JP2019535001A - 細胞あたりのpd−l1発現量を検出する方法及びその使用 - Google Patents
細胞あたりのpd−l1発現量を検出する方法及びその使用 Download PDFInfo
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Abstract
Description
上記に要約されるように、本発明の実施形態は、所定の閾値を上回るプログラム死リガンド1(PD−L1)を発現する細胞を検出する方法を対象とする。本発明の方法において、PD−L1発現を検出することができる細胞は、さまざまな例において、新生細胞及び免疫細胞を含む。その結果、いくつかの例において、本発明の方法において検出される、細胞、たとえば、新生細胞などは、所定の閾値を超えるPD−L1タンパク質の細胞あたりの発現レベルを有する。
上記に要約されるように、本開示は、所定の閾値を上回るPD−L1を発現する細胞を検出する方法を提供する。PD−L1発現は、PD−L1タンパク質発現レベル、またはPD−L1符号化転写物(すなわち、mRNA)発現レベルに基づき、細胞あたりの基準で定量化されることができる。いくつかの例において、本発明の方法は、細胞あたりのPD−L1タンパク質発現量のみを定量化し、細胞あたりのPD−L1転写物発現量を定量化しない。いくつかの例において、PD−L1タンパク質レベル及びPD−L1転写レベルの両方を定量化する組み合わされた方法を用いることができる。
上記に要約されるように、本開示の方法は、サイトメトリーによってアッセイする標識細胞懸濁液を含む。標識細胞懸濁液をサイトメトリーによってアッセイするさまざまな方法は、たとえば、フローサイトメーターを使用してフローサイトメトリーによってアッセイすること、たとえば、細胞サイトメーターを使用することなどによって、標識細胞懸濁液を細胞サイトメトリーによってアッセイすること、及び同様のことを備えるが、これらに限定されない、本明細書に記載された方法における有用性を見出すことができる。標識細胞懸濁液試料を、細胞あたりのPD−L1発現量についてアッセイすることができる。いくつかの事例において、サイトメトリーによってアッセイされる、追加の細胞パラメータは、本開示の新生細胞を検出する際に有用性を見出すこともできる。その結果、さまざまな細胞パラメータを測定するために、標識細胞懸濁液をサイトメトリーによってアッセイするさまざまな方法を用いることができる。
上記に要約されるように、本開示の方法は、所定の閾値を上回るPD−L1を発現する新生細胞が新生物試料中に存在するか否かを検出することを備える。本明細書に記載された方法は、さまざまな新生物試料に適用可能であり、そこで新生物試料は、いずれかの新生物(すなわち、異常に増殖する)組織または細胞集団または細胞の試料を含むことができる。異常な組織増殖は、たとえば、対象組織の増殖を適切で正常な、または健常な組織の増殖と比較することなどによるまざまな手段によって、決定されることができる。新生物は、良性新生物、原位置新生物、悪性新生物、及び不確定または未知の挙動の新生物を含む。悪性新生物は、がんを含み、その結果、本発明の方法は、所定の閾値を上回るPD−L1を発現するがん細胞ががん試料中に存在するか否かを検出することを備えることができる。
上記に要約されるように、本発明の方法は、所定の閾値を上回る細胞あたりのPD−L1レベルを発現する細胞の検出を備えるため、これらの方法を実施する際に有用なさまざまな試薬を含む。たとえば、本発明の方法は、サイトメトリーアッセイが実施されることを可能にするために、細胞を標識特異的結着メンバーの試薬と接触させることによって、所定の閾値を上回るPD−L1を発現する細胞を検出することを一般的に備える。
ホモサピエンスCD274分子(CD274)、転写バリアント1、mRNA(NM_014143.3):
GGCGCAACGCTGAGCAGCTGGCGCGTCCCGCGCGGCCCCAGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAATCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCTCATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCATCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGGACAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGTGAATGATTTCTTTTGAAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTATACATTGGAAGCATAAAGATCAAACCGTTGGTTGCATAGGATGTCACCTTTATTTAACCCATTAATACTCTGGTTGACCTAATCTTATTCTCAGACCTCAAGTGTCTGTGCAGTATCTGTTCCATTTAAATATCAGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCCACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTTATTCAAAAACCATTTATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATTGAATCTACAGATGTGAGCAAGACAAAGTACCTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAAACCCGAGCAGTGTTGCCAAGAGGAGGAAATAGGCCAATGTGGTCTGGGACGGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTTACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAGCATTATATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACTGCTACTGCCTTTCATTCATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTATTTATTTTAGTGTTTCTTATATAGCAGATGGAATGAATTTGAAGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGTTTCTAAGTTATCTTTCCCATAGCTTTTCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATATACATTTAGACAACCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTATGTTTGCTCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCCTGTGCCAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTACATGTGCATTTGTACAGTAATTGGTGTGACAGTGTTCTTTGTGTGAATTACAGGCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCTCCTTGTGGTGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAATGGCATAGGCAGAGATGATACCTAATTCTGCATTTGATTGTCACTTTTTGTACCTGCATTAATTTAATAAAATATTCTTATTTATTTTGTTACTTGGTACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCAGTGGAGAAAGTTAAAAAA(配列番号:3)、または、
ホモサピエンスCD274分子(CD274)、転写バリアント2、mRNA(NM_001267706.1):
GGCGCAACGCTGAGCAGCTGGCGCGTCCCGCGCGGCCCCAGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAATCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCTCATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCATCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGGACAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGTGAATGATTTCTTTTGAAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTATACATTGGAAGCATAAAGATCAAACCGTTGGTTGCATAGGATGTCACCTTTATTTAACCCATTAATACTCTGGTTGACCTAATCTTATTCTCAGACCTCAAGTGTCTGTGCAGTATCTGTTCCATTTAAATATCAGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCCACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTTATTCAAAAACCATTTATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATTGAATCTACAGATGTGAGCAAGACAAAGTACCTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAAACCCGAGCAGTGTTGCCAAGAGGAGGAAATAGGCCAATGTGGTCTGGGACGGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTTACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAGCATTATATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACTGCTACTGCCTTTCATTCATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTATTTATTTTAGTGTTTCTTATATAGCAGATGGAATGAATTTGAAGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGTTTCTAAGTTATCTTTCCCATAGCTTTTCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATATACATTTAGACAACCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTATGTTTGCTCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCCTGTGCCAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTACATGTGCATTTGTACAGTAATTGGTGTGACAGTGTTCTTTGTGTGAATTACAGGCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCTCCTTGTGGTGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAATGGCATAGGCAGAGATGATACCTAATTCTGCATTTGATTGTCACTTTTTGTACCTGCATTAATTTAATAAAATATTCTTATTTATTTTGTTACTTGGTACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCAGTGGAGAAAGTTAAAAAA(配列番号:4)
を含むが、これらに限定されない。
上記に要約されるように、本開示は、新生物について被検体を処置する方法を備える。用語「被検体」、「個体」、「宿主」、及び「患者」は、本明細書で互換的に使用され、診断、処置、または治療が望まれる、いずれかの哺乳類被検体、特にヒトを指す。
また、提供されるものは、上述された方法のうちの1つ以上を実施するためのキットである。本発明のキットは、大きく異なることができる。本発明のキットに含まれる試薬及び装置は、所定の閾値を上回るPD−L1を発現する新生細胞を検出する方法に関して、上記に言及されるものを含む。
1.所定の閾値を上回るプログラム死リガンド1(PD−L1)を発現する新生細胞が新生物試料中に存在するか否かを検出する方法であって、
前記新生物試料をPD−L1に特異的な標識結着メンバーと接触させ、標識細胞懸濁液を生成すること、
前記標識細胞懸濁液をサイトメトリーによってアッセイし、細胞あたりのPD−L1発現量を定量化し、所定の閾値を上回るPD−L1を発現する新生細胞が前記新生物試料中に存在するか否かを検出すること
を備える、方法。
2.前記サイトメトリーによってアッセイすることは、細胞周期をアッセイすることをさらに有する、付記1に記載の方法。
3.細胞周期をアッセイすることは、細胞あたりのDNA量を定量化することを有する、付記2に記載の方法。
4.細胞周期をアッセイすることは、発現した細胞周期マーカーを検出することを有する、付記2または3に記載の方法。
5.前記細胞懸濁液の試料をDNA標識試薬と接触させることをさらに備える、付記1〜4のいずれか一つに記載の方法。
7.前記サイトメトリーによってアッセイすることは、異数性をアッセイすることをさらに有する、付記1〜6のいずれか一つに記載の方法。
8.検出された細胞は、増殖性である、付記1〜7のいずれか一つに記載の方法。
9.検出された細胞は、異数体である、付記1〜8のいずれか一つに記載の方法。
10.前記標識は、前記新生物試料を免疫細胞に特異的な少なくとも1個の標識結着メンバーと接触させることをさらに有する、付記1〜9のいずれか一つに記載の方法。
12.免疫細胞に特異的な前記少なくとも1個の標識結着メンバーは、リンパ球マーカーCD8に特異的な標識結着メンバーを含む、付記10または11に記載の方法。
13免疫細胞の少なくとも1個のマーカーに陰性である細胞を検出することを備える、付記10〜12のいずれか一つに記載の方法。
14.前記新生物試料は、体液試料である、付記1〜13のいずれか一つに記載の方法。
15.前記体液試料は、血液試料である、付記14に記載の方法。
17.検出された細胞は、造血器癌細胞である、付記14〜16のいずれか一つに記載の方法。
18.前記新生物試料は、固形腫瘍試料である、付記1〜13のいずれか一つに記載の方法。
19.前記固形腫瘍は、上皮性腫瘍である、付記18に記載の方法。
20.前記固形腫瘍は、肺癌腫瘍である、付記18または19に記載の方法。
22.前記固形腫瘍は、乳癌腫瘍である、付記18または19に記載の方法。
23.検出された細胞は、扁平上皮癌細胞を含む、付記18〜22のいずれか一つに記載の方法。
24.検出された細胞は、腺癌細胞を含む、付記18〜22のいずれか一つに記載の方法。
25.検出された細胞は、腺扁平上皮癌細胞を含む、付記18〜22のいずれか一つに記載の方法。
27.前記生検は、固形組織診である、付記26に記載の方法。
28.前記新生物試料を前記固形組織診から調製する、付記27に記載の方法。
29.前記新生物試料を前記固形組織診から調製することは、前記固形組織診の組織をホモジナイズすることを有する、付記28に記載の方法。
30.前記生検は、液体生検である、付記26に記載の方法。
32.前記生検は、穿刺吸引(FNA)生検である、付記26に記載の方法。
33.前記新生物試料を前記FNA生検から調製することをさらに備える、付記32に記載の方法。
34.前記標識細胞懸濁液試料の前記細胞は、固定される、付記1〜33のいずれか一つに記載の方法。
35.前記新生物試料の前記細胞を固定することをさらに備える、付記1〜34のいずれか一つに記載の方法。
37.前記新生物試料の前記細胞を固定することは、前記接触中に実施される、付記35に記載の方法。
38.前記新生物試料の前記細胞を固定することは、前記接触後に実施される、付記35に記載の方法。
39.前記細胞を固定することは、前記新生物試料の前記細胞を穏和な架橋剤と接触させることを有する、付記35〜38のいずれか一つに記載の方法。
40.前記穏和な架橋剤は、ホルムアルデヒドに基づく固定液を含む、付記39に記載の方法。
42.前記所定の閾値は、1個の細胞あたり500個以上のPD−L1分子である、付記41に記載の方法。
43.前記所定の閾値は、1000個以上のPD−L1分子である、付記42に記載の方法。
44.被検体中の新生物が抗プログラム死リガンド1(PD−L1)免疫療法応答性であるか否かかを識別する方法であって、
前記新生物から調製される細胞懸濁液試料をPD−L1に特異的な標識結着メンバーと接触させ、標識細胞懸濁液を生成すること、
前記標識細胞懸濁液をサイトメトリーによってアッセイし、所定の閾値を超えるPD−L1レベルを各発現する細胞集団が存在するか否かを検出し、前記新生物が抗PD−1/PD−L1免疫療法応答性であるか否かを識別すること
を備える、方法。
45.前記サイトメトリーによってアッセイすることは、細胞周期をアッセイすることをさらに有する、付記44に記載の方法。
47.細胞周期をアッセイすることは、発現した細胞周期マーカーを検出することを有する、付記45または46に記載の方法。
48.前記細胞懸濁液試料をDNA標識試薬と接触させることをさらに備える、付記44〜47のいずれか一つに記載の方法。
49.前記細胞懸濁液試料を発現した細胞周期マーカーに特異的な標識結着メンバーと接触させることをさらに備える、付記44〜48のいずれか一つに記載の方法。
50.前記サイトメトリーによってアッセイすることは、異数性をアッセイすることをさらに有する、付記44〜49のいずれか一つに記載の方法。
52.前記細胞集団は、異数体である、付記44〜51のいずれか一つに記載の方法。
53.前記異数体細胞は、前記被検体中の循環腫瘍細胞の存在を示す、付記52に記載の方法。
54.前記標識は、前記細胞懸濁液試料を免疫細胞に特異的な少なくとも1個の標識結着メンバーと接触させることをさらに有する、付記44〜53のいずれか一つに記載の方法。
55.免疫細胞に特異的な前記少なくとも1個の標識結着メンバーは、リンパ球マーカーCD45に特異的な標識結着メンバーを含む、付記54に記載の方法。
57.前記標識細胞懸濁液をサイトメトリーによってアッセイし、増殖性免疫細胞が存在するか否かを検出することをさらに備える、付記54〜56のいずれか一つに記載の方法。
58.増殖性免疫細胞量を定量化することをさらに備える、付記57に記載の方法。
59.前記細胞集団が免疫細胞マーカーに陰性であるか否かを識別することを備える、付記54〜58のいずれか一つに記載の方法。
60.前記細胞集団は、循環腫瘍細胞を含む、付記44〜59のいずれか一つに記載の方法。
62.前記新生物は、固形腫瘍である、付記44〜60のいずれか一つに記載の方法。
63.前記固形腫瘍は、上皮性腫瘍である、付記62に記載の方法。
64.前記固形腫瘍は、肺癌腫瘍である、付記62または61に記載の方法。
65.前記肺癌腫瘍は、非小細胞肺癌(NSCLC)腫瘍である、付記64に記載の方法。
67.前記細胞集団は、扁平上皮癌細胞を含む、付記63〜66のいずれか一つに記載の方法。
68.前記細胞集団は、腺癌細胞を含む、付記63〜66のいずれか一つに記載の方法。
69.前記細胞集団は、腺扁平上皮癌細胞を含む、付記63〜66のいずれか一つに記載の方法。
70.前記細胞懸濁液試料は、生検から調製される、付記44〜69のいずれか一つに記載の方法。
72.前記細胞懸濁液試料を前記固形組織診から調製することをさらに備える、付記71に記載の方法。
73.前記細胞懸濁液試料を前記固形組織診から調製することは、前記固形組織診の組織をホモジナイズすることを有する、付記72に記載の方法。
74.前記生検は、液体生検である、付記70に記載の方法。
75.前記細胞懸濁液試料を前記液体生検から調製することをさらに備える、付記74に記載の方法。
77.前記細胞懸濁液試料を前記FNA生検から調製することをさらに備える、付記76に記載の方法。
78.前記標識細胞懸濁液試料の前記細胞は、固定される、付記44〜77のいずれか一つに記載の方法。
79.前記細胞懸濁液試料の前記細胞を固定することをさらに備える、付記44〜78のいずれか一つに記載の方法。
80.前記細胞懸濁液の前記細胞を固定することは、前記接触前に実施される、付記79に記載の方法。
82.前記細胞懸濁液の前記細胞を固定することは、前記接触後に実施される、付記79に記載の方法。
83.前記細胞を固定することは、前記細胞懸濁液試料の前記細胞を穏和な架橋剤と接触させることを有する、付記79〜82のいずれか一つに記載の方法。
84.前記穏和な架橋剤は、ホルムアルデヒドに基づく固定液を含む、付記83に記載の方法。
85.前記所定の閾値は、1個の細胞あたり100個以上のPD−L1分子である、付記44〜84のいずれか一つに記載の方法。
87.前記所定の閾値は、1000個以上のPD−L1分子である、付記86に記載の方法。
88.前記サイトメトリーによってアッセイすることは、前記細胞集団のサイズを定量化することをさらに有する、付記44〜87のいずれか一つに記載の方法。
89.前記細胞集団の前記サイズが前記細胞懸濁液試料中の前記新生細胞の1%を超える場合に、前記新生物は、抗PD−1/PD−L1免疫療法応答性と識別される、付記88に記載の方法。
90.前記新生物は、免疫組織化学的検査によってPD−L1陽性と以前に識別されている、付記44〜89のいずれか一つに記載の方法。
抗PD−1/PD−L1免疫療法を、抗PD−1/PD−L1免疫療法応答性の新生物を含む被検体に投与することを備え、
前記新生物は付記44〜90のいずれか一つに記載の方法に従って、抗PD−1/PD−L1免疫療法応答性と識別される、
方法。
92.前記被検体は、化学療法によって以前に治療されている、付記91に記載の方法。
93.前記被検体は、放射線療法によって以前に治療されている、付記91または92に記載の方法。
94.前記被検体は、免疫療法によって以前に治療されている、付記91〜93のいずれか一つに記載の方法。
95.前記被検体は、免疫関連疾患を有するか、または免疫関連疾患を発症する増加したリスクにある、付記91〜94のいずれか一つに記載の方法。
97.前記治療中に前記新生物の前記抗PD−1/PD−L1免疫療法応答性を監視すること、及び、前記新生物が抗PD−1/PD−L1免疫療法応答性と識別されるときにのみ前記治療を継続することをさらに備える、付記91〜96のいずれか一つに記載の方法。
98.前記監視することは、
前記新生物からの細胞懸濁液試料をPD−L1に特異的な標識結着メンバーと接触させ、標識細胞懸濁液を生成すること、
前記標識細胞懸濁液をサイトメトリーによってアッセイし、所定の閾値を超えるPD−L1レベルを各発現する細胞集団が存在するか否かを検出し、前記腫瘍が抗PD−1/PD−L1免疫療法応答性であるか否かを識別すること
を有する、付記97に記載の方法。
99.PD−L1に特異的な標識結着メンバー、及び
固定試薬を含む細胞懸濁液固定液
を備える、キット。
100.前記固定試薬は、穏和な架橋剤である、付記99に記載のキット。
102.透過処理試薬をさらに含む、付記99〜101のいずれか一つに記載のキット。
103.前記細胞懸濁液固定液は、前記透過処理試薬を含む、付記102に記載のキット。
104.細胞破砕装置をさらに備える、付記99〜103のいずれか一つに記載のキット。
105.DNA標識試薬をさらに備える、付記99〜104のいずれか一つに記載のキット。
107.免疫細胞に特異的な少なくとも1個の標識結着メンバーをさらに備える、付記99〜106のいずれか一つに記載のキット。
108.免疫細胞に特異的な前記少なくとも1個の標識結着メンバーは、リンパ球マーカーCD45に特異的な標識結着メンバーを含む、付記107に記載のキット。
109.免疫細胞に特異的な前記少なくとも1個の標識結着メンバーは、リンパ球マーカーCD8に特異的な標識結着メンバーを含む、付記107または108に記載のキット。
110.生検採取装置をさらに備える、付記99〜109のいずれか一つに記載のキット。
細胞懸濁液を、新たに採取された肺組織、またはBrifford et al.(Acta Cytol.1982 Mar−Apr;26(2):195−200)に記載されるような非吸引穿刺細胞診(FNNAC)方法(別名、「穿刺吸引細胞診(Cytopuncture)」方法)に従って穿刺吸引(FNA)によって採取された試料から調製した。試料を健常な肺を含む被検体から、また非小細胞肺癌(NSCLC)を含む患者から採取した。
さらに腫瘍及び免疫細胞について、臨床肺試料を調製し、フローサイトメトリーによって解析し、PD−L1及び細胞周期色素データを取得した。取得されたデータの例を図5に提供される表2に示す。また表2における比較のために提供されるものは、免疫組織化学的検査(IHC)によって評価されるような、PD−L1+腫瘍データの割合である。
上記に要約されるように、本明細書に記載される方法をさらに用いて、直接的に、または間接的に、循環腫瘍細胞(CTC)について組織試料を解析することが可能である。例として、フローサイトメトリーによって、細胞周期色素及びCTC検出を使用して、肺組織試料を免疫細胞マーカー、PD−L1及び細胞周期パラメータについて解析した。表3は、以下のように、このような評価からのデータを提供する。
35U.S.C.§119(e)に従い、本出願は、2016年9月6日に出願された、米国仮特許出願第62/384,037号の出願日に優先権を主張し、その本開示は、本明細書に参照により援用される。
Claims (15)
- 所定の閾値を上回るプログラム死リガンド1(PD−L1)を発現する新生細胞が新生物試料中に存在するか否かを検出する方法であって、
前記新生物試料をPD−L1に特異的な標識結着メンバーと接触させ、標識細胞懸濁液を生成すること、
前記標識細胞懸濁液をサイトメトリーによってアッセイし、細胞あたりのPD−L1発現量を定量化し、所定の閾値を上回るPD−L1を発現する新生細胞が前記新生物試料中に存在するか否かを検出すること
を備える、方法。 - 前記サイトメトリーによってアッセイすることは、細胞周期をアッセイすることをさらに有する、請求項1に記載の方法。
- 前記サイトメトリーによってアッセイすることは、異数性をアッセイすることをさらに有する、請求項1または2に記載の方法。
- 検出された細胞は、増殖性である、請求項1〜3のいずれか一項に記載の方法。
- 前記接触させることは、前記新生物試料を免疫細胞に特異的な少なくとも1個の標識結着メンバーと接触させることをさらに有する、請求項1〜4のいずれか一項に記載の方法。
- 検出された細胞は、循環腫瘍細胞、造血器癌細胞、または固形腫瘍の細胞である、請求項1〜5のいずれか一項に記載の方法。
- 前記所定の閾値は、1個の細胞あたり100個以上のPD−L1分子である、請求項1〜6のいずれか一項に記載の方法。
- 被検体中の新生物が抗プログラム死リガンド1(PD−L1)免疫療法応答性であるか否かを識別する方法であって、
前記新生物から調製される細胞懸濁液試料をPD−L1に特異的な標識結着メンバーと接触させ、標識細胞懸濁液を生成すること、
前記標識細胞懸濁液をサイトメトリーによってアッセイし、所定の閾値を超えるPD−L1レベルを各発現する細胞集団が存在するか否かを検出し、前記新生物が抗PD−1/PD−L1免疫療法応答性であるか否かを識別すること
を備える、方法。 - 前記細胞集団は、異数体である、請求項8に記載の方法。
- 前記異数体の細胞は、前記被検体中の循環腫瘍細胞の存在を示す、請求項9に記載の方法。
- 前記標識細胞懸濁液をサイトメトリーによってアッセイし、増殖性免疫細胞が存在するか否かを検出することをさらに備える、請求項8〜10のいずれか一項に記載の方法。
- 被検体を新生物について処置する方法であって、
抗PD−1/PD−L1免疫療法を、抗PD−1/PD−L1免疫療法応答性新生物を含む被検体に投与することを備え、
前記新生物は請求項8〜11のいずれか一項に記載の方法に従って抗PD−1/PD−L1免疫療法応答性と識別される、
方法。 - PD−L1に特異的な標識結着メンバー、及び
固定試薬を含む細胞懸濁液固定液
を備える、キット。 - 前記細胞懸濁液固定液は、透過処理試薬を含む、請求項13に記載のキット。
- 細胞破砕装置をさらに備える、請求項13または14に記載のキット。
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US10782298B2 (en) | 2020-09-22 |
JP2022133307A (ja) | 2022-09-13 |
CR20190172A (es) | 2019-06-13 |
EP3510378A4 (en) | 2020-07-08 |
CN109906367A (zh) | 2019-06-18 |
MX2019002578A (es) | 2019-09-18 |
US20180136214A1 (en) | 2018-05-17 |
IL265175A (en) | 2019-05-30 |
WO2018048936A1 (en) | 2018-03-15 |
WO2019136082A1 (en) | 2019-07-11 |
SG11201901991QA (en) | 2019-04-29 |
EP3510378A1 (en) | 2019-07-17 |
CA3036278A1 (en) | 2018-03-15 |
CN112088309A (zh) | 2020-12-15 |
BR112019004470A2 (pt) | 2019-09-03 |
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