JPWO2019237070A5

JPWO2019237070A5 -

Info

Publication number: JPWO2019237070A5
Application number: JP2020567526A
Authority: JP
Publication date: 2022-06-16

Description

ある態様において、多量体タンパク質は、配列番号：7または配列番号：8を含むポリペプチドを含む。ある態様において、多量体タンパク質は、配列番号：9、配列番号：10、配列番号：11、配列番号：12、配列番号：53、配列番号：55、配列番号：57、配列番号：59、配列番号：60、配列番号：61、配列番号：62、配列番号：63、配列番号：64もしくは配列番号：67、またはそれらに対して80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%もしくは99%の配列同一性を有するアミノ酸配列を含むポリペプチドを含む。ある態様において、多量体タンパク質は、配列番号：53、配列番号：55、配列番号：57、配列番号：59、配列番号：60、配列番号：61、配列番号：62、配列番号：63、配列番号：64または配列番号：67を含むポリペプチドを含み、ここで最初の19アミノ酸(MGWSCIILFLVATATGVHS（配列番号：73）、リーダー配列)は存在しない。 In some embodiments, the multimeric protein comprises a polypeptide comprising SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the multimeric protein is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: Number: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 67, or 80%, 85%, 86%, 87%, 88%, 89 for them. Includes polypeptides containing amino acid sequences having a%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the multimeric protein is SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: It contains a polypeptide containing number: 64 or SEQ ID NO: 67, where the first 19 amino acids (MGWSCIILFLVATATGVHS (SEQ ID NO: 73) , leader sequence) are absent.

配列表

Sequence listing

Claims

a) Lectin domain;
b) Trimeric domain; and
c) An isolated polypeptide containing a dimerization domain.

(a) Lectin domains, trimerization domains and dimerization domains are covalently linked from the N-terminus to the C-terminus, or together.
(b) A polypeptide in which the lectin domain, dimerization domain and trimerization domain are covalently linked together in the direction from the N-terminus to the C-terminus , where optionally the polypeptide. Further comprising a linker, preferably
(i) The polypeptide of (a) further comprises a linker between the lectin domain and the trimerization domain, or
(ii) The polypeptide according to claim 1, wherein the polypeptide of (b) further comprises a linker between the dimerization domain and the trimerization domain .

The poly according to claim 1 or 2 , wherein the lectin domain comprises a Siglec sialic acid-bound V-set immunoglobulin-like domain, or a variant thereof, and optionally, the lectin domain comprises a Siglec extracellular domain, or a variant thereof. peptide.

Siglec is a mammalian Siglec and optionally,
(a) Siglec is human Siglec, optionally,
(i) Siglec chooses from Siglec-3, Siglec-7 and Siglec-9
Selected and / or
(ii) The lectin domain contains or contains SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 51.
(b) Siglec is Mouse Siglec,
The polypeptide according to claim 3 .

The polypeptide according to any one of claims 1 to 3 , wherein the lectin domain comprises a C-type lectin domain.

(i) The trimerization domain is a natural or synthetic trimerization domain, and optionally the trimerization domain is the T4 phage fibritin (foldon) trimerization domain. Yes, preferably the trimerization domain comprises SEQ ID NO: 5 and / or
(ii) The dimerization domain is a natural dimerization domain or a synthetic dimerization domain, and optionally the dimerization domain is an immunoglobulin Fc domain, preferably an immunoglobulin Fc domain. Contains SEQ ID NO: 6,
The polypeptide according to any one of claims 1 to 5 .

The polypeptide according to any one of claims 2 to 6 , wherein the linker comprises SEQ ID NO: 69.

The polypeptide according to any one of claims 1 to 6 , wherein the polypeptide comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 57 or SEQ ID NO: 67.

A multimeric protein comprising the polypeptide according to any one of claims 1 to 8 .

A multimeric protein comprising six separate polypeptides according to any one of claims 1-8 that are complexed to yield a hexamer protein.

(i) Multimeric proteins bind to carbohydrate ligands at K _D of .01 nM-100 nM and optionally K _D of 10 nM or less when measured by surface plasmon resonance or bio-layer interferometry. And preferably less than or equal to 1nM and / or
(ii) The multimeric protein according to claim 9 or 10 , wherein the carbohydrate ligand is a Siglec ligand and optionally the Siglec ligand is selected from Siglec-3, Siglec-7 and Siglec-9 ligands .

A multimeric protein containing 6 polypeptides, each polypeptide in the N-terminal to C-terminal direction, the first siglec-7 sialic acid binding V-set immunoglobulin-like domain, the first siglec. -7 C2-set domain, second Siglec -7 C2-set domain, T4 phage fibritin (foldon) trimerization domain and Fc domain included:
( a) The first, second and third polypeptides are trimericized in their respective trimerization domains;
( b) The fourth, fifth and sixth polypeptides are trimericized in their respective trimerization domains;
( c) The first and second polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( d) The third and fourth polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( e) A multimeric protein in which the 5th and 6th polypeptides are dimerized (eg, covalently bonded) in their respective Fc domains.

A multimeric protein containing 6 polypeptides, each of which is the first Siglec-9 sialic acid-bound V-set immunoglobulin-like domain, the first Siglec, from N-terminus to C-terminus. Includes -9 C2-set domain, second Siglec -9 C2-set domain, T4 phage fibritin (foldon) trimerization domain and Fc domain:
( a) The first, second and third polypeptides are trimericized in their respective trimerization domains;
( b) The fourth, fifth and sixth polypeptides are trimericized in their respective trimerization domains;
( c) The first and second polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( d) The third and fourth polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( e) A multimeric protein in which the 5th and 6th polypeptides are dimerized (eg, covalently bonded) in their respective Fc domains.

A multimeric protein containing 6 polypeptides, each of which is a Siglec-3 sialic acid-binding V-set immunoglobulin-like domain, a Siglec-3 C2-set domain, from the N-terminus to the C-terminus. , Fc domain and T4 phage fibritin (foldon) trimerization domain:
( a) The first, second and third polypeptides are trimericized in their respective trimerization domains;
( b) The fourth, fifth and sixth polypeptides are trimericized in their respective trimerization domains;
( c) The first and second polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( d) The third and fourth polypeptides are dimerized (eg, covalently) in their respective Fc domains;
( e) A multimeric protein in which the 5th and 6th polypeptides are dimerized (eg, covalently bonded) in their respective Fc domains.

The multimeric protein according to any one of claims 9 to 14 , wherein the multimeric protein is treated with sialidase.

A pharmaceutical composition comprising the multimeric protein according to any one of claims 9 to 15 .

The multimeric protein according to any one of claims 9 to 15 or the pharmaceutical composition according to claim 16 for use in treating a Siglec-mediated disorder in a subject .

A method for detecting carbohydrates in a sample,
(a) A step of contacting the sample with the multimeric protein according to any one of claims 9 to 15 under the condition that the multimeric protein forms a multimeric protein-carbohydrate complex when carbohydrates are present in the sample. ;and
(b) A method comprising detecting the presence of a complex, if any, resulting in step (a).

A method of detecting carbohydrates in a subject with cancer.
(a) When carbohydrates are present in the sample, the sample obtained from the subject and the multimeric protein according to any one of claims 9 to 15 are used, and the multimeric protein forms a multimeric protein-carbohydrate complex. The process of contacting under conditions; and
(b) A method comprising detecting the presence of a complex, if any, resulting in step (a).

A method of identifying a subject with cancer who may respond to treatment with a Siglec inhibitor, wherein the method is:
(a) When a sigrec ligand is present in the sample, the sample obtained from the subject and the multimeric protein according to any one of claims 9 to 15 are combined, and the multimeric protein is a multimeric protein-cigrec ligand complex. The process of contacting under conditions that form the body; and
(b) Including the step of detecting the presence of the complex generated in step (a), if any.
A method in which the presence of a complex indicates that a subject responds to treatment with a Siglec inhibitor.

A Siglec inhibitor for use in the treatment of cancer in a subject , wherein the cancer is identified by the method of claim 19 as comprising cancerous cells expressing one or more ligands for Siglec. There is a Siglec inhibitor .

The cygrec inhibitor according to any one of claims 18 to 20 , wherein the sample used in the method is selected from a tissue sample, a body fluid sample, or a cell sample.