JPWO2020173897A5 - - Google Patents
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- JPWO2020173897A5 JPWO2020173897A5 JP2021549919A JP2021549919A JPWO2020173897A5 JP WO2020173897 A5 JPWO2020173897 A5 JP WO2020173897A5 JP 2021549919 A JP2021549919 A JP 2021549919A JP 2021549919 A JP2021549919 A JP 2021549919A JP WO2020173897 A5 JPWO2020173897 A5 JP WO2020173897A5
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- 102000037865 fusion proteins Human genes 0.000 claims description 60
- 108020001507 fusion proteins Proteins 0.000 claims description 60
- 102100032530 Glypican-3 Human genes 0.000 claims description 26
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 claims description 26
- 102000019298 Lipocalin Human genes 0.000 claims description 19
- 108050006654 Lipocalin Proteins 0.000 claims description 19
- 150000001413 amino acids Chemical class 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 14
- 206010028980 Neoplasm Diseases 0.000 claims description 13
- 125000000539 amino acid group Chemical group 0.000 claims description 11
- 210000004881 tumor cell Anatomy 0.000 claims description 11
- 101001023833 Homo sapiens Neutrophil gelatinase-associated lipocalin Proteins 0.000 claims description 10
- 102000047202 human LCN2 Human genes 0.000 claims description 10
- 108060003951 Immunoglobulin Proteins 0.000 claims description 8
- 102000018358 immunoglobulin Human genes 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- 210000004698 lymphocyte Anatomy 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 5
- 230000035772 mutation Effects 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 102000000588 Interleukin-2 Human genes 0.000 claims description 4
- 108010002350 Interleukin-2 Proteins 0.000 claims description 4
- 241000699670 Mus sp. Species 0.000 claims description 4
- 230000005867 T cell response Effects 0.000 claims description 4
- 230000003013 cytotoxicity Effects 0.000 claims description 4
- 231100000135 cytotoxicity Toxicity 0.000 claims description 4
- 230000028327 secretion Effects 0.000 claims description 4
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 238000012286 ELISA Assay Methods 0.000 claims description 2
- 101000946124 Homo sapiens Lipocalin-1 Proteins 0.000 claims description 2
- 230000006044 T cell activation Effects 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 201000011510 cancer Diseases 0.000 claims description 2
- 230000001419 dependent effect Effects 0.000 claims description 2
- 230000007783 downstream signaling Effects 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000004044 response Effects 0.000 claims description 2
- 230000001568 sexual effect Effects 0.000 claims description 2
- 210000004602 germ cell Anatomy 0.000 description 4
- 206010057248 Cell death Diseases 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
Description
本明細書にいう「ヒト抗体」には、フレームワーク領域とCDR領域がどちらもヒト生殖細胞系免疫グロブリン配列に由来する可変領域を有する抗体が含まれる。さらにまた、抗体が定常領域を含有する場合、その定常領域もヒト生殖細胞系免疫グロブリン配列に由来する。本発明のヒト抗体は、ヒト生殖細胞系免疫グロブリン配列によってコードされていないアミノ酸残基（例えばインビトロでのランダム突然変異導入もしくは部位特異的突然変異導入によって導入される突然変異またはインビボでの体細胞突然変異によって導入される突然変異）を含みうる。ただし、本明細書において使用される「ヒト抗体」という用語は、マウスなど別の哺乳動物種の生殖細胞系に由来するCDR配列がヒトフレームワーク配列上に移植されている抗体を包含しないものとする。
[本発明1001]
CD137とGPC3との両方に結合する能力を有する融合タンパク質であって、該融合タンパク質は少なくとも2つのサブユニットを任意の順序で含み、第1サブユニットは完全長免疫グロブリンまたはその抗原結合ドメインを含みかつGPC3に特異的であり、第2サブユニットはリポカリンムテインを含みかつCD137に特異的である、前記融合タンパク質。
[本発明1002]
少なくとも2つのサブユニットを含む融合タンパク質であって、第1サブユニットは完全長免疫グロブリンまたはその抗原結合ドメインを含みかつGPC3に特異的であり、第2サブユニットはリポカリンムテインを含みかつCD137に特異的であり、第2サブユニットは、N末端において、第1サブユニットの各重鎖のC末端に、任意でリンカーを介して連結されている、前記融合タンパク質。
[本発明1003]
（a）最大でも約1nMのK
D
値で、または第1サブユニットに含まれる免疫グロブリンもしくはその抗原結合ドメインの単独でのK
D
値と同等かそれより低いK
D
値で、GPC3に結合する能力を有するか、
（b）最大でも約3nMのEC
50
値で、または第2サブユニットに含まれるCD137に特異的なリポカリンムテインのEC
50
値と同等かそれより低いEC
50
値で、CD137に結合する能力を有するか、
（c）カニクイザルGPC3と交差反応するか、
（d）ELISAアッセイにおいて融合タンパク質を測定した場合に、最大でも約10nMのEC
50
値でCD137とGPC3とに同時に結合する能力を有するか、
（e）GPC3発現腫瘍細胞に結合する能力を有するか、
（f）IL-2分泌の増加を誘導する能力を有するか、
（g）SEQ ID NO:83より高レベルに、および/またはSEQ ID NO:83と比較してより良い効率で、IL-2分泌の増加を誘導する能力を有するか、
（h）リンパ球媒介性細胞傷害性を誘導する能力を有するか、
（i）SEQ ID NO:83よりも強化された、T細胞によって媒介されるGPC3発現腫瘍細胞の殺滅を誘導する能力、および/またはSEQ ID NO:83と比較してより良い効力で細胞傷害性T細胞活性化を誘導する能力を有するか、
（j）GPC3依存的にT細胞応答を共刺激する能力を有するか、または
（k）腫瘍微小環境におけるT細胞応答を共刺激する能力を有する、
本発明1001または本発明1002の融合タンパク質。
[本発明1004]
マウスにおいて少なくとも50時間、少なくとも75時間、少なくとも100時間、少なくとも125時間、少なくとも150時間、少なくとも175時間、少なくとも200時間、少なくとも250時間の、もしくはさらに長い半減期を有し、および/またはマウスにおいてSEQ ID NO:83の半減期よりも長い半減期を有する、本発明1001～1003のいずれかの融合タンパク質。
[本発明1005]
少なくとも6.5、少なくとも6.8、少なくとも7.1、少なくとも7.4、少なくとも7.5、少なくとも7.7の、もしくはさらに高い等電点を有し、および/またはSEQ ID NO:83の等電点よりも高い等電点を有する、本発明1001～1004のいずれかの融合タンパク質。
[本発明1006]
リポカリンムテインが、成熟ヒト好中球ゼラチナーゼ関連リポカリン（hNGAL）の直鎖ポリペプチド配列（SEQ ID NO:2）の位置28、36、40～41、49、52、65、68、70、72～73、77、79、81、83、87、94、96、100、103、106、125、127、132および134に対応する位置に、1つまたは複数の変異アミノ酸残基を含む、本発明1001～1005のいずれかの融合タンパク質。
[本発明1007]
リポカリンムテインが、成熟ヒト好中球ゼラチナーゼ関連リポカリン（hNGAL）の直鎖ポリペプチド配列（SEQ ID NO:2）の位置20、25、28、33、36、40～41、44、49、52、59、68、70～73、77～82、87、92、96、98、100、101、103、122、125、127、132および134に対応する位置に、1つまたは複数の変異アミノ酸残基を含む、本発明1001～1005のいずれかの融合タンパク質。
[本発明1008]
成熟hNGALの直鎖ポリペプチド配列（SEQ ID NO:2）と比較して、リポカリンムテインのアミノ酸配列が、以下の変異アミノ酸残基のセットのうちの1つを含む、本発明1001～1007のいずれかの融合タンパク質:
[本発明1009]
リポカリンムテインのアミノ酸配列が、SEQ ID NO:39～57からなる群より選択されるアミノ酸配列に対して、少なくとも85％の配列同一性を有する、本発明1001～1008のいずれかの融合タンパク質。
[本発明1010]
リポカリンムテインが、成熟ヒト涙液リポカリンの直鎖ポリペプチド配列（SEQ ID NO:1）の位置5、26～31、33～34、42、46、52、56、58、60～61、65、71、85、94、101、104～106、108、111、114、121、133、148、150および153に対応する位置に、1つまたは複数の変異アミノ酸残基を含む、本発明1001～1005のいずれかの融合タンパク質。
[本発明1011]
成熟ヒト涙液リポカリンの直鎖ポリペプチド配列（SEQ ID NO:1）と比較して、リポカリンムテインのアミノ酸配列が、以下の変異アミノ酸残基のセットのうちの1つを含む、本発明1001～1005および本発明1010のいずれかの融合タンパク質:
[本発明1012]
リポカリンムテインのアミノ酸配列が、SEQ ID NO:32～38からなる群より選択されるアミノ酸配列に対して、少なくとも85％の配列同一性を有する、本発明1001～1005および本発明1010～1011のいずれかの融合タンパク質。
[本発明1013]
第2サブユニットが、N末端において、リンカーを介して、第1サブユニットの各重鎖定常領域（CH）のN末端もしくはC末端または第1サブユニットの各軽鎖定常領域（CL）のN末端もしくはC末端に連結されている、本発明1001～1012のいずれかの融合タンパク質。
[本発明1014]
抗体が、それぞれSEQ ID NO:78とSEQ ID NO:79、SEQ ID NO:129とSEQ ID NO:79、SEQ ID NO:114とSEQ ID NO:115、もしくはSEQ ID NO:126とSEQ ID NO:127である重鎖可変領域と軽鎖可変領域を含むか、または、SEQ ID NO:78とSEQ ID NO:79、SEQ ID NO:129とSEQ ID NO:79、SEQ ID NO:114とSEQ ID NO:115、もしくはSEQ ID NO:126とSEQ ID NO:127に示されるアミノ酸配列に対して少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、少なくとも92％、少なくとも95％、少なくとも97％、少なくとも98％もしくはそれ以上の配列同一性を有する配列を有する重鎖可変領域と軽鎖可変領域を含む、本発明1001～1013のいずれかの融合タンパク質。
[本発明1015]
抗体の重鎖が以下のCDR配列のセット:
[本発明1016]
抗体がIgG4バックボーンを有する、本発明1001～1015のいずれかの融合タンパク質。
[本発明1017]
IgG4バックボーンが以下の突然変異のうちの1つまたは複数を有する、本発明1016の融合タンパク質:
S228P、N297A、F234A、L235A、M428L、N434S、M252Y、S254TおよびT256E。
[本発明1018]
SEQ ID NO:87とSEQ ID NO:82に示されるアミノ酸、SEQ ID NO:88とSEQ ID NO:82に示されるアミノ酸、SEQ ID NO:81とSEQ ID NO:89に示されるアミノ酸、SEQ ID NO:81とSEQ ID NO:90に示されるアミノ酸、SEQ ID NO:91とSEQ ID NO:82に示されるアミノ酸、SEQ ID NO:92とSEQ ID NO:82に示されるアミノ酸、SEQ ID NO:81とSEQ ID NO:93に示されるアミノ酸、SEQ ID NO:81とSEQ ID NO:94に示されるアミノ酸、SEQ ID NO:95とSEQ ID NO:82に示されるアミノ酸、またはSEQ ID NO:96とSEQ ID NO:82に示されるアミノ酸を含む、本発明1001～1017のいずれかの融合タンパク質。
[本発明1019]
SEQ ID NO:87とSEQ ID NO:82、SEQ ID NO:88とSEQ ID NO:82、SEQ ID NO:81とSEQ ID NO:89、SEQ ID NO:81とSEQ ID NO:90、SEQ ID NO:91とSEQ ID NO:82、SEQ ID NO:92とSEQ ID NO:82、SEQ ID NO:81とSEQ ID NO:93、SEQ ID NO:81とSEQ ID NO:94、SEQ ID NO:95とSEQ ID NO:82、またはSEQ ID NO:96とSEQ ID NO:82に示されるアミノ酸配列に対して、少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、少なくとも92％、少なくとも95％、少なくとも97％、少なくとも98％またはそれ以上の配列同一性を有するアミノ酸配列を含む、本発明1001～1018のいずれかの融合タンパク質。
[本発明1020]
本発明1001～1019のいずれかの融合タンパク質をコードするヌクレオチド配列を含む、核酸分子。
[本発明1021]
本発明1001～1019のいずれかの融合タンパク質を生産する方法であって、該融合タンパク質が、該融合タンパク質をコードする核酸から出発して生産される、前記方法。
[本発明1022]
CD137の下流シグナリング経路の活性化とGPC3陽性腫瘍細胞の会合とを同時に行う方法であって、腫瘍を含む組織に、本発明1001～1019のいずれかの1つもしくは複数の融合タンパク質または該融合タンパク質を含む1つもしくは複数の組成物を適用する工程を含む、前記方法。
[本発明1023]
T細胞の共刺激とGPC3陽性腫瘍細胞の会合とを同時に行う方法であって、腫瘍を含む組織に、本発明1001～1019のいずれかの1つもしくは複数の融合タンパク質または該融合タンパク質を含む1つまたは複数の組成物を適用する工程を含む、前記方法。
[本発明1024]
GPC3陽性腫瘍細胞の近傍において限局的リンパ球応答を誘導する方法であって、腫瘍を含む組織に、本発明1001～1019のいずれかの1つもしくは複数の融合タンパク質または該融合タンパク質を含む1つまたは複数の組成物を適用する工程を含む、前記方法。
[本発明1025]
GPC3陽性腫瘍細胞のリンパ球媒介性細胞溶解の増加を誘導する方法であって、腫瘍を含む組織に、本発明1001～1019のいずれかの1つもしくは複数の融合タンパク質または該融合タンパク質を含む1つもしくは複数の組成物を適用する工程を含む、前記方法。
[本発明1026]
本発明1001～1019のいずれかの1つまたは複数の融合タンパク質を含む、薬学的組成物。
[本発明1027]
GPC3陽性がんを予防、改善または処置する方法であって、腫瘍を含む組織に、本発明1001～1019のいずれかの融合タンパク質または該融合タンパク質を含む1つまたは複数の組成物を適用する工程を含む、前記方法。
As used herein, "human antibodies" include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may have amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic in vivo). Mutations introduced by mutation). However, the term "human antibody" as used herein is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. do.
[Invention 1001]
A fusion protein capable of binding both CD137 and GPC3, said fusion protein comprising at least two subunits in any order, the first subunit comprising a full-length immunoglobulin or antigen binding domain thereof and specific for GPC3, wherein the second subunit contains a lipocalin mutein and is specific for CD137.
[Invention 1002]
A fusion protein comprising at least two subunits, the first subunit comprising a full-length immunoglobulin or antigen-binding domain thereof and specific for GPC3 and the second subunit comprising a lipocalin mutein and specific for CD137 and the second subunit is linked at its N-terminus to the C-terminus of each heavy chain of the first subunit, optionally via a linker.
[Invention 1003]
(a) binds to GPC3 with a KD value of at most about 1 nM, or with a KD value equal to or lower than the KD value of the immunoglobulin contained in the first subunit or its antigen-binding domain alone; have the ability to
(b) having the ability to bind CD137 with an EC50 value of at most about 3 nM or an EC50 value equal to or lower than that of a CD137- specific lipocalin mutein contained in the second subunit; mosquito,
(c) cross-reacts with cynomolgus GPC3, or
(d) have the ability to simultaneously bind CD137 and GPC3 with an EC50 value of at most about 10 nM when the fusion protein is measured in an ELISA assay ;
(e) has the ability to bind to GPC3-expressing tumor cells;
(f) has the ability to induce an increase in IL-2 secretion;
(g) have the ability to induce increased IL-2 secretion to a higher level than SEQ ID NO:83 and/or with a better efficiency compared to SEQ ID NO:83;
(h) has the ability to induce lymphocyte-mediated cytotoxicity;
(i) enhanced ability to induce T cell-mediated killing of GPC3-expressing tumor cells over SEQ ID NO:83 and/or cytotoxicity with better potency compared to SEQ ID NO:83; have the ability to induce sexual T cell activation;
(j) have the ability to co-stimulate T cell responses in a GPC3-dependent manner, or
(k) having the ability to co-stimulate T cell responses in the tumor microenvironment;
A fusion protein of the invention 1001 or invention 1002.
[Invention 1004]
has a half-life of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, at least 250 hours, or even longer in mice and/or SEQ in mice The fusion protein of any of invention 1001-1003, which has a half-life greater than that of ID NO:83.
[Invention 1005]
have an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7, or even higher, and/or have an isoelectric point higher than that of SEQ ID NO:83; A fusion protein of any of the inventions 1001-1004.
[Invention 1006]
The lipocalin mutein is from position 28, 36, 40 to 41, 49, 52, 65, 68, 70, 72 of the linear polypeptide sequence (SEQ ID NO:2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL). 1001 of the invention comprising one or more mutated amino acid residues at positions corresponding to 73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 Any fusion protein of ~1005.
[Invention 1007]
The lipocalin muteins are at positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 44, 49, 52 of the linear polypeptide sequence (SEQ ID NO:2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL). one or more mutated amino acid residues at positions corresponding to 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 The fusion protein of any of the inventions 1001-1005, comprising:
[Invention 1008]
Any of invention 1001-1007, wherein the amino acid sequence of the lipocalin mutein comprises one of the following set of mutated amino acid residues as compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO:2): or fusion protein:
[Invention 1009]
1008. The fusion protein of any of the inventions 1001-1008, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:39-57.
[Invention 1010]
The lipocalin muteins are at positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 1001-1005 of the invention comprising one or more mutated amino acid residues at positions corresponding to 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 any of the fusion proteins of
[Invention 1011]
Invention 1001- wherein the amino acid sequence of the lipocalin mutein comprises one of the following set of mutated amino acid residues, as compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO:1): 1005 and any fusion protein of the invention 1010:
[Invention 1012]
Any of Inventions 1001-1005 and Inventions 1010-1011, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:32-38. fusion protein.
[Invention 1013]
At the N-terminus of the second subunit, via a linker, the N-terminus or C-terminus of each heavy-chain constant region (CH) of the first subunit or the N-terminus of each light-chain constant region (CL) of the first subunit The fusion protein of any of invention 1001-1012, linked terminally or C-terminally.
[Invention 1014]
The antibody is SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:129 and SEQ ID NO:79, SEQ ID NO:114 and SEQ ID NO:115, or SEQ ID NO:126 and SEQ ID NO, respectively :127, or SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:129 and SEQ ID NO:79, SEQ ID NO:114 and SEQ ID NO:114. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least The fusion protein of any of inventions 1001-1013, comprising a heavy chain variable region and a light chain variable region having sequences with 95%, at least 97%, at least 98% or more sequence identity.
[Invention 1015]
The heavy chain of the antibody has the following set of CDR sequences:
[Invention 1016]
The fusion protein of any of invention 1001-1015, wherein the antibody has an IgG4 backbone.
[Invention 1017]
A fusion protein of the invention 1016, wherein the IgG4 backbone has one or more of the following mutations:
S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T and T256E.
[Invention 1018]
amino acids set forth in SEQ ID NO:87 and SEQ ID NO:82; amino acids set forth in SEQ ID NO:88 and SEQ ID NO:82; amino acids set forth in SEQ ID NO:81 and SEQ ID NO:89; amino acids set forth in NO:81 and SEQ ID NO:90, amino acids set forth in SEQ ID NO:91 and SEQ ID NO:82, amino acids set forth in SEQ ID NO:92 and SEQ ID NO:82, SEQ ID NO: the amino acids set forth in 81 and SEQ ID NO:93, the amino acids set forth in SEQ ID NO:81 and SEQ ID NO:94, the amino acids set forth in SEQ ID NO:95 and SEQ ID NO:82, or SEQ ID NO:96 and the amino acids set forth in SEQ ID NO:82.
[Invention 1019]
SEQ ID NO:87 and SEQ ID NO:82, SEQ ID NO:88 and SEQ ID NO:82, SEQ ID NO:81 and SEQ ID NO:89, SEQ ID NO:81 and SEQ ID NO:90, SEQ ID NO:81 and SEQ ID NO:90 NO:91 and SEQ ID NO:82, SEQ ID NO:92 and SEQ ID NO:82, SEQ ID NO:81 and SEQ ID NO:93, SEQ ID NO:81 and SEQ ID NO:94, SEQ ID NO: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least The fusion protein of any of inventions 1001-1018, comprising amino acid sequences having 92%, at least 95%, at least 97%, at least 98% or more sequence identity.
[Invention 1020]
A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of any of the inventions 1001-1019.
[Invention 1021]
A method of producing a fusion protein according to any of the inventions 1001-1019, wherein said fusion protein is produced starting from a nucleic acid encoding said fusion protein.
[Invention 1022]
A method for simultaneously activating the downstream signaling pathway of CD137 and the association of GPC3-positive tumor cells, wherein one or more of the fusion proteins of any one of 1001 to 1019 of the present invention or the fusion proteins are applied to a tissue containing a tumor. the method comprising applying one or more compositions comprising
[Invention 1023]
A method for co-stimulation of T cells and association of GPC3-positive tumor cells at the same time, wherein a tissue containing a tumor contains one or more fusion proteins of any one of the present inventions 1001 to 1019 or the fusion proteins1 The above method, comprising applying one or more compositions.
[Invention 1024]
A method of inducing a focal lymphocyte response in the vicinity of GPC3-positive tumor cells, comprising one or more fusion proteins of any of the inventions 1001 to 1019 or one containing such fusion proteins in a tissue containing the tumor Or the method comprising applying a plurality of compositions.
[Invention 1025]
1. A method of inducing increased lymphocyte-mediated cytolysis of GPC3-positive tumor cells, comprising in a tissue comprising a tumor one or more fusion proteins of any of the inventions 1001-1019 or said fusion proteins. The above method, comprising applying one or more compositions.
[Invention 1026]
A pharmaceutical composition comprising one or more fusion proteins of any of the inventions 1001-1019.
[Invention 1027]
A method for preventing, ameliorating or treating GPC3-positive cancer, the step of applying the fusion protein of any of the present inventions 1001 to 1019 or one or more compositions comprising the fusion protein to a tissue containing a tumor. The above method, comprising
Claims (16)
（b）最大でも約3nMのEC50値で、または第2サブユニットに含まれるCD137に特異的なリポカリンムテインの単独でのEC50値と同等かそれより低いEC50値で、CD137に結合する能力を有するか、
（c）カニクイザルGPC3と交差反応するか、
（d）ELISAアッセイにおいて融合タンパク質を測定した場合に、最大でも約10nMのEC50値でCD137とGPC3とに同時に結合する能力を有するか、
（e）GPC3発現腫瘍細胞に結合する能力を有するか、
（f）IL-2分泌の増加を誘導する能力を有するか、
（g）SEQ ID NO:83より高レベルに、および/またはSEQ ID NO:83と比較してより良い効率で、IL-2分泌の増加を誘導する能力を有するか、
（h）リンパ球媒介性細胞傷害性を誘導する能力を有するか、
（i）SEQ ID NO:83よりも強化された、T細胞によって媒介されるGPC3発現腫瘍細胞の殺滅を誘導する能力、および/またはSEQ ID NO:83と比較してより良い効力で細胞傷害性T細胞活性化を誘導する能力を有するか、
（j）GPC3依存的にT細胞応答を共刺激する能力を有するか、または
（k）腫瘍微小環境におけるT細胞応答を共刺激する能力を有する、
請求項1または請求項2記載の融合タンパク質。 (a) binds to GPC3 with a KD value of at most about 1 nM, or with a KD value equal to or lower than the KD value of the immunoglobulin contained in the first subunit or its antigen-binding domain alone; have the ability to
(b) binds CD137 with an EC50 value of at most about 3 nM or with an EC50 value equal to or lower than the EC50 value of the CD137-specific lipocalin mutein alone contained in the second subunit; have the ability to
(c) cross-reacts with cynomolgus GPC3, or
(d) have the ability to simultaneously bind CD137 and GPC3 with an EC50 value of at most about 10 nM when the fusion protein is measured in an ELISA assay;
(e) has the ability to bind to GPC3-expressing tumor cells;
(f) has the ability to induce an increase in IL-2 secretion;
(g) have the ability to induce increased IL-2 secretion to a higher level than SEQ ID NO:83 and/or with a better efficiency compared to SEQ ID NO:83;
(h) has the ability to induce lymphocyte-mediated cytotoxicity;
(i) enhanced ability to induce T cell-mediated killing of GPC3-expressing tumor cells over SEQ ID NO:83 and/or cytotoxicity with better potency compared to SEQ ID NO:83; have the ability to induce sexual T cell activation;
(j) capable of costimulating T cell responses in a GPC3-dependent manner, or (k) capable of costimulating T cell responses in the tumor microenvironment,
3. The fusion protein of claim 1 or claim 2.
少なくとも6.5、少なくとも6.8、少なくとも7.1、少なくとも7.4、少なくとも7.5、少なくとも7.7の、もしくはさらに高い等電点を有し、および/もしくはSEQ ID NO:83の等電点よりも高い等電点を有する、
請求項1～3のいずれか一項記載の融合タンパク質。 has a half-life of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, at least 250 hours, or even longer in mice and/ or SEQ in mice have a half-life longer than that of ID NO:83 and /or
having an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7, or even higher, and/or having an isoelectric point higher than that of SEQ ID NO:83;
A fusion protein according to any one of claims 1-3.
リポカリンムテインが、成熟ヒト好中球ゼラチナーゼ関連リポカリン（hNGAL）の直鎖ポリペプチド配列（SEQ ID NO:2）の位置20、25、28、33、36、40～41、44、49、52、59、68、70～73、77～82、87、92、96、98、100、101、103、122、125、127、132および134に対応する位置に、1つもしくは複数の変異アミノ酸残基を含む、または
リポカリンムテインが、成熟ヒト涙液リポカリンの直鎖ポリペプチド配列（SEQ ID NO:1）の位置5、26～31、33～34、42、46、52、56、58、60～61、65、71、85、94、101、104～106、108、111、114、121、133、148、150および153に対応する位置に、1つもしくは複数の変異アミノ酸残基を含む、
請求項1～4のいずれか一項記載の融合タンパク質。 The lipocalin mutein is from position 28, 36, 40 to 41, 49, 52, 65, 68, 70, 72 of the linear polypeptide sequence (SEQ ID NO:2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL). contains one or more mutated amino acid residues at positions corresponding to 73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134, or
The lipocalin muteins are at positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 44, 49, 52 of the linear polypeptide sequence (SEQ ID NO:2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL). one or more mutated amino acid residues at positions corresponding to 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 contains or
The lipocalin muteins are at positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, containing one or more mutated amino acid residues at positions corresponding to 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153;
A fusion protein according to any one of claims 1-4 .
リポカリンムテインのアミノ酸配列が、SEQ ID NO:39～57からなる群より選択されるアミノ酸配列に対して、少なくとも85％の配列同一性を有する、
請求項1～7のいずれか一項記載の融合タンパク質。 The amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:32-38, or the amino acid sequence of the lipocalin mutein is SEQ ID NO:39 having at least 85% sequence identity to an amino acid sequence selected from the group consisting of -57;
A fusion protein according to any one of claims 1-7 .
抗体の重鎖が以下のCDR配列のセット:
請求項1～9のいずれか一項記載の融合タンパク質。 The antibody is SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:129 and SEQ ID NO:79, SEQ ID NO:114 and SEQ ID NO:115, or SEQ ID NO:126 and SEQ ID NO, respectively :127, or SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:129 and SEQ ID NO:79, SEQ ID NO:114 and SEQ ID NO:114. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least comprising a heavy chain variable region and a light chain variable region having sequences with 95%, at least 97%, at least 98% or more sequence identity; and/or
The heavy chain of the antibody has the following set of CDR sequences:
A fusion protein according to any one of claims 1-9 .
S228P、N297A、F234A、L235A、M428L、N434S、M252Y、S254TおよびT256E。 A fusion protein according to any one of claims 1 to 10 , wherein the antibody has an IgG4 backbone, in particular one or more of the following mutations :
S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T and T256E .
請求項1～11のいずれか一項記載の融合タンパク質。 SEQ ID NO:87 and SEQ ID NO:82, SEQ ID NO:88 and SEQ ID NO:82, SEQ ID NO:81 and SEQ ID NO:89, SEQ ID NO:81 and SEQ ID NO:90, SEQ ID NO:81 and SEQ ID NO:90 NO:91 and SEQ ID NO:82, SEQ ID NO:92 and SEQ ID NO:82, SEQ ID NO:81 and SEQ ID NO:93, SEQ ID NO:81 and SEQ ID NO:94, SEQ ID NO: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least comprising amino acid sequences having 92%, at least 95%, at least 97%, at least 98% or more sequence identity , especially the amino acids shown in SEQ ID NO:87 and SEQ ID NO:82, SEQ ID NO: 88 and SEQ ID NO:82, SEQ ID NO:81 and SEQ ID NO:89, SEQ ID NO:81 and SEQ ID NO:90, SEQ ID NO:91 and amino acids set forth in SEQ ID NO:82, amino acids set forth in SEQ ID NO:92 and SEQ ID NO:82, amino acids set forth in SEQ ID NO:81 and SEQ ID NO:93, SEQ ID NO:81 and SEQ ID comprising the amino acids set forth in NO:94, the amino acids set forth in SEQ ID NO:95 and SEQ ID NO:82, or the amino acids set forth in SEQ ID NO:96 and SEQ ID NO:82;
A fusion protein according to any one of claims 1-11 .
（i）腫瘍を含む組織に、請求項1～12のいずれか一項記載の1つもしくは複数の融合タンパク質もしくは該融合タンパク質を含む1つもしくは複数の組成物を適用する工程を含む、CD137の下流シグナリング経路の活性化とGPC3陽性腫瘍細胞の会合とを同時に行う方法、
（ii）腫瘍を含む組織に、請求項1～12のいずれか一項記載の1つもしくは複数の融合タンパク質もしくは該融合タンパク質を含む1つもしくは複数の組成物を適用する工程を含む、T細胞の共刺激とGPC3陽性腫瘍細胞の会合とを同時に行う方法、または
（iii）腫瘍を含む組織に、請求項1～12のいずれか一項記載の1つもしくは複数の融合タンパク質もしくは該融合タンパク質を含む1つもしくは複数の組成物を適用する工程を含む、GPC3陽性腫瘍細胞の近傍において限局的リンパ球応答を誘導する方法。 a method,
(i) applying one or more fusion proteins according to any one of claims 1 to 12 or one or more compositions comprising said fusion proteins to a tissue comprising a tumor. a method of simultaneously activating downstream signaling pathways and engaging GPC3-positive tumor cells;
(ii) applying one or more fusion proteins according to any one of claims 1 to 12 or one or more compositions comprising said fusion proteins to a tissue comprising a tumor. co-stimulation and association of GPC3-positive tumor cells at the same time, or
(iii) GPC3-positive, comprising the step of applying one or more fusion proteins according to any one of claims 1 to 12 or one or more compositions comprising said fusion proteins to a tissue containing a tumor A method of inducing a focal lymphocyte response in the vicinity of tumor cells .
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| WO2022216744A2 (en) * | 2021-04-05 | 2022-10-13 | Cytovia Therapeutics, Llc. | Bispecific antibodies targeting nkp46 and gpc3 and methods of use thereof |
| WO2022223019A1 (en) * | 2021-04-23 | 2022-10-27 | Shanghai Henlius Biotech, Inc. | Anti-gpc3 antibodies, multispecific antibodies and methods of use |
| JP2024522078A (en) * | 2021-05-21 | 2024-06-11 | ベイジーン スウィッツァーランド ゲーエムベーハー | Anti-GPC3 and anti-CD137 multispecific antibodies and methods of use thereof |
| CN115960242B (en) * | 2021-09-09 | 2023-10-17 | 广东东阳光药业股份有限公司 | Anticancer binding molecules and uses thereof |
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