WO2018168949A1 - がん免疫療法の新規バイオマーカ - Google Patents
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Definitions
- the present invention relates to the field of cancer immunotherapy. More particularly, the present invention relates to prediction of a subject's responsiveness to cancer immunotherapy and treatment using cancer immunotherapy based on the prediction. In another aspect, the present invention relates to a novel application for large-scale, high-efficiency repertoire analysis. More specifically, the present invention relates to predicting a subject's responsiveness to cancer immunotherapy using a diversity index obtained by large-scale high-efficiency repertoire analysis.
- immune checkpoint inhibitors such as the anti-PD-1 antibody nivolumab
- results that are far superior to docetaxel, which was the conventional standard treatment for non-small cell lung cancer, in overall survival and have become standard treatment.
- a method uses T cell receptor (TCR) diversity of T cells as an indicator of responsiveness to cancer immunotherapy.
- TCR T cell receptor
- cancer immunotherapy uses a bio-defense mechanism, and therefore there are individual differences in response to treatment, and biomarkers that can determine the individual difference before treatment (for example, , The proportion of cells expressing specific surface markers in T cells, surface proteins expressed in tumors, etc.) have been explored.
- the composition of cells considered to be attacking the tumor eg, the ratio of CD8 + PD-1 + cells
- Fig. 4 Cannot be used as a biomarker.
- the inventors have surprisingly found that the TCR diversity of a subject's T cells can be used to predict a subject's response to treatment. For example, 20-30% of patients are effective against anti-PD-1 antibody (Nivolumab) against lung cancer patients. Cancer immunotherapy including immune checkpoint inhibitors is often very expensive, and if effective patients can be predicted before treatment with anti-PD-1 antibodies, etc., more effective treatment will be realized. In addition, medical costs are wasted, and soaring social security costs can be reduced. Therefore, the present invention provides a novel biomarker for predicting responsiveness to cancer immunotherapy.
- the cancer immunotherapy comprises the administration of an immune checkpoint inhibitor.
- the immune checkpoint inhibitor can be a PD-1 inhibitor.
- the PD-1 inhibitor can be an anti-PD-1 antibody including nivolumab or pembrolizumab.
- TCR T cell receptor
- a plurality of indices are used as the TCR diversity, for example, Shannon index, Simpson index, inverse Simpson index, standardized Shannon index, DE index (for example, DE50 index, DE30 index, DE80 index) or Unique index (for example, , Unique 30 index, Unique 50 index, Unique 80 index).
- the TCR diversity is a DE index.
- the TCR diversity is the DE50 index.
- the TCR diversity is the TCR diversity of a subject's T cells.
- T cells that are positive for T cell suppressor cell surface markers can be used.
- T cells that are positive for T cell stimulating cell surface markers can be used.
- T cells CD8, PD-1, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA-4), CD366 (TIM-3) TCR diversity of T cells positive for one or more cell surface markers selected from the group consisting of: CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1), TIGIT and CD367 (GITR) Use.
- T cells are preferably CD8 + .
- the T cell is CD8 + and is positive for a T cell suppressor cell surface marker.
- the T cell is CD8 + and is positive for a T cell stimulatory cell surface marker.
- the T cell is CD8 + and is positive for a T cell suppressor cell surface marker and a T cell stimulator cell surface marker.
- the T cell is CD8 + and PD-1, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA ⁇ 4) one or more cell surface markers selected from the group consisting of CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1), TIGIT and CD367 (GITR) are positive It is.
- the T cells are selected from the group consisting of CD8 + PD1 + , CD8 + 4-1BB + , CD8 + TIM3 + , CD8 + OX40 + , CD8 + TIGIT + , and CD8 + CTLA4 + T cells.
- the T cell may be a CD8 + PD-1 + T cell.
- the T cell can be a T cell in peripheral blood.
- TCR diversity can be TCR ⁇ diversity or TCR ⁇ diversity. In one embodiment of the invention, such high TCR diversity indicates that the subject is a responding patient.
- Another embodiment of the invention involves administering cancer immunotherapy to a subject with high TCR diversity.
- a composition comprising an immune checkpoint inhibitor is provided for treating cancer in a subject with high T cell TCR diversity.
- a composition comprising an immune checkpoint inhibitor, the composition for treating cancer in a subject with a high TCR diversity of CD8 + PD-1 + T cells in peripheral blood Provided.
- the subject's TCR diversity is higher than a threshold, the subject is shown to be a patient who has responded to cancer immunotherapy. In another embodiment, a subject is indicated to be a non-responder to cancer immunotherapy if the subject's TCR diversity is below a threshold. If the DE index is used, use a standardized DE index for the number of leads, or indicate that the subject is a patient with cancer immunotherapy by comparison with a threshold adjusted for the number of leads. Can do. In one embodiment, a subject having a DE50 index normalized to a TCR ⁇ 30000 lead of 0.39% or greater indicates that the subject is a responding patient.
- a subject having a DE50 index normalized to a TCR ⁇ 30000 lead of 0.24% or greater indicates that the subject is a responding patient.
- a comparison of the DE50 index normalized to any number of leads and a threshold corresponding to the number of leads can indicate that the subject is a responding patient or that the subject is a non-responding patient. Examples of combinations of lead counts and thresholds are provided herein.
- the threshold is determined based on ROC analysis. In some embodiments, the threshold is determined based on specificity, for example, a threshold higher than the maximum value of non-response. In some embodiments, the threshold is determined based on sensitivity, for example, a threshold lower than the responder's lowest line. In the present invention, the step of determining such a threshold value can be included, and the threshold value thus determined can be used.
- T cells are CD8, PD-1, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA-4), CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), Those that are positive for one or more cell surface markers selected from the group consisting of CD226 (DNAM-1), TIGIT, and CD367 (GITR) can be isolated.
- T cells are preferably CD8 + .
- the T cell is CD8 + and is positive for a T cell suppressor cell surface marker.
- the T cells are selected from the group consisting of CD8 + PD1 + , CD8 + 4-1BB + , CD8 + TIM3 + , CD8 + OX40 + , CD8 + TIGIT + , and CD8 + CTLA4 + T cells.
- the T cell is CD8 + and is positive for a T cell stimulatory cell surface marker.
- Particularly preferred is a method comprising isolating CD8 + PD-1 + T cells from a peripheral blood sample of a subject and measuring TCR diversity of CD8 + PD-1 + T cells.
- One embodiment of the present invention includes determining TCR diversity by a method that includes large scale high efficiency TCR repertoire analysis.
- One embodiment of the present invention is a method of using TCR diversity as determined by a large scale, high efficiency TCR repertoire analysis as an indicator of a subject's medical condition, particularly responsiveness to treatment.
- One embodiment of the present invention is a method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising measuring TCR diversity of a subject's T cells in vitro, and high TCR diversity. And determining that the subject is responsive to cancer immunotherapy. Alternatively, if the TCR diversity is low, it can be determined that the subject is poorly responsive to cancer immunotherapy.
- T cells can be CD8 + PD-1 + . Furthermore, the T cells can be derived from the peripheral blood of the subject.
- Another embodiment of the present invention is a method of diagnosing a subject's responsiveness to cancer immunotherapy comprising obtaining a peripheral blood sample from the subject, and TCR diversity of T cells in the peripheral blood of the subject.
- a method of measuring gender by a method that includes large-scale high-efficiency TCR repertoire analysis and, if TCR diversity is high, determining that the subject is responsive to cancer immunotherapy .
- TCR diversity is low, it can be determined that the subject is poorly responsive to cancer immunotherapy.
- T cells can be CD8 + PD-1 + .
- a method for diagnosing a subject's responsiveness to cancer immunotherapy and treating a subject's cancer comprising obtaining a peripheral blood sample from the subject, A method is provided comprising the steps of measuring TCR diversity of T cells in the blood and administering cancer immunotherapy to a subject if the TCR diversity is higher than a reference value.
- T cells can be CD8 + PD-1 + .
- the present invention also provides a technique for predicting the responsiveness of a subject to cancer immunotherapy using the diversity index obtained by large-scale high-efficiency repertoire analysis.
- the present invention provides the following items.
- (Item 1) A method of using T cell receptor (TCR) diversity of a subject's T cells as an index of responsiveness of the subject to cancer immunotherapy.
- (Item 2) The method according to the preceding item, wherein the T cell is CD8 + and one or more T cell suppressor cell surface markers are positive.
- (Item 3) The method according to any of the preceding items, wherein the T cell is CD8 + and one or more T cell stimulating cell surface markers are positive.
- the T cells are CD8 + and PD-1, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA-4) ), One or more cell surface markers selected from the group consisting of CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1), TIGIT and CD367 (GITR)
- TIM-3 TIM-3
- CD223 LAG-3
- DNAM-1 CD226
- TIGIT TIGIT
- GITR CD367
- the cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
- the immune checkpoint inhibitor is a PD-1 inhibitor.
- the PD-1 inhibitor is nivolumab or pembrolizumab.
- the TCR diversity is represented by a Shannon index, a Simpson index, a reverse Simpson index, a standardized Shannon index, a Unique50 index, a DE30 index, a DE80 index, or a DE50 index.
- (Item 18) A composition comprising an immune checkpoint inhibitor, which is used for treating cancer in a subject having a high TCR TCR diversity.
- (Item 18A) The composition according to any of the preceding items, having the characteristics described in any one or more of the items.
- (Item 19) The composition according to any of the preceding items, wherein the T cells are CD8 + and one or more T cell suppressor cell surface markers are positive.
- (Item 20) The composition according to any of the preceding items, wherein the T cell is CD8 + and one or more T cell stimulating cell surface markers are positive.
- the T cell is CD8 + , and PD-1, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA-4) ), One or more cell surface markers selected from the group consisting of CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1), TIGIT and CD367 (GITR) A composition according to any of the preceding items. (Item 22) The composition according to any one of the preceding items, wherein the T cell is a CD8 + PD-1 + T cell.
- composition according to any of the preceding items, wherein the T cell is a T cell in the peripheral blood of the subject.
- the immune checkpoint inhibitor is a PD-1 inhibitor.
- the composition according to any one of the preceding items, wherein the PD-1 inhibitor is nivolumab or pembrolizumab.
- the TCR diversity of the subject's T cells is expressed as a Shannon index, a Simpson index, a reverse Simpson index, a standardized Shannon index, a Unique50 index, a DE30 index, a DE80 index, or a DE50 index.
- (Item 33) A method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising: measuring the TCR diversity of the subject's T cells in vitro; If the TCR diversity is high, the subject is determined to be responsive to cancer immunotherapy, or if the TCR diversity is low, the subject is responsive to cancer immunotherapy And determining that is bad. (Item 33A) The method according to any of the preceding items, having the characteristics described in any one or more of the items.
- (Item 34) A method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising: Obtaining a peripheral blood sample from the subject; Measuring TCR diversity of T cells in the peripheral blood of the subject by a method comprising large-scale high-efficiency TCR repertoire analysis; If the TCR diversity is high, the subject is determined to be responsive to cancer immunotherapy, or if the TCR diversity is low, the subject is responsive to cancer immunotherapy And determining that is bad. (Item 34A) A method according to any of the preceding items, having the features described in any one or more of the items.
- (Item 35) A method for diagnosing the responsiveness of a subject to cancer immunotherapy and treating cancer in the subject, Obtaining a peripheral blood sample from the subject; Measuring TCR diversity of T cells in the peripheral blood of the subject; Subjecting the subject to cancer immunotherapy if the TCR diversity is higher than a reference value.
- (Item 35A) The method according to any of the preceding items, having the features described in any one or more of the items.
- (Item 36) A method of using repertoire diversity determined by a method including large-scale high-efficiency repertoire analysis as an indicator of responsiveness of a subject to treatment.
- (Item 36A) A method according to any of the preceding items, having the features described in any one or more of the items.
- the method according to any of the preceding items, wherein the treatment is a treatment associated with an immune response.
- the repertoire analysis is a TCR repertoire analysis.
- the diversity index representing the TCR diversity of the subject being equal to or greater than a threshold is an indicator that the subject is a responding patient, or the diversity index is less than the threshold.
- the subject is an indicator that the subject is a non-responder, wherein the threshold is determined based on ROC analysis or based on sensitivity Or a method determined based on specificity.
- the diversity index representing the TCR diversity of the subject being equal to or greater than a threshold is an indicator that the subject is a responding patient, or the diversity index is less than the threshold.
- the diversity index obtained by performing the TCR repertoire analysis on peripheral blood cells that can be easily sampled can be used as a biomarker for predicting the effect of cancer immunotherapy.
- companion treatment and individual improvement become possible, social insurance premiums can be reduced, and it becomes possible for individuals to receive accurate treatment.
- FIG. 1 is a diagram illustrating an exemplary procedure for TCR repertoire analysis to predict the therapeutic effect of a patient receiving treatment with an anti-PD-1 antibody.
- FIG. 2A is a diagram showing clinical evaluation of patients # 1 and # 2 treated with anti-PD-1 antibody by CT imaging before and after the start of treatment.
- FIG. 2B shows clinical evaluation by CT image diagnosis before and after the start of treatment for patient # 3 treated with anti-PD-1 antibody, and FDG-PET images before and after the start of treatment for patient # 4. It is a figure which shows the clinical evaluation by a diagnosis.
- FIG. 3 is a diagram showing the results of FACS analysis for patient # 1.
- FIG. 4 is a diagram showing a comparison of PD1-positive cells between patients who responded to anti-PD-1 antibody and patients who did not respond.
- FIG. 5 is a diagram showing a comparison of TCR ⁇ diversity indices between pre-treatment PD-1 antibody responders and non-responders.
- TCR ⁇ diversity index of Shannon index
- B standardized Shannon index
- C Simpson index
- D inverse Simpson index
- E DE50 index
- FIG. 6 is a diagram showing a comparison of TCR ⁇ diversity indices between patients with pre-treatment PD-1 antibody response and non-response patients.
- RNA was extracted from CD8 + PD1 + cells before treatment of anti-PD-1 antibody treated patients, and large-scale high-efficiency TCR repertoire analysis was performed to calculate the diversity index (TCR ⁇ ).
- TCR ⁇ diversity index
- FIG. 7 is a plot of sensitivity and 1-specificity when the threshold value is variously changed when each of the diversity indexes of TCR ⁇ and TCR ⁇ of each patient used in Example 1 is used as an index.
- ROC curve The upper figure uses the TCR ⁇ diversity index, and the lower figure uses the TCR ⁇ diversity index.
- FIG. 8 depends on the number of reads of each diversity index (Shannon index, standardized Shannon index, Simpson index, inverse Simpson index, DE30 index, DE50 index, DE80 index, Unique30 index, Unique50 index, and Unique80 index) for TCR ⁇ . It is a figure which shows a change.
- Example 1 By randomly resampling various numbers of leads from the data obtained in Example 1, a diversity index corresponding to each number of leads was calculated, and the median value of 100 random resamplings was plotted for each subject.
- the horizontal axis is the number of resample reads (logarithm), and the vertical axis is the value of the diversity index.
- the vertical axis is also represented by a logarithmic axis. Each individual was displayed using the same color in each index.
- FIG. 10 is a diagram showing a comparison of diversity indices standardized to 30000 leads of TCR ⁇ between pre-treatment PD-1 antibody response patients and non-response patients.
- FIG. 10 is a diagram showing a comparison of diversity indices standardized to 30000 leads of TCR ⁇ between pre-treatment PD-1 antibody response patients and non-response patients.
- FIG. 11 is a diagram showing a comparison of diversity indexes standardized to 30000 leads of TCR ⁇ between pre-treatment PD-1 antibody response patients and non-response patients.
- FIG. 11 is a diagram showing a comparison of diversity indexes standardized to 30000 leads of TCR ⁇ between pre-treatment PD-1 antibody response patients and non-response patients.
- each diversity index (Shannon index, standardized Shannon index, Simpson index, inverse Simpson index, DE30 index, DE50 index, DE80 index, Unique30 index, Unique50 index and Unique80 index) for TCR ⁇ . It is a figure which shows the change according to the number of leads.
- the horizontal axis indicates the number of resample leads (logarithmic axis), and the vertical axis indicates the value of each index.
- the DE index threshold is indicated by the logarithmic axis as well as the vertical axis. It is understood that the DE index threshold has a linear relationship with the number of leads on the logarithmic axis.
- each diversity index (Shannon index, standardized Shannon index, Simpson index, inverse Simpson index, DE30 index, DE50 index, DE80 index, Unique30 index, Unique50 index and Unique80 index) for TCR ⁇ . It is a figure which shows the change according to the number of leads.
- the horizontal axis indicates the number of resample leads (logarithmic axis), and the vertical axis indicates the value of each index.
- the DE index threshold is indicated by the logarithmic axis as well as the vertical axis. It is understood that the DE index threshold has a linear relationship with the number of leads on the logarithmic axis.
- FIG. 14 is a diagram showing an estimated value by linear regression on the logarithmic axis of a change in the threshold value of the DE50 index of TCR ⁇ and TCR ⁇ depending on the number of reads.
- FIG. 15 is a diagram showing a correlation analysis of the number of reads between T cell fractions.
- X axis is the number of reads of each TCR ⁇ clone in CD8 + PD-1 + fraction
- Y axis is in each cell fraction (CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, CD8 + CTLA4 +) Indicates the number of leads. Dots indicate individual TCR ⁇ clones.
- R indicates Pearson's correlation coefficient.
- FIG. 16 shows the TCR ⁇ chain in CD8 + PD-1 +, CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, and CD8 + CTLA4 + fractions isolated from PBMCs of patients with therapeutic response by FACS sorting.
- FIG. 17 shows TCR ⁇ chain in CD8 + PD-1 +, CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, and CD8 + CTLA4 + fractions isolated from PBMCs of patients with therapeutic response by FACS sorting.
- the values calculated for the Shannon index, Normalized Shannon index, Inverse Simpson index, and% DE50 index are shown (middle).
- cancer immunotherapy refers to a method of treating cancer using an immune function of an organism. Cancer immunotherapy is broadly divided into cancer immunotherapy by strengthening the immune function against cancer and cancer immunotherapy by inhibiting the immune avoidance function of cancer. Furthermore, cancer immunotherapy includes active immunotherapy that activates the immune function in the body and passive immunotherapy that returns immune cells that have been activated or proliferated outside the body. is there.
- cancer immunotherapy examples include nonspecific immunostimulants, cytokine therapy, cancer vaccine therapy, dendritic cell therapy, adoptive immunotherapy, nonspecific lymphocyte therapy, cancer antigen-specific T cell therapy, antibody Therapy, immune checkpoint inhibition therapy, CAR-T therapy and the like.
- Immunity checkpoint (inhibition) therapy using immunity checkpoint inhibitors has received much attention in recent years (Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012 Mar 22; 12 (4): 252-64.). Cancer cells express various proteins on the surface, but this has led to avoidance of attacks by immune cells such as T cells, so in normal conditions, only the immune function of the body eliminates cancer tissue. It is considered impossible. Immune checkpoint inhibitors prevent efficient cancers by the immune function of the living body by inhibiting ligand-receptor interactions, etc., which control the transmission of suppressive signals from such cancer tissues to the immune function. It makes exclusion possible.
- TCR T cell receptor
- TCR T cell receptor
- CD8 + PD-1 + T cells T cells
- TCR T cell receptor
- Another embodiment of the invention is a method of administering an immune checkpoint inhibitor, as shown below, to a selected (responsive) subject based on T cell receptor (TCR) diversity.
- a method is provided for interrupting, discontinuing or avoiding an immune checkpoint inhibitor to a subject found to be non-responsive based on T cell receptor (TCR) diversity.
- a typical example of an immune checkpoint inhibitor is a PD-1 inhibitor.
- the PD-1 inhibitors, anti-PD-1 antibody nivolumab (Nivolumab; sold as Obujibo TM) and Pemuburorizumabu (Pembrolizumab; sold as Kiitoruda TM) include, but are not limited to.
- nivolumab may be selected as the subject.
- nivolumab therapy is that using the diversity index calculated by the large-scale, high-efficiency TCR repertoire analysis of the present invention. It has been shown in the Examples that it is possible to clearly distinguish non-responsive subjects, and it has been found that responsiveness and non-responsiveness can be clearly distinguished by specific thresholds, especially using the DE50 index. Because. Of course, it is considered that the diversity index can be used to the same extent for other PD-1 inhibitors.
- the anti-PD-1 antibody is considered to exert an anticancer effect by releasing the suppression of T cell activation by the PD-1 signal.
- PD-1 programmed death 1
- SHP-2 a type of tyrosine phosphatase
- ZAP70 a transfer protein
- PD-L1 is also thought to interact with CD80 and suppress T cell activation (Butte, M.J., Keir, M.E., Phamduy, T.B.etal .: PD -L1 interacts specifically with B7-1 to inhibit T cell proliferation. Immunity, 27, 111-122 (2007)).
- PD-1 is highly expressed in killer T cells and natural killer cells infiltrating cancer tissues, and it is considered that the immune response is attenuated by PD-L1 on the tumor.
- an anti-PD-1 antibody an effect of enhancing the anti-tumor immune response can be obtained.
- immune checkpoint inhibitors include PD-L1 inhibitors (for example, anti-PD-L1 antibodies averumab, durvalumab or atezolizumab).
- the PD-L1 inhibitor binds the PD-1 pathway to the PD-L1 side and inhibits it to produce an antitumor immune response.
- immune checkpoint inhibitors include CTLA-4 inhibitors (for example, anti-CTLA-4 antibodies ipilimumab or tremerylmab).
- CTLA-4 inhibitors activate T cells through a different pathway than PD-1 inhibition and produce an anti-tumor immune response.
- T cells are activated by the interaction of surface CD28 with CD80 or CD86.
- CTLA-4 cytotoxic T-lymphocyte-associated antigen 4
- the surface expressed CTLA-4 interacts preferentially with CD80 or CD86 with higher affinity than CD20.
- CTLA-4 inhibitors produce an anti-tumor immune response by inhibiting the interaction between CD20 and CD80 or CD86 by inhibiting CTLA-4.
- the immune checkpoint inhibitor is TIM-3 (T-cell immunoglobulin and mucin containing protein-3), LAG-3 (lymphocyte activation gene-3), B7-H3, B7-H4, B7-H5. (VISTA) or immune checkpoint proteins such as TIGIT (T cell immunoreceptor with Ig and ITIM domain) may be targeted.
- TIM-3 T-cell immunoglobulin and mucin containing protein-3
- LAG-3 lymphocyte activation gene-3
- TIGIT T cell immunoreceptor with Ig and ITIM domain
- the above immune checkpoint is considered to suppress the immune response to the self tissue, the immune checkpoint increases in T cells even when an antigen such as a virus is present in the living body for a long period of time. Since tumor tissues are also antigens that exist in the body for a long time, it is thought that anti-tumor immunity is avoided by these immune checkpoints. Disables the avoidance function and has an anti-tumor effect.
- an index for predicting responsiveness of a subject having cancer to cancer immunotherapy is provided.
- the cancers targeted in the present invention include lung cancer, non-small cell lung cancer, kidney (renal cell) cancer, prostate cancer, stomach cancer, testicular cancer, liver (sternal) cancer, skin cancer, and esophageal cancer.
- Examples include, but are not limited to, cancer, uterine cancer (body / neck), head and neck cancer, ovarian cancer, and breast cancer.
- a method is provided that uses the TCR diversity of a subject having lung cancer as an indicator of the subject's responsiveness to cancer immunotherapy.
- TCR diversity The biological defense mechanism by the immune system largely depends on the specific immunity carried mainly by T cells and B cells. T cells and B cells do not react with their own cells or molecules, but can specifically recognize and attack foreign pathogens such as viruses and bacteria. Therefore, T cells and B cells have a mechanism capable of recognizing and distinguishing various antigens derived from other organisms together with self-antigens by receptor molecules expressed on the cell surface. In T cells, a T cell receptor (TCR) acts as an antigen receptor. Intracellular signals are transmitted by stimulation from these antigen receptors, production of inflammatory cytokines and chemokines is enhanced, cell proliferation is enhanced, and various immune responses are initiated.
- TCR T cell receptor
- TCR recognizes a peptide (peptide-MHC complex, pMHC) bound to the peptide binding groove of a major histocompatibility complex (MHC) expressed on an antigen-presenting cell, and thereby recognizes self and non-self. It recognizes and recognizes the antigenic peptide (Cell 1994, 76, 287-299).
- TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains, and there are an ⁇ type TCR expressed by normal T cells and a ⁇ type TCR having a special function.
- ⁇ and ⁇ chain TCR molecules form a complex with a plurality of CD3 molecules (CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses.
- Endogenous antigens such as viral antigens that have proliferated in cells with viral infection and cancer antigens derived from cancer cells are presented as antigenic peptides on MHC class I molecules.
- an antigen derived from a foreign microorganism is taken up into an antigen-presenting cell by endocytosis, processed and then presented on an MHC class II molecule.
- These antigens are recognized by TCRs expressed by CD8 + T cells or CD4 + T cells, respectively.
- costimulatory molecules such as CD28, ICOS and OX40 molecules are important for stimulation via TCR molecules.
- the TCR gene has a large number of V regions (variable regions, V), J regions (joining region, J), D regions (diversity region, D) and constant regions C regions (constant regions) encoded in different regions on the genome. , C).
- V variable regions
- J jointing region
- D diversity region
- C constant regions
- C constant regions
- the ⁇ -chain and ⁇ -chain TCR are VJ-C genes
- the ⁇ -chain and ⁇ -chain TCRs are VDJ-.
- a gene consisting of C is expressed. Reconstruction of these gene fragments creates diversity, and insertion or deletion of one or more bases between V and D or D and J gene fragments results in the formation of random amino acid sequences. More diverse TCR gene sequences have been created.
- the region where the TCR molecule and the pMHC complex surface directly bind is composed of the complementarity determining region (CDR) CDR1, CDR2 and CDR3 regions rich in diversity within the V region.
- CDR3 region includes a part of the V region, a VDJ region formed by a random sequence, and a part of the J region, and forms the most diverse antigen recognition site.
- the other region is called FR (framework region) and plays a role of forming a structure that becomes a skeleton of the TCR molecule.
- the ⁇ chain TCR is first reconstituted and associates with the pT ⁇ molecule to form a pre-TCR complex molecule.
- the ⁇ chain TCR is then reconstituted to form an ⁇ TCR molecule and, if no functional ⁇ TCR is formed, reconstitution occurs in the other ⁇ chain TCR gene allele. It is known that positive and negative selections in the thymus are performed, and TCRs with appropriate affinity are selected to acquire antigen specificity (Annual Review, Immunology, 1993, 6, 309-326).
- T cells produce one type of TCR with high specificity for a particular antigen. Since a large number of antigen-specific T cells are present in a living body, a variety of TCR repertoires (repertoires) are formed and can effectively function as a defense mechanism against various pathogens.
- TCR diversity refers to the diversity of the T cell receptor repertoire (repertoire) of a subject, and can be measured by those skilled in the art using various means known in the art. .
- An index indicating TCR diversity is referred to as a “TCR diversity index”.
- TCR diversity index any known index in the field can be used, for example, Shannon-Weaver index, Simpson index, and inverse Simpson index.
- TCR for diversity index such as Normalized Shannon-Weaver index, DE index (eg DE50 index, DE30 index, DE80 index) or Unique index (eg Unique50 index, Unique30 index, Unique80 index) It can be used by applying to.
- TCR repertoire analysis by molecular biological techniques has been devised based on TCR gene information obtained from human genome sequences.
- RNA is extracted from a cell sample, complementary DNA is synthesized, and then the TCR gene is PCR amplified and quantified.
- RNA can be extracted and purified from cells dissolved in TRIzol LS reagent using RNeasy Plus Universal Mini Kit (QIAGEN).
- Complementary DNA synthesis from the extracted RNA can be performed using any reverse transcriptase known in the art, such as Superscript III TM (Invitrogen).
- PCR amplification of the TCR gene can be appropriately performed by those skilled in the art using any polymerase known in the art. However, in the amplification of genes with large fluctuations such as the TCR gene, it can be said that there is an advantageous effect for accurate measurement if it can be amplified “unbiased”.
- all isotypes and subtypes are composed of one set of primers consisting of one forward primer and one reverse primer as described in WO2015 / 075939 (Repertoire Genesis Inc.).
- TCR genes including genes, are amplified without changing the frequency of presence to determine TCR diversity.
- the following primer design is advantageous for unbiased amplification.
- a gene containing all V regions is amplified by adding an adapter sequence to its 5 ′ end without setting a primer in the highly diverse V region.
- This adapter has an arbitrary length and sequence on the base sequence, and about 20 base pairs is optimal, but a sequence of 10 to 100 bases can be used.
- the adapter added to the 3 'end is removed by a restriction enzyme, and all TCR genes are amplified by amplifying with an adapter primer of the same sequence as the 20 base pair adapter and a reverse primer specific to the C region which is a common sequence. To do.
- Complementary strand DNA is synthesized from the TCR or BCR gene messenger RNA by reverse transcriptase, and then double-stranded complementary DNA is synthesized. Double-stranded complementary DNAs containing V regions of different lengths are synthesized by reverse transcription reaction or double-stranded synthesis reaction, and an adapter consisting of 20 base pairs and 10 base pairs at the 5 ′ end of these genes is subjected to DNA ligase reaction. Add by.
- These genes can be amplified by setting reverse primers in the C region of the ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain of the TCR.
- a primer having a mismatch that matches the sequences of C ⁇ , C ⁇ , C ⁇ , and C ⁇ of the TCR and does not prime to other C region sequences is set.
- the reverse primer in the C region is optimally prepared in consideration of the base sequence, base composition, DNA melting temperature (Tm), and the presence or absence of a self-complementary sequence so that amplification with the adapter primer is possible.
- Tm DNA melting temperature
- By setting a primer in a region excluding a base sequence that differs between allele sequences in the C region sequence all alleles can be uniformly amplified. In order to increase the specificity of the amplification reaction, multiple stages of nested PCR are performed.
- the length (number of bases) of the candidate primer sequence is not particularly limited with respect to a sequence in which none of the primers includes a sequence that differs between allele sequences, but is 10 to 100 bases, preferably 15 to 50 bases. The number is more preferably 20 to 30 bases.
- TCR diversity can be determined from the obtained read data.
- TCR repertoire analysis By PCR amplification of TCR genes from human samples and using next-generation sequencing analysis technology, conventional TCR repertoire analysis, which obtains limited information such as V-chain usage frequency, more detailed gene information at the clone level A large-scale high-efficiency TCR repertoire analysis can be realized.
- the sequencing method is not limited as long as the sequence of the nucleic acid sample can be determined. Any sequence known in the art can be used, but next-generation sequencing (NGS) is preferably used. . Examples of the next-generation sequencing include, but are not limited to, pyrosequencing, sequencing by synthesis (sequencing by synthesis), sequencing by ligation, and ion semiconductor sequencing.
- the number of unique reads can be derived to determine TCR diversity.
- a reference database to be used is prepared for each V, D, and J gene region.
- nucleic acid sequence data sets for each region and allele published by IMGT are used, but not limited thereto, any data set in which a unique ID is assigned to each sequence can be used.
- a homology search is performed with a reference database for each gene region, and the closest reference allele and its sequence are compared. Record the alignment.
- an algorithm with high mismatch tolerance is used for the homology search. For example, when general BLAST is used as a homology search program, settings such as a reduction in window size, a reduction in mismatch penalty, and a reduction in gap penalty are performed for each region.
- the homology score, alignment length, kernel length (length of consecutively matched base sequences) and the number of matching bases are used as indices, and these are applied according to a predetermined priority order.
- the CDR3 sequences are extracted with the CDR3 head on the reference V and the CDR3 end on the reference J as marks. By translating this into an amino acid sequence, it is used for classification of the D region.
- the reference database for the D region is prepared, a combination of the homology search result and the amino acid sequence translation result is set as the classification result.
- alleles V, D, and J are assigned to each sequence in the input set. Subsequently, the TCR repertoire is derived by calculating the appearance frequency of each of V, D, and J, or the appearance frequency of the combination of the entire input set. Depending on the accuracy required for classification, the appearance frequency is calculated in units of alleles or gene names. The latter is possible by translating each allele into a gene name.
- the matching leads are aggregated, and the number of leads detected in the sample and all the leads for each unique lead (the other lead that does not have the same sequence) The ratio (frequency) to the number can be calculated.
- the diversity index or similarity index can be calculated using statistical analysis software such as ESTIMATES or R (vegan) using data such as the number of samples, lead type, and number of leads.
- statistical analysis software such as ESTIMATES or R (vegan) using data such as the number of samples, lead type, and number of leads.
- TCR repertoire analysis software Repertoire Genesis Inc.
- the diversity index can be obtained from the number-of-reads data of each unique lead obtained as described above.
- Shannon-Weaver index also simply referred to as Shannon index
- Simpson index Normalized Shannon-Weaver index
- DE50 index are calculated according to the following formulas: be able to.
- N Total number of reads
- n i Number of reads for the i-th unique lead
- S Number of unique reads
- S 50 Number of top unique reads that account for 50% of all leads
- the DE index can also be expressed as a percentage or a percentage (%), and those skilled in the art will understand the meaning of the numerical values displayed clearly and appropriately, convert the threshold value, etc. It is possible to carry out the invention.
- the DE index can be calculated as the number of upper unique reads occupying an arbitrary ratio (1 to 99%) of all reads / the number of unique reads, and can be used as a diversity index in the present invention.
- S 50 the number of upper unique reads that occupy 50% of all leads.
- DE30 index and DE80 index using S 30 (the number of top unique reads that occupy 30% of all leads) and S 80 (the number of top unique reads that occupy 80% of all leads).
- the DE index can be a value standardized with respect to the number of leads (eg, standardized with respect to 80000 leads, 30000 leads, 10000 leads, etc.).
- a molecule of DE index can also be used UniqueX index using S x directly.
- Examples of the Unique index include the Unique 30, the Unique 50, or the Unique 80 index.
- TCR diversity is measured using large scale, high efficiency TCR repertoire analysis.
- large-scale high-efficiency repertoire analysis is described in WO2015 / 075939 (the disclosure of this document is incorporated herein by reference in its entirety as necessary). Is TCR, it is referred to as “large-scale high-efficiency TCR repertoire analysis”.
- the large-scale high-efficiency repertoire analysis is a method of quantitatively analyzing a subject's repertoire (T cell receptor (TCR) or B cell receptor (BCR) variable region) using a database.
- the first additional adapter nucleic acid sequence is suitable for binding to DNA capture beads and emPCR reaction
- the second additional adapter nucleic acid sequence is emPCR reaction
- the molecular identification (MID Tag) sequence includes a sequence for imparting uniqueness so that an amplification product can be identified. Specific details of this method are described in WO2015 / 075939, and those skilled in the art can carry out an analysis by referring to this document and the examples of this specification as appropriate.
- a method uses TCR diversity of T cell subpopulations.
- T cells that are positive for T cell suppressor cell surface markers can be used.
- T cells that are positive for T cell stimulating cell surface markers can be used.
- the T cells are selected from the group consisting of CD8 + PD1 + , CD8 + 4-1BB + , CD8 + TIM3 + , CD8 + OX40 + , CD8 + TIGIT + , and CD8 + CTLA4 + T cells.
- the “T cell stimulation system cell surface marker” refers to a cell surface molecule that transmits a signal for activating T cells.
- Examples of the “T cell stimulating cell surface marker” include, but are not limited to, CD28, CD154 (CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) or CD27.
- T cell suppressor cell surface marker refers to a cell surface molecule that transmits a signal that suppresses T cells.
- T cell inhibitory cell surface markers include PD-1, CD152 (CTLA-4), CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1) , TIGIT or CD367 (GITR), but not limited thereto.
- the high TCR subpopulation of T cell subpopulations expressing such cell surface markers indicates that TCRs that recognize cancer tissue surface antigens in that subpopulation. It can be considered that it is likely to benefit from treatment with an immune checkpoint inhibitor because there is definitely what it has.
- a subpopulation of T cells is, for example, a population of CD8 + T cells.
- a CD8 +, and a T cell subpopulations which express one or more immune checkpoint molecules e.g., CD8 + and PD-1, CD28, CD154 ( CD40L), CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), CD27, CD152 (CTLA-4), CD366 (TIM-3), CD223 (LAG-3), CD272 (BTLA), CD226 (DNAM-1), TIGIT and CD367
- the T cell is CD8 + .
- the TCR diversity of a T cell subpopulation that is positive for a T cell stimulatory cell surface marker the TCR diversity of a subpopulation of T cells that are positive for a T cell suppressor cell surface marker
- the TCR diversity of subpopulations of T cells that are positive for T cell stimulatory cell surface markers and T cell suppressor cell surface markers can be used.
- the T cell subpopulation may be a PD-1 + T cell population.
- TCR diversity can be determined for each subpopulation of T cells, and in a preferred embodiment of the invention, the subpopulation of T cells is a population of CD8 + PD-1 + T cells. TCR diversity for the appropriate subpopulation may be used as a more accurate indicator when used as an indicator of a subject's medical condition.
- Such methods of separating subpopulations of T cells are known in the art and can be performed using an appropriate cell sorter (eg, BD FACSAria III cell sorter (BD Bioscience)). Those skilled in the art can appropriately use labeled antibodies against cell surface markers that distinguish the subpopulations to be separated. TCR repertoire analysis as described above using nucleic acid samples extracted from the separated subpopulations can determine the TCR diversity for a particular T cell subpopulation.
- BD FACSAria III cell sorter BD Bioscience
- T cells obtained from any tissue can be used.
- T cells can be obtained from, for example, peripheral blood, tumor sites, normal tissues, bone marrow, or thymus.
- the TCR diversity of T cells in the peripheral blood of the subject is determined.
- the collection of T cells from peripheral blood is non-invasive and simple.
- TCR chain for measuring TCR is ⁇ chain, ⁇ chain, ⁇ chain and / or ⁇ chain.
- TCR ⁇ diversity is used.
- TCR ⁇ is used.
- the response to cancer immunotherapy can be determined based on RECIST v1.1 (Revised RECIST guideline (version 1.1)).
- the effect of cancer treatment is determined as complete response (CR), partial response (PR), progression (progressive disease: PD), or stable (stable disease: SD) based on changes in tumor size. can do.
- a “non-responder” refers to a subject that has progressed or stabilized with respect to cancer treatment.
- the responsiveness of a subject to cancer treatment includes that the subject is a “response patient” or that the subject is a “non-response patient”.
- determining a subject's responsiveness to cancer treatment includes determining whether the subject is a responder or non-responder.
- One aspect of the present invention uses TCR diversity to predict or determine that a subject is a “response patient” or that a subject is a “non-response patient”.
- the timing of determination is preferably predicted before the start of treatment, but may be after the start of treatment. It is because it has medical usefulness also to judge whether the treatment currently performed is appropriate.
- prognosis can be determined using the TCR diversity of the present invention. For example, using the TCR diversity of the present invention, it is possible to predict that a response patient will be non-responsive, that is, recurrence.
- the time of judgment can be determined after performing cancer immunotherapy (for example, after administration of an immune checkpoint inhibitor) and then performing a repertoire analysis over time, and determining the prognosis from the diversity index. is there.
- the present invention provides a method of using T cell receptor (TCR) diversity of a subject's T cells as a responsiveness or diagnostic indicator of the subject's cancer immunotherapy.
- TCR T cell receptor
- the TCR diversity can be provided as a diversity index.
- T cells can be CD8 + PD-1 + in peripheral blood.
- the present invention provides a method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising measuring the TCR diversity of a T cell of the subject in vitro, and the TCR diversity.
- the subject is determined to be responsive to cancer immunotherapy.
- the TCR diversity is low, it can be determined that the subject is poorly responsive to cancer immunotherapy.
- the T cell can be a CD8 + PD-1 + T cell in peripheral blood.
- the present invention relates to a method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising obtaining a peripheral blood sample from the subject, and T cells in the peripheral blood of the subject. Measuring a TCR diversity of the subject by a method comprising a large-scale high-efficiency TCR repertoire analysis, and determining that the subject is responsive to cancer immunotherapy if the TCR diversity is high Providing a method. Alternatively, if the TCR diversity is low, it can be determined that the subject is poorly responsive to cancer immunotherapy.
- the T cell can be a CD8 + PD-1 + T cell in peripheral blood.
- TCR diversity of CD8 + PD-1 + T cells is useful as an advantageous indicator of responsiveness to treatment with immune checkpoint inhibitors, particularly PD-1 inhibitors (eg, nivolumab or pembrolizumab) .
- cancer is composed of a plurality of cell populations rather than a uniform cell population. These various cancer cells are thought to express different cancer antigens for each cell. Therefore, it is predicted from the results of the examples of the present invention that immune cells recognizing more diverse antigens are required to suppress cancer cells, and the effects of immune checkpoint inhibitors in patients with diverse T cells. Can be considered to be easily demonstrated.
- the subject in one embodiment of the invention, the subject's TCR Shannon-Weaver index, Inverse Simpson index, Simpson index, Normalized Shannon-Weaver index Weaver index), DEX index (X is 0 to 100, for example, DE30 index, DE50 index, DE80 index, etc.) and / or UniqueX (X is 0 to 100, for example, Unique30 index, Unique50 index, Unique80 index, etc.) Based on the value, it is determined that the subject is a responder, is not a responder, or is a non-responder for any of the cancer immunotherapy described herein Or decide not to be a non-responder.
- numerical values based on multiple analyzes described herein can be used as a threshold.
- the threshold values described in the present specification are merely examples, and those skilled in the art can determine and use further threshold values based on the determination results in the further subject population.
- a method for determining a threshold is also disclosed herein, and the method of the present invention may include the step of determining a threshold, and may use a predetermined threshold according to such a method.
- the threshold of the diversity index can be set by calculating a diversity index of a certain number of responding patients and non-responsive patients and determining a numerical value that distinguishes the responding patients from the non-responsive patients.
- the minimum value of the responding patient population is used (the response patient is reliably determined to be effective, the sensitivity is 100%), and the maximum value of the non-responsive patient population is used (non-response)
- the patient is not determined to be a responding patient, the specificity is 100%), or ROC analysis is used (maximizing the effectiveness of determination based on the balance between sensitivity and specificity).
- the reference range may be a range such as an average value ⁇ standard deviation (SD) or an average value ⁇ 2SD, for example, and an upper limit or a lower limit of the reference range may be a reference value.
- SD standard deviation
- ⁇ 2SD average value
- the threshold value can be determined by calculating an average value -SD or an average value -2SD from the values of the response group.
- a subject's CD8 + PD-1 + T cell TCR ⁇ Shannon-Weaver index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is The patient can be determined by conducting a retrospective or prospective clinical trial, and in one specific embodiment, the threshold is about 3.2 to 4.4, preferably about 3.3 to about 4.2. For example, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9 can be set as a range. , About 4.0, about 4.1, about 4.2, etc. (any other particular number between these particular numbers may be used).
- the threshold when the subject's CD8 + PD-1 + T cell TCR ⁇ reverse Simpson index is greater than or equal to a threshold, the subject is shown to be a responding patient, and the threshold is Can be determined by conducting a retrospective or prospective clinical trial for the threshold value, and in one specific embodiment, the threshold can be set in the range of about 9 to about 19, preferably about 10 to about 18. Specific threshold values include, for example, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, etc. (any other specific value between these specific numbers) May be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ Simpson index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is Retrospective or prospective clinical trials can be performed and determined, and in one specific embodiment, the threshold is about 0.86 to about 0.96, preferably about 0.88 to about 0.94. For example, about 0.86, about 0.87, about 0.88, about 0.89, about 0.90, about 0.91, about 0.92 can be set. , About 0.93, about 0.94, about 0.95, about 0.96, etc. (any other specific number between these specific numbers may be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ standardized Shannon-Weaver index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is: Retrospective or prospective clinical trials can be performed on the subject patient, and in one specific embodiment, the threshold is about 0.41 to about 0.51, preferably about 0.42 to about 0.00.
- the threshold value can be set in a range of 49. Specific threshold values include, for example, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, and about 0. .48, about 0.49, etc. (any other specific number between these specific numbers may be used).
- the threshold can be set in the range of about 0.0007 to about 0.0015, and preferably about 0.00. 0008 to about 0.0014, about 0.0009 to about 0.0013, about 0.0010 to about 0.0011, and the like can be appropriately determined.
- Specific threshold values include, for example, about 0.0007, About 0.0008, about 0.0009, about 0.0010, about 0.0011, about 0.0012, about 0.0013, about 0.0014, about 0.0015, etc. Any of It can be mentioned may also be) using a constant numerical values.
- the subject's CD8 + PD-1 + T cell TCR ⁇ Shannon-Weaver index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is The patient can be determined by conducting a retrospective or prospective clinical trial, and in one specific embodiment, the threshold is from about 3.2 to about 4.3, preferably from about 3.4 to about 4.1. As specific numerical values, for example, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4. 0, about 4.1, etc. (any other specific numerical value between these specific numerical values may be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ reverse Simpson index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is about 8
- a threshold is about 8
- a subject's CD8 + PD-1 + T cell TCR ⁇ Simpson index is greater than or equal to a threshold value, indicating that the subject is a responding patient
- the threshold value for the subject patient Retrospective or prospective clinical trials can be performed and determined, and in one specific embodiment, the threshold is about 0.90 to about 0.96, preferably about 0.92 to about 0.95.
- the threshold is about 0.90, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96 can be set. (Any other specific numerical value between these specific numerical values may be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ standardized Shannon-Weaver index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is: It can be set in the range of about 0.37 to about 0.48, preferably about 0.38 to about 0.47. Specific values include, for example, about 0.38, about 0.39, and about 0. .40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, etc. (any other between these specific numbers May be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ DE50 index is greater than or equal to a threshold, indicating that the subject is a responding patient, and the threshold is about 0. 0004 to about 0.0012, preferably about 0.0005 to about 0.0012, and specific numerical values are, for example, about 0.0005, about 0.0006, about 0.0007. , About 0.0008, about 0.0009, about 0.0010, about 0.0011, about 0.0012, etc. (any other specific number between these specific numbers may be used). be able to.
- the method for diagnosing the responsiveness of a subject to cancer immunotherapy of the present invention can include a step of specifying a threshold for determining that the subject is responsive to cancer immunotherapy.
- a method for specifying can be specified by conducting a clinical test as exemplified in Examples and the like, calculating a diversity index, and performing statistical processing as necessary.
- the index value calculated by measuring the TCR diversity of the subject is rounded off as appropriate (for example, if it is DE50, it is rounded off to five decimal places) and compared with the threshold value. be able to.
- As the effective number two digits or one digit can be adopted in consideration of the detection limit.
- the threshold value computed from the numerical value which made the lower limit the average value-SD of the Responder group and average value-2SD, and ROC analysis is employable.
- the average value -SD of the Responder group for some diversity indices in the examples of the present specification is as follows.
- the average value ⁇ 2SD of the Responder group is as follows.
- the threshold value can be set using ROC analysis (Receiver Operating Characteristic analysis).
- ROC analysis Receiveiver Operating Characteristic analysis
- the cut-off value determined using the ROC analysis can be used to predict the effect before treatment in, for example, an anti-PD-1 antibody-treated patient.
- a ROC curve is created by plotting the positive rate when the cut-off value is changed as sensitivity on the vertical axis and the false positive rate (1-specificity) on the horizontal axis.
- the method of setting the cut-off value at the point where the distance from the upper left corner of the ROC curve is the minimum, and the farthest from the diagonal line where the area under the curve (AUC) in the ROC curve is 0.500 In other words, there is a method of calculating a new point, that is, (sensitivity + specificity-1) and setting the maximum value (youden index) as a cutoff value.
- the ROC curve for each diversity index described herein is shown in FIG.
- the cut-off values calculated from the Youden index based on Example 1 of this specification are shown in Table 12, and the numerical values exemplified in this way can be used as threshold values.
- sensitivity refers to the probability of correctly determining what should be determined as positive, and there is a relationship in which false negatives decrease when sensitivity is high. High sensitivity is useful for exclusion diagnosis (rule out).
- specificity refers to the probability that a negative one is correctly determined as negative, and there is a relationship that false positives decrease when the specificity is high. High specificity is useful for definitive diagnosis.
- the cutoff value of the Shannon-Weaver index of TCR ⁇ is about 3.7
- the cutoff value of the reverse Simpson index of TCR ⁇ is about 13
- the cutoff value of the standardized Shannon-Weaver index of TCR ⁇ is about 0.43
- TCR ⁇ Shannon-Weaver index cutoff value of 3.8 TCR ⁇ reverse Simpson index cutoff value of about 17
- TCR ⁇ standardized Shannon-Weaver index cutoff A value of about 0.42 and a cut-off value of the DE50 index of TCR ⁇ of about 0.0007 can be used.
- ROC analysis can be performed using the information about the further subject, and a cutoff value can be determined.
- Diversity indicators can be affected by the amount sampled. That is, some TCR diversity indices vary depending on the number of reads for sequencing. When such a diversity index is used, it is possible to evaluate the responsiveness more accurately by performing standardization corresponding to the number of specific leads.
- the Shannon, Simpson, and Reverse Simpson indices are almost constant regardless of the number of leads. In particular, the actual analysis level of 10,000 leads or more is considered to be hardly affected by the number of leads. It is not always necessary to standardize the index.
- the DE index generally tends to decrease as the number of leads increases. If the difference in the number of sequencing reads between subjects or between the subject and the reference subject is large (for example, when there is a variation of 10 times or more), use a DE index that is standardized for a certain number of leads. Therefore, it is considered possible to evaluate the responsiveness more accurately.
- the DE index can be approximated to a linear relationship between the number of leads and the logarithmic axis, and the index can be standardized based on this relationship. Therefore, it is possible to evaluate the responsiveness by comparing the DE index at a certain number of leads with a threshold value adjusted according to a linear relationship.
- the linear relationship between the DE index threshold and the number of leads described in the examples of the present specification is expressed by the following formula:
- the threshold linear relationship can be treated as having a width.
- the value of the diversity index is on the vertical axis and the number of leads is on the horizontal axis, it can be expressed in a band shape. If it is above the upper limit, it is determined to be a responder, if it is below the lower limit, it is determined to be non-response, and if it is in the middle, it is possible to use an indicator that determines administration at the discretion of the doctor. For example, a 95% reliability interval of a fitting curve can be used as the fluctuation range, and an example is shown in the examples of the present specification. By performing such calculations, specificity or sensitivity can be maximized.
- Resampling can be performed by randomly acquiring leads from the obtained leads.
- resampling may be performed a plurality of times. In such a case, a representative value (median value, average value, etc.) of the diversity index for each trial can be used as a standardized diversity index.
- the number of leads used as a standard for standardization is not limited. For example, 1000, 10000, 20000, 40000, 80000, 100000, or 200000, etc. (any other specific numerical value between these specific numerical values is used. May be used). In some embodiments, a DE50 index standardized for 30000 reads is used.
- a subject is a responder if the Shannon-Weaver index, normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells, is above a threshold.
- the threshold is shown and can be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment the threshold is about 2.8-4.1, preferably about 3.
- the threshold value can be set in the range of 9 to about 4.1.
- Specific threshold values include, for example, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3 .3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, etc. (these specific numerical values Any other specific numerical value in between may be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ 30000 lead normalized Simpson index is greater than or equal to a threshold
- the subject is shown to be a responding patient.
- the threshold can be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is about 8 to about 16, preferably about 13 to about 15. For example, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, etc. (for these specific numerical values) Any other specific numerical value in between may be used).
- a subject is shown to be a responder if the Simpson index normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells is above a threshold.
- the threshold can be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is from about 0.89 to about 0.94, preferably about 0.1.
- the threshold value can be set in the range of 92 to about 0.94. Specific threshold values include, for example, about 0.89, about 0.90, about 0.91, about 0.92, about 0.93, and about 0. .94, etc. (any other specific number between these specific numbers may be used).
- a subject is a responder if the normalized Shannon-Weaver index, normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells, is greater than or equal to a threshold.
- the threshold can be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is from about 0.41 to about 0.54, preferably about The threshold value can be set in the range of 0.50 to about 0.52.
- Specific threshold values include, for example, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, About 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, about 0.53, about 0.54, etc. (these specific Any other specific number between numbers may be used)
- the subject is a responder if the DE50 index (%) normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells is above a threshold.
- the threshold may be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is suitably in the range of about 0.36 to 0.40, etc. Specific thresholds that can be determined include, for example, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, etc. (other values between these specific numbers Any specific numerical value may be used).
- a subject is a responder if the Shannon-Weaver index, normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells, is above a threshold.
- the threshold is shown and can be determined by conducting a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is from about 3.2 to about 4.0, preferably about 3 7 to about 3.9, and specific numerical values include, for example, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, etc. (any other specific number between these specific numbers may be used).
- a subject's CD8 + PD-1 + T cell TCR ⁇ 30000 lead normalized reverse Simpson index is greater than or equal to a threshold
- the subject is shown to be a responding patient.
- the threshold value can be set in a range of about 10 to 23, preferably about 12 to about 22. Specific values include, for example, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, etc. (any other specific number between these specific numbers may be used) be able to.
- a subject is shown to be a responder if the Simpson index normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells is above a threshold.
- the threshold can be determined by performing a retrospective or prospective clinical trial on the subject patient, and in one specific embodiment, the threshold is from about 0.90 to about 0.97, preferably about 0.
- the threshold value can be set in the range of 92 to about 0.96. Specific threshold values include, for example, about 0.90, about 0.91, about 0.92, about 0.93, about 0.94, and about 0. .95, about 0.96, about 0.97, etc. (any other specific number between these specific numbers may be used).
- the subject is a responder if the normalized Shannon-Weaver index, normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells, is greater than or equal to a threshold value.
- the threshold value can be set in the range of about 0.42 to about 0.53, preferably about 0.47 to about 0.52, and specific values include, for example, about 0.42, About 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, About 0.53, etc. (any other specific number between these specific numbers may be used).
- the subject is a responder if the DE50 index (%) normalized to the TCR ⁇ 30000 lead of the subject's CD8 + PD-1 + T cells is above a threshold.
- the threshold value can be set in the range of about 0.22 to about 0.26, preferably about 0.23 to about 0.25. Specific values include, for example, about 0.22 , About 0.23, about 0.24, about 0.25, about 0.26, etc. (any other specific number between these specific numbers may be used).
- the threshold value calculated based on the ROC analysis can be used in the same manner as described above for the standardized diversity index.
- the threshold value exemplified in this specification is used for the diversity index standardized for 30000 reads. It is possible.
- the cut-off value of the Shannon-Weaver index normalized to 30,000 leads of TCR ⁇ is about 3.9
- the cut-off value of the reverse Simpson index standardized to 30000 leads of TCR ⁇ is about 30,000 leads of TCR ⁇
- the standardized Simpson index cut-off value is about 0.92
- the standardized Shannon-Weaver index cut-off value is about 0.51 for the TCR ⁇ 30000 lead
- the DE50 is standardized for the TCR ⁇ 30000 lead.
- TCR ⁇ As an index cut-off value of about 0.39, as a cutoff value of Shannon-Weaver index normalized to 30000 lead of TCR ⁇ , 3.8 as a cut-off value of reverse Simpson index normalized to 30000 lead of TCR ⁇ About 22, TCR ⁇
- the standardized Simpson index cut-off value for the 30000 lead is about 0.95
- the standardized Shannon-Weaver index cut-off value is about 0.51 for the TCR ⁇ 30000 lead
- the TCR ⁇ 30000 lead is standardized.
- a standardized DE50 index cutoff value of about 0.24 can be used.
- the cut-off value can be selected and adjusted according to the purpose. For example, (i) exclude non-responding patients (in terms of social security costs) or (ii) leakage of responding patients. It is possible to decide according to the purpose of erasing (from the viewpoint of a doctor / treatment). For (i), this can be achieved by setting the cutoff value to a value higher than the highest non-response line, and for (ii), the cutoff value is set to a value lower than the lowest responder line. Can be achieved. These values can be determined by one skilled in the art based on the diversity index of the subject population, or the maximum or minimum of the diversity index exhibited by non-responding and responding subjects as described herein. It is possible to set a threshold based on an example value.
- a threshold value of normal value is set from a large number of data and it can be discriminated whether it is an abnormal value or not, it can be used as a marker.
- keytruder PD-L1 antibody
- PD-1 high positivity is considered as a marker for application, the actual response rate is about 50%.
- a marker for example, DE50 index of TCR diversity
- the TCR is TCR ⁇ . In another preferred embodiment, the TCR is TCR ⁇ . TCR ⁇ may be preferred. Although not wishing to be bound by theory, the diversity index of TCR ⁇ does not appear to overlap in the numbers shown by responsive and non-responsive subjects. However, the present invention is not limited to this, and may be TCR ⁇ . Although it is not desired to be bound by theory, it is shown that, for example, it is possible to make a distinction by using the DE50 index.
- the diversity utilized in the present invention includes isolating CD8 + PD-1 + T cells from a subject's peripheral blood sample and TCR diversity of CD8 + PD-1 + T cells. Can be calculated using the process of measuring, determining or calculating.
- One embodiment of the invention is a method of diagnosing a subject's responsiveness to cancer immunotherapy, wherein the subject is responsive to cancer immunotherapy when the TCR diversity is high It is a method including the process to determine.
- the TCR diversity calculated here is advantageous to use a large-scale high-efficiency TCR repertoire analysis (WO2015 / 075939) detailed in this specification.
- WO2015 / 075939 large-scale high-efficiency TCR repertoire analysis
- the diversity index calculated by large-scale high-efficiency TCR repertoire analysis is more elaborate and more accurately reflects the condition of the subject.
- the diversity index in large-scale high-efficiency TCR repertoire analysis can clearly distinguish between responsiveness and non-responsiveness. In repertoire analysis other than high-efficiency TCR repertoire analysis, it is considered that the distinction between responsiveness and non-responsiveness is not sufficient. Therefore, when TCR diversity measured using large-scale high-efficiency repertoire analysis is used, more accurate evaluation results can be given than with conventional analysis.
- the TCR diversity of a subject is measured by a method including large-scale high-efficiency TCR repertoire analysis. If higher, the subject is determined to be responsive to cancer immunotherapy. Whether TCR diversity is high may be determined relatively, or whether diversity is high compared to a predetermined diversity index threshold (for example, those described in this specification). Can be determined. And when the diversity index is high, it can be judged that the response to cancer immunotherapy is good or there, and the subsequent treatment can be performed as necessary.
- the T cells that can be used can be any type of one or more T cells described herein, preferably CD8 + PD-1 + T cells in peripheral blood.
- a further embodiment of the present invention is a method of diagnosing a subject's responsiveness to cancer immunotherapy and treating the subject's cancer, comprising measuring TCR diversity of the subject's T cells;
- a method (so-called companion diagnosis or companion treatment), comprising the step of applying cancer immunotherapy to the subject when the TCR diversity is higher than a reference value.
- the standard value or threshold value of the TCR diversity can be appropriately determined by those skilled in the art based on the description of the present specification, and specific numerical values of the diversity index are exemplified in the present specification. Can be adopted.
- the T cells that can be used can be any type of one or more T cells described herein, and preferably the T cells can be CD8 + PD-1 + T cells in peripheral blood.
- the invention provides a composition comprising an immune checkpoint inhibitor for treating cancer in a subject with high T cell TCR diversity. It has been found by the inventors that such immune checkpoint inhibitors are advantageously administered to subjects with high T cell TCR diversity.
- a subject with low T cell TCR diversity can be determined to be a non-responder, and can also be determined not to administer an immune checkpoint inhibitor, or to interrupt or discontinue administration.
- the T cell that measures TCR diversity can be any type of one or more T cells described herein, and preferably CD8 + PD-1 + T cells in peripheral blood.
- composition of the present invention is preferably a pharmaceutical composition, and examples of the immune checkpoint inhibitor contained as an active ingredient thereof include PD-1 inhibitors.
- PD-1 inhibitors include nivolumab or pembrolizumab, which are anti-PD-1 antibodies.
- composition can be formulated into any dosage form such as aerosol, liquid, extract, elixir, capsule, granule, pill, ointment, powder, tablet, solution, suspension, emulsion and the like.
- the composition may include any pharmaceutically acceptable additive and / or excipient known in the art.
- compositions of the present invention can be administered by any suitable route determined by those skilled in the art and include, but are not limited to, intravenous injection, infusion, oral, parenteral, transdermal and the like. it can.
- a composition for treating cancer in a subject having a high T cell TCR Shannon index, Simpson index, standardized Shannon index, or DE50 index.
- a composition for treating cancer in a subject having a high T cell TCR DE50 index is provided.
- composition for treating cancer in a subject having a DE50 index normalized to 30000 reads of 0.24% or higher for TCR ⁇ of CD8 + PD-1 + T cells in peripheral blood having a DE50 index normalized to 30000 reads of 0.24% or higher for TCR ⁇ of CD8 + PD-1 + T cells in peripheral blood.
- a method for using repertoire diversity determined by a method that includes large-scale, high-efficiency TCR repertoire analysis as an indicator of responsiveness of a subject to treatment.
- This method uses a set of primers consisting of one forward primer and one reverse primer to amplify the TCR gene or BCR gene including all isotypes and subtype genes without changing the frequency of repertoire. To determine the diversity.
- This primer design is advantageous for unbiased amplification, as described herein and in WO2015 / 075939.
- the diversity index calculated by large-scale high-efficiency repertoire analysis is more precise and reflects the condition of the subject more accurately.
- the diversity index in large-scale high-efficiency repertoire analysis can clearly distinguish between responsiveness and non-responsiveness to treatment, whereas It is considered that repertoire analysis other than large-scale high-efficiency repertoire analysis does not sufficiently distinguish between responsiveness and non-responsiveness to the treatment.
- the treatment of interest is a treatment associated with an immune response.
- the repertoire analysis utilized is a TCR repertoire analysis.
- reagents described in the examples were used specifically for the reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.
- PBMC peripheral blood mononuclear cells
- nivolumab, Opdivo anti-PD-1 antibody
- Whole blood was separated from PBMC by specific gravity centrifugation using Ficoll-Hypaque, and the number of cells was counted with a hemocytometer.
- the isolated PBMC were either directly immunostained or suspended in cell cryopreservation solution STEM-CELLBANKER and stored in liquid nitrogen.
- Double staining with anti-CD8 antibody and anti-PD-1 antibody PBMC were immunostained according to the following procedure. 1. In order to remove STEM-CELLBANKER, the frozen cells were suspended in Stain Buffer (PBS, 0.1% BSA, 0.1% Sodium Azide) and washed twice by centrifuging and discarding the supernatant. 2. Fresh or stored PBMC were suspended in Stain buffer to 1 ⁇ 10 6 / tube, and centrifuged at 1,000 rpm for 5 minutes at 4 ° C. 3. The cells were stained in 100 ⁇ L of antibody diluent (5 ⁇ l / test anti-human CD8 antibody, 2.0 ⁇ g anti-human PD-1 antibody) at room temperature for 30 minutes under light shielding. 4).
- Stain Buffer PBS, 0.1% BSA, 0.1% Sodium Azide
- the suspension was suspended in 2 mL of Stain buffer, centrifuged at 1,000 rpm for 5 minutes at 4 ° C., and then washed twice by discarding the supernatant. 5). After washing, the suspension was suspended in 100 ⁇ L of Stain buffer, 5 ⁇ L of 7-AAD was added, and the mixture was reacted at room temperature for 10 minutes under light shielding. 6). 500 ⁇ L of Stain buffer was added to the cells and filtration was performed. 7). The stained cells were subjected to sorting separation of 7AAD-CD8 + PD-1 + cell population using a BD FACSAria III cell sorter (BD Bioscience). 8).
- Sorted cells were transferred to a 1.5 mL Eppendorf tube and centrifuged at 1,000 rpm for 5 minutes at 4 ° C. 9. After removing the Stain buffer leaving 50 ⁇ L of the supernatant, 750 ⁇ L of TRIzol LS reagent (Invitrogen) was added and the cells were lysed by pipetting. 10. 200 ⁇ L of DEPC Water was added to the dissolved TRIzol solution to adjust to 1000 ⁇ L, and the mixture was frozen and stored at ⁇ 80 ° C. after mixing with Vortex.
- TRIzol LS reagent Invitrogen
- RNA extraction Total RNA from cells dissolved in TRIzol LS reagent was extracted and purified using the RNeasy Plus Universal Mini Kit (QIAGEN). The purified RNA was quantified using a Nanodrop absorbance meter (Thermo Scientific) or a TapeStation2200 (Agilent).
- RNAsin RNase inhibitor
- E. coli DNA polymerase I E. coli DNA Ligase
- RNase H RNase H
- Double-stranded DNA is purified by column using MiniElute Reaction Cleanup Kit (QIAGEN), and then kept at 16 ° C overnight in the presence of P20EA / 10EA adapter (Table 4) and T4 ligase in the following T4 ligase buffer. A ligation reaction was performed.
- the adapter-added double-stranded DNA purified by the column as described above was digested with the following composition using Not I restriction enzyme (50 U / ⁇ L, Takara) in order to remove the adapter added to the 3 ′ end.
- Not I restriction enzyme 50 U / ⁇ L, Takara
- the digestion time can be changed as appropriate.
- PCR From double-stranded complementary DNA, using a common adapter primer P20EA and C region-specific primers shown in Table 2 (CG1, CK1 or CL1), it was performed 1 st PCR amplification. PCR had the following composition, and 20 cycles of 95 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute were performed.
- PCR was performed using the P20EA primer and the C region specific primer (CA2 or CB2) with the following reaction composition.
- PCR had the following composition, and 20 cycles of 95 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute were performed.
- the obtained 2nd PCR amplification product (10 ⁇ L) was purified using Agencourt AMPure XP (Beckman Coulter).
- Tag addition PCR was performed using 5 ⁇ L of 30 ⁇ L of the final eluate as a template.
- P22EA-ST1-R and mCG-ST1-R, mCK-ST1-R or mCL-ST1-R primers shown in FIG. 1 were used.
- the PCR cycle was 20 cycles of 95 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute.
- Tag PCR amplification product (10 ⁇ L) was purified using Agencourt AMPure XP (Beckman Coulter). INDEX was added using Nextera XT Index Kit v2 SetA (Illumina) using 2 ⁇ L of 30 ⁇ L of the final eluate as a template. PCR cycles were 95 ° C for 20 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds for 12 cycles. To confirm PCR amplification, 10 ⁇ L of the amplified product was confirmed by 2% agarose gel electrophoresis.
- the obtained indexed PCR amplification product was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), diluted to an appropriate concentration, and then sequenced using a MiSeq sequencer (Illumina). The operation and procedure of the sequencer followed the MiSeq instructions and manual.
- FIG. 3 shows a part of the result of FACS analysis. FSC / SSC lymphocyte gate (upper) and double staining of CD8 antibody and PD-1 antibody (lower) were shown. For the lymphocyte fraction, 7AAD ⁇ CD8 + PD-1 + cell fraction (P3) was sorted by FACS sorting (FIG. 3).
- TCR repertoire analysis of CD8 + PD-1 + T cells Using CD8 + PD-1 + T cells collected by FACS sorting, a comprehensive nucleotide sequence of the TCR gene was determined by a next-generation sequencer according to the method described in the method.
- Table 10 shows the number of cells and the amount of RNA collected by FACS sorting.
- Table 11 shows the number of TCR leads acquired from each sample, the number of assigned leads, the number of in-frame leads, and the number of unique leads.
- the point where the distance to the upper left corner of the ROC curve is the minimum is the cut-off value, and the point farthest from the diagonal line where the area under the curve (AUC) in the ROC curve is 0.500 That is, there is a method of calculating (sensitivity + specificity-1) and setting the maximum value (Youden index) as a cutoff value.
- the ROC curve for each diversity index is shown in FIG. DE50 showed the highest AUC value for both TCR ⁇ and TCR ⁇ compared to other diversity indicators, suggesting the best predictive ability.
- the cut-off value for each diversity index was calculated from the Youden index using the R program (ROCRpackage) and shown in Table 12. Below these cut-off values, a high therapeutic effect with anti-PD-1 antibody cannot be expected.
- TCR ⁇ repertoire diversity tends to be more clearly distinguished than TCR ⁇ repertoire diversity.
- DE50 index tends to be more clearly distinguished at any repertoire.
- CD8 + PD-1 + T cells are known to be released from immunosuppression by anti-PD-1 antibodies and exert antitumor effects. From this experiment, it was found that patients with higher diversity of CD8 + PD-1 + T cells in the peripheral blood of lung cancer patients have higher therapeutic effects on anti-PD-1 antibodies. Tumor infiltrating T cells recognize tumor specific antigens and exert antitumor effects. Tumor cells accumulate many gene mutations during the process of oncogenesis and produce nascent antigens (neoantigens) that are not expressed in normal cells. Immunotherapy such as immune checkpoint inhibitors is known to be highly effective against tumors that accumulate more gene mutations.
- the diversity index may vary depending on the number of samples, that is, the number of reads obtained by sequencing. Therefore, a fixed number of leads (100, 300, 1000, 3000, 10000, 30000, 80000) is obtained by random sampling from the data of each subject obtained in Example 1, and the diversity index ( Shannon-Weaver index, Simpson index, standardized Shannon index, inverse Simpson index, DE30 index, DE50 index, DE80 index, Unique30 index, Unique50 index, Unique80 index) were calculated and plotted. Resampling was attempted 100 times, and the median value of each diversity index was used as a standardized value for each number of leads. The changes with the number of reads for the diversity index of TCR ⁇ and TCR ⁇ are shown in FIGS. 8 and 9, respectively.
- FIGS. 13-33 The values normalized for each diversity index to 100, 300, 1000, 3000, 10000, 30000 and 80000 reads for each subject are shown in Tables 13-33 below.
- the Clinical column shows the therapeutic effect of each subject.
- Res_min indicates the minimum value of the index in the response group
- Non_max indicates the maximum value of the index in the non-response group.
- Discrim indicates whether or not the minimum value of the exponent in the response group is larger than the maximum value of the exponent in the non-response group.
- the t statistic of the diversity index between the response group and the non-response group is shown in the ttest column.
- the diversity index can be standardized and compared with the number of leads.
- the comparison with a specific threshold value is standardized with respect to the number of leads. It can be said that it is advantageous.
- Example 1 it is known from the data of Example 1 that the DE50 value has good separation between the response group and the non-response group.
- whether or not the distinction is good is examined even when standardized to a certain number of leads (100, 300, 1000, 3000, 10000, 30000, 80000). Exploring the index and the number of reads that show a “clear” separation (minimum AUC of the ROC curve is 1) such that the minimum value in the response group exceeds the maximum value in the non-response group. It is shown as Yes in the 32 Discrim column. In DE50 it was demonstrated that such complete separation was possible.
- the threshold value used when evaluating responsiveness using such a standardized diversity index was examined.
- ROC analysis was performed based on the value when each diversity index was standardized to each number of leads, and a threshold value was obtained.
- the cut-off values of each diversity index calculated based on the ROC analysis are shown in Table 33 (TCR ⁇ ) and Table 34 (TCR ⁇ ) below.
- TCR ⁇ Table 33
- TCR ⁇ Table 34
- a threshold value to be used according to the purpose of (i) eliminating non-responding patients (in terms of social security costs) or (ii) eliminating leakage of responding patients (in terms of doctors / treatment)
- the threshold of each diversity index when standardized to 30000 reads was further examined. Examples of maximum or minimum values of diversity index for non-responding and responding subjects normalized to 30000 reads and threshold values based on ROC analysis are shown in Table 35 (TCRa) and Table 36 (TCR ⁇ ) below.
- threshold values calculated from ROC analysis based on values standardized for each number of leads were plotted (FIGS. 12 and 13).
- the threshold values for Shannon, Simpson, reverse Simpson index, and Unique index are almost constant regardless of the number of reads. It was found that the DE index threshold, which tends to decrease with increasing number of reads, approximates a linear function in the log-log plot (log-log) (FIGS. 12 and 13). In particular, the correlation coefficient is very high in plots of 3000 leads or more that are likely to be used in actual analysis.
- the minimum value of responding patients and / or the maximum value of non-responding patients can be used as threshold values, and the linear variation of such values with respect to the number of leads was also examined. It is possible to use the response more than Res_Min and non-response in NonRes_Max or less. The results of the variation are shown in Table 39 below.
- PBMC peripheral blood mononuclear cells
- PBMC PBMC Antibody staining of PBMC PBMC were immunostained according to the following procedure. 2.1 Lyophilized PBMCs were lysed and the number of cells shown in Table 41 was suspended in Stain Buffer. 2.2 To remove STEM-CELLBANKER, it was suspended in Stain Buffer, centrifuged at 800 xg for 5 minutes at 4 ° C, and washed twice. 2.3 The cells were suspended in Stain buffer, and the antibodies shown in Table 40 below were added according to the instructions in the attached instructions, and allowed to react with the cells at room temperature for 30 minutes in the dark.
- RNA extraction was performed according to “1.3. RNA extraction”, “1.4. Synthesis of complementary DNA and double-stranded complementary DNA” and “1.5. PCR” in Example 1. Performed according to the method. 2.9 After antibody staining, the suspension was suspended in 2 mL of Stain buffer, centrifuged at 800 ⁇ g for 5 minutes at 4 ° C., and the supernatant was discarded and washed twice.
- TCR repertoire between T cell fractions Regarding the sequence of TCR clones obtained by TCR repertoire analysis, clones that are common between CD8P + PD1 +, CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, and CD8 + CTLA4 + T cell fractions Compared.
- Table 43 shows TCR clones that exist in common among all the fractions or among the plurality of fractions.
- TCR clones that are frequently present in CD8 + PD-1 + fractions are also frequently present in CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, or CD8 + CTLA4 + fractions Became clear.
- CD8 + PD-1 + T cells may co-express 4-1BB, TIM3, OX40, TIGIT, or CTLA4 molecules.
- the correlation between each T cell fraction was investigated about the read number of the TCR clone (FIG. 15). High-frequency TCR clones were commonly present in each T cell fraction and showed high correlation.
- examine the proportion of TCR clone reads common between CD8 + PD-1 + T cells and CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, or CD8 + CTLA4 + T cells Table 44.
- the percentage of reads occupied by CD8 + PD-1 + TCR clones in each fraction was significantly higher than that of control CD8 + T cells, CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, Or higher in CD8 + CTLA4 + T cells.
- This means that the tumor-specific TCR of the tumor-specific T cells contained in CD8 + PD-1 + T cells is also 4-1BB, TIM3, OX40, TIGIT, or CTLA-4 positive T cell fractions. It was suggested to be included in high frequency.
- any T cell fraction of CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, CD8 + CTLA4 + in peripheral blood can be used as a biomarker based on TCR diversity. Is done.
- the CD8 + PD1 + T cell diversity index is similar to the CD8 + 4-1BB +, CD8 + TIM3 +, CD8 + OX40 +, CD8 + TIGIT +, or CD8 + CTLA4 + T cells of the same patient. Indicated.
- the diversity index obtained by performing TCR repertoire analysis on peripheral blood cells that can be easily sampled can be used as a biomarker for predicting the effect of cancer immunotherapy.
- SEQ ID NO: 1 BSL-18E primer
- SEQ ID NO: 2 P20EA primer
- SEQ ID NO: 3 P10EA primer
- SEQ ID NO: 4 P22EA-ST1-R primer
- SEQ ID NO: 5 CA1 primer
- SEQ ID NO: 6 CA2 primer
- SEQ ID NO: 7 CA-ST1- R primer sequence number 8: CB1 primer sequence number 9: CB2 primer sequence number 10: CB-ST1-R primer sequence number 11-60: CDR3 sequence of each TCR ⁇ chain clone in Example 3
Abstract
Description
(項目1) 被験体のT細胞のT細胞受容体(TCR)多様性を、該被験体のがん免疫療法に対する応答性の指標として用いる方法。
(項目2) 前記T細胞が、CD8+であり、かつ1以上のT細胞抑制系細胞表面マーカが陽性である、前記項目に記載の方法。
(項目3) 前記T細胞が、CD8+であり、かつ1以上のT細胞刺激系細胞表面マーカが陽性である、前記項目のいずれかに記載の方法。
(項目4) 前記T細胞が、CD8+であり、かつPD-1、CD28、CD154(CD40L)、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、CD27、CD152(CTLA-4)、CD366(TIM-3)、CD223(LAG-3)、CD272(BTLA)、CD226(DNAM-1)、TIGITおよびCD367(GITR)からなる群から選択される1以上の細胞表面マーカが陽性である、前記項目のいずれかに記載の方法。
(項目5) 前記T細胞が、CD8+PD-1+T細胞である、前記項目のいずれかに記載の方法。
(項目6) 前記T細胞が、前記被験体の末梢血中のT細胞である、前記項目のいずれかに記載の方法。
(項目7) 前記がん免疫療法が、免疫チェックポイント阻害剤の投与を含む、前記項目のいずれかに記載の方法。
(項目8) 前記免疫チェックポイント阻害剤が、PD-1阻害剤である、前記項目のいずれかに記載の方法。
(項目9) 前記PD-1阻害剤が、ニボルマブまたはペムブロリズマブである、前記項目のいずれかに記載の方法。
(項目10) 前記TCR多様性が、Shannon指数、Simpson指数、逆Simpson指数、標準化Shannon指数、Unique50指数、DE30指数、DE80指数またはDE50指数であらわされる、前記項目のいずれかに記載の方法。
(項目11) 前記TCR多様性が、DE50指数であらわされる、前記項目のいずれかに記載の方法。
(項目12) 前記TCRがTCRαである、前記項目のいずれかに記載の方法。
(項目13) 以下の表:
に記載されるリード数のいずれかに標準化した前記被験体のDE50指数が、該表に記載される該リード数に対応する閾値以上である場合、該被験体が奏効患者であることが示されるか、または該閾値未満である場合、該被験体が非奏効患者であることが示される、前記項目のいずれかに記載の方法。
(項目14) 前記TCRがTCRβである、前記項目のいずれかに記載の方法。
(項目15) 以下の表:
に記載されるリード数のいずれかに標準化した前記被験体のDE50指数が、該表に記載される該リード数に対応する閾値以上である場合、該被験体が奏効患者であることが示されるか、または該閾値未満である場合、該被験体が非奏効患者であることが示される、前記項目のいずれかに記載の方法。
(項目16) 該被験体の末梢血サンプルからCD8+PD-1+T細胞を単離する工程と、
該CD8+PD-1+T細胞のTCR多様性を決定する工程と
をさらに含む、前記項目のいずれかに記載の方法。
(項目17) 前記TCR多様性が、大規模高効率TCRレパトア解析を含む方法によって決定される、前記項目のいずれかに記載の方法。
(項目18) 免疫チェックポイント阻害剤を含む組成物であって、T細胞のTCR多様性が高い被験体においてがんを治療するための、組成物。
(項目18A) 前記項目のいずれか1つまたは複数に記載される特徴を有する、前記項目に記載の組成物。
(項目19) 前記T細胞が、CD8+であり、かつ1以上のT細胞抑制系細胞表面マーカが陽性である、前記項目のいずれかに記載の組成物。
(項目20) 前記T細胞が、CD8+であり、かつ1以上のT細胞刺激系細胞表面マーカが陽性である、前記項目のいずれかに記載の組成物。
(項目21) 前記T細胞が、CD8+であり、かつPD-1、CD28、CD154(CD40L)、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、CD27、CD152(CTLA-4)、CD366(TIM-3)、CD223(LAG-3)、CD272(BTLA)、CD226(DNAM-1)、TIGITおよびCD367(GITR)からなる群から選択される1以上の細胞表面マーカが陽性である、前記項目のいずれかに記載の組成物。
(項目22) 前記T細胞が、CD8+PD-1+T細胞である、前記項目のいずれかに記載の組成物。
(項目23) 前記T細胞が、前記被験体の末梢血中のT細胞である、前記項目のいずれかに記載の組成物。
(項目24) 前記免疫チェックポイント阻害剤が、PD-1阻害剤である、前記項目のいずれかに記載の組成物。
(項目25) 前記PD-1阻害剤が、ニボルマブまたはペムブロリズマブである、前記項目のいずれかに記載の組成物。
(項目26) 前記被験体のT細胞のTCR多様性が、Shannon指数、Simpson指数、逆Simpson指数、標準化Shannon指数、Unique50指数、DE30指数、DE80指数またはDE50指数であらわされる、前記項目のいずれかに記載の組成物。
(項目27) 前記被験体のT細胞のTCR多様性が、DE50指数であらわされる、前記項目のいずれかに記載の組成物。
(項目28) 前記TCRがTCRαである、前記項目のいずれかに記載の組成物。
(項目29) 以下の表:
に記載されるリード数のいずれかに標準化した前記被験体のDE50指数が、該表に記載される該リード数に対応する閾値以上である、前記項目のいずれかに記載の組成物。
(項目30) 前記TCRがTCRβである、前記項目のいずれかに記載の組成物。
(項目31) 以下の表:
に記載されるリード数のいずれかに標準化した前記被験体のDE50指数が、該表に記載される該リード数に対応する閾値以上である、前記項目のいずれかに記載の組成物。
(項目32) 前記被験体のTCR多様性が、大規模高効率TCRレパトア解析を含む方法によって決定される、前記項目のいずれかに記載の組成物。
(項目33) 被験体のがん免疫療法に対する応答性を診断する方法であって、
in vitroで該被験体のT細胞のTCR多様性を測定する工程と、
該TCR多様性が高い場合、該被験体ががん免疫療法に対して応答性が良いと判定するか、または該TCR多様性が低い場合、該被験体ががん免疫療法に対して応答性が悪いと判定する工程と
を含む、方法。
(項目33A) 前記項目のいずれか1つまたは複数に記載の特徴を有する、前記項目に記載の方法。
(項目34) 被験体のがん免疫療法に対する応答性を診断する方法であって、
該被験体から末梢血サンプルを得る工程と、
該被験体の末梢血中のT細胞のTCR多様性を、大規模高効率TCRレパトア解析を含む方法によって測定する工程と、
該TCR多様性が高い場合、該被験体ががん免疫療法に対して応答性が良いと判定するか、または該TCR多様性が低い場合、該被験体ががん免疫療法に対して応答性が悪いと判定する工程と
を含む、方法。
(項目34A) 前記項目のいずれか1つまたは複数に記載の特徴を有する、前記項目に記載の方法。
(項目35) 被験体のがん免疫療法に対する応答性を診断し、該被験体のがんを治療する方法であって、
該被験体から末梢血サンプルを得る工程と、
該被験体の末梢血中のT細胞のTCR多様性を測定する工程と、
該TCR多様性が基準値より高い場合、該被験体にがん免疫療法を施す工程と
を含む、方法。
(項目35A) 前記項目のいずれか1つまたは複数に記載の特徴を有する、前記項目に記載の方法。
(項目36) 大規模高効率レパトア解析を含む方法によって決定されたレパトアの多様性を、被験体の治療への応答性の指標として用いる方法。
(項目36A) 前記項目のいずれか1つまたは複数に記載の特徴を有する、前記項目に記載の方法。
(項目37) 前記治療は免疫反応に関連する治療である、前記項目のいずれかに記載の方法。
(項目38) 前記レパトア解析はTCRレパトア解析である、前記項目のいずれかに記載の方法。
(項目39) 前記被験体のTCR多様性をあらわす多様性指数が閾値以上であることは、該被験体が奏効患者であることの指標であるか、または、該多様性指数が閾値未満であることは、該被験体が非奏効患者であることの指標である、前記項目のいずれかに記載の方法であって、該閾値が、ROC解析に基づいて定められるか、感度に基づいて定められるか、または特異度に基づいて決定されたものである、方法。
(項目40) 前記被験体のTCR多様性をあらわす多様性指数が閾値以上であることは、該被験体が奏効患者であることの指標であるか、または、該多様性指数が閾値未満であることは、該被験体が非奏効患者であることの指標である、前記項目のいずれかに記載の方法であって、該閾値が、該被験体の多様性指数の算出に用いられたリード数に対して標準化されたものである、方法。
本明細書において、「がん免疫療法」とは、生物の有する免疫機能を用いてがんを治療する方法をいう。がん免疫療法には、大きく分けて、がんに対する免疫機能を強化することによるがん免疫療法と、がんの免疫回避機能を阻害することによるがん免疫療法が存在する。さらに、がん免疫療法には、体内での免疫機能を賦活化する能動免疫療法と、体外で免疫機能を賦活化させた、または増殖させた免疫細胞を体内に戻すことによる受動免疫療法とがある。
免疫システムによる生体防御機構は、主にT細胞やB細胞によって担われる特異的免疫に大きく依存している。T細胞やB細胞は自己の細胞や分子には反応せず、ウイルスや細菌などの外来性の病原体を特異的に認識して攻撃することができる。そのために、T細胞やB細胞は細胞表面上に発現した受容体分子によって自己抗原とともに他の生物由来の多様な抗原を認識し、識別できる機構を有している。T細胞ではT細胞受容体(T cell receptor, TCR)が抗原受容体として働く。それら抗原受容体からの刺激によって細胞内シグナルが伝達され、炎症性サイトカインやケモカインなどの産生が亢進し、細胞増殖が増進され、様々な免疫応答が開始される。
本発明の好ましい実施形態では、大規模高効率TCRレパトア解析を用いてTCR多様性を測定する。本明細書において、「大規模高効率レパトア解析」とは、WO2015/075939(この文献の開示は本明細書においてその全体が必要に応じて参考として援用される。)に記載されており、対象がTCRの場合「大規模高効率TCRレパトア解析」と称する。大規模高効率レパトア解析では、データベースを用いて被験体のレパトア(Repertoire)(T細胞レセプター(TCR)またはB細胞レセプター(BCR)の可変領域)を定量的に解析する方法であり、この方法は、(1)該被験者から非バイアス的に増幅した、T細胞レセプター(TCR)またはB細胞レセプター(BCR)の核酸配列を含む核酸試料を提供する工程;(2)該核酸試料に含まれる該核酸配列を決定する工程;および(3)決定された該核酸配列にもとづいて、各遺伝子の出現頻度またはその組み合わせを算出し、該被験体のレパトアを導出する工程を包含し、前記(1)は、以下の工程(1-1)標的となる細胞に由来するRNA試料を鋳型として相補的DNAを合成する工程;(1-2)該相補的DNAを鋳型として二本鎖相補的DNAを合成する工程;(1-3)該二本鎖相補的DNAに共通アダプタープライマー配列を付加してアダプター付加二本鎖相補的DNAを合成する工程;(1-4)該アダプター付加二本鎖相補的DNAと、該共通アダプタープライマー配列からなる共通アダプタープライマーと、第1のTCRまたはBCRのC領域特異的プライマーとを用いて第1のPCR増幅反応を行う工程であって、該第1のTCRまたはBCRのC領域特異的プライマーは、該TCRまたはBCRの目的とするC領域に十分に特異的であり、かつ、他の遺伝子配列に相同性のない配列を含み、かつ、増幅された場合に下流にサブタイプ間に不一致塩基を含むよう設計される、工程;(1-5)(1-4)のPCR増幅産物と、該共通アダプタープライマーと、第2のTCRまたはBCRのC領域特異的プライマーとを用いて第2のPCR増幅反応を行う工程であって、該第2のTCRまたはBCRのC領域特異的プライマーは、該第1のTCRのC領域特異的プライマーの配列より下流の配列において該TCRまたはBCRのC領域に完全マッチの配列を有するが他の遺伝子配列に相同性のない配列を含み、かつ、増幅された場合に下流にサブタイプ間に不一致塩基を含むよう設計される、工程;および(1-6)(1-5)のPCR増幅産物と、該共通アダプタープライマーの核酸配列に第1の追加アダプター核酸配列を含む付加共通アダプタープライマーと、第2の追加アダプター核酸配列および分子同定(MID Tag)配列を第3のTCRまたはBCRのC領域特異的配列に付加したアダプター付の第3のTCRのC領域特異的プライマーとを用いて第3のPCR増幅反応を行う工程であって、該第3のTCRのC領域特異的プライマーは、該第2のTCRまたはBCRのC領域特異的プライマーの配列より下流の配列において該TCRまたはBCRのC領域に完全マッチの配列を有するが他の遺伝子配列に相同性のない配列を含み、かつ、増幅された場合に下流にサブタイプ間に不一致塩基を含むよう設計され、該第1の追加アダプター核酸配列は、DNA捕捉ビーズへの結合およびemPCR反応に適切な配列であり、該第2の追加アダプター核酸配列は、emPCR反応に適切な配列であり、該分子同定(MID Tag)配列は、増幅産物が同定できるように、ユニークさを付与するための配列である、工程を包含する。この方法の具体的な詳細はWO2015/075939に記載されており、当業者はこの文献および本明細書の実施例等を適宜参照して解析を実施することができる。
がん免疫療法に対する応答は、RECIST v1.1(New response evaluation criteria in solid tumours:Revised RECIST guideline (version 1.1))に基づいて決定することができる。
以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。また、本発明の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。
1つの局面において本発明は、被験体のT細胞のT細胞受容体(TCR)多様性を、該被験体のがん免疫療法に対する応答性の、または当該応答性の診断の指標として用いる方法を提供する。ここで、TCR多様性は、多様性指数として提供され得る。T細胞は、末梢血中のCD8+PD-1+であり得る。
TCRαのシャノン-ウィーバー指数: 3.37
TCRαの逆シンプソン指数: 9.83
TCRαの標準化シャノン-ウィーバー指数: 0.422
TCRαのDE50指数: 0.0013605
TCRβのシャノン-ウィーバー指数: 3.69
TCRβの逆シンプソン指数: 15.84
TCRβの標準化シャノン-ウィーバー指数: 0.433
TCRβのDE50指数: 0.0009545
TCRαのシャノン-ウィーバー指数: 2.66
TCRαの逆シンプソン指数: -4.19
TCRαの標準化シャノン-ウィーバー指数: 0.366
TCRαのDE50指数: 0.0007697
TCRβのシャノン-ウィーバー指数: 3.03
TCRβの逆シンプソン指数: 1.43
TCRβの標準化シャノン-ウィーバー指数: 0.385
TCRβのDE50指数: 0.0005064
直線関係の一例として、本明細書の実施例に記載されるDE指数の閾値とリード数との直線関係は、以下の式:
本発明のさらなる局面において、本発明は、免疫チェックポイント阻害剤を含む組成物であって、T細胞のTCR多様性が高い被験体においてがんを治療するための、組成物を提供する。このような免疫チェックポイント阻害剤が、T細胞のTCR多様性が高い被験体に投与することが有利であることが、本発明者らによって見出された。そして、T細胞のTCR多様性が低い被験体には、非奏効患者であると判断することができ、免疫チェックポイント阻害剤を投与しない、または投与を中断もしくは中止するという判断も行うことができる。TCR多様性を測定するT細胞は、本明細書において記載される任意の種類の1または複数のT細胞であり得、好ましくは末梢血中のCD8+PD-1+T細胞であり得る。
1つの局面では、大規模高効率TCRレパトア解析を含む方法によって決定されたレパトアの多様性を、被験体の治療への応答性の指標として用いる方法を提供する。この手法は、1種のフォーワードプライマーと1種のリバースプライマーからなる1セットのプライマーですべてのアイソタイプやサブタイプ遺伝子を含むTCR遺伝子またはBCR遺伝子を、存在頻度を変えることなく増幅して、レパトアの多様性を決定する。本明細書に記載され、WO2015/075939でも記載されるようにこのプライマー設計は、非バイアス的な増幅に有利である。
本明細書において「または」は、文章中に列挙されている事項の「少なくとも1つ以上」を採用できるときに使用される。「もしくは」も同様である。本明細書において「2つの値」の「範囲内」と明記した場合、その範囲には2つの値自体も含む。
(1.材料と方法)
1.1.末梢血単核球細胞(PBMC)の分離
12例の肺癌患者における抗PD-1抗体(nivolumab、オプジーボ)治療開始前において、全血をヘパリン含採血管に8mL採取した。全血はFicoll-Hypaqueを用いた比重遠心分離によりPBMCを分離し、血球計数盤にて細胞数をカウントした。単離されたPBMCは直接免疫細胞染色するか、あるいは細胞用凍結保存液STEM-CELLBANKERに懸濁して液体窒素中にて保管された。
PBMCは下記の手順に従って免疫染色した。
1.凍結保管した細胞はSTEM-CELLBANKERを取り除くため、Stain Buffer(PBS、0.1% BSA、0.1% Sodium Azide)に懸濁し、遠心後に上清を捨てることで2回洗浄した。
2.新鮮あるいは保管PBMCを1×106/tubeになるようにStain bufferに懸濁し、1,000rpmで5分間、4℃で遠心した。
3.細胞は100μLの抗体希釈液(5μl/test抗ヒトCD8抗体、2.0μg 抗ヒトPD-1抗体)中で、室温、30分間、遮光下で染色した。
4.抗体染色後、2mLのStain bufferに懸濁し、1,000rpm、5分間4℃で遠心後、上清を捨てることで2回洗浄した。
5.洗浄後100μLのStain bufferに懸濁して、5μLの7-AADを添加し、遮光下室温で10分間反応させた。
6.細胞に500μLのStain bufferを加え、フィルトレーションを行った。
7.染色細胞は、BD FACSAria III セルソーター(BD Bioscience)を用いて、7AAD-CD8+PD-1+細胞集団をソーティング分離した。
8.ソーティングした細胞は、1.5mLエッペンチューブに移し、1,000rpm、5分間4℃で遠心した。
9.50μLの上清を残してStain bufferを除去後、750μLのTRIzol LS試薬(Invitrogen)を加えてピペッティングすることで細胞を溶解した。
10.溶解したTRIzol液に200μLのDEPC Waterを加え、1000μLに調整した後、Vortexによる混和後に-80℃にて凍結保管した。
TRIzol LS試薬に溶解した細胞からの全RNAの抽出と精製は、RNeasy Plus Universal Mini Kit (QIAGEN)を用いて行った。精製されたRNAを、Nanodrop吸光度計(Thermo Scientific)あるいはTapeStation2200(Agilent)を用いて定量した。
抽出されたRNAを用いて、相補的DNAを合成するため、1.25μLのBSL-18Eプライマー(表1)と3.75μLのRNAを混和して、70℃で8分間アニーリングした。
二本鎖相補的DNAから、表2に示す共通アダプタープライマーP20EAとC領域特異的プライマー(CG1、CK1またはCL1)を用いて、1st PCR増幅を行った。PCRは次に示す組成で、95℃ 20秒、60 ℃30秒、72℃ 1分のサイクルを20サイクル行った。
得られた各ユニークリードのリード数データから多様性指数を求めた。シャノン-ウィーバー指数(Shannon-Weaver index)、シンプソン指数(Simpson index)、逆シンプソン指数(Inverse Simpson index)(1/λ)およびDE50指数は次の数式に従って算出された。N:全リード数、ni:i番目ユニークリードのリード数、S:ユニークリード数、S50:全リードの50%を占める上位ユニークリードの数
2.1.臨床評価
抗PD-1抗体治療開始前および治療後3カ月後において患者胸部CTもしくはPET画像診断を行い、腫瘍量の形態的評価および腫瘍量の変化に基づいて治療効果を評価した(図1)。一部の患者の効果判定結果(完全奏効、部分奏効、安定、進行)および主な治療経過は表9に示した。一部の患者の胸部CT画像を図2に示した。図2に示される4例のうち2例の患者が治療後3か月の時点で部分奏効(PR)と判定され、他の2例は非奏効と判定された。
12例の肺癌患者治療前において採取されたPBMCを用いてFACS解析を行った。PBMCはPE-Cy7標識抗ヒトCD8抗体およびFITC標識抗PD-1抗体で染色された。FSC/SSCゲーティングによりリンパ球分画を、さらに7AADにより死細胞を除去した。FACS Aria IIIセルソーターにより7AAD-CD8+PD-1+T細胞を集め、TRIzol LS RNA抽出試薬に溶解した。図3にFACS解析の結果の一部を示す。FSC/SSCによるリンパ球ゲート(上段)およびCD8抗体およびPD-1抗体の二重染色(下段)を示した。リンパ球分画について、7AAD-CD8+PD-1+細胞分画(P3)をFACSソーティングにより分取した(図3)。
FACSソーティングにより回収したCD8+PD-1+T細胞を用いて、方法に記載された方法に従って次世代シーケンサによるTCR遺伝子の網羅的塩基配列の決定を行った。FACSソーティングにより回収された細胞数およびRNA量を表10に示す。各サンプルより獲得したTCRリード数、アサインされたリード数、In-frameリード数およびユニークリード数を表11に示す。
抗PD-1抗体治療患者の間でCD8+PD-1+T細胞の多様性を比較するため、12例の肺癌患者試料から配列決定されたTCRリードデータを用いて多様性指標を算出し、比較した。個々のユニークリードとそのリード数(コピー数)を用いて、方法に記載の数式に従って多様性指標を算出した。多様性指標には、Shannon-Weaver index、Normalized Shannon-Weaver index、Simpson index、逆Simpson indexおよびDE50 indexを用いた(図5および6)。有意差検定は、ノンパラメトリックなマン・ホイットニー検定(両側検定)を用いた。TCRα鎖において、Shannon-Weaver indexはNon-Responderに比してResponderで有意に高い値を示した(平均値±標準偏差、Non-Responder vs. Responder、2.796±0.9519 vs. 4.081±0.7124、P=0.0411)。同様に、Normalized Shannon-Weaver index、逆Simpson indexおよびDE50の多様性指標は、それぞれ0.3327±0.1018 vs. 0.4771±0.05547(P=0.0260)、7.530±4.906 vs. 23.85±14.02(P=0.0152)、0.0006220±0.0003472 vs. 0.001951±0.0005909(P=0.0022)であり、いずれもNon-Responderに比してResponderで有意に高い値を示した。また、同様にTCRβ鎖においてもすべての指標においてNon-Responderに比べResponderで有意に高い値を示した。平均値±標準偏差は、それぞれ3.129±0.6742 vs. 4.345±0.6555、P=0.0087(Shannon-Weaver index)、0.3528±0.0612 vs. 0.4815±0.04832、P=0.0087(Normalized Shannon-Weaver index)、8.198±3.551 vs. 30.25±14.41、P=0.0087(逆Simpson index)、0.0003910±0.00007243 vs. 0.001403±0.0004480、P=0.0022(DE50)であった。これらの結果から、CD8+PD-1+T細胞の多様性はNon-Responderに比べResponderの方が明らかに高いことが明らかになった。
抗PD-1抗体治療患者における治療前の効果予測をするために、各多様性指標のカットオフ値の設定をROC解析(Receiver Operating Characteristic analysis)を用いて行った。ROC解析では、カットオフ値を変化させていった場合の陽性率を感度として縦軸に、偽陽性率(1-特異度)を横軸にプロットしたROC曲線が作成される。カットオフ値を設定する場合、ROC曲線の左上隅との距離が最小になる点をカットオフ値とする方法と、ROC曲線における曲線下面積(AUC)が0.500となる斜点線から最も離れたポイント、すなわち(感度+特異度-1)を計算して、その最大値となるポイント(Youden index)をカットオフ値にする方法がある。各多様性指標におけるROC曲線を図7に示した。DE50は他の多様性指標と比較して、TCRα、TCRβともにAUCが最も高い値を示し、予測能が最も優れていると示唆された。各多様性指標におけるカットオフ値は、Rプログラム(ROCRpackage)を用いてYouden指数より算出され、表12に示された。これらカットオフ値未満では抗PD-1抗体による高い治療効果が見込めないと予測されることになる。
CD8+PD-1+T細胞は抗PD-1抗体により免疫抑制を解除され、抗腫瘍効果を発揮することが知られる。本実験から肺癌患者の末梢血中のCD8+PD-1+T細胞の多様性が高い患者ほど抗PD-1抗体に対する治療効果が高いことが分かった。腫瘍浸潤T細胞は腫瘍特異的抗原を認識し、抗腫瘍効果を発揮する。腫瘍細胞は腫瘍化の過程で多くの遺伝子変異を蓄積し、正常細胞には発現していない新生抗原(ネオアンチゲン)を産生するようになる。免疫チェックポイント阻害薬などの免疫療法はより多くの遺伝子変異を蓄積する腫瘍に対する効果が高いことが知られている。より多数の新生抗原がT細胞のターゲットとなることが腫瘍を抑制するために重要であると考えられる。抗PD-1抗体に有効な患者には、治療前に免疫抑制された多数の新生抗原に反応する多様なT細胞が存在すると推測される。それらが抗PD-1抗体により抑制解除され、より高い治療効果をもたらすと推測される。肺がん患者に対して抗PD-1抗体(Nivolumab)に有効な患者は20~30%である。抗PD-1抗体治療前に有効患者を予測できればより効果的な治療を実現し、医療費の無駄をなくすことができる。試料採取の容易な末梢血細胞におけるTCRレパトア解析を行い、多様性指標をバイオマーカとして利用すれば、これまで不可能であった抗PD-1抗体治療の効果予測が可能になると期待される。
多様性指数は、サンプル数、すなわち、シーケンシングによって得られたリード数の影響を受け変動する可能性がある。そのため、実施例1において得られた各被験体のデータから、ランダムサンプリングにより一定リード数(100、300、1000、3000、10000、30000、80000)を取得し、当該リードに基づいた多様性指数(Shannon-Weaver指数、Simpson指数、標準化Shannon指数、逆Simpson指数、DE30指数、DE50指数、DE80指数、Unique30指数、Unique50指数、Unique80指数)をそれぞれ算出し、プロットした。リサンプリングは100回試行し、各多様性指標の中央値をそれぞれのリード数に対して標準化された値として用いた。TCRαおよびTCRβの多様性指数についてのリード数による変化は、図8および図9にそれぞれ示される。
(1.材料と方法)
1.末梢血単核球細胞(PBMC)の分離
1例の抗PD-1抗体(nivolumab、オプジーボ)治療奏効患者の全血をヘパリン含採血管に20mL採取した。Ficoll-Paque PLUS(GE Healthcare)を用いた比重遠心分離によりPBMCを分離し、血球計数盤にて細胞数をカウントした。単離されたPBMCは細胞用凍結保存液STEM-CELLBANKER(TaKaRa Bio)に懸濁し、-80℃超低温冷凍庫に保管した。
PBMCは下記の手順に従って免疫染色した。
2.1 凍結保管したPBMCを溶解し、表41に示される細胞数をStain Bufferに懸濁した。
2.2 STEM-CELLBANKERを取り除くため、Stain Bufferで懸濁後、800×gで5分間、4℃で遠心し、2回洗浄した。
2.3 細胞をStain bufferに懸濁し、下記表40の抗体を添付説明書の指示に従い添加し、遮光、室温で30分間細胞と反応させた。
2.5 細胞に500μLのStain bufferを加え、フィルトレーションを行った後、BD FACSAria IIIセルソーター(BD Bioscience)、あるいはFACSMelodyセルソーター(BD Bioscience)を用いて、上記ソート分画をソーティング分離した。
2.6 ソーティング分画は、800×g、5分間、4℃で遠心することで回収した。
2.7 上清を250μl残して除去後、Trizol LS試薬(Invitrogen)を750μl加えてピペッティングし、細胞を溶解した。
2.8 RNA抽出後のTCRレパトア解析は、実施例1の「1.3.RNA抽出」、「1.4.相補的DNAおよび二本鎖相補的DNAの合成」および「1.5. PCR」の方法に従って実施した。
2.9 抗体染色後、2mLのStain bufferに懸濁し、800×g、5分間4℃で遠心後、上清を捨てることで2回洗浄した。
CD8+PD1+、CD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、およびCD8+CTLA4+T細胞分画のリンパ球に占める割合(%)とFACSソーティングにより回収された細胞数を表41に示した。CD8と各分子マーカを共発現する約1 x 104~1 x 105のT細胞を回収し、これらのT細胞分画からRNAを抽出し、定法に従いTCRレパトア解析を行った。TCRシーケンス解析の結果、各試料より得られた全リード数、ユニークリード数、In-frameリード数を表42aおよび表42bに示した。いずれの試料においても10万リード以上のリードを獲得することができた。
TCRレパトア解析によって獲得されたTCRクローンの配列について、CD8P+PD1+、CD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、およびCD8+CTLA4+T細胞分画間で共通するクローンを比較した。すべての分画間、あるいは複数の分画間に共通して存在するTCRクローンを表43に示した。CD8+PD-1+分画に高頻度に存在するTCRクローンは、CD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、あるいはCD8+CTLA4+分画にも高頻度に存在することが明らかになった。このことは、CD8+PD-1+T細胞が、4-1BB、TIM3、OX40、TIGIT、あるいはCTLA4分子を共発現している可能性を示唆していた。また、TCRクローンのリード数について各T細胞分画間の相関を調べた(図15)。高頻度に存在するTCRクローンは、共通して各T細胞分画に存在し、高い相関を示した。次に、CD8+PD-1+T細胞とCD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、あるいはCD8+CTLA4+T細胞の間で共通するTCRクローンのリードの割合を調べた(表44)。CD8+PD-1+のTCRクローンが各分画に占めるリードの割合は、対照のCD8+T細胞と比べ、著明にCD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、あるいはCD8+CTLA4+T細胞で高かった。このことは、CD8+PD-1+T細胞に含有される腫瘍特異的T細胞の腫瘍特異的TCRが、4-1BB、TIM3、OX40、TIGIT、またはCTLA-4陽性のT細胞分画にも高頻度に含まれることを示唆していた。これらのことから、末梢血中のCD8+4-1BB+、CD8+TIM3+、CD8+OX40+、CD8+TIGIT+、CD8+CTLA4+のいずれのT細胞分画も、TCR多様性によるバイオマーカとして利用できることと期待される。
以上のように、本発明の好ましい実施形態を用いて本発明を例示してきたが、本発明は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。
配列番号2:P20EA プライマー
配列番号3:P10EA プライマー
配列番号4:P22EA-ST1-R プライマー
配列番号5:CA1 プライマー
配列番号6:CA2 プライマー
配列番号7:CA-ST1-R プライマー
配列番号8:CB1 プライマー
配列番号9:CB2 プライマー
配列番号10:CB-ST1-R プライマー
配列番号11~60:実施例3における各TCRβ鎖クローンのCDR3配列
Claims (40)
- 被験体のT細胞のT細胞受容体(TCR)多様性を、該被験体のがん免疫療法に対する応答性の指標として用いる方法。
- 前記T細胞が、CD8+であり、かつ1以上のT細胞抑制系細胞表面マーカが陽性である、請求項1に記載の方法。
- 前記T細胞が、CD8+であり、かつ1以上のT細胞刺激系細胞表面マーカが陽性である、請求項1に記載の方法。
- 前記T細胞が、CD8+であり、かつPD-1、CD28、CD154(CD40L)、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、CD27、CD152(CTLA-4)、CD366(TIM-3)、CD223(LAG-3)、CD272(BTLA)、CD226(DNAM-1)、TIGITおよびCD367(GITR)からなる群から選択される1以上の細胞表面マーカが陽性である、請求項1に記載の方法。
- 前記T細胞が、CD8+PD-1+T細胞である、請求項1に記載の方法。
- 前記T細胞が、前記被験体の末梢血中のT細胞である、請求項1~5のいずれか1項に記載の方法。
- 前記がん免疫療法が、免疫チェックポイント阻害剤の投与を含む、請求項1~6のいずれか1項に記載の方法。
- 前記免疫チェックポイント阻害剤が、PD-1阻害剤である、請求項7に記載の方法。
- 前記PD-1阻害剤が、ニボルマブまたはペムブロリズマブである、請求項8に記載の方法。
- 前記TCR多様性が、Shannon指数、Simpson指数、逆Simpson指数、標準化Shannon指数、Unique50指数、DE30指数、DE80指数またはDE50指数であらわされる、請求項1~9のいずれか1項に記載の方法。
- 前記TCR多様性が、DE50指数であらわされる、請求項1~10のいずれか1項に記載の方法。
- 前記TCRがTCRαである、請求項1~11のいずれか1項に記載の方法。
- 前記TCRがTCRβである、請求項1~11のいずれか1項に記載の方法。
- 該被験体の末梢血サンプルからCD8+PD-1+T細胞を単離する工程と、
該CD8+PD-1+T細胞のTCR多様性を決定する工程と
をさらに含む、請求項1~15のいずれか1項に記載の方法。 - 前記TCR多様性が、大規模高効率TCRレパトア解析を含む方法によって決定される、請求項1~16のいずれか1項に記載の方法。
- 免疫チェックポイント阻害剤を含む組成物であって、T細胞のTCR多様性が高い被験体においてがんを治療するための、組成物。
- 前記T細胞が、CD8+であり、かつ1以上のT細胞抑制系細胞表面マーカが陽性である、請求項18に記載の組成物。
- 前記T細胞が、CD8+であり、かつ1以上のT細胞刺激系細胞表面マーカが陽性である、請求項18に記載の組成物。
- 前記T細胞が、CD8+であり、かつPD-1、CD28、CD154(CD40L)、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、CD27、CD152(CTLA-4)、CD366(TIM-3)、CD223(LAG-3)、CD272(BTLA)、CD226(DNAM-1)、TIGITおよびCD367(GITR)からなる群から選択される1以上の細胞表面マーカが陽性である、請求項18に記載の組成物。
- 前記T細胞が、CD8+PD-1+T細胞である、請求項18に記載の組成物。
- 前記T細胞が、前記被験体の末梢血中のT細胞である、請求項18~22のいずれか1項に記載の組成物。
- 前記免疫チェックポイント阻害剤が、PD-1阻害剤である、請求項18~23のいずれか1項に記載の組成物。
- 前記PD-1阻害剤が、ニボルマブまたはペムブロリズマブである、請求項24に記載の組成物。
- 前記被験体のT細胞のTCR多様性が、Shannon指数、Simpson指数、逆Simpson指数、標準化Shannon指数、Unique50指数、DE30指数、DE80指数またはDE50指数であらわされる、請求項18~25のいずれか1項に記載の組成物。
- 前記被験体のT細胞のTCR多様性が、DE50指数であらわされる、請求項18~26のいずれか1項に記載の組成物。
- 前記TCRがTCRαである、請求項18~27のいずれか1項に記載の組成物。
- 前記TCRがTCRβである、請求項18~27のいずれか1項に記載の組成物。
- 前記被験体のTCR多様性が、大規模高効率TCRレパトア解析を含む方法によって決定される、請求項18~31のいずれか1項に記載の組成物。
- 被験体のがん免疫療法に対する応答性を診断する方法であって、
in vitroで該被験体のT細胞のTCR多様性を測定する工程と、
該TCR多様性が高い場合、該被験体ががん免疫療法に対して応答性が良いと判定するか、または該TCR多様性が低い場合、該被験体ががん免疫療法に対して応答性が悪いと判定する工程と
を含む、方法。 - 被験体のがん免疫療法に対する応答性を診断する方法であって、
該被験体から末梢血サンプルを得る工程と、
該被験体の末梢血中のT細胞のTCR多様性を、大規模高効率TCRレパトア解析を含む方法によって測定する工程と、
該TCR多様性が高い場合、該被験体ががん免疫療法に対して応答性が良いと判定するか、または該TCR多様性が低い場合、該被験体ががん免疫療法に対して応答性が悪いと判定する工程とを含む、方法。 - 被験体のがん免疫療法に対する応答性を診断し、該被験体のがんを治療する方法であって、
該被験体から末梢血サンプルを得る工程と、
該被験体の末梢血中のT細胞のTCR多様性を測定する工程と、
該TCR多様性が基準値より高い場合、該被験体にがん免疫療法を施す工程と
を含む、方法。 - 大規模高効率レパトア解析を含む方法によって決定されたレパトアの多様性を、被験体の治療への応答性の指標として用いる方法。
- 前記治療は免疫反応に関連する治療である、請求項36に記載の方法。
- 前記レパトア解析はTCRレパトア解析である、請求項36または37に記載の方法。
- 前記被験体のTCR多様性をあらわす多様性指数が閾値以上であることは、該被験体が奏効患者であることの指標であるか、または、該多様性指数が閾値未満であることは、該被験体が非奏効患者であることの指標である、請求項1~17のいずれか1項に記載の方法であって、該閾値が、ROC解析に基づいて定められるか、感度に基づいて定められるか、または特異度に基づいて決定されたものである、方法。
- 前記被験体のTCR多様性をあらわす多様性指数が閾値以上であることは、該被験体が奏効患者であることの指標であるか、または、該多様性指数が閾値未満であることは、該被験体が非奏効患者であることの指標である、請求項1~17または39のいずれか1項に記載の方法であって、該閾値が、該被験体の多様性指数の算出に用いられたリード数に対して標準化されたものである、方法。
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TW201839128A (zh) | 2018-11-01 |
CA3057041A1 (en) | 2018-09-20 |
EP3597766A1 (en) | 2020-01-22 |
JP7150280B2 (ja) | 2022-10-11 |
TWI786096B (zh) | 2022-12-11 |
EP3597766A4 (en) | 2020-11-25 |
US11858994B2 (en) | 2024-01-02 |
AU2018235623A1 (en) | 2019-08-22 |
US20200024349A1 (en) | 2020-01-23 |
KR20190129926A (ko) | 2019-11-20 |
CN110546269A (zh) | 2019-12-06 |
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