region of the lung; adenocarcinoma accounts for approximately 40% of all lung cancer cases, a type of cancer that usually occurs in the outer regions of the lung; LCC accounts for approximately 10-15% of all lung cancer cases, and LCC patients often have rapid tumor growth and poor prognosis. Other less common types of lung cancer include carcinoid tumors, adenoid cystic carcinoma, hamartomas, lymphomas, and sarcomas.
"programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo and binds to both ligands PD-L1 and PD-L2. The term "PD-1" as used herein includes variants, isoforms, and species homologs of human PD-1(hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1.
As described herein, a therapeutic anti-human PD-1 antibody or anti-hPD-1 antibody refers to a monoclonal antibody that specifically binds to mature human PD-1.
As used herein, "RECIST 1.1 standard of efficacy" refers to Eisenhauver et al, e.a. et al, eur.j Cancer 45: 228-247(2009) is defined for target damage or non-target damage based on the context of the measured response. Prior to immunotherapy, it is the most common standard for efficacy assessment of solid tumors. However, with the advent of the immune age, many problems which have not been found in the tumor evaluation before are presented, so that based on the newly appeared phenomenon caused by immunotherapy, in 2016, the RECIST working group provides a new judgment standard after correcting the existing "RECIST v.1.1", namely the "irRECIST standard" described herein, aiming at better evaluating the curative effect of immunotherapy drugs.
The term "ECOG" score is an indicator of a patient's general health and ability to tolerate treatment, as measured by their physical strength. ECOG physical performance scoring criteria score: 0 minute, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes. A score of 0 means that the motility was completely normal and had no difference from the motility before onset. A score of 1 means that the person is free to walk and engage in light physical activities, including general housework or office work, but not heavy physical activities.
As used herein, "treating" cancer refers to employing a treatment regimen described herein (e.g., administration of an anti-PD-1 antibody) to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject having or diagnosed with cancer. Positive treatment effects in cancer can be measured in a variety of ways (see w.a. weber, j.nucl.med., 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, T/C.ltoreq.42% is the minimum level of anti-tumor activity according to the NCI standard. T/C (%) is considered median treated/median control tumor volume x 100. In some embodiments, the therapeutic effect achieved by the combination of the invention is any of PR, CR, OR, PFS, DFS and OS. PFS (also called "time to tumor progression") refers to the length of time during and after treatment during which cancer does not grow and includes the amount of time a patient experiences CR or PR and the amount of time a patient experiences SD. DFS refers to the length of time during and after treatment that a patient is still disease free. OS refers to an extension of life expectancy compared to an initial or untreated individual or patient. In some embodiments, the response to a combination of the invention is any of PR, CR, PFS, DFS, OR OS, assessed using RECIST 1.1 efficacy criteria. The treatment regimen for a combination of the invention effective in treating a cancer patient can vary depending on a variety of factors such as the disease state, age, weight of the patient and the ability of the therapy to elicit an anti-cancer response in the subject. Although embodiments of the invention may not achieve an effective positive therapeutic effect in each subject, a positive therapeutic effect should be effective and achieved in a statistically significant number of subjects.
The terms "mode of administration", "dosing regimen", which are used interchangeably, refer to the dosage and time of use of each therapeutic agent in the combination of the invention.
In the following paragraphs, various aspects of the present invention are described in further detail.
anti-PD-1 antibodies
Herein, a "PD-1 antibody" refers to any chemical compound or biomolecule that binds to the PD-1 receptor, blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. Alternative nouns or synonyms for PD-1 and its ligands include: for PD-1, PDCD1, PD1, CD279, and SLEB 2; for PD-L1, there are PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274 and B7-H; and for PD-L2 there are PDCD1L2, PDL2, B7-DC and CD 273. In any of the inventive methods of treatment, medicaments and uses for treating a human subject, the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD 1. The human PD-1 amino acid sequence can be found at NCBI locus number: NP _ 005009. Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP-054862 and NP-079515.
Herein, when referring to an "anti-PD-1 antibody," unless otherwise indicated or described, the term includes antigen-binding fragments thereof.
The anti-PD-1 antibody applicable to any application, therapy, medicament and kit disclosed by the invention is combined with PD-1 with high specificity and high affinity, blocks the combination of PD-L1/2 and PD-1 and inhibits PD-1 signal transduction, thereby achieving an immunosuppressive effect. In any of the uses, therapies, medicaments and kits disclosed herein, the anti-PD-1 antibody includes the full-length antibody itself, as well as antigen-binding portions or fragments that bind to the PD-1 receptor and exhibit functional properties similar to those of an intact Ab in inhibiting ligand binding and upregulating the immune system. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof that cross-competes for binding to human PD-1 with terieprinimab. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a chimeric, humanized, or human Ab or antigen-binding fragment thereof. In certain embodiments for treating a human subject, the Ab is a humanized Ab.
In some embodiments, the anti-PD-1 antibodies for any of the uses, therapies, medicaments and kits described herein include monoclonal antibodies (mabs) or antigen-binding fragments thereof that specifically bind to PD-1, and preferably specifically bind to human PD-1. The mAb may be a human, humanized or chimeric antibody and may include human constant regions. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; preferably, the anti-PD-1 antibodies or antigen-binding fragments thereof suitable for use in any of the uses, therapies, medicaments and kits described herein comprise a heavy chain constant region of human IgG1 or IgG4 isotype, more preferably a human IgG4 constant region. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof comprises the S228P mutation that replaces a serine residue in the hinge region with a proline residue that is typically present at the corresponding position of an IgG4 isotype antibody.
Examples of anti-PD-1 antibodies that bind to human PD-1 and that can be used in the uses, therapies, medicaments and kits described in the present invention are described in WO 2014206107. Human PD-1 mabs that may be used as anti-PD-1 antibodies in the uses, therapies, medicaments and kits described in this invention include any of the anti-PD-1 antibodies described in WO2014206107, including: teraprimab (Toripalimab), a humanized IgG4 mAb having the structure described in WHO Drug Information (Vol.32, phase 2, p.372-373 (2018)), and comprising the light and heavy chain amino acid sequences shown in sequences SEQ ID NO. 9 and 10 in a preferred embodiment, the anti-PD-1 antibody useful in any of the uses, therapies, drugs, and kits described herein is selected from the humanized antibodies 38, 39, 41, and 48 described in WO 206107. in a particularly preferred embodiment, the anti-PD-1 antibody useful in any of the uses, therapies, drugs, and kits described herein is Teraprimab.
anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits described herein also include Nivolumab and Pembrolizumab, which have been approved by the FDA.
In certain embodiments, anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits described herein also include anti-PD-L1 monoclonal antibodies that specifically bind to PD-L1 to block the binding of PD-L1 to PD-1, such as nivolumab, pembrolizumab, toriplalimab, sinilimab, Camrelizumab, tislellizumab, cemipimab.
In some embodiments, the expression level of PD-L1 by malignant cells and/or by infiltrating immune cells within the tumor is determined to be "overexpressed" or "elevated" based on comparison to the expression level of PD-L1 by an appropriate control. For example, the protein or mRNA expression level of control PD-L1 can be a level quantified in non-malignant cells of the same type or in sections from matched normal tissue.
Cytotoxic anticancer drugs
In some embodiments of the invention for use in the treatment of non-small cell lung cancer, cytotoxic anticancer agents include agents that disrupt DNA structure and function, agents that affect nucleic acid biosynthesis, agents that interfere with the transcriptional process to inhibit RNA synthesis, agents that act on DNA replication topoisomerase inhibitors, and agents that affect protein synthesis and function. Wherein, the drugs for destroying the structure and the function of the DNA comprise nitrogen mustard, cyclophosphamide and platinum anticancer drugs. Drugs that affect nucleic acid biosynthesis include thymidylate synthase inhibitors, DNA polymerase inhibitors, antifolate metabolism anticancer drugs, ribonucleotide reductase inhibitors, and purine nucleotide synthase inhibitors. In some embodiments, the cytotoxic anticancer drug of the present invention is selected from the group consisting of an antifolate metabolizing anticancer drug and a platinum anticancer drug.
Anti-folate metabolism anticancer drugs
In some embodiments of the invention for use in the treatment of non-small cell lung cancer, the anti-folate metabolism anticancer drug is selected from the group consisting of methotrexate and pemetrexed, preferably pemetrexed. Pemetrexed (pemetrexed) is an antifolate preparation containing a core containing a pyrrolopyrimidine group, which inhibits cell replication by disrupting normal intracellular folate-dependent metabolic processes, thereby inhibiting tumor growth. Pemetrexed is a compound having a structure represented by formula (I).
(I)
In some embodiments of the present invention, pemetrexed may also refer to a composition comprising a therapeutically effective amount of a compound of formula (I), its free base, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
Platinum group anti-cancer drugs
In some embodiments of the invention for use in the treatment of non-small cell lung cancer, the platinum anticancer agent is selected from cisplatin, carboplatin, and oxaliplatin; carboplatin is preferred. Carboplatin, was discovered by Clear et al in 1980, first marketed in the uk in 1986, approved for marketing by FDA in the united states in 1989, and gradually popularized in use. The production of carboplatin powder and injection is approved in 1990 of China. Carboplatin is a second-generation platinum compound, has a biochemical characteristic similar to that of cisplatin, is a new medicine which is widely regarded as important in recent years, and belongs to a cell cycle nonspecific medicine. It mainly acts on N7 and O6 atoms of guanine of DNA to cause interchain and intrachain cross-linking of DNA, destroy DNA molecule, prevent its spiral melting, interfere with DNA synthesis and produce cytotoxicity. Carboplatin is a compound having the structure shown in formula (II).
In some embodiments of the present invention, carboplatin may also refer to a composition comprising a therapeutically effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
Pharmaceutical composition
The invention provides a pharmaceutical composition comprising the anti-PD-1 antibody, the anti-folate metabolism anticancer drug, the platinum anticancer drug and other pharmaceutically acceptable carriers. In some embodiments, the invention also provides a pharmaceutical composition comprising an anti-PD-1 antibody described herein and an anti-folate metabolism anticancer agent and other pharmaceutically acceptable carriers.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carriers suitable for use in the composition comprising the anti-PD-1 antibody are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration, such as by injection or infusion, while the carriers for the composition comprising the other anti-cancer agent are suitable for parenteral administration, such as oral administration. The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts, antioxidants, water, non-aqueous carriers, and/or adjuvants such as preserving, wetting, emulsifying, and dispersing agents.
The content of the anticancer active ingredients (the anti-PD-1 antibody, the anti-folate metabolism anticancer drug and the platinum anticancer drug as described herein) in each pharmaceutical composition of the present invention is generally the amount of each of these anticancer active ingredients at the time of single administration. For example, for a fixed dose of 240mg of the anti-PD-1 antibody described herein, 240mg of the anti-PD-1 antibody can be contained per dose of the pharmaceutical composition. Of course, for example, in the case of an oral tablet, the 240mg of the anti-PD-1 antibody may be divided into 2 or more tablets, as long as all of the tablets are taken at the time of administration to achieve an administration dose of 240 mg.
Dosage and dosing regimen
The choice of a dosing regimen (also referred to herein as an administration regimen) for a pharmaceutical combination of the invention depends on several factors, including the rate of solid serum or tissue turnover, the level of symptoms, the overall immunogenicity, and the degree of accessibility of the target cells, tissues, or organs of the individual being treated. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Thus, the dosage and frequency of administration of each of the biotherapeutic and chemotherapeutic agents will depend in part on the particular therapeutic agent, the severity of the cancer being treated and the patient's characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules may be obtained. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific pub. ltd, Oxfordshire, UK; kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; baert et al (2003) New engl.j.med.348: 601-608; milgrom et al (1999) New engl.j.med.341: 1966-; slamon et al (2001) New Engl. J. Med.344: 783-792; beniaminovitz et al (2000) New engl.j.med.342: 613-619; ghosh et al (2003) New engl.j.med.348: 24-32; lipsky et al (2000) New engl.j.med.343: 1594-; physicians 'Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); medical Economics Company; ISBN: 1563634457, respectively; 57th edition (11 months 2002). Determination of an appropriate dosage regimen may be made by a clinician, for example, with reference to parameters or factors known or suspected to affect treatment or expected to affect treatment in the art, and will depend on, for example, the clinical history of the patient (e.g., previous treatment), the type and stage of cancer being treated, and biomarkers responsive to one or more therapeutic agents in the combination therapy.
Each therapeutic agent of the pharmaceutical combination of the invention may be administered simultaneously (i.e., in the same pharmaceutical composition), concurrently (i.e., in separate pharmaceutical formulations, administered one after the other in any order), or sequentially in any order. Sequential administration is particularly useful where the therapeutic agents in the pharmaceutical combination can be in different dosage forms (one drug is a tablet or capsule and the other drug is a sterile liquid formulation) and/or on different dosing schedules (e.g., the chemotherapeutic agent is administered at least daily and the biologic therapeutic agent is administered less frequently (e.g., once per week, once every two weeks, or once every three weeks)).
In some embodiments, the therapeutic agents in at least one drug combination are administered using the same dosage regimen (therapeutic dose, frequency and duration) as is typically used when the agents are used in monotherapy to treat the same tumor. In other embodiments, the patient receives a lesser total amount of the at least one therapeutic agent in the combination therapy than when the agents are used as monotherapy, e.g., a smaller dose, a less frequent dose, and/or a shorter duration of treatment.
Each therapeutic agent in the pharmaceutical combination of the present invention may be administered orally or parenterally, which includes intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical and transdermal routes of administration.
The anti-PD-1 antibodies of the invention may be administered in a single dose ranging from about 0.01 to about 20mg/kg of individual body weight, from about 0.1 to about 10mg/kg of individual body weight, or from about 120mg to about 480mg as a fixed dose, by continuous infusion or by intermittent dosing. For example, the dose may be about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10mg/kg of the body weight of the individual, or a fixed dose of about 120mg, 240mg, 360mg, or 480 mg. Dosing regimens are generally designed to achieve such exposure, which results in sustained Receptor Occupancy (RO) based on the typical pharmacokinetic properties of abs. A representative dosing regimen may be about once per week, about once every two weeks, about once every three weeks, about once every four weeks, about once a month, or longer. In some embodiments, the anti-PD-1 antibody is administered to the individual about once every three weeks.
In some embodiments, the anti-PD-1 antibody of the invention is tereprinimab in a single administered dose selected from about 1 to about 5mg/kg of individual body weight. In some embodiments, a single administered dose of tereprinimab selected from a dose of about 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, and 5mg/kg of an individual's body weight, or a fixed dose of 120mg, 240mg, and 360mg is administered intravenously. In some preferred embodiments, the tereprinimab is administered as a liquid drug, and the selected dose of the drug is administered by intravenous infusion over a period of 30-60 minutes. In some embodiments, the tereprimab is administered by intravenous infusion at a fixed dose of about 3mg/kg or about 240mg once every three weeks (Q3W) over a period of 30 minutes. In some embodiments, the tereprimab is administered by intravenous infusion at a fixed dose of about 4.5mg/kg or about 360mg once every three weeks (Q3W) over a period of 30 minutes.
The anti-folate metabolizing anticancer agent of the present invention is administered at its approved or recommended dose, and treatment is continued until clinical effect is observed or until unacceptable toxicity or disease progression occurs. In some embodiments, the anti-folate metabolism anticancer agent of the present invention is pemetrexed, which is administered in a single dose selected from about 200mg to about 800mg/m2Body surface area. In some embodiments, a single administered dose of pemetrexed is selected from about 300mg/m2、400mg/m2、500mg/m2、600mg/m2And 700mg/m2Any dosage in body surface area. A representative dosing regimen may be about once every week, once every two weeks, once every three weeks, once every four weeks, or oneOnce a month. In some embodiments, the pemetrexed is administered to the individual once every three weeks. In some embodiments, the pemetrexed is at about 500mg/m2Body surface area, once every three weeks (Q3W).
Herein, the Body Surface Area (BSA) is defined by the Dubois formula: BSA (m)2)＝0.20247x height(m)0.725x weight(kg)0.425。
The platinum anticancer agents of the invention are administered at their approved or recommended doses, with continued treatment until the maintenance phase of the disease is entered, or until unacceptable toxicity or disease progression occurs. In some embodiments, the platinum anticancer agent of the present invention is carboplatin and the single dose thereof is selected from about AUC 4, AUC 5, AUC 6, and AUC 7. In some embodiments, the single administration dose of carboplatin is about AUC 5. A representative dosing regimen may be about once every week, once every two weeks, once every three weeks, once every four weeks, or once a month. In some embodiments, the carboplatin is administered to the individual once every three weeks. In some embodiments, the carboplatin is administered at about AUC 5 once every three weeks.
In some embodiments, the tereprinimab is administered at a fixed dose of about 240mg, Q3W, and the pemetrexed is at about 500mg/m2Body surface area, Q3W administration, carboplatin AUC 5, Q3W administration. In some embodiments, the tereprinimab is administered at a fixed dose of about 360mg, Q3W, and the pemetrexed is at about 500mg/m2Body surface area, Q3W administration, carboplatin was administered at about AUC 5, Q3W. In some embodiments, the tereprinimab is administered at a fixed dose of about 240mg, Q3W, and the pemetrexed is administered at a fixed dose of about 200mg, Q3W. In some embodiments, the tereprinimab is administered at about 360mg fixed dose, Q3W, and the pemetrexed is administered at about 200mg fixed dose, Q3W.
In some embodiments, on the day of tereprimab administration, pemetrexed may be administered before or after tereprimab administration, and carboplatin may be administered before or after tereprimab administration.
The anti-PD-1 antibody and the cytotoxic anticancer drug of the present invention may be administered in a cycleThe same or different, for one week, two weeks, three weeks, one month, two months, three months, four months, five months, half a year or longer, optionally, the time of each administration cycle may be the same or different, and the interval between each administration cycle may be the same or different. For example, in some embodiments, the tereprinimab is administered at a fixed dose of about 240mg once every three weeks, and the pemetrexed is at about 500mg/m2Body surface area, administered once every three weeks, carboplatin was administered at about AUC 5 once every three weeks for three weeks.
Prediction
The invention provides an application of a kit for predicting the effect of an anti-PD-1 antibody and chemotherapy on an EGFR mutant advanced non-small cell lung cancer patient, wherein the kit comprises a reagent for detecting DSPP and/or TP53 gene mutation in peripheral blood or tumor tissues of an individual. Preferably, the kit further comprises one or more of the reagents and/or devices for the following uses: (1) reagents and/or devices for detecting the number of M1 macrophages and M2 macrophages in the patient; (2) reagents and/or devices for detecting the expression levels of the following genes in the patient: RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB. In some preferred embodiments, the kit comprises reagents for detecting mutations in the DSPP and/or TP53 genes, whether and what EGFR mutation the patient has, and reagents for detecting the number of M1 macrophages and M2 macrophages in the patient.
The invention also provides application of the reagent and/or the device for detecting the number of M1 macrophages and M2 macrophages of the patient in preparing a kit for predicting the effect of the anti-PD-1 antibody and chemotherapy on the combination treatment of EGFR mutant advanced non-small cell lung cancer patients, and the reagent and/or the device for detecting the expression level of the following genes of the patient: use of RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB in the manufacture of a kit for predicting the effect of a medicament comprising a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof on the treatment of a non-small cell lung cancer patient.
The present invention also provides a method of predicting the effect of an anti-PD-1 antibody in combination with chemotherapy on an EGFR mutant advanced non-small cell lung cancer patient, said method comprising detecting the presence or absence of a mutation in the DSPP and/or TP53 gene in the patient, wherein the presence of a mutation in the DSPP and/or TP53 gene, preferably a double mutation in DSPP and TP53, or a mutation in DSPP indicates that the patient responds better to the treatment than in a patient in which said mutation is absent; preferably, the patient is tested for tumor cells and/or peripheral blood. Preferably, the method further comprises one or more of the following detections: (1) detecting the number of M1 macrophages and M2 macrophages in the patient; wherein a patient having a higher than mean M1 macrophage ratio, a lower than mean M2 macrophage ratio, and/or a higher than mean M1/M2 macrophage ratio as compared to a non-small cell lung cancer patient's average M1 macrophage to M2 macrophage ratio indicates that the patient responds better to the treatment than a patient having a lower than mean M1 macrophage ratio, a higher than mean M2 macrophage ratio, and/or a lower than mean M1/M2 macrophage ratio; (2) detecting whether the patient has an EGFR mutation and what EGFR mutation the patient has; wherein a patient having an EGFR mutation selected from the group consisting of an exon 19 deletion and an exon 21L858R mutation but not having an EGFR T790M mutation responds to treatment more favorably than a patient not having an EGFR mutation selected from the group consisting of an exon 19 deletion and an exon 21L858R mutation but having an EGFR T790M mutation.
Herein, the reagents and devices include one or more of primers, probes, reagents required for PCR amplification, reagents required for cell isolation and purification, reagents required for immunohistochemical analysis, and reagents required for sequencing, which are required for detecting the corresponding subjects, and corresponding devices. For example, when detecting the presence or absence of a mutation in a gene, the detection reagents typically include reagents required for detecting the mutation in the gene, including but not limited to primers, probes, reagents required for PCR amplification, and the like. Depending on the method of detection, the reagents and/or devices required may vary. The above mutations, cell number, density and concentration can be detected by a known method. For example, with respect to DSPP mutations, EGFR mutations, and TP53 mutations, detection can be performed using whole exome and transcriptome sequencing, and thus, the reagents described herein above for detecting these mutations include reagents required for whole exome and transcriptome sequencing. For M1 and M2 macrophages, flow cytometry or transcriptome sequencing may be used for analysis, and thus the reagents and/or devices may include reagents (e.g., labeled specific antibodies) and devices necessary to perform flow cytometry.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1: clinical study on non-small cell lung cancer treatment by anti-PD-1 antibody in combination with chemotherapy
Grouping standard: eligible subjects must (1) be 18-75 years of age, (2) have advanced or recurrent, EGFR sensitive mutant non-small cell lung cancer, (3) have failed prior first-line EGFR-TKI treatment, (4) have no EGFR T790M mutation, 5) have an ECOG score of 0 or 1, (6) have no history of autoimmune disease, and (7) have not previously received any anti-PD-1/or anti-PD-L1 immunotherapy.
The subject must have an assessable lesion according to RECIST v1.1 criteria, not allow for a combined small cell lung cancer or squamous cell carcinoma, not allow for other mutations that could be targeted for treatment, not allow for prior systemic chemotherapy, not allow for long-term systemic immunosuppressive therapy.
From 4 months 2018 to 3 months 2019, 65 EGFR + NSCLC patients were screened, and a total of 40 patients enrolled in the present study. The median age of the subjects was 58 years (range: 19 to 73 years), including 21 (52.5%) female and 19 (47.5%) male patients. Exon 19 of EGFR was deleted in 23 (57.5%) patients, while exon 21L858R mutation was present in 17 (42.5%) patients. 20 (50.0%) patients received gefitinib (gefitinib) as first line therapy, 16 (40.0%) patients received icotinib (icotinib) and 4 (10.0%) patients received erlotinib (erlotinib) as first line therapy. None of the patients in the group had the EGFR T790M mutation found in the tumor tissue samples from the second biopsy. 30 subjects participated in the study group with 360mg group of combined chemotherapy of Tereprinimab, and 10 subjects participated in the study group with 240mg group of combined chemotherapy of Tereprinimab. Demographic data for the enrolled subjects are shown in table 1.
Table 1: demographic data of the subjects in the cohort
Note: PD-L1 positive was defined as greater than or equal to 1% expression of PD-L1 staining tumor cells with JS311 IHC.
The tested drugs are: anti-PD-1 antibodies terlipril mab (toriplalimab, WO2014206107), Pemetrexed (commercially available), and Carboplatin (commercially available).
In this study, the safety and clinical efficacy of two dosing regimens of terepril mab were compared. Subjects in the cohort received 240mg or 360mg of Terepril mab intravenously every 3 weeks until disease progression or intolerable toxicity was confirmed. In the induction phase, the subject also received six cycles of 500mg/m2Pemetrexed plus carboplatin AUC 5 treatment was given intravenously every three weeks. In the maintenance phase, the subject receives 240mg or 360mg of Terepril mab in combination with 500mg/m2Pemetrexed.
Evaluation was performed every 6 weeks according to the solid tumor Response Evaluation Criteria (RECIST), version 1.1, and irRECIST, an evaluation criterion for efficacy of immune-related solid tumors. Treatment with a second disease progression was not allowed in this study.
And (3) clinical design:
this is a multicenter, non-blind clinical phase II trial. This study was conducted to evaluate the safety and antitumor activity of the anti-PD-1 antibody in combination with pemetrexed and carboplatin for advanced or recurrent non-small cell lung cancer with EGFR sensitive mutation (without EGFR T790M mutation) after failure of EGFR-TKI treatment.
1.1 safety study:
by 22 days 6/2020, 39 of 40 patients (97.5%) experienced treatment-related adverse effects (TRAE). The most common (≧ 20%) TRAE observed included 33 cases (82.5%) of leukopenia, 28 cases (70.0%) of neutropenia, 27 cases (67.5%) of anemia, 21 cases (52.5%) of elevated AST, 20 cases (50.0%) of elevated ALT, 19 cases (47.5%) of nausea, 19 cases (47.5%) of thrombocytopenia, 15 cases (37.5%) of reduced appetite, 11 cases (27.5%) of constipation and 10 cases (25.0%) of debilitation (see Table 2). Grade 3 and above TRAE occurred in 26 patients (65.0%). 4 (10%) patients were permanently deprived of Tereprimab due to TRAE, while 15 (37.5%) patients received Tereprimab with a delay due to TRAE. There was no significant difference in the incidence and severity of Adverse Effects (AE) in the comparison of the 360mg (n-30) and 240mg (n-10) terepril mab groups.
Table 2: common (> 10%) adverse effects associated with therapy (n ═ 40)
AE parameters
Total incidence (%)
Grade 3-5 incidence (%)
Patients with at least one TRAE
39(97.5)
26(65.0)
Leukopenia
33(82.5)
9(22.5)
Neutropenia
28(70.0)
16(40.0)
Anemia (anemia)
27(67.5)
2(5.0)
Increased aspartate aminotransferase
21(52.5)
2(5.0)
Increased alanine aminotransferase
20(50.0)
2(5.0)
Nausea
19(47.5)
0
Thrombocytopenia
19(47.5)
4(10.0)
Decrease of appetite
15(37.5)
1(2.5)
Constipation
11(27.5)
0
Debilitation
10(25.0)
0
Vomiting
7(17.5)
0
Rash
6(15.0)
0
Urinary tract infection
5(12.5)
0
Increased glutamyl transferase
4(10.0)
0
Influenza and other diseases
4(10.0)
0
Pulmonary infection
4(10.0)
1(2.5)
Generate heat
4(10.0)
0
Upper respiratory tract infection
4(10.0)
1(2.5)
1.2 antitumor Activity Studies:
by 22 days 10 months in 2020, the median follow-up time is 10.5 months. Of all 40 patients, 18 (45.0%) died, 22 (55.0%) discontinued treatment due to disease progression, 3 (7.5%) discontinued treatment due to adverse reactions, and 2 (5.0%) patients remained in the study. The overall confirmed Objective Remission Rate (ORR) was 50.0% (95% CI: 33.8 to 66.2) and the Disease Control Rate (DCR) was 87.5% (95% CI: 73.2 to 95.8). Tumor shrinkage was observed in 36 (90.0%) patients (fig. 1 a). Median duration of remission (DOR) was 7.0 months (fig. 1b), median progression-free survival (PFS) was 7.0 months (95% CI: 4.8 to 8.4), and median total survival (OS) was 23.5 (95% CI: 18.0 to NR months) (fig. 1c and 1 d). In patients who completed induction therapy (n-29), ORR was 69.0%.
Example 2: correlation study of biomarker and clinical curative effect
2.1WES
In 40 patients of all cohorts, we performed Whole Exome Sequencing (WES) on tumor biopsies and paired peripheral blood from 34 of the patients. WES identified 7048 gene alterations, including 3505 missense mutations, 84 gene deletions, 123 rearrangements, 119 splice site substitutions, 349 truncations, and 2748 gene amplifications. Noting the significant difference in gene changes between Partial Response (PR) patients and non-PR patients (fig. 2a), we concluded that gene mutations might be used as predictive markers for this protocol. Using Maftools: : surfgroup (v2.6.05) genomic predictions related to efficacy were made for the first 100 most frequently mutated genes. Considering the small data set, we set a relatively stringent criterion (P.ltoreq.0.01) to screen for genes or combinations of genes with predictive power. We have found that the prediction model will work well when the number of components reaches two. To ensure the validity of the model, the minimum sample size of each group should exceed 25% of the population (fig. 2 b).
After screening, the double mutation of DSPP and TP53(DT) was found to be the most potent combination, with median PFS significantly longer than wild type (9.3vs 4.9 months, P ═ 0.008; fig. 2 c). Furthermore, immunoinfiltration analysis of 354 EGFR mutant patients from 18 TCGA dataset items using CIBERSORTx showed that the DT double mutation had significantly increased CD8 compared to wild type tumors+T cells, and decreased M2 macrophage infiltration (fig. 2 d). DSPP alone was used as a potential predictive biomarker (8.1vs 5.3 months, P ═ 0.054; fig. 2e), while TP5 alone was used as a biomarker3 cannot be used. The similarity of immunoinfiltration characteristics of the DSPP mutation and the DT double mutation compared to TP53 (fig. 2f) indicates the potential predictive value of the DSPP mutation rather than TP 53. However, neither DT single mutation nor double mutation predicted OS.
2.2 transcriptome sequencing
Total RNA was extracted from available tumor tissue samples and the quality and quantity of RNA was determined by capillary electrophoresis on eukaryotic total RNA Pico chips (Agilent technologies). The prepared library was sequenced on an Illumina HiSeq 2000 sequencer.
To find differential gene expression and immunoinfiltration profiles corresponding to different therapeutic effects, we performed RNA-seq on 18 patients with sufficient tissue samples. In general, the similarity between samples is stronger (fig. 3 a). Only 13 Differentially Expressed Genes (DEGs) were found between the two groups (P.ltoreq.0.05, Log)2Fold Change ≧ 2), including 8 up-regulated genes and 5 down-regulated genes (FIG. 3 b).
Furthermore, we found that the expression values of these 13 genes cannot be directly used to distinguish two different treatment groups. Considering the small number of DEG and similar expression values between the two groups, we tried to select candidate marker genes using a Support Vector Machine (SVM) and then identify genes with high classification performance (fig. 4 a). The results show that six components in a group can have precise discriminative power (fig. 4 b). The gene set has the best grouping capability and consists of six genes including RNF220, MAPK8IP2, PTGER2, P2RX4, ACADVL and SPTSSB (fig. 4 c). After 1000 independent random grouping experiments on our dataset we found that 5 out of 6 test samples could be correctly grouped 985 times using the predictor described above, of which 951 could be grouped as all pairs. Notably, differential expression analysis of the Sample Specific Networks (SSNs) between PR and non-PR groups at the network level also validated the grouping capability of the six gene sets (fig. 4 c).
We then focused on analyzing the different levels of immune cell infiltration between PR and non-PR groups. We observed CD8 in PR patients+T cell abundance was significantly lower than non-PR patients (fig. 3 c). In addition, PR patients had significantly higher M1 macrophage abundance ratios than non-PR patients (fig. 3 d).
Although there was no significant difference in the ratio of M2 macrophage abundance between the two groups, the M1/M2 macrophage abundance ratio was found to be significantly higher for the PR group (P ═ 0.025; fig. 3d), indicating that macrophage abundance has a key role in determining the anti-tumor activity of anti-PD-1 antibody in combination with chemotherapy in EGFR mutant non-small cell lung cancer. In addition, the DSPP mutant tumors had significantly higher M1/M2 macrophage abundance ratios (P < 0.001; FIG. 3e) compared to wild-type tumors.
2.3 Integrated analysis of Whole exome and transcriptome sequencing
This is a phase II trial of a single stage design. At a unilateral significance level of 0.05, a total of 39 patients could provide 80% efficacy to demonstrate efficacy of the tropialimab combination chemotherapy in a two-line setting with a target ORR of 50% versus 30% for standard chemotherapy using the capper-Pearson method.
Safety and efficacy assays included all patients receiving > 1 dose of study drug treatment. ORR and its 95% accurate Confidence Interval (CI) were determined by the method of cloner and Pearson. PFS and OS were plotted using the Kaplan-Meier method and reported median and corresponding bilateral 95% CIs. Response duration was analyzed using the Kaplan-Meier method and data from all responders was used. The data analyzed had a cutoff date of 10 months and 22 days in 2020. Statistical analysis was performed using SAS or GraphPad Prism software.
With attention to the potential predictive significance of DSPP (dentin sialophosphoprotein) mutations, we performed comprehensive analysis of whole exome and transcriptome sequencing data from the TCGA database to elucidate the biological mechanisms of the association between DSPP mutations and therapeutic effects. First, we performed differential expression analysis and pathway enrichment. 1612 DEGs (1536 up-and 76 down-regulates; P is less than or equal to 0.05, Log)2FoldChange ≧ 2, FIG. 5 a). After ranking the Enrichment Scores (ES) estimated from the GSEA data, the top 20 up and down pathways were selected. We found that the mitogen-activated protein kinase (MAPK) signaling pathway genome was significantly down-regulated in the DSPP mutant group (ES ═ 0.309) (fig. 5 b). Analysis of the protein-protein interaction network (score. gtoreq.900) downloaded from the STRING database showed that DSPP could be usedTo establish a link with MAPK1 and MAPK3 through the interaction of ITGAV, an important regulatory gene in the MAPK signaling pathway (fig. 5 c). The relationship between these genes suggests that DSPP can influence the efficacy of immunotherapy through MAPK signaling pathways. In addition, DSPP mutations were associated with increased IFN- γ signature gene expression in our cohort (average score: 0.12 vs. 0.08, P ═ 0.182; fig. 6a) and TCGANSCLC cohort (average score: 0.49 vs. 0.30, P < 0.001; fig. 6 b).
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.