Engineered TAM receptor ligand polypeptide and uses thereof

Abstract

The present invention relates to an engineered TAM receptor ligand polypeptide (e.g., Gas6 or ProS1 polypeptide) that binds to the TAM receptor family (the three types of receptor tyrosine kinases Tyro3, Axl or MerTK), a fusion protein comprising same, or uses thereof. Specifically, the present invention relates to mutations in a laminin G-like domain of a TAM receptor ligand polypeptide for increasing productivity and / or structural safety.

Description

Engineered TAM receptor ligand polypeptides and uses thereof

[0001] This application claims priority to Korean Patent Application No. 2024-0032187, filed March 6, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence list

[0003] This application includes a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of said sequence listing, dated March 6, 2025, is named KC24186.SEQ.xml and is 668.0 kilobytes in size.

[0004] The present invention relates to engineered TAM receptor ligand polypeptides (e.g., Gas6 or ProS1 polypeptides) that bind to the TAM receptor family (three receptor tyrosine kinases: Tyro3, ​​Axl, or MerTK), fusion proteins comprising the same, or uses thereof. In particular, the present invention relates to amino acid mutations within the laminin G-like domain of TAM receptor ligand polypeptides to increase productivity and / or structural stability.

[0005] If substances that should be broken down within cells continue to accumulate without being broken down, this can cause various problems. In particular, if endogenous proteins misfold and form aggregates, they lose their normal function and become more resistant to degradation. This can lead to accumulation in tissues such as the brain or heart, potentially causing organ damage. Diseases caused by these protein aggregates include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease.

[0006] For example, Alzheimer's disease is the leading cause of dementia and a fatal disease accompanied by learning disabilities and memory impairment. It is characterized by the deposition of beta-amyloid (Aβ) outside the brain cell membrane, which is created by the abnormal breakdown of amyloid precursor protein (APP). Oligomers and fibrils formed by the aggregation of beta-amyloid cause synaptic dysfunction and cytotoxicity through various pathways, and are reported to negatively affect neurons by altering the function of astrocytes and microglia, which are responsible for immunity in the brain. Recently, a study using a monoclonal antibody that specifically binds to Aβ oligomers and fibrils reported that this monoclonal antibody induced the removal of Aβ and restored cognitive function in Alzheimer's disease patients, and thus, the treatment strategy of Alzheimer's disease using anti-Aβ antibodies is emerging as a new hope. Aducanumab (Aduhelm), developed by Biogen, was approved by the U.S. FDA in 2020 as the world's first treatment for Alzheimer's disease. However, despite this progress in the development of Alzheimer's disease treatments, it has been reported that 55% of patients receiving anti-Aβ antibody treatment show amyloid-related imaging abnormalities (ARIA) accompanied by severe edema. It is known that ARIA is due to synaptic and cytotoxicity caused by an inflammatory response activated by anti-Aβ antibodies stimulating Fc receptors (Hampel et al. Brain 2023: 146; 4414-4424 and Taylor et al. Molecular Neurodegeneration (2023) 18:59).Because synapses and neurons in the brain are sensitive to inflammatory cytokines, even if treatment with anti-Aβ antibodies removes Aβ, synaptic and neuronal damage is unavoidable. Therefore, a critical challenge in treating Alzheimer's disease is developing treatments that effectively remove Aβ oligomers and fibrils without causing inflammation or synaptic and neuronal damage.

[0007] Because the specific removal of protein aggregates largely relies on antibody drugs, other diseases caused by protein aggregates also suffer from the same problems described above. Furthermore, antibodies stimulate Fc receptors, which trigger an inflammatory response and can cause adverse effects when used for immune-related diseases. Therefore, the development of technologies capable of suppressing inflammatory responses while simultaneously removing only target components, such as protein aggregates or proteins associated with immune diseases, is necessary.

[0008] To solve these technical challenges, the inventors of the present invention have developed a novel antibody-like fusion molecule that recognizes a target substance and induces phagocytosis without an inflammatory response by using a fragment containing a laminin G-like domain, in which the Gla domain is essentially deleted from Gas6 or ProS1 proteins that bind to the TAM receptor and induce phagocytosis, for a purpose similar to an antibody Fc region (see Korean Patent No. 10-2549520 and International Publication No. WO2022 / 164288A1, the entire contents of which are incorporated herein by reference).

[0009] There is still a need to develop engineered TAM receptor ligand molecules with improved properties, particularly those that are advantageous and improve production, purification and storage, considering not only pharmacological effects but also productivity, physical properties and / or structural stability.

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] (Patent Document 1) Korean Patent No. 10-2549520 (Korean counterpart to International Publication No. WO2022 / 164288A1)

[0013] (Patent Document 2) Korean Patent Publication No. 10-2020-0044023 (Korean counterpart to International Publication No. WO2019 / 040612A1)

[0014] (Patent Document 3) International Publication No. WO2011 / 107591

[0015] [Non-patent literature]

[0016] (Non-patent literature 1) Hampel et al. Brain. 2023. 146: 4414-4424

[0017] (Non-patent literature 2) Taylor et al. Molecular Neurodegeneration. 2023. 18:59

[0018] (Non-patent Document 3) Greg Lemke. Cold Spring Harb Perspective Biol. 2013; 5:a009076

[0019] (Non-patent Document 4) Sasaki et al. The Journal of Biological Chemistry. 2002. 277(46):44164-44170

[0020] (Non-patent literature 5) Sasaki et al. The EMBO Journal. 2006. 25:80-87

[0021] The present invention aims to solve one or more of the above-described problems.

[0022] The present invention aims to provide a laminin G-like domain variant having improved productivity and / or structural stability.

[0023] Another object of the present invention is to provide engineered TAM receptor ligand polypeptides that have improved productivity and / or structural stability and are capable of inducing phagocytic activity without accompanying an inflammatory response.

[0024] Another object of the present invention is to provide a fusion molecule capable of inducing phagocytic activity without accompanying an inflammatory response and with improved productivity and / or structural stability.

[0025] Another object of the present invention is to provide a pharmaceutical composition or method for removing a target substance from an object.

[0026] Another object of the present invention is to provide a pharmaceutical composition or method for preventing or treating a disease or disorder characterized by or related to the accumulation of a target substance.

[0027] Another object of the present invention is to provide a pharmaceutical composition or method for preventing or treating an immune disease.

[0028] Another object of the present invention is to provide the use of laminin G-like domain variants, engineered TAM receptor ligand polypeptides, or fusion molecules for the prevention or treatment of diseases or conditions characterized by or associated with accumulation of target substances.

[0029] Another object of the present invention is to provide a use of a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, or a fusion molecule for the prevention or treatment of an immune disease.

[0030] Another object of the present invention is to provide a nucleic acid encoding a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, or a fusion molecule, a vector for expressing them, or a cell producing them.

[0031] The purpose of the present invention is not limited to the purposes mentioned above. The purpose of the present invention will become more apparent from the following description and may be realized by the means and combinations thereof set forth in the claims.

[0032] A representative configuration of the present invention to achieve the above purpose is as follows.

[0033] In one aspect of the present invention, a laminin G-like domain variant is provided which comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain.

[0034] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (i) to (x):

[0035] (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1;

[0036] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272, and Gln273 based on sequence number 1;

[0037] (iii) addition of one, two or three amino acids between residues corresponding to Thr178 and Thr179 based on sequence number 1;

[0038] (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0039] (v) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on SEQ ID NO: 1;

[0040] (vi) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO: 1;

[0041] (vii) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO: 1;

[0042] (viii) Substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 based on sequence number 1 with a hydrophilic amino acid;

[0043] (ix) The amino acid of the residue corresponding to Thr90 in sequence number 1 is replaced with Tyr, Ile, Leu or Phe; and

[0044] (x) An amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on sequence number 1.

[0045] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (i) to (x):

[0046] (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1;

[0047] (ii) one or more amino acid substitutions selected from the group consisting of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His, based on SEQ ID NO: 1;

[0048] (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179 based on sequence number 1;

[0049] (iv) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 based on SEQ ID NO: 1;

[0050] (v) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on SEQ ID NO: 1;

[0051] (vi) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO: 1;

[0052] (vii) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO: 1;

[0053] (viii) Substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 based on sequence number 1 with a hydrophilic amino acid;

[0054] (ix) The amino acid of the residue corresponding to Thr90 in sequence number 1 is replaced with Tyr, Ile, Leu or Phe; and

[0055] (x) An amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on sequence number 1.

[0056] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (i) to (ix) described herein.

[0057] In one embodiment, the one or more amino acid mutations may further comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381 and Ala389 based on SEQ ID NO: 1, in addition to one or more selected from the group consisting of (i) to (ix) described herein.

[0058] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (ii), (iv), (v), and (vi):

[0059] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Met22, Phe23, Lys272, and Gln273 based on sequence number 1;

[0060] (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0061] (v) a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on sequence number 1; and

[0062] (vi) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO: 1.

[0063] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (ii) and (iii):

[0064] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Gly175 and Asp177 based on sequence number 1; and

[0065] (iii) Addition of one, two or three amino acids between residues corresponding to Thr178 and Thr179 based on sequence number 1.

[0066] In one embodiment, a laminin G-like domain variant is provided, wherein the variant comprises one or more amino acid modifications compared to the amino acid sequence of a wild-type laminin G-like domain (e.g., SEQ ID NO: 1), wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (i), (ii), and (vii):

[0067] (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1;

[0068] (ii) amino acid substitution at the residue corresponding to the Leu3 residue based on sequence number 1; and

[0069] (vii) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO: 1.

[0070] In one embodiment, the one or more amino acid mutations may comprise (i) a deletion of an amino acid at a residue corresponding to Asp1, Ile2, or Asp1 and Ile2 based on SEQ ID NO: 1.

[0071] In one embodiment, the one or more amino acid mutations may comprise (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272, and Gln273 based on SEQ ID NO: 1.

[0072] In one embodiment, the one or more amino acid mutations may comprise (iii) the addition of one or two amino acids between residues corresponding to Thr178 and Thr179 of SEQ ID NO: 1.

[0073] In one embodiment, the one or more amino acid mutations may comprise (iv) the addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 of SEQ ID NO: 1.

[0074] In one embodiment, the one or more amino acid mutations may comprise (v) a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) of SEQ ID NO: 1.

[0075] In one embodiment, the one or more amino acid mutations may comprise (vi) a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) of SEQ ID NO: 1.

[0076] In one embodiment, the one or more amino acid mutations may comprise a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) of SEQ ID NO: 1 (vii).

[0077] In one embodiment, the one or more amino acid mutations may include (viii) a substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 of SEQ ID NO: 1 with a hydrophilic amino acid.

[0078] In one embodiment, the one or more amino acid mutations may include (ix) a substitution of the amino acid at the residue corresponding to Thr90 in SEQ ID NO: 1 with Tyr, Ile, Leu or Phe.

[0079] In one embodiment, the one or more amino acid mutations may comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 of (x) SEQ ID NO: 1.

[0080] In one embodiment, the one or more amino acid mutations may comprise (i) a deletion of amino acids at residues corresponding to Asp1 and Ile2 based on SEQ ID NO: 1.

[0081] In one embodiment, the one or more amino acid mutations may comprise (ii) one or more amino acid substitutions selected from the group consisting of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His, based on SEQ ID NO: 1.

[0082] In one embodiment, the one or more amino acid mutations may comprise (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179 based on SEQ ID NO: 1.

[0083] In one embodiment, the one or more amino acid mutations may comprise (iv) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 of SEQ ID NO: 1.

[0084] In one embodiment, the one or more amino acid mutations may comprise (v) a deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224 of SEQ ID NO: 1.

[0085] In one embodiment, the one or more amino acid mutations may comprise (vi) a deletion of amino acids in the nine consecutive residues corresponding to Val262 to Leu270 of SEQ ID NO: 1.

[0086] In one embodiment, the one or more amino acid mutations may comprise (vii) a deletion of amino acids at three consecutive residues corresponding to Ala398 to Ala400 of SEQ ID NO: 1.

[0087] In one embodiment, the at least one amino acid mutation comprises a substitution of an amino acid corresponding to at least one residue selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353 and Phe361 of SEQ ID NO: 1 with a hydrophilic amino acid, wherein the hydrophilic amino acid may be selected from the group consisting of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro and Tyr.

[0088] In one embodiment, the one or more amino acid mutations may comprise one or more amino acid substitutions selected from the group consisting of (viii) Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr.

[0089] In one embodiment, the one or more amino acid mutations may comprise (ix) an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu and Thr90Phe based on SEQ ID NO: 1.

[0090] In one embodiment, the one or more amino acid mutations may comprise one or more amino acid substitutions selected from the group consisting of (x) Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser.

[0091] In one embodiment, the laminin G-like domain variant may comprise (1) an amino acid substitution of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln and an amino acid substitution of Asp177Glu, Asp177Gln or Asp177Asn, and (2) an addition of Asn and Thr or an addition of Glu and Val between residues corresponding to Thr178 and Thr179, based on SEQ ID NO: 1.

[0092] In one embodiment, the laminin G-like domain variant can comprise (1) amino acid substitutions of Met22Leu, Met22Ile or Met22Val; and Phe23Tyr, based on SEQ ID NO: 1, (2) deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224, (3) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217, (4) deletion of amino acids in nine residues corresponding to Val262 to Leu270, and (5) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His.

[0093] In one embodiment, the laminin G-like domain variant may comprise (1) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (2) amino acid deletions of nine residues corresponding to Val262 to Leu270; and (3) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu, or Lys272Gln; and Gln273Arg, Gln273Lys, or Gln273His, based on SEQ ID NO: 1.

[0094] In one embodiment, the laminin G-like domain variant can comprise (1) a deletion of an amino acid at a residue corresponding to Asp1, Ile2, or Asp1 and Ile2, (2) an amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu, or Leu3Gln, and (3) a deletion of an amino acid at any one residue, any two consecutive or discontinuous residues, or three residues corresponding to Ala398 to Ala400, based on SEQ ID NO: 1.

[0095] In one embodiment, the laminin G-like domain variant comprises a sequence of SEQ ID NO: 1.

[0096] (i) deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2;

[0097] (ii) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His;

[0098] (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179;

[0099] (iv) addition of Gly between residues corresponding to Arg216 and Thr217, addition of Gly and Ser, or addition of Gly and any other amino acid;

[0100] (v) Deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224;

[0101] (vi) Deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270;

[0102] (vii) Deletion of amino acids in three consecutive residues corresponding to Ala398 to Ala400;

[0103] (viii) a substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 with a hydrophilic amino acid (e.g., Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, or Tyr); and

[0104] (ix) may include an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu, and Thr90Phe.

[0105] In one embodiment, the laminin G-like domain variant comprises a sequence of SEQ ID NO: 1.

[0106] (i) Deletion of amino acids at residues corresponding to Asp1 and Ile2;

[0107] (ii) amino acid substitutions of Leu3Asp; Met22Leu; Phe23Tyr; Gly175Asp; Asp177Glu; Lys272Asp; and Gln273Arg;

[0108] (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179;

[0109] (iv) addition of Gly between residues corresponding to Arg216 and Thr217, addition of Gly and Ser, or addition of Gly and any other amino acid;

[0110] (v) Deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224;

[0111] (vi) Deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270;

[0112] (vii) Deletion of amino acids in three consecutive residues corresponding to Ala398 to Ala400;

[0113] (viii) a substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353 and Phe361 with a hydrophilic amino acid; and

[0114] (ix) may include an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu, and Thr90Phe.

[0115] In one embodiment, the laminin G-like domain variant can comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31, SEQ ID NOs: 115 to 384, and SEQ ID NOs: 385 to 397.

[0116] In one embodiment, the laminin G-like domain can be derived from Gas6 or ProS1.

[0117] In one embodiment, the laminin G-like domain can be part of a fusion protein.

[0118] In one embodiment, the laminin G-like domain variant can comprise an amino acid sequence having greater than 70% sequence identity to the amino acid sequence of a wild-type laminin G-like domain.

[0119] In another aspect of the present invention, an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant described herein is provided.

[0120] In one embodiment, the engineered TAM receptor ligand polypeptide can be an engineered Gas6 polypeptide or an engineered ProS1 polypeptide.

[0121] In one embodiment, the engineered TAM receptor ligand polypeptide can be an engineered TAM receptor ligand polypeptide in which the Gla domain is deleted.

[0122] In one embodiment, the engineered TAM receptor ligand polypeptide may further lack one or more selected from the group consisting of a thrombin-sensitive loop domain and an EGF-like domain.

[0123] In another aspect of the present invention, an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant as described herein and not comprising a Gla domain and (ii) a binding molecule that specifically binds to a target substance is provided.

[0124] In another aspect of the present invention, a fusion molecule is provided comprising (i) an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant as described herein and (ii) a binding molecule that specifically binds to a target substance.

[0125] In one embodiment, the binding molecule can be an antibody, an antigen-binding fragment, an antibody-like protein, a peptide, an aptamer, or a soluble receptor.

[0126] In one embodiment, the binding molecule can be an antibody, antigen-binding fragment, antibody-like protein, peptide, aptamer, or soluble receptor that binds to a Gla domain or phosphatidylserine (PS).

[0127] In one embodiment, the binding molecule can be an antibody or antigen-binding fragment thereof that binds to amyloid beta or TNF alpha.

[0128] In one embodiment, the target substance may be a substance that accumulates in biological tissue and causes a disease.

[0129] In one embodiment, the target substance can be an autoantigen, an autoantibody, a complex of an autoantigen and an autoantibody, a cytokine, a chemokine, complement, a receptor, an immune cell specific marker, a cell adhesion molecule, or a combination thereof.

[0130] In one embodiment, the engineered TAM receptor ligand polypeptide can have phagocytosis-inducing activity through interaction with the TAM receptor.

[0131] In one embodiment, the induction of phagocytosis may not be accompanied by an inflammatory response.

[0132] In another aspect of the present invention, a nucleic acid molecule is provided that encodes a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, or a fusion molecule described herein.

[0133] In another aspect of the present invention, an expression vector comprising a nucleic acid molecule described herein is provided.

[0134] In another aspect of the present invention, a host cell is provided that produces a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, or a fusion molecule described herein.

[0135] In another aspect of the present invention, a pharmaceutical composition is provided comprising an effective amount of a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, a fusion molecule described herein, a nucleic acid molecule described herein, or an expression vector described herein.

[0136] In one embodiment, a use and pharmaceutical composition are provided for the prevention or treatment of a disease or condition characterized by abnormal accumulation of a target substance in a living body.

[0137] In one embodiment, a use and pharmaceutical composition for preventing or treating an immune disease are provided.

[0138] In another aspect of the present invention, a method is provided for preventing or treating a disease or disorder caused by abnormal accumulation or aggregation of a target substance in a subject, comprising administering to the subject a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, a fusion molecule described herein, a nucleic acid molecule described herein, or an expression vector described herein.

[0139] In another aspect of the present invention, a method is provided for reducing or eliminating a target substance to a normal level or inhibiting the production or formation of a target substance in a subject, comprising administering to the subject a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, a fusion molecule described herein, a nucleic acid molecule described herein, or an expression vector described herein.

[0140] In another aspect of the present invention, a method is provided for preventing or treating an immune disorder, delaying the progression of a symptom associated with an immune disorder, or alleviating a symptom of an immune disorder in a subject, comprising administering to the subject a laminin G-like domain variant described herein, an engineered TAM receptor ligand polypeptide described herein, a fusion molecule described herein, a nucleic acid molecule described herein, or an expression vector described herein.

[0141] Engineered TAM receptor ligand polypeptides comprising a laminin G-like domain variant described herein exhibit TAM receptor binding ability or phagocytosis induction ability equivalent to or improved than a (native or engineered) TAM receptor ligand polypeptide comprising a wild-type laminin G-like domain, while exhibiting improved productivity and / or structural stability compared to a (native or engineered) TAM receptor ligand polypeptide comprising a wild-type laminin G-like domain.

[0142] In one embodiment, the engineered Gas6 fusion (e.g., Adu-eGas6) exhibits improved productivity and structural stability compared to a wild-type Gas6 fusion (e.g., Adu-Gas6(WT)), as well as pharmacological efficacy that is higher than or comparable to the wild-type Gas6 fusion (e.g., Adu-Gas6(WT)). In one embodiment, the engineered Gas6 fusion (e.g., Adu-eGas6, Gan-eGas6, or Ada-eGas6) exhibits at least a 3- to 6-fold improvement in productivity compared to the wild-type Gas6 fusion (e.g., Adu-Gas6(WT) or Gan-Gas6(WT)) when produced in an Expi239F cell line, and at least a 5- to 20-fold improvement in productivity compared to the wild-type Gas6 fusion (e.g., Adu-Gas6(WT)) when produced in an ExpiCHO cell line. In another embodiment, the engineered Gas6 fusion (e.g., Adu-eGas6) was obtained with a final purity of 90% or greater and exhibited improved physical properties. In yet another embodiment, the engineered Gas6 fusion (e.g., Adu-eGas6) exhibited improved stability compared to the wild-type Gas6 fusion (e.g., Adu-Gas6(WT)) in a room temperature (25°C) accelerated stability test. At the same time, the engineered Gas6 fusion (e.g., Adu-eGas6) has TAM receptor binding ability, phagocytosis-inducing ability, and TAM signaling-inducing ability that are higher than or comparable to the wild-type Gas6 fusion (e.g., Adu-Gas6(WT)), and thus can effectively induce cells expressing the TAM receptor to phagocytose.

[0143] Figure 1a schematically illustrates the structures of the TAM receptor and TAM receptor ligands. In the structure of the TAM receptor, the N-terminus begins with two Ig domains, followed by two fibronectin type 3 domains, and the C-terminus is followed by a single-pass transmembrane domain and a protein tyrosine kinase. In the structures of the TAM receptor ligands, protein S (ProS1) and Gas6, from the N-terminus to the C-terminus, they contain a Gla domain, a thrombin-sensitive region (TSR), four EGF-like domains, and an SHBG-like domain (laminin G-like domain) consisting of two LG repeats. In Figure 1a, EGF stands for epidermal growth factor, Ig stands for immunoglobulin, LG stands for laminin G, and SHBG stands for sex hormone-binding globulin.

[0144] Figure 1b schematically illustrates an amyloid beta- and FITC-conjugated phagocytosis-inducing fusion molecule comprising Gas6 as a non-limiting example of a TAM receptor ligand of the fusion molecule.

[0145] Figure 2 is a three-dimensional representation of the major structural differences between the wild-type LG-like domain (left) and the engineered LG-like domain (right). In the engineered LG-like domain, position 1 represents alpha-helix stabilization, positions 2-1 and 2-2 represent loop optimization, and position 3 represents N-terminal and C-terminal optimization.

[0146] Figure 3 is a schematic diagram of the human Gas6 protein and the Adu-Gas6 polypeptide in which the Gla domain or the Gla domain and the EGF-like domain are deleted and the Adu binding fragment is fused.

[0147] Figure 4a is a graph showing the expression levels of engineered Adu-Gas6 variants transiently expressed in HEK293F cells using an anti-Gas6 antibody.

[0148] Figure 4b is a graph showing the expression levels of engineered Adu-Gas6 variants transiently expressed in HEK293F cells using an anti-histidine antibody.

[0149] Figure 5a is a graph showing the productivity of engineered Adu-Gas6 variants fused with aducanumab.

[0150] Figure 5b is a graph showing the productivity of engineered Adu-Gas6 V300 variants fused with aducanumab.

[0151] Figure 6a is a gel photograph showing the expression levels of engineered Gas6 variants containing major mutations confirmed by electrophoresis.

[0152] Figure 6b is a drawing showing the culture medium of engineered Gas6 variants containing major mutations loaded onto a gel, stained with Coomassie blue, and observed with a laser wavelength of 700 nm.

[0153] Figure 6c is a graph quantifying the band thickness of Figure 6b based on the wild type.

[0154] Figure 7a is a graph showing the productivity of engineered Gas6 variants containing only secondary mutations.

[0155] Figure 7b is a graph showing the productivity of engineered Gas6 variants containing only one secondary mutation.

[0156] Figure 8a is a graph showing the productivity of engineered Gas6 variants (Adu-eGas6) in Expi293F cells.

[0157] Figure 8b is a graph showing the productivity of engineered Gas6 variants (Adu-eGas6) in ExpiCHO cells.

[0158] Figure 9 is a graph showing the binding activity of an engineered Gas6 variant (Adu-eGas6) to human Axl confirmed by ELISA.

[0159] Figure 10 is a graph showing the binding activity of an engineered Gas6 mutant (Adu-eGas6) to human Axl using flow cytometry (FACS).

[0160] Figure 11a is a graph showing the activity of the Axl receptor according to the concentration of the engineered Gas6 variant (Adu-eGas6) when not treated with amyloid beta aggregates (Aβ aggregates).

[0161] Figure 11b is a graph showing the activity of the Axl receptor according to the concentration of the engineered Gas6 variant (Adu-eGas6) when treated with amyloid beta aggregates (Aβ aggregates).

[0162] Figure 12a shows THP-1 treated with engineered Gas6 variant (Adu-eGas6) over time. Axl This is a graph showing changes in the phagocytosis activity of amyloid beta aggregates.

[0163] Figure 12b shows THP-1 treated with engineered Gas6 variant (Adu-eGas6). Axl This is a graph showing the phagocytic activity against amyloid beta aggregates.

[0164] Figure 13a is a graph showing the change in purity of an engineered Gas6 variant according to a room temperature accelerated stability test confirmed through SE-HPLC analysis.

[0165] Figure 13b is a graph showing changes in protein concentration of engineered Gas6 variants according to room temperature accelerated stability tests, confirmed through protein quantification.

[0166] The detailed description of the present invention, which follows, will be described with reference to specific drawings (where drawings exist) regarding specific embodiments in which the present invention may be practiced; however, the present invention is not limited thereto, but is defined solely by the appended claims to the full scope equivalent to or equivalent to what the claims describe. It should be understood that the various embodiments / embodiments of the present invention, while different from each other, are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment / embodiment to another, or multiple embodiments / embodiments may be combined, without departing from the spirit and scope of the present invention. Technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly used in the art to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and a term expressed in the singular should be construed to also refer to the plural (i.e., at least one), unless the context makes it inappropriate. Any references cited in this specification are incorporated by reference in their entirety.

[0167] I. Definition

[0168] The term "about" refers to the typical error range for each value known to those of ordinary skill in the art. Furthermore, unless otherwise specified, all numbers, values, and / or expressions expressing ingredients, conditions, compositions, amounts, and so forth used herein should be understood to be modified by the term "about" because such numbers are approximations that inherently reflect, among other things, the various uncertainties of measurement that arise in obtaining such values. As examples, this may include ±10% or less, ±9% or less, ±8% or less, ±7% or less, ±6% or less, ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, or ±0.5% or less of a given numerical value.

[0169] The “laminin G-like domain,” “LG-like domain,” “laminin globular (G)-like domain,” or “LNS (Laminin-alpha, Neurexin, and Sex hormone-binding globulin) domain” has an average length of 177 amino acids and can be found in one to six copies in various laminin family members as well as in numerous other extracellular proteins. For example, all laminin alpha chains have five laminin G domains, all collagen family proteins have one laminin G domain, the CNTNAP protein has four laminin G domains, while neurexin 1 and 2 each have six laminin G domains. Various binding functions are attributed to the different laminin G modules. Laminin G-containing proteins are thought to play diverse roles in cell adhesion, signaling, migration, assembly, and differentiation.Various human proteins containing laminin G-like domains include laminin alpha chains (LAMA1, LAMA2, LAMA3, LAMA4, LAMA5), cadherin EGF LAG seven-pass G type receptors (CELSR1, CELSR2, CELSR3), contactin-related proteins (CNTNAP1, CNTNAP2, CNTNAP3, CNTNAP3B, CNTNAP4, CNTNAP5), some collagens (COL5A1, COL5A3, COL9A1, COL11A1, COL11A2, COL12A1, COL14A1, COL15A1, COL16A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL24A1, COL27A1), crumbs homolog 1, and 2 (CRB1, CRB2), fat homologs (FAT1, FAT2, FAT3, FAT4), NEL-like proteins (NELL1, NELL2), neurexins (NRXN1, NRXN2, NRXN3), slit homologs (SLIT1, SLIT2, SLIT3), thrombospondins (THBS1, THBS2, THBS3, THBS4, TSPEAR), agrin (AGRIN), chondroitin sulfate proteoglycan 4 (CSPG4), eyes shut homolog (EYS), growth arrest-specific protein 6 (GAS6), perlecan (HSPG2), pikachurin (EGFLAM), protein S (PROS1), sex hormone-binding globulin (SHBG), and usherin (USH2A).

[0170] The term "variant" or "engineered" refers to a protein or (poly)peptide that has a sequence that differs by one or more amino acid residues from the original amino acid sequence of the protein or (poly)peptide, e.g., the sequence of the wild-type or naturally occurring protein or (poly)peptide, and any truncation, deletion, insertion, substitution, etc. and any combination thereof are possible in the final structure as long as the activity of the protein or (poly)peptide is maintained. An example of a variant is a form in which an amino acid residue in a site that is not essential for activity is truncated or deleted, or a form in which an amino acid residue in a site that is important for function or properties is substituted. In addition, in some cases, modifications such as phosphorylation, glycosylation, methylation, and farnesylation may be performed. Such sequence mutations and modifications are desirable when the productivity, function, and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility of the protein are increased by the mutation in the amino acid sequence.

[0171] As used herein, the term "increased productivity" or "increased productivity" means an "increase" in the expression amount and / or yield after purification compared to a wild-type protein or (poly)peptide when expressed and purified under the same conditions. In one embodiment, the wild-type protein or (poly)peptide that serves as a reference value may be a wild-type laminin G-like domain or a wild-type laminin G-like domain-containing protein. In addition, in one specific embodiment, the wild-type laminin G-like domain may be a polypeptide consisting of SEQ ID NO: 1 or a polypeptide comprising SEQ ID NO: 1. An example of a polypeptide comprising the laminin G-like domain of SEQ ID NO: 1 may be a wild-type Gas6 protein having a sequence of SEQ ID NO: 63 or a fusion protein in which the wild-type Gas6 protein is fused with another protein. Thus, according to one embodiment, the productivity of a fusion protein comprising a wild-type laminin G-like domain-containing wild-type Gas6 and another polypeptide (the same polypeptide as the other polypeptide included in the fusion protein providing the baseline) can be compared using a fusion protein comprising a wild-type laminin G-like domain-containing wild-type Gas6 as the baseline. According to one specific example, an “increase” may be an increase of at least about 1%, an increase of at least about 2%, an increase of at least about 3%, an increase of at least about 4%, an increase of at least about 5%, an increase of at least about 6%, an increase of at least about 7%, an increase of at least about 8%, an increase of at least about 9%, an increase of at least about 10%, an increase of at least about 15%, an increase of at least about 20%, an increase of at least about 30%, an increase of at least about 40%, an increase of at least about 50%, an increase of at least about 60%, or an increase of at least about 70% relative to a baseline value.Evaluation of expression level, purity, or productivity can be performed by methods known in the art, for example, by the non-limiting and exemplary methods described in the examples herein.

[0172] As used herein, the term "improved stability" or "increased stability" means that the change in concentration and / or purity of the protein, for example, "loss" of concentration and / or purity, is "less" compared to the wild-type protein or (poly)peptide when stored under the same conditions at room temperature (about 25°C) for 4 weeks. In one embodiment, the wild-type protein or (poly)peptide may be a wild-type laminin G-like domain. In addition, in one embodiment, the wild-type laminin G-like domain may be a polypeptide consisting of SEQ ID NO: 1 or a polypeptide comprising SEQ ID NO: 1. An example of a polypeptide comprising the laminin G-like domain of SEQ ID NO: 1 may be a wild-type Gas6 protein having the sequence of SEQ ID NO: 63 or a fusion protein in which the wild-type Gas6 protein is fused with another protein. Thus, according to one embodiment, the stability of a fusion protein comprising a wild-type laminin G-like domain-containing wild-type Gas6 as a baseline and another polypeptide (the same polypeptide as the other polypeptide included in the fusion protein providing the baseline) can be compared using a fusion protein comprising a wild-type laminin G-like domain-containing wild-type Gas6 as a baseline.According to one specific example, “improvement in stability” or “increase in stability” may include cases where, when a sample containing the same concentration of protein is stored under the same conditions, for example, at room temperature for 4 weeks, and the concentration and / or purity is measured, the reduction ratio in the laminin G-like variant-containing protein sample of the present invention is about 1% or less less, about 2% or less less, about 3% or less less, about 4% or less less, about 5% or less less, about 6% or less less, about 7% or less less, about 8% or less less, about 9% or less less, about 10% or less less, about 15% or less less, about 20% or less less, about 30% or less less, about 40% or less less, about 50% or less less, about 60% or less less, or about 70% or less less than the reduction ratio in the wild-type laminin G-like domain-containing protein sample. The stability can be assessed by methods known in the art, for example, by the non-limiting and exemplary methods described in the examples herein.

[0173] A method for inducing mutations in an amino acid sequence is to use a method for producing a nucleic acid molecule containing a nucleotide sequence corresponding to the amino acid sequence to be changed by mutating the nucleotide sequence encoding the protein, and a method for obtaining a gene encoding the same can be mutated in vivo or in vitro using any mutagenesis technique well known in the art. For example, site-directed mutagenesis (Hutchinson et al., J. Biol. Chem., 253:6551, 1978; Zoller and Smith, DNA, 3:479-488, 1984; Oliphant et al., Gene, 44:177, 1986; Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83:710, 1986), TAB linkers (Pharmacia), PCR technology (Higuchi, 1989, "Using PCR to Engineer DNA" in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70) can be used.

[0174] The term "antibody" is used in the broadest sense and includes various antibody structures, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), antibody fragments having antigen-binding activity, and antibody fusions (e.g., fusions of an antibody and a (poly)peptide or of an antibody and a compound). As used herein, the prefix "anti-", when associated with an antigen, means that the antibody is reactive with that antigen. Antibodies reactive with a particular antigen can be produced by, but are not limited to, synthetic and / or recombinant methods, such as screening of recombinant antibody libraries in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid. A typical IgG antibody consists of two identical heavy chains and two identical light chains joined by disulfide bonds. Each heavy and light chain comprises a constant region and a variable region. The heavy chain variable region (HVR) and light chain variable region (LVR) each contain three segments, called "complementarity determining regions" ("CDRs") or "hypervariable regions," which are primarily involved in binding to antigenic epitopes. These are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. The more conserved regions of the variable regions outside the CDRs are referred to as "framework regions" ("FRs"). As used herein, an antibody may be, for example, an animal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0175] Additionally, antibodies can be used as antibody-binding fragments as long as their function is maintained. The term "antibody-binding fragment" refers to a portion of an antibody or a polypeptide comprising the same that has specific binding ability to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments and single-domain antibodies.

[0176] The term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin are covalently linked to form a VH-VL heterodimer. The heavy (VH) and light (VL) chains may be linked directly, by a peptide linker that links the N-terminus of the VH to the C-terminus of the VL, or by a peptide linker that links the C-terminus of the VH to the N-terminus of the VL.

[0177] The term "antibody-like protein" refers to a protein scaffold capable of specifically binding to a target substance, much like an antibody. Antibody-like proteins are smaller, measuring 2-20 kDa, compared to antibodies (which average about 150 kDa), and can be designed to target binding sites that antibodies cannot reach. They are known to be more stable at higher temperatures than antibodies and are much easier to synthesize using non-mammalian cells such as viruses and yeast, as well as chemically synthesize. Examples of antibody-like proteins include, but are not limited to, affibodies, adnectins, affimers, DARPin scaffolds, and aptamers.

[0178] The term "aptamer" refers to single-stranded DNA (ssDNA) or RNA that has high specificity and affinity for a specific substance. Aptamers have very high affinity for a specific substance and are stable, can be synthesized through a relatively simple method, can be modified in various ways to increase binding affinity, and can target cells, proteins, and even small organic substances. Therefore, their specificity and stability are much higher than those of antibodies that have already been developed. In addition, aptamers can be manufactured using the known SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Such aptamers, for example, aptamers that specifically bind to beta-amyloid, tau, and alpha-synuclein can be manufactured using the known SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method.

[0179] The term "available receptor" refers to a domain having an activity capable of binding a target substance, i.e. an endogenous ligand, wherein said domain may be derived from or a derivative of an endogenous membrane receptor or an intracellular receptor.

[0180] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, a nucleic acid molecule of the invention comprises a contiguous open reading frame encoding an LG-like domain variant, a TAM receptor ligand polypeptide, or a fusion molecule, antibody, or fragment, derivative, fusion, or variant thereof.

[0181] The term "conservative amino acid substitution" refers to the replacement of one amino acid of a class with another amino acid of the same class. A conservative amino acid substitution does not alter the structure, function, or both of the polypeptide. Conservative amino acid substitutions are provided in Table 1 under the heading "Preferred Substitutions" and are further described below with respect to amino acid side chain classes (1) through (6). Amino acid substitutions can be introduced into a molecule of interest and into a product screened for a desired activity, such as maintained / improved antigen binding, reduced immunogenicity, or improved physical properties.

[0182]

[0183] Amino acids can be generally grouped according to their common side chain properties as follows, except where otherwise noted herein:

[0184] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

[0185] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0186] (3) Acidic: Asp, Glu;

[0187] (4) Basic: His, Lys, Arg;

[0188] (5) Residues affecting chain orientation: Gly, Pro;

[0189] (6) Aromatic: Trp, Tyr, Phe.

[0190] Non-conservative substitutions involve exchanging members of one of these classes for members of another class.

[0191] The term "subject" is used interchangeably with "subject," "subject," and "patient," and can be any mammal, such as a primate (e.g., a human), a companion animal (e.g., a dog, a cat, etc.), a livestock animal (e.g., a cow, a pig, a horse, a sheep, a goat, etc.), and a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.), in need of prevention or treatment of a disease or condition. In one embodiment, the subject is a human.

[0192] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such effect is therapeutic in that it partially or completely cures a disease and / or other unwanted or undesirable condition. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, improving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and remission or improving prognosis. Preferably, "treatment" may refer to medical intervention for an existing disease or disorder.

[0193] The term "prevention" means obtaining a desired preventive pharmacological and / or physiological effect with a view to partially or completely preventing a disease or its symptoms.

[0194] The term "administration" means providing an active ingredient to a subject to achieve a preventive or therapeutic purpose.

[0195] II. Laminin G-like domain variants / engineered TAM receptor ligand polypeptides

[0196] TAM receptors (three receptor tyrosine kinases, Tyro3, ​​Axl or MerTK) are mainly expressed in macrophages and can be activated by their ligands (e.g., Gas6, ProS1, Tubby, Tulp1 or Gal3) to induce phagocytosis.

[0197] Gas6 protein (growth arrest specific 6 protein) is a ligand protein of approximately 75 kDa that can interact with the TAM receptor family, and contains four domains from the N-terminus to the C-terminus: a Gla domain, a thrombin-sensitive loop domain (TSR), an EGF-like domain, and a laminin G-like domain (LG-like domain). It is known that the Gla domain at the N-terminus recognizes phosphatidylserine (PS) on the surface of apoptotic cells and signals the induction of phagocytosis, and the LG-like domain at the C-terminus interacts with the TAM receptor to induce phagocytosis by macrophages (refer to the literature [Greg Lemke, Cold Spring Harb Perspect Biol 2013; 5:a009076]). Another ligand of the TAM receptor, ProS1 (Protein S), contains four domains: a Gla domain, a thrombin-sensitive loop region, an EGF-like domain, and a laminin G-like domain (also called SHBG-like domain), and has high structural and functional similarity to the Gas6 protein.

[0198] Through previous research, the present inventors have developed a novel chimeric protein-based phagocytosis derivative that can effectively remove only a target protein (e.g., Aβ) while suppressing the inflammatory response. Specifically, the novel chimeric protein-based phagocytosis derivative is obtained by fusing an antigen-binding molecule that specifically binds to the target protein in place of the Gla domain to a Gas6 protein engineered to delete the Gla domain and include an LG-like domain. For example, a chimeric protein named Adu-Gas6, which targets Aβ, was shown in in vitro experiments to very effectively remove Aβ not only from microglia but also from astrocytes present in the brain, unlike existing anti-Aβ antibodies, and it was confirmed to dramatically reduce excessive inflammatory responses, which were a side effect of antibody treatment (see Korean Patent No. 10-2549520).

[0199] Through further studies of protein design based on the tertiary structure of the Gas6 protein, we found that introducing one or more amino acid modifications into the LG-like domain of the TAM receptor ligand protein significantly improved protein productivity and structural stability while maintaining pharmacological effects.

[0200] In this specification, the terms “engineered TAM receptor ligand,” “engineered TAM receptor ligand protein,” or “engineered TAM receptor ligand polypeptide” are used interchangeably.

[0201] As used herein, an LG-like domain engineered to contain one or more amino acid mutations is referred to as an “LG-like domain variant.” As exemplified in FIGS. 1A and 1B , a TAM receptor ligand protein can be engineered to have the Gla domain deleted, or the Gla domain and the EGF-like domain deleted. In this regard, an engineered TAM receptor ligand protein can refer to a TAM receptor ligand protein modified to include an LG-like domain variant in place of a wild-type LG-like domain, or a TAM receptor ligand protein modified to include an LG-like domain variant in place of a wild-type LG-like domain and the Gla domain deleted, or a TAM receptor ligand protein in which the Gla domain and the EGF-like domain are deleted. When the Gla domain and the EGF-like domain are deleted and the LG-like domain variant of the present invention is included, the engineered TAM receptor ligand protein can refer to an LG-like domain variant. Depending on the context, an engineered TAM receptor ligand protein may refer to a TAM receptor ligand protein comprising a wild-type LG-like domain and a deletion of the Gla domain or the Gla domain and the EGF-like domain.

[0202] In one aspect of the present invention, a method is provided for improving protein productivity and / or structural stability of a fusion molecule comprising an LG-like domain and a domain that binds to a targeting component, the method comprising introducing one or more amino acid mutations into the LG-like domain of a TAM receptor ligand protein.

[0203] In order to engineer the Gas6 LG-like domain (SEQ ID NO: 1), the existing known protein structure-related theories, protein structure design deep learning model Inpainting, and Rosetta (Rosetta Commons) software are used to perform calculations on the design and stability of the protein structure, thereby selecting or determining the desired amino acid mutation. The modules used to create the protein structure within the Rosetta software are the Remodel module and the Fast Design module. The Remodel module is a program that customizes loops according to the design, and the Fast Design module is a module that designs the protein structure while performing FastRelax, which optimizes and stabilizes the protein structure. According to one implementation example, Rosetta simulations can be performed using the ref2015 score function.

[0204] Additionally or alternatively, you can use AlphaFold (Google DeepMind) to filter and select desired mutations based on designed structure prediction and stability. For example, you can perform structure prediction using AlphaFold for the sequence containing each designed mutation, and prioritize selecting the top 30 designs with a pLDDT value of 80 or higher. The pLDDT value is the reliability of the structural model predicted from the protein sequence. A value of 90 or higher can be considered a very reliable region, a value of 70 to 90 a somewhat reliable region, a value of 50 to 70 a low reliable region, and a value of 50 or less a nearly disordered region.

[0205] In another aspect of the present invention, LG-like domain variants and methods for preparing the same are provided, which comprise one or more amino acid modifications that enhance the productivity and / or structural stability of the LG-like domain, a TAM receptor ligand protein comprising the LG-like domain, or a fusion molecule comprising the LG-like domain and a domain that binds to a target component, compared to the amino acid sequence of a wild-type LG-like domain.

[0206] In another aspect, an LG-like domain variant comprising one or more amino acid mutations that improve the productivity and / or structural stability of an LG-like domain, a TAM receptor ligand polypeptide comprising an LG-like domain, or a polypeptide comprising an LG-like domain and a domain that binds a target component, compared to an amino acid sequence of a wild-type LG-like domain, wherein the LG-like domain variant has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least LG-like domain variants having 99% but less than 100% identity are provided.

[0207] In one embodiment, the one or more amino acid mutations introduced into the wild-type LG-like domain may comprise one or more selected from the group consisting of (i) to (x):

[0208] (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1;

[0209] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272, and Gln273 based on sequence number 1;

[0210] (iii) addition of one, two or three amino acids between residues corresponding to Thr178 and Thr179 based on sequence number 1;

[0211] (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0212] (v) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on SEQ ID NO: 1;

[0213] (vi) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO: 1;

[0214] (vii) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO: 1;

[0215] (viii) Substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 based on sequence number 1 with a hydrophilic amino acid;

[0216] (ix) The amino acid of the residue corresponding to Thr90 in sequence number 1 is replaced with Tyr, Ile, Leu or Phe; and

[0217] (x) An amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on sequence number 1.

[0218] In one embodiment, the laminin G-like domain variant can comprise one or more amino acid mutations selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), and (vii) described herein (e.g., amino acid mutation(s) belonging to any one, any two, any three, any four, any five, any six, or seven of (i), (ii), (iii), (iv), (v), (vi), and (vii)).

[0219] In one embodiment, the laminin G-like domain variant can comprise one or more amino acid mutations selected from the group consisting of (i), (ii) and (vii) described herein (e.g., amino acid mutation(s) belonging to any one, any two or all three categories of (i), (ii) and (vii)).

[0220] In one embodiment, the laminin G-like domain variant may comprise one or more amino acid mutations selected from the group consisting of (ii) and (iii) described herein (e.g., amino acid mutation(s) belonging to any one or both categories of (ii) and (iii)).

[0221] In one embodiment, the laminin G-like domain variant can comprise one or more amino acid mutations selected from the group consisting of (ii), (iii), (v) and (vi) described herein (e.g., amino acid mutation(s) belonging to any one, any two, any three, or all four categories of (ii), (iii), (v) and (vi)).

[0222] In one embodiment, the laminin G-like domain variant can comprise one or more amino acid mutations selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii) and (ix) described herein (e.g., amino acid mutation(s) belonging to any one category, any two categories, any three categories, any four categories, any five categories, any six categories, any seven categories or any eight categories of (i), (ii), (iii), (iv), (v), (vi), (vii) and (ix)).

[0223] In one embodiment, the laminin G-like domain variant can comprise one or more amino acid mutations selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii) and (ix) described herein (e.g., amino acid mutation(s) belonging to any one category, any two categories, any three categories, any four categories, any five categories, any six categories, any seven categories, any eight categories or any nine categories of (i), (ii), (iii), (iv), (v), (vi), (vii) and (ix)).

[0224] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (i) as described herein, and optionally one or more amino acid mutations selected from the group consisting of (ii) to (x) as described herein.

[0225] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (ii) described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) and (iii) to (x) described herein.

[0226] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (iii) described herein, and optionally one or more amino acid mutations selected from the group consisting of (i), (ii) and (iv) to (x) described herein.

[0227] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (iv) as described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (iii) and (v) to (x) as described herein.

[0228] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (v) as described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (iv) and (vi) to (x) as described herein.

[0229] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (vi) as described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (v) and (vii) to (x) as described herein.

[0230] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (vii) as described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (vi) and (viii) to (x) as described herein.

[0231] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (viii) described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (vii), (ix) and (x) described herein.

[0232] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (ix) described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (viii) and (x) described herein.

[0233] In one embodiment, the laminin G-like domain variant comprises one or more amino acid mutations selected from the group consisting of (x) described herein, and optionally one or more amino acid mutations selected from the group consisting of (i) to (ix) described herein.

[0234] In one embodiment, the laminin G-like domain variant can comprise (i) a deletion of an amino acid at a residue corresponding to Asp1, Ile2, or Asp1 and Ile2 based on SEQ ID NO: 1. Specifically, the laminin G-like domain variant can comprise a deletion of an amino acid at a residue corresponding to Asp1 and Ile2 based on SEQ ID NO: 1.

[0235] In one embodiment, the laminin G-like domain variant may comprise an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272, and Gln273 based on SEQ ID NO: 1. Specifically, the laminin G-like domain variant may comprise, based on SEQ ID NO: 1,

[0236] Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln;

[0237] Met22Leu, Met22Ile, or Met22Val;

[0238] Phe23Tyr;

[0239] Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln;

[0240] Asp177Glu, Asp177Gln or Asp177Asn;

[0241] Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and

[0242] It may comprise one or more amino acid substitutions selected from the group consisting of Gln273Arg, Gln273Lys or Gln273His. More specifically, the laminin G-like domain variant may comprise, based on SEQ ID NO: 1,

[0243] Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln;

[0244] Met22Leu, Met22Ile, or Met22Val;

[0245] Phe23Tyr;

[0246] Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln;

[0247] Asp177Glu, Asp177Gln or Asp177Asn;

[0248] Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and

[0249] The laminin G-like domain variant may comprise an amino acid substitution of Gln273Arg, Gln273Lys, or Gln273His. As another example, the laminin G-like domain variant may comprise one or more amino acid substitutions selected from the group consisting of Leu3Asp, Met22Leu, Phe23Tyr, Gly175Asp, Asp177Glu, Lys272Asp, and Gln273Arg based on SEQ ID NO: 1. More specifically, the laminin G-like domain variant may comprise an amino acid substitution of Leu3Asp, Met22Leu, Phe23Tyr, Gly175Asp, Asp177Glu, Lys272Asp, and Gln273Arg based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise one or more amino acid substitutions selected from the group consisting of Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His based on SEQ ID NO: 1. More specifically, the laminin G-like domain variant may comprise the amino acid substitutions Met22Leu, Met22Ile or Met22Val; and Phe23Tyr based on SEQ ID NO: 1. More specifically, the laminin G-like domain variant may comprise an amino acid substitution of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His based on SEQ ID NO: 1.As another example, the laminin G-like domain variant may comprise one or more amino acid substitutions selected from the group consisting of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn, based on SEQ ID NO: 1. More specifically, the laminin G-like domain variant may comprise an amino acid substitution of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn, based on SEQ ID NO: 1.

[0250] In one embodiment, the laminin G-like domain variant can comprise (iii) an addition of one, two, or three amino acids between residues corresponding to Thr178 and Thr179 of SEQ ID NO: 1. Specifically, the laminin G-like domain variant can comprise an addition of two amino acids between residues corresponding to Thr178 and Thr179 of SEQ ID NO: 1. The added amino acids can be any amino acids capable of reconstructing the alpha helical structure of the region comprising Thr178 and Thr179. More specifically, the laminin G-like domain variant can further comprise Asn and Thr, or Glu and Val, between residues corresponding to Thr178 and Thr179 of SEQ ID NO: 1.

[0251] In one embodiment, the laminin G-like domain variant can comprise (iv) an addition of one, two or three amino acids, preferably one or two amino acids, between residues corresponding to Arg216 and Thr217 of SEQ ID NO: 1. Specifically, the laminin G-like domain variant can comprise an addition of Gly, an addition of Gly and Ser, or an addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 of SEQ ID NO: 1. Any other amino acid means, for example, any one of the 21 amino acids set forth in Table 1. More specifically, the laminin G-like domain variant can comprise Gly between residues corresponding to Arg216 and Thr217 of SEQ ID NO: 1.

[0252] In one embodiment, the laminin G-like domain variant can comprise a deletion of an amino acid at a residue corresponding to one or more residues selected from Thr217 to Glu224 of SEQ ID NO: 1. For example, one, two, three, four, five, six, seven, or eight amino acids can be deleted from Thr217 to Glu224 of SEQ ID NO: 1. The two or more deleted amino acids can be consecutive or discontinuous. For example, the laminin G-like domain variant can comprise a deletion of an amino acid at eight consecutive residues corresponding to Thr217 to Glu224 of SEQ ID NO: 1.

[0253] In one embodiment, the laminin G-like domain variant can comprise a deletion of an amino acid at a residue corresponding to one or more residues selected from Val262 to Leu270 of SEQ ID NO: 1. For example, one, two, three, four, five, six, seven, eight, or nine amino acids can be deleted from Val262 to Leu270 of SEQ ID NO: 1. The two or more deleted amino acids can be consecutive or discontinuous. For example, the laminin G-like domain variant can comprise a deletion of an amino acid at nine consecutive residues corresponding to Val262 to Leu270 of SEQ ID NO: 1.

[0254] In one embodiment, the laminin G-like domain variant can comprise a deletion of an amino acid at a residue corresponding to one or more residues selected from Ala398 to Ala400 of SEQ ID NO: 1. For example, one, two, or three amino acids can be deleted from Ala398 to Ala400 of SEQ ID NO: 1. The two or more deleted amino acids can be consecutive or discontinuous. For example, the laminin G-like domain variant can comprise a deletion of an amino acid at three consecutive residues corresponding to Ala398 to Ala400.

[0255] In one embodiment, the laminin G-like domain variant may comprise (viii) a substitution of an amino acid at a residue corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 of SEQ ID NO: 1 with a hydrophilic amino acid. In the present specification, the hydrophilic amino acid may be selected from the group consisting of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, and Tyr. It may be preferable that each amino acid be substituted with an amino acid having a similar structure and relatively high hydrophilicity among hydrophilic amino acids. Preferred amino acid substitutions at residues corresponding to Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 of SEQ ID NO: 1 are presented in Table 4. More preferably, the laminin G-like domain variant may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more or eight amino acid substitutions selected from the group consisting of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser based on SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise the amino acid substitution(s) set forth in Table 10 based on SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise the amino acid substitution(s) of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr based on SEQ ID NO: 1.

[0256] In one embodiment, the laminin G-like domain variant may comprise (ix) a substitution of an amino acid residue corresponding to Thr90 in SEQ ID NO: 1 with Tyr, Ile, Leu or Phe. Specifically, the laminin G-like domain variant may comprise an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu and Thr90Phe in SEQ ID NO: 1. More specifically, the laminin G-like domain variant may comprise an amino acid substitution of Thr90Tyr in SEQ ID NO: 1.

[0257] In one embodiment, the laminin G-like domain variant may comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 of SEQ ID NO: 1 (x). The substituted amino acid may be selected from the group consisting of hydrophilic amino acids Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, and Tyr. It may be preferable that each amino acid be substituted with an amino acid having a similar structure and relatively greater hydrophilicity among hydrophilic amino acids. Preferred amino acid substitutions at residues corresponding to Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 of SEQ ID NO: 1 are presented in Table 9. More preferably, the laminin G-like domain variant may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight amino acid substitutions selected from the group consisting of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu, and Ala389Ser based on SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise the amino acid substitution(s) set forth in Table 10 based on SEQ ID NO: 1. For example, the laminin G-like domain variant may include amino acid substitutions of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu, and Ala389Ser relative to SEQ ID NO: 1.

[0258] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0259] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Met22, Phe23, Lys272, and Gln273 based on sequence number 1;

[0260] (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0261] (v) deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 based on sequence number 1; and

[0262] (vi) A laminin G-like domain variant is provided comprising at least one selected from the group consisting of deletions of amino acids at residues corresponding to at least one consecutive or discontinuous residue selected from Val262 to Leu270 of SEQ ID NO: 1. (Stabilization of ring structure)

[0263] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0264] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Gly175 and Asp177 based on sequence number 1; and

[0265] (iii) A laminin G-like domain variant is provided comprising at least one selected from the group consisting of an addition of one, two or three amino acids between residues corresponding to Thr178 and Thr179 of SEQ ID NO: 1. (Stabilization of alpha helix structure)

[0266] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0267] (i) deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1; and

[0268] (ii) A laminin G-like domain variant is provided comprising at least one amino acid substitution selected from the group consisting of a residue corresponding to the Leu3 residue based on sequence number 1. (N-terminal stabilization)

[0269] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0270] (vii) A laminin G-like domain variant is provided comprising a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 based on SEQ ID NO: 1. (C-terminal stabilization)

[0271] In one embodiment, the laminin G-like domain variant may comprise all of the amino acid mutations (i) to (ix) disclosed herein.

[0272] In one embodiment, the laminin G-like domain variant comprises all of the amino acid mutations (i) to (ix) disclosed herein, and may further comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on SEQ ID NO: 1 disclosed herein (x).

[0273] In another aspect, a laminin G-like domain variant is provided, which comprises one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications comprise one or more selected from the group consisting of (i) to (ix):

[0274] (i) Deletion of one or more amino acids at residues corresponding to Asp1 and Ile2 based on sequence number 1;

[0275] (ii) one or more amino acid substitutions selected from the group consisting of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His based on SEQ ID NO: 1;

[0276] (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179 based on sequence number 1;

[0277] (iv) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0278] (v) deletion of amino acid(s) at residues corresponding to one or more consecutive or discontinuous residues corresponding to Thr217 to Glu224 based on SEQ ID NO: 1;

[0279] (vi) Deletion of amino acid(s) at residues corresponding to one or more consecutive or discontinuous residues corresponding to Val262 to Leu270 of SEQ ID NO: 1;

[0280] (vii) Deletion of amino acid(s) at residues corresponding to one or more consecutive or discontinuous residues corresponding to Ala398 to Ala400 based on SEQ ID NO: 1;

[0281] (viii) an amino acid of a residue corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353 and Phe361 based on sequence number 1 is replaced with an amino acid selected from the group consisting of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro and Tyr; and

[0282] (ix) One or more amino acid substitutions selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu and Thr90Phe based on sequence number 1.

[0283] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0284] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His based on SEQ ID NO: 1;

[0285] (iv) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0286] (v) deletion of amino acids at residues corresponding to one or more consecutive or discontinuous residues (e.g., eight consecutive residues) selected from Thr217 to Glu224 based on SEQ ID NO: 1; and

[0287] (vi) A laminin G-like domain variant is provided comprising at least one selected from the group consisting of deletions of amino acids at residues corresponding to one or more consecutive or discontinuous residues (e.g., nine consecutive residues) selected from Val262 to Leu270 of SEQ ID NO: 1. (Stabilization of ring structure)

[0288] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0289] (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn based on sequence number 1; and

[0290] (iii) A laminin G-like domain variant is provided comprising at least one selected from the group consisting of addition of Asn and Thr, or addition of Glu and Val between residues corresponding to Thr178 and Thr179 based on sequence number 1. (Stabilization of alpha helix structure)

[0291] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0292] (i) deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1; and

[0293] (ii) A laminin G-like domain variant comprising an amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln based on SEQ ID NO: 1 is provided. (N-terminal stabilization)

[0294] In one embodiment, the laminin G-like domain variant is a laminin G-like domain variant comprising one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications

[0295] (vii) A laminin G-like domain variant is provided comprising a deletion of an amino acid at a contiguous or discontinuous residue (e.g., three contiguous residues) corresponding to one or more residues selected from Ala398 to Ala400 of SEQ ID NO: 1. (C-terminal stabilization)

[0296] For example, a laminin G-like domain variant may include (1) an amino acid substitution of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln and an amino acid substitution of Asp177Glu, Asp177Gln or Asp177Asn, and (2) an addition of Asn and Thr or an addition of Glu and Val between residues corresponding to Thr178 and Thr179, based on SEQ ID NO: 1.

[0297] For example, a laminin G-like domain variant can comprise (1) an amino acid substitution of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr, based on SEQ ID NO: 1, (2) a deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224, (3) an addition of Gly, an addition of Gly and Ser, or an addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217, (4) a deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270, and (5) an amino acid substitution of Lys272Asp, Lys272Asn, Lys272Glu, or Lys272Gln; and Gln273Arg, Gln273Lys, or Gln273His.

[0298] For example, a laminin G-like domain variant can comprise (1) an amino acid deletion at a residue corresponding to Asp1, Ile2, or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2), (2) an amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu, or Leu3Gln, and (3) a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400, based on SEQ ID NO: 1.

[0299] For example, the laminin G-like domain variants may comprise, based on SEQ ID NO: 1, (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn; (2) amino acid additions of Asn and Thr, or additions of Glu and Val, between residues corresponding to Thr178 and Thr179; (3) amino acid substitutions of Met22Leu, Met22Ile or Met22Val; and Phe23Tyr; (4) amino acid deletions of eight consecutive residues corresponding to Thr217 to Glu224; (5) addition of the amino acids Gly, Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (6) amino acid deletion in nine consecutive residues corresponding to Val262 to Leu270; and (7) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His.

[0300] For example, a laminin G-like domain variant can comprise (1) an amino acid substitution of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn; (2) an addition of the amino acids Asn and Thr, or an addition of Glu and Val, between the residues corresponding to Thr178 and Thr179; (3) an amino acid deletion at a residue corresponding to Asp1, Ile2 or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2); (4) an amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; and (5) an amino acid deletion at a residue corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400.

[0301] For example, laminin G-like domain variants can include (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn; (2) addition of the amino acids Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179; (3) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; and (4) substitution of the amino acid of the residue corresponding to Thr90 with Tyr, Ile, Leu or Phe.

[0302] For example, laminin G-like domain variants include (1) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (2) amino acid deletions in eight consecutive residues corresponding to Thr217 to Glu224; (3) addition of the amino acids Gly, Gly and Ser, or Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (4) amino acid deletions in nine consecutive residues corresponding to Val262 to Leu270; (5) amino acid substitutions of Lys272Asp and Gln273Arg; (6) amino acid deletions in residues corresponding to Asp1, Ile2, or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2); (7) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; and (8) amino acid deletions at residues corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400.

[0303] For example, laminin G-like domain variants include (1) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (2) amino acid deletions in eight consecutive residues corresponding to Thr217 to Glu224; (3) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (4) amino acid deletions in nine consecutive residues corresponding to Val262 to Leu270; (5) amino acid substitutions of Lys272Asp, Gln273Arg; (6) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp, and Phe361Tyr; And (7) the amino acid of the residue corresponding to Thr90 may include a substitution with Tyr, Ile, Leu or Phe.

[0304] For example, laminin G-like domain variants include (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn; (2) addition of the amino acids Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179; (3) amino acid substitutions of Met22Leu, Met22Ile or Met22Val; and Phe23Tyr; (4) amino acid deletions of eight consecutive residues corresponding to Thr217 to Glu224; (5) addition of the amino acids Gly, Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (6) amino acid deletions of nine consecutive residues corresponding to Val262 to Leu270; (7) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln and Gln273Arg, Gln273Lys or Gln273His; (8) amino acid deletions at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 (e.g., residues corresponding to Asp1 and Ile2); (9) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; and (10) amino acid deletions at residues corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400.

[0305] For example, laminin G-like domain variants include (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu, or Gly175Gln; and Asp177Glu, Asp177Gln, or Asp177Asn; (2) addition of the amino acids Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179; (3) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (4) amino acid deletions of eight consecutive residues corresponding to Thr217 to Glu224; (5) addition of the amino acids Gly, Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (6) amino acid deletions of nine consecutive residues corresponding to Val262 to Leu270; (7) amino acid substitutions of Lys272Asp and Gln273Arg; (8) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; (9) amino acid substitutions of residues corresponding to Thr90 may include substitutions with Tyr, Ile, Leu or Phe.

[0306] For example, laminin G-like domain variants include (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; and Asp177Glu, Asp177Gln or Asp177Asn; (2) addition of the amino acids Asn and Thr or addition of Glu and Val between residues corresponding to Thr178 and Thr179; (3) amino acid deletions at residues corresponding to Asp1, Ile2 or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2); (4) amino acid substitutions of Leu3Asp; (5) amino acid deletions at residues corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400; (6) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; and (7) amino acid substitutions of residues corresponding to Thr90 may include substitutions with Tyr, Ile, Leu or Phe.

[0307] For example, laminin G-like domain variants include (1) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (2) amino acid deletions in eight consecutive residues corresponding to Thr217 to Glu224; (3) addition of the amino acid Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (4) amino acid deletions in nine consecutive residues corresponding to Val262 to Leu270; (5) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu, or Lys272Gln; and Gln273Arg, Gln273Lys, or Gln273His; (6) amino acid deletion at a residue corresponding to Asp1, Ile2 or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2); (7) amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; (8) amino acid deletion at a residue corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400; (9) amino acid substitution of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; and (10) amino acid substitution of a residue corresponding to Thr90 to Tyr, Ile, Leu or Phe.

[0308] For example, laminin G-like domain variants include (1) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln and Asp177Glu, Asp177Gln or Asp177Asn; (2) amino acid additions of Asn and Thr, or additions of Glu and Val, between residues corresponding to Thr178 and Thr179; (3) amino acid substitutions of Met22Leu, Met22Ile or Met22Val; and Phe23Tyr; (4) amino acid deletions of eight consecutive residues corresponding to Thr217 to Glu224; (5) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217; (6) amino acid deletions of nine consecutive residues corresponding to Val262 to Leu270; (7) amino acid substitutions of Lys272Asp and Gln273Arg; (8) amino acid deletions at residues corresponding to Asp1, Ile2 or Asp1 and Ile2 (e.g., two residues corresponding to Asp1 and Ile2); (9) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; (10) amino acid deletions at residues corresponding to one or more consecutive or discontinuous residues (e.g., three consecutive residues) from Ala398 to Ala400; (11) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; and (12) amino acid substitutions of the residue corresponding to Thr90 to Tyr, Ile, Leu or Phe.

[0309] For example, laminin G-like domain variants can include (1) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (2) amino acid deletions of nine consecutive residues corresponding to Val262 to Leu270; and (3) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu, or Lys272Gln; and Gln273Arg, Gln273Lys, or Gln273His.

[0310] For example, laminin G-like domain variants may include (1) deletions of amino acids at residues corresponding to Asp1 and Ile2; (2) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu, or Leu3Gln; (3) amino acid substitutions of Met22Leu, Met22Ile, or Met22Val; and Phe23Tyr; (4) amino acid substitutions of Gly175Asp, Gly175Asn, Gly175Glu, or Gly175Gln; and Asp177Glu, Asp177Gln, or Asp177Asn; (5) amino acid additions of Asn and Thr, or of Glu and Val, between residues corresponding to Thr178 and Thr179; (6) additions of the amino acids Gly, Gly and Ser, or of Gly and any other one amino acid between residues corresponding to Arg216 and Thr217; (7) amino acid deletions in eight consecutive residues corresponding to Thr217 to Glu224; (8) amino acid deletions in nine consecutive residues corresponding to Val262 to Leu270; (9) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His; (10) amino acid deletions in three residues corresponding to Ala398 to Ala400; (11) amino acid substitutions of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr; and (12) amino acid substitutions of the residue corresponding to Thr90 to Tyr, Ile, Leu or Phe.

[0311] In another aspect, a laminin G-like domain variant is provided, which comprises one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications comprise:

[0312] (i) Deletion of amino acids at residues corresponding to Asp1 and Ile2 based on sequence number 1;

[0313] (ii) one or more amino acid substitutions selected from the group consisting of Leu3Asp, Met22Leu, Phe23Tyr, Gly175Asp, Asp177Glu, Lys272Asp and Gln273Arg based on sequence number 1;

[0314] (iii) Addition of Asn and Thr between residues corresponding to Thr178 and Thr179 based on sequence number 1;

[0315] (iv) Addition of Gly between residues corresponding to Arg216 and Thr217 based on sequence number 1;

[0316] (v) Deletion of amino acids from eight residues corresponding to Thr217 to Glu224 based on sequence number 1;

[0317] (vi) Deletion of amino acids in 9 residues corresponding to Val262 to Leu270 based on sequence number 1;

[0318] (vii) Deletion of amino acids at three residues corresponding to Ala398 to Ala400 based on sequence number 1;

[0319] (viii) one or more amino acid substitutions selected from the group consisting of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr based on sequence number 1; and

[0320] (ix) Amino acid substitution of Thr90Tyr based on sequence number 1.

[0321] In one embodiment, the laminin G-like domain variant described herein may further comprise, in addition to the mutations described above, an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on SEQ ID NO: 1 (x). The substituted amino acid may be selected from the group consisting of hydrophilic amino acids Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, and Tyr. It may be preferable that each amino acid be substituted with an amino acid having a similar structure and relatively greater hydrophilicity among hydrophilic amino acids. Preferred amino acid substitutions at residues corresponding to Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381 and Ala389 with respect to SEQ ID NO: 1 are presented in Table 9. More preferably, the laminin G-like domain variant may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more or eight amino acid substitutions selected from the group consisting of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser with respect to SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise the amino acid substitution(s) presented in Table 5 or the amino acid substitution(s) presented in Table 10 with respect to SEQ ID NO: 1. For example, the laminin G-like domain variant may further comprise amino acid substitutions of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser based on SEQ ID NO: 1.

[0322] In another aspect, a laminin G-like domain variant is provided, which comprises one or more amino acid modifications that improve productivity or structural stability compared to the amino acid sequence of a wild-type laminin G-like domain, wherein the one or more amino acid modifications comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on SEQ ID NO: 1. The substituted amino acid may be selected from the group consisting of hydrophilic amino acids Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, and Tyr. It may be preferable that each amino acid be substituted with an amino acid having a similar structure and relatively greater hydrophilicity among hydrophilic amino acids. More preferably, the laminin G-like domain variant may comprise one or more amino acid substitutions selected from the group consisting of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser based on SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise an amino acid substitution of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu or Ala389Ser based on SEQ ID NO: 1. For example, the laminin G-like domain variant may comprise amino acid substitutions of Leu36Gln, Ala110Asp, Leu138Gln, and Ala381Glu relative to SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise amino acid substitutions of Leu36Gln, Ala110Asp, Leu138Gln, and Ala249Asn relative to SEQ ID NO: 1.As another example, the laminin G-like domain variant may comprise amino acid substitutions of Leu36Gln, Leu138Gln, and Phe193Tyr based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise amino acid substitutions of Leu36Gln, Ala110Asp, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu, and Ala389Ser based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise amino acid substitutions of Leu36Gln, Ala110Asp, Ala249Asn, Ala254Thr, Ala381Glu, and Ala389Ser based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Leu36Gln based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Ala110Asp based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Leu138Gln based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Phe193Tyr based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Ala249Asn based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Ala254Thr based on SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Ala381Glu relative to SEQ ID NO: 1. As another example, the laminin G-like domain variant may comprise an amino acid substitution of Ala389Ser relative to SEQ ID NO: 1. For example.The laminin G-like domain variant may include amino acid substitutions of Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser based on SEQ ID NO: 1.

[0323] The expression "based on SEQ ID NO: 1" in this specification is used to specify the amino acid mutation position of the present invention and should not be construed as limiting the amino acid sequence of the laminin G-like domain or polypeptide described herein. In addition, the expression is used to determine the corresponding amino acid residue in a laminin G-like domain having the amino acid sequence of SEQ ID NO: 1 and a laminin G-like domain of a different species or protein. For example, in another laminin G-like domain, the amino acid residue or position corresponding to the amino acid mutation residue or position described herein can be determined by aligning the amino acid sequence of the other laminin G-like domain with SEQ ID NO: 1. Any method known in the art can be used to align the two sequences.

[0324] For example, for an LG-like domain comprising a sequence of SEQ ID NO: 66, amino acids corresponding to amino acids Asp1, Ile2, Leu3, Met22, Phe23, Gly175, Asp177, Lys272, Gln273, Thr178, Thr179, Arg216, Thr217 to Glu224, Val262 to Leu270, Ala398 to Ala400, Gly85, Val86, Ala239, Ala283, Val353 and Phe361 in the amino acid sequence of SEQ ID NO: 1 are Asp1, Ile2, Leu3, Met22, Phe23, Gly175, Asp177, Lys272, Gln273, Thr178, Thr179, Arg216, Thr217 to Glu224, Val262 to Leu270, Ala398 to Ala400, Gly85, Val86, Ala239, Ala283, Val353, and Phe361. As another example, for the LG-like domain comprising the sequence of SEQ ID NO: 68, the amino acids corresponding to amino acids Asp1, Ile2, Leu3, Met22, Phe23, Gly175, Asp177, Lys272, Gln273, Thr178, Thr179, Arg216, Thr217 to Glu224, Val262 to Leu270, Ala398 to Ala400, Gly85, Val86, Ala239, Ala283, Val353 and Phe361 in the amino acid sequence of SEQ ID NO: 1 are Met2, Phe3, Gly155, Asp157, Lys252, Gln253, Thr158, Thr159, Arg196, Thr197 to Glu204, Val242 to Leu250, Ala378 to Ala380, Gly65, Val66, Ala219, Ala263, Val333, and Phe341.As another example, for the LG-like domain comprising the sequence of SEQ ID NO: 76, the amino acids corresponding to amino acids Asp1, Ile2, Leu3, Met22, Phe23, Gly175, Asp177, Lys272, Gln273, Thr178, Thr179, Arg216, Thr217 to Glu224, Val262 to Leu270, Ala398 to Ala400, Gly85, Val86, Ala239, Ala283, Val353 and Phe361 in the amino acid sequence of SEQ ID NO: 1 are Asp1, Ile2, Leu3, Met22, Phe23, Lys212, Gln213, Arg156, Thr157 to Glu164, Val202 to Leu210, Ala338 to Ala340, Gly85, Val86, Ala179, Ala223, Val293, and Phe301.

[0325] It is understood that the descriptions of variations of items (i) to (x) described herein may be applied to the same items in different implementations to the extent that they do not contradict each other.

[0326] In one embodiment, the laminin G-like domain variant comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31 and SEQ ID NOs: 115 to 397.

[0327] In one embodiment, the laminin G-like domain variant comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31.

[0328] In one embodiment, the laminin G-like domain variant comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 115 to 384.

[0329] In one embodiment, the laminin G-like domain variant comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 385 to 397.

[0330] In one embodiment, the laminin G-like domain variant comprises, consists of, or consists essentially of a sequence that is, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31 and SEQ ID NOs: 115 to 397.

[0331] In one aspect of the present invention, a polypeptide is provided that comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31 and SEQ ID NOs: 115 to 397, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence.

[0332] SEQ ID NO: 1 represents the wild-type laminin G-like domain, and SEQ ID NOs: 2 to 31 represent 30 exemplary laminin G-like domain variants. A description of the major regions of these variants is provided in Table 4. The amino acid sequences of each of these are provided in Table 6. The nucleotide sequences for these amino acid sequences are provided in SEQ ID NOs: 32 to 62, respectively.

[0333] SEQ ID NOs: 115 to 384 represent other exemplary laminin G-like domain variants. Descriptions of key regions of these variants are provided in Table 7. Representative amino acid sequences of these variants are provided in Table 8. The nucleotide sequences for these amino acid sequences are readily available and can be readily obtained by those skilled in the art.

[0334] SEQ ID NOs: 385 to 397 represent other exemplary laminin G-like domain variants. Descriptions of the key regions of these variants are provided in Table 9. The amino acid sequences of these variants are provided in Table 10. The respective nucleotide sequences for these amino acid sequences can be readily ascertained and prepared by those skilled in the art.

[0335] In the above table, underlined letters in bold indicate amino acid residues that are altered compared to the wild-type (SEQ ID NO: 1) laminin G-like domain, and deletions in the mutant are not indicated.

[0336] In one embodiment, the wild-type laminin G-like domain capable of binding to a TAM receptor can be derived from a Gas6 protein or a ProS1 protein. The ProS1 protein, like the Gas6 protein, contains a laminin G-like domain and can activate the TAM receptor, and is a member of the vitamin K-dependent protein family. For example, the wild-type laminin G-like domain can comprise, consist essentially of, or consist essentially of any one sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NOs: 64 to 88. For example, the wild-type laminin G-like domain can comprise, consist essentially of, or consist essentially of any one sequence selected from the group consisting of SEQ ID NOs: 89 to 114.

[0337] In one embodiment, the LG-like domain variant described herein can be, for example, at least 70%, at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of a wild-type LG-like domain.

[0338] In one embodiment, a laminin G-like domain variant described herein comprises one or more mutations described herein and can have, for example, at least 70%, at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NOs: 64 to 88.

[0339] In one embodiment, the laminin G-like domain variant may be part of a fusion molecule, specifically a fusion protein. A specific description of the fusion molecule or fusion protein is provided below.

[0340] In another aspect, an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant described herein is provided.

[0341] In one embodiment, the engineered TAM receptor ligand polypeptide can be an engineered Gas6 polypeptide or an engineered ProS1 polypeptide. Specifically, an engineered Gas6 polypeptide comprising a laminin G-like domain variant as described herein can be provided.

[0342] In one embodiment, the engineered TAM receptor ligand polypeptide may have a deletion of the Gla domain.

[0343] In one embodiment, the engineered TAM receptor ligand polypeptide may further have a deletion of the thrombin-sensitive loop region and / or the EGF-like domain in addition to the Gla domain.

[0344] In one embodiment, the engineered TAM receptor ligand polypeptide can induce phagocytosis.

[0345] In one embodiment, the engineered TAM receptor ligand polypeptide can induce phagocytosis without an inflammatory response (compared to an antibody having an Fc region). By "without an inflammatory response" is meant a statistically significantly lower inflammatory response or no detectable inflammatory response compared to an antibody having an Fc region. A statistically significantly lower inflammatory response can be at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% lower inflammatory response compared to an antibody having an Fc region. Inflammatory responses can be measured in vitro or in vivo. For example, inflammatory responses can be measured in vitro by analyzing inflammatory cytokine expression, inflammatory enzyme activity, and cell activation markers in cells and culture media. Furthermore, inflammatory responses can be measured in vivo by analyzing inflammation-related gene expression, protein distribution, or physiological markers in tissues and fluids.

[0346] In one embodiment, the engineered TAM receptor ligand polypeptide may have improved productivity and / or structural stability compared to a TAM receptor ligand polypeptide having a wild-type LG-like domain.

[0347] In one embodiment, the engineered TAM receptor ligand polypeptide may be part of a fusion molecule, specifically a fusion protein. Specific descriptions of fusion molecules or fusion proteins are provided below.

[0348] III. Fusion Molecules

[0349] In another aspect of the present invention, a fusion molecule is provided comprising (i) an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant as described herein and not comprising a Gla domain, and (ii) a binding molecule that specifically binds to a target substance.

[0350] In one embodiment, the engineered TAM receptor ligand polypeptide can be an engineered Gas6 polypeptide or an engineered ProS1 polypeptide.

[0351] In one embodiment, the engineered TAM receptor ligand polypeptide may further have a deletion of the thrombin-sensitive loop region and / or the EGF-like domain.

[0352] In one embodiment, the binding molecule may be an antibody, an antigen-binding fragment, an antibody-like protein, a peptide, an aptamer, or a soluble receptor, but there is no particular limitation as long as it is in a form that can specifically bind to the target substance.

[0353] The antibody or antigen-binding fragment thereof may be selected from, for example, i) an immunoglobulin selected from IgG1, IgG2, IgG3 and IgG4; ii) a native antibody fragment such as Fv, Fab, Fab', F(ab')2, VHH, VNAR and the like; iii) an engineered antibody such as scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, pentabody and the like. The antibody or active fragment thereof may be, for example, a Mab, a Fab or a single-chain Fv fragment (scFv) thereof based on six complementarity-determining regions (CDRs) derived from an antibody or antibody that specifically binds to the target substance. The protein or active fragment thereof that specifically binds to a target substance is not particularly limited in form or scope as long as it contains a portion essential for the activity of specifically binding to the target substance, is linked to an engineered TAM receptor ligand polypeptide, and exhibits an effect that does not cause synaptic damage and does not involve an inflammatory response. For example, the target substance may be beta-amyloid or TNF-alpha, and the protein or active fragment thereof that specifically binds to the target substance may include aducanumab, gantenerumab, donanemab, lecanemab, adalimumab, or a single-chain Fv fragment of one of these. For example, the CDR sequences and heavy / light chain sequences of aducanumab, donanemab, lecanemab, and adalimumab are as follows.

[0354]

[0355]

[0356]

[0357] The aptamer is not limited as long as it can specifically bind to a target substance such as beta-amyloid, tau, alpha-synuclein, etc., and the base used in the aptamer may be selected from the group consisting of deoxy bases of A, G, C, and U, unless otherwise specified.

[0358] In addition, the aptamer may be modified to enhance stability by binding one or more selected from the group consisting of polyethylene glycol (PEG), inverted deoxythymidine (idT), locked nucleic acid (LNA), 2'-methoxy nucleoside, 2'-amino nucleoside, 2'F-nucleoside, amine linker, thiol linker, and cholesterol to the 5' terminal region, middle region, 3' terminal region, or both terminal regions. idT (inverted deoxythymidine) is one of the molecules generally used to prevent nuclease-induced degradation of aptamers that have low resistance to nucleases. Nucleic acid units form a chain by combining with the 3'-OH of the previous unit and the 5'-OH of the next unit, but idT is a molecule that causes an artificial change by combining the 3'-OH of the previous unit and the 3'-OH of the next unit so that the 5'-OH, not the 3'-OH, is exposed, thereby inhibiting degradation by 3'exonuclease, a type of nuclease.

[0359] In one embodiment, the target substance may be a substance that accumulates in biological tissue and causes a disease. For example, the target substance may be an autoantigen, an autoantibody, a complex of an autoantigen and an autoantibody, a cytokine, a chemokine, a complement, a receptor, an immune cell-specific marker, a cell adhesion molecule, or a combination thereof.

[0360] Specifically, the target substances are amyloid beta, amyloid beta derived from amyloid precursor protein, alpha-synuclein, and PrP. Sc, microtubule-associated protein tau, Huntington exon 1, TAR DNA-binding protein 43, superoxide dismutase 1, ABri peptide, ADan peptide, immunoglobulin light chain fragment, immunoglobulin heavy chain fragment, N-terminal fragment of serum amyloid A protein, transthyretin, β-2 microglobulin, N-terminal fragment of apolipoprotein AI, C-terminal fragment of apolipoprotein AII, N-terminal fragment of apolipoprotein AIV, apolipoprotein C-II, apolipoprotein C-III, gelsolin fragment, lysozyme, fibrinogen alpha chain fragment, N-terminal truncated cystatin C, amylin, calcitonin, artial natriuretic factor, Prolactin, insulin, Lactadherin or Medin, Lactotransferrin or Lactoferrin, Odontogenic ameloblast-associated protein, Pulmonary surfactant-associated protein C, Leukocyte cell-derived chemotaxin-2, Galectin-7, Corneodesmosin, C-terminal fragment of TGFBI (or keratoepithelin), Semenogelin-1, S100 protein (A8 or A9), Enfuvirtide, Apolipoprotein E, Apoptosis-associated Spec-like protein containing a Capase Activating Supplementary Domain The target substance may include one or more selected from the group consisting of the Recruitment Domain. Exemplary target substances and related diseases or symptoms are shown in Table 2 below.

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368] In a non-limiting exemplary embodiment, the sequence information of the target agent can be obtained from a public database, and the target agent-binding sequence can be obtained from a publication or a public database. As an exemplary embodiment, some sequences from the public database are exemplified below. Those skilled in the art will understand that the sequences of the target agent or the domain capable of binding the target agent are not limited to the specific sequences exemplified below, but include isomers, orthologues, variants, and mutants. For example, if amyloid beta derived from amyloid precursor protein is the target agent, the target agent can have a sequence available under GenBank Accession No. AAB29908.1 or a fragment thereof (e.g., amyloid beta (29-40) or Chain A (Accession No. 1BJC_A)), and the domain capable of binding the target agent can be the sequences disclosed in U.S. Patent No. 7,794,719. If the target substance is alpha-synuclein, it may have a sequence available as UniProtKB / Swiss-Prot: P37840.1 or a fragment thereof, and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Patent No. 8,609,820. If the target substance is microtubule-associated protein tau, it may have a sequence available as UniProtKB / Swiss-Prot: P10636.5 or a fragment thereof, and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Patent No. 1,028,7343. The target substance is PrP ScIf the target sequence is Huntington's disease exon 1, it may have a sequence available as GenBank Accession No. NP_001073592.1 or a fragment thereof, and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Published Patent Application No. 2021 / 0070870. If the target sequence is Huntington's disease exon 1, it may have a sequence available as GenBank Accession No. NP_001375421.1 or a fragment thereof, and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Published Patent Application No. 2022 / 0332808. If the target substance is TAR DNA binding protein 43 (TDP43), it may have a sequence available as UniProtKB / Swiss-Prot: Q13148.1 or a fragment thereof, and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Patent No. 9,587,014. If the target substance is superoxide dismutase 1 (SOD1), it may have a sequence available as GenBank: CAG46542.1 or a fragment thereof, and the domain capable of binding to the target substance may be a sequence disclosed in U.S. Patent No. 9,283,271. If the target substance is an immunoglobulin light chain fragment, it may have a sequence available as PDB: 6Z1O_A or a fragment thereof, and the domain capable of binding to the target substance may be a sequence disclosed in U.S. Patent No. 8,268,973. If the target substance is an N-terminal fragment of serum amyloid A protein, it may have a sequence available as GenBank: AAB24060.1, GenBank: AAA85338.1, NCBI NP_001372595.1 or NCBI Reference Sequence: NP_110381.2 or a fragment thereof, and the domain capable of binding to the target sequence may be the sequences disclosed in U.S. Patent No. 8,268,973. If the target substance is transthyretin, it may have a sequence available as UniProtKB / Swiss-Prot:P02766.The domain capable of binding to a target substance may have a sequence available as 1 or a fragment thereof, and the domain may be the sequences disclosed in U.S. Patent No. 11,267,877. If the target substance is amylin, IAPP (AIAPP), it may have a sequence available as UniProtKB / Swiss-Prot:P10997 or a fragment thereof, and the domain capable of binding to a target substance may be the sequences disclosed in U.S. Patent No. 10,882,902. If the target substance is APOE, it may have a sequence available as UniProtKB / Swiss-Prot:P02649.1 or a fragment thereof, and the domain capable of binding to a target substance may be the sequences disclosed in U.S. Published Patent Application No. 2022 / 0411485. If the target substance is an apoptosis-related Spec-like protein containing a caspase-activating complement domain, it may have a sequence available as UniProtKB / Swiss-Prot:Q9ULZ3.2 or a fragment thereof (e.g., U.S. Published Patent Application No. 2021 / 0079075), and the domain capable of binding to the target substance may be the sequences disclosed in U.S. Published Patent Application No. 2021 / 0079075. The entire contents of the references listed above are incorporated herein by reference.

[0369] In one embodiment, the target substance to which the binding molecule specifically binds and which is to be removed or reduced may be a substance whose amount or expression, when increased or elevated, induces, induces, or results in an undesirable or pathological immune response, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response. The target substance may be an inflammatory substance.The target substance is a self-antigen, an auto-antibody thereof, or a complex of an auto-antigen and an auto-antibody, for example, a substance listed in Table 3; Immune cell surface molecules including costimulatory molecules and receptors, such as CD20, CD19, CD52, CD80 / 86, CD28, CD40, CD40L, OX40, OX40L, C5 alpha receptor 1, FCGRT, IL1A, IL-1R, IL-6R, IL-17R, IL-4R, IL-5R (including IL5RA), IL-13R, IFN-gamma receptor, IL-12R, IL-21R, IL-22R, TGF-beta receptor, IL-23R, thymic stromal lymphopoietin receptor (TSLPR), IL-31R, IL-33R, IGF-1R, TNFR, FcRn large subunit p51, integrin alpha-D (ITGAD), Toll-like receptors (TLRs) including TLR3, TLR4, TLR5, and TLR7; Complement, such as complement C1q, complement C3, complement C5, etc.; Chemokines, such as CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, etc.; Cytokines, such as IL-1, TNF-alpha (TNFA), IL-6, IL-17, IL-4, IL-5, IL-13, IFN-gamma, IL-12, IL-21, IL-22, TGF-beta, IL-23, thymic stromal lymphopoietin (TSLP), IL-31, IL-33, etc.; It may be one or more selected from cell adhesion molecules, such as ICAM1, VCAM1, MADCAM1, integrin alpha 4, integrin beta 7, LFA-1 (or MAC-1), VLA-4, etc. Table 3 below lists exemplary references that disclose the amino acid sequences of self-antigens as well as antibodies that bind to related diseases and target substances.Those skilled in the art will appreciate that ligands, receptors or autoantibodies that bind to the listed target autoantigen substances may also be included as binding molecules of the present invention.

[0370] Antibodies that bind to immune cell surface molecules, including costimulatory molecules and receptors, may include, for example, Rituximab for CD20, Rozanolixizumab for FCGRT, Bermechimab for IL1A, Adalimumab for TNFA, Benralizumab for IL5RA, and Nemolizumab for IL31RA, and the specific sequences of such antibodies can be found in the following exemplary references: Critical Reviews in Oncology / Hematology 64 (2007) 210-225, US11618788B2, WO2021159939A1, US 9932395 B2, WO2023129870A2, US9708407B2, US10233243B2, KR20230004638A, US7531166B2, AU2008207483B2, US8536308B2, US20220280532A1, US20210017286A1, US 10738112 B2, US10259869B2, US9073999B2, CN113817058B, CN113896793B, Schroeter et al., (2015) A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display, mAbs, 7:1, 138-151], US8859739B2, US20210301028A1, US7186809B2, EP 2292665 B1, US020220081485A1, US9834600B2, WO2023027177A1, US9856317B2, US20050154192A1, EP1449851A1, US7700098B2, KR101380570B1, CN114573713A, WO2022031890A1, US8765918B2,US20220177567A1, WO2022031942A2, US9394362B2, US 7495085 B2, US7811567B2, EP2327423B1, US20230088269A1, US20030064069A1, US8012482B2, US5262319A, US20070065436A1, WO2006050949A2, EP2171060B1, US20170275356A1, US20110158992A1, US20120219565A1, US 9908941B2, US10273297B2, US20200385476A1, US10442866B1, US20100272738A1, US20200308272A1, WO2013173761A2, US 9994640 B2, US 11014990 B2, US20120149879A1, US20220002432A1, US20230212295A1, US9328169B2, EP2177537A2, US20170327584A1, EP3581585A1, US20180086833A1, KR20170120601A, US20080069777A1, CA2554965C, WO2016197974A1, US20220111045A1, US7122185B2, US11739142B2, US9005617B2, US9005617B2, US9005617B2, US20210277103A1, US9005617B2, US9005617B2, US9005617B2, CN110117329A, JP6289520B2, WO2022047125A1, US9005617B2, US8658377B2, US7601815B2, US10047156B2, US7964194B2, US10647766B2, US 20210087263A1, EP1325031A2, US11505612B2, CN101440130A, CN102665759B, US 8147834B2, US 11246883B2, 문헌[Sci Transl Med.(2019) 11(506):eaau8217], US8153583B2,US7312320B2,References [Cable Gonzalez, Communications biology (2023) 6(31)], US8703146B2 and US20210040225A1.,

[0371] The references listed in Table 3 and the entire contents of the references listed above are incorporated herein by reference.

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381] As another example, the target substance may be phosphatidylserine (PS). As an example, PS can be exposed on the surface of apoptotic cells. Molecules that bind to PS may include a Gla domain and a PS-binding molecule. PS is associated with diseases or conditions such as systemic lupus erythematosus (SLE), rheumatoid arthritis, cystic fibrosis, or Scott's syndrome (see US 7,247,303 B2, US 2011 / 0318360 A1).

[0382] In one embodiment, the LG-like domain derivative, engineered TAM receptor ligand, or fusion molecule may have phagocytosis-inducing activity through interaction with the TAM receptor. Specifically, the induction of phagocytosis may not be accompanied by an inflammatory response.

[0383] Since the fusion molecule described here induces phagocytosis through interaction with the TAM receptor, phagocytosis may be induced in cells expressing the TAM receptor. Phagocytosis generally involves engulfing cells or particles larger than 0.5 μm, including the process of tethering, engulfing, and then degrading the cells or particles. Phagocytosis may involve the formation of a phagosome that surrounds the internalized cell or particle, and may also involve the process of degrading the phagosome within the phagolysosome through the fusion of the phagosome and the lysosome. Among phagocytosis, efferocytosis is also used for cells that are dying or dying by apoptosis or necrosis.

[0384] Cells expressing TAM receptors may be one or more types of professional phagocytes, one or more types of non-professional phagocytes, or a combination thereof. Professional phagocytes are cells whose primary role is to remove dead cells and accumulated debris through phagocytosis, and include macrophages, neutrophils, dendritic cells, and mast cells. Macrophages generally reside in each tissue that can be a gateway for infection, and in many cases are called by different names depending on the tissue: adipose tissue macrophages in adipose tissue, monocytes in bone marrow or blood, Kupffer cells in the liver, sinus histiocytes in lymph nodes, alveolar macrophages in the alveoli, histiocytes or giant cells that connect them in connective tissue, microglia in the central nervous system, Hopfbauer cells in the placenta, intraglomerular mesangial cells in the kidney, osteoclasts in the bone, epithelioid cells in granulomas, red pulp macrophages in the red pulp of the spleen, and macrophages in the peritoneal cavity. Examples include peritoneal macrophages and LysoMac cells of Peyer's patches.

[0385] On the other hand, non-professional phagocytes mainly have a function specific to the tissue in which they reside, but are capable of phagocytosis when necessary, and include epithelial cells, endothelial cells, fibroblasts, mesenchymal cells, etc., and some tissue-specific cells, such as astrocytes or oligodendrocytes in the central nervous system, Muller glia in the retina, hepatocytes in the liver, satellite cells in the muscles, Sertoli cells in the testis, etc., and some lymphocytes, such as natural killer cells, large granular lymphocytes, eosinophils, basophils, and B cells. The fusion molecule according to the present invention can induce phagocytosis in phagocytes specific to the tissue in which the target substance to be removed has accumulated. For example, when removing abnormal proteins accumulated within the brain, phagocytosis can be induced in astrocytes, microglia, oligodendrocytes, or a combination thereof. This can be induced, for example, by local administration of the fusion molecule described herein to such tissues or by manipulating cells within such tissues to express and secrete the fusion molecule.

[0386] In one embodiment, the induction of phagocytosis by an LG-like domain derivative, an engineered TAM receptor ligand, or a fusion molecule may be non-inflammatory. This approach allows for the safer treatment of tissue dysfunction caused by the accumulation of target substances compared to existing techniques, as it removes the target substance without inducing an inflammatory response and suppresses tissue damage caused by the inflammatory response.

[0387] The fusion molecule described herein may additionally include a tag. Adding such a tag to the fusion molecule can be used to purify the fusion molecule, or to confirm its expression, activity, or activity process.

[0388] For example, the tags include His-tag, T7-tag, S-tag, FLAG-tag, Strep-tag, Trx (thioredoxin)-tag, His-patch thioredoxin-tag, lacZ(-Galactosidase)-tag, chloramphenicol acetyltransferase-tag, trpE-tag, avidin / streptavidin / Strep-tag, T7gene10-tag, staphylococcal protein A-tag, staphylococcal protein G-tag, GST (glutathione-S-transferase)-tag, DHFR (dihydrofolate reductase)-tag, CBD's(cellulose binding domains)-tag, MBP(maltose binding protein)-tag, galactose-binding protein (GBP)-tag, calmodulin binding protein (CBP)-tag, HAI (hemagglutinin influenza virus)-tag, HSV-tag, B-(VP7 protein region of bluetongue virus)-tag, polycysteine-tag, polyphenyalanine-tag, (Ala-Trp-Trp-Pro)n-tag, polyaspartic acid-tag, c-myc-tag, lac-repressor-tag, etc., but are not limited thereto. The tag may be located at the N-terminus, C-terminus, or internally of the protein of interest.

[0389] The fusion molecule described herein may additionally include a signal peptide or leader sequence at the N-terminus. A signal peptide is a short peptide present at the N-terminus during the early stage of synthesis of a protein destined for the secretory pathway, and is known to specify the intracellular location of the protein and (in the case of membrane proteins) membrane topology. The signal peptide may be cleaved off during the process of expression of the fusion molecule and secretion into the extracellular space.

[0390] The components included in the fusion molecule described herein may be directly linked to each other or may be linked by a linker comprising a short oligopeptide or polypeptide. Typically, the linker may comprise 2 to 500 amino acid residues. The linker is not particularly limited in length or type, as long as it is a linker that can connect each component so that it can have the intended activity, thereby forming the fusion molecule. A commonly used oligopeptide linker is, for example, a linker in the form of (GGGGS)n, i.e., one or more Gly-Gly-Gly-Gly-Ser units repeated (SEQ ID NO: 398). In addition, (GSSGGS)n (SEQ ID NO: 399), KESGSVSSEQLAQFRSLD (SEQ ID NO: 400), EGKSSGSGSESKST (SEQ ID NO: 401), GSAGSAAGSGEF (SEQ ID NO: 402), (EAAAK)n (SEQ ID NO: 403), CRRRRRREAEAC (SEQ ID NO: 404), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 405), GGGGGGGG (SEQ ID NO: 406), GGGGGG (SEQ ID NO: 407), AEAAAKEAAAAKA (SEQ ID NO: 408), PAPAP (SEQ ID NO: 409), (Ala-Pro)n (SEQ ID NO: 410), VSQTSKLTRAETVFPDV (SEQ ID NO: 411), PLGLWA (SEQ ID NO: 412), TRHRQPRGWE (SEQ ID NO: 413), AGNRVRRSVG (SEQ ID NO: 414), RRRRRRRR (SEQ ID NO: 415), GFLG (SEQ ID NO: 416), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 417) can also be used as a linker, but are not limited thereto. n, which represents the number of repetitions of the linker unit, is, for example, an integer of 1 to 20, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0391] The fusion molecule can have the following structure from N-terminus to C-terminus: [binding molecule]-[engineered TAM receptor ligand polypeptide] or [engineered TAM receptor ligand polypeptide]-[binding molecule]. When including a linker, the fusion molecule can have the following structure from N-terminus to C-terminus: [binding molecule]-[linker]-[engineered TAM receptor ligand polypeptide] or [engineered TAM receptor ligand polypeptide]-[linker]-[binding molecule]. In these structures, the engineered TAM receptor ligand polypeptide can comprise the LG-like domain variant together with other domains, or can be the LG-like domain variant alone.

[0392] The fusion molecule may further comprise a scaffold, wherein the TAM receptor ligand polypeptide or the binding molecule is linked to the scaffold, or wherein both the TAM receptor ligand polypeptide and the binding molecule are linked to the scaffold at different locations on the scaffold. The scaffold may include, but is not limited to, a single chain Fc region with reduced or eliminated Fc receptor binding affinity, a multimeric Fc region with reduced or eliminated Fc receptor binding affinity, an antibody without a variable region, or an Fc-hinge region with reduced or eliminated Fc receptor binding affinity. The TAM receptor ligand polypeptide may be linked or fused to one location on the scaffold, and the binding molecule may be linked or fused to another location on the scaffold. The linkage or fusion between the TAM receptor ligand polypeptide / binding molecule and the scaffold may be a direct linkage or may be via a linker.

[0393] IV. Nucleic Acids, Expression Vectors, and Cells

[0394] In another aspect, provided herein is a nucleic acid molecule encoding a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide or a fusion molecule, or an expression vector comprising the same.

[0395] The nucleic acid sequence encoding the laminin G-like domain variant, engineered TAM receptor ligand polypeptide or fusion molecule described herein may be mutated by substitution, deletion, insertion or a combination thereof of one or more nucleic acid bases, as long as it encodes a protein or peptide having equivalent activity.

[0396] All or part of the nucleic acid sequence encoding the laminin G-like domain variant, engineered TAM receptor ligand polypeptide, or fusion molecule described herein may be isolated from nature, artificially synthesized, or produced by genetic recombination methods. The nucleic acid sequence encoding the laminin G-like domain variant, engineered TAM receptor ligand polypeptide, or fusion molecule is provided operably linked to an expression vector capable of expressing the same.

[0397] An "expression vector" is a vector capable of expressing a target protein or target RNA by introducing a nucleic acid sequence encoding a target gene into a suitable host cell, and refers to a genetic construct that includes essential regulatory elements operably linked to allow the gene insert to be expressed. The term "operably linked" refers to the functional linkage of two or more components arranged so as to allow the described construct to function in its intended manner. For example, when a promoter sequence is operably linked to a sequence encoding a protein A, this means that the promoter is linked to the sequence encoding the protein A so that the sequence encoding the protein A is transcribed and / or expressed in the cell. In addition, the term includes all other meanings generally recognized by those skilled in the art and can be appropriately interpreted according to the context. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[0398] Suitable expression vectors have expression control elements such as a promoter, initiation codon, termination codon, polyadenylation signal, and enhancer. The initiation codon and termination codon are generally considered to be part of the nucleic acid sequence encoding the protein, and the protein coding sequence is designed to be in-frame in the vector to enable operation. The promoter may be constitutive or inducible. Additionally, typical expression vectors include a selection marker. Operable linkage with the expression vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0399] The expression vector may be preferably constructed to express and subsequently purify and isolate the laminin G-like domain variant, engineered TAM receptor ligand polypeptide or fusion molecule described herein in a host cell, or to be introduced into a cell in vivo so that the cell can express and secrete said laminin G-like domain variant, engineered Gas6 polypeptide or fusion molecule. When the purpose is to introduce cells in vivo, the vector may preferably be a non-integrating vector, i.e., a vector that does not integrate into the genome of the host cell.

[0400] In another aspect, cells producing a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, or a fusion molecule described herein are provided. In one embodiment, a cell producing a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, or a fusion molecule described herein may be transformed to include a nucleic acid molecule described herein or an expression vector comprising the same. "Transformation" includes any method for introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as is known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0401] Host cells include, but are not limited to, prokaryotic host cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus). Additionally, fungi such as Aspergillus, yeasts including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa, and the like, other lower eukaryotic cells, or cells derived from higher eukaryotes including insect cells, plant cells, mammals, etc., can be used as host cells.

[0402] V. Manufacturing Method

[0403] In another aspect of the present invention, a method of making a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide or a fusion molecule is provided, comprising culturing a host cell under conditions suitable for expression of the laminin G-like domain variant, engineered TAM receptor ligand polypeptide or fusion molecule described herein, and recovering the laminin G-like domain variant, engineered TAM receptor ligand polypeptide or fusion molecule from the culture.

[0404] For recombinant production of the laminin G-like domain variants, engineered TAM receptor ligand polypeptides or fusion molecules described herein, a polynucleotide encoding the laminin G-like domain variants, engineered TAM receptor ligand polypeptides or fusion molecules described herein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be isolated or sequenced using conventional methods known in the art.

[0405] Culturing transformed cells can be performed using methods widely known in the art. For example, laminin G-like domain variants, engineered TAM receptor ligand polypeptides, or fusion molecules can be mass-produced by culturing transformants in nutrient media. The media and culture conditions can be appropriately selected based on host cell tolerance. During culture, conditions such as temperature, media pH, and culture time can be appropriately adjusted to ensure cell growth and mass protein production.

[0406] To recover the laminin G-like domain variants, engineered TAM receptor ligand polypeptides or fusion molecules described herein, conventional biochemical separation techniques for separation and purification, such as treatment with protein precipitants (salting out), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and various chromatographies, can be used, and these are usually used in combination to isolate highly pure proteins (Sambrook et al., Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)).

[0407] VI. Pharmaceutical Composition

[0408] In another aspect of the present invention, a pharmaceutical composition is provided comprising an effective amount of a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, a fusion molecule, a nucleic acid molecule, or an expression vector described herein. The laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, a fusion molecule, a nucleic acid molecule, or an expression vector described herein can be formulated into a pharmaceutical composition by mixing it with a pharmaceutically acceptable carrier. Accordingly, a pharmaceutical composition comprising a laminin G-like domain variant, an engineered TAM receptor ligand polypeptide, a fusion molecule, a nucleic acid molecule, or an expression vector described herein and a pharmaceutically acceptable carrier can be provided.

[0409] The pharmaceutical composition may be used to prevent or treat diseases or conditions characterized by or associated with abnormal accumulation of a target substance within a living body. The pharmaceutical composition may be used to remove the target substance within a living body. Furthermore, the pharmaceutical composition may be used to prevent or treat immune diseases in a subject. The target substance is defined as described above.

[0410] In one embodiment, the target agent is amyloid beta, amyloid beta derived from amyloid precursor protein, alpha-synuclein, PrP Sc, microtubule-associated protein tau, Huntington exon 1, TAR DNA-binding protein 43, superoxide dismutase 1, ABri peptide, ADan peptide, immunoglobulin light chain fragment, immunoglobulin heavy chain fragment, N-terminal fragment of serum amyloid A protein, transthyretin, β-2 microglobulin, N-terminal fragment of apolipoprotein AI, C-terminal fragment of apolipoprotein AII, N-terminal fragment of apolipoprotein AIV, apolipoprotein C-II, apolipoprotein C-III, gelsolin fragment, lysozyme, fibrinogen alpha chain fragment, N-terminally truncated cystatin C, amylin, calcitonin, artial natriuretic factor, prolactin, Insulin, Lactadherin or Medin, Lactotransferrin or Lactoferrin, Odontogenic ameloblast-associated protein, Pulmonary surfactant-associated protein C, Leukocyte cell-derived chemotaxin-2, Galectin-7, Corneodesmosin, C-terminal fragment of TGFBI (or keratoepithelin), Semenogelin-1, S100 protein (A8 or A9), Enfuvirtide, Apolipoprotein E, Apoptosis-associated Spec-like protein containing a Capase Activating Recruitment Domain It may include one or more selected from the group consisting of:

[0411] Preferably, the target substance may be amyloid beta or amyloid beta derived from amyloid precursor protein.

[0412] In one embodiment, the target substance is a self-antigen, an auto-antibody thereof, or a complex of an auto-antigen and an auto-antibody, for example, a substance listed in Table 3; Immune cell surface molecules including costimulatory molecules and receptors, such as CD20, CD19, CD52, CD80 / 86, CD28, CD40, CD40L, OX40, OX40L, C5 alpha receptor 1, FCGRT, IL1A, IL-1R, IL-6R, IL-17R, IL-4R, IL-5R (including IL5RA), IL-13R, IFN-gamma receptor, IL-12R, IL-21R, IL-22R, TGF-beta receptor, IL-23R, thymic stromal lymphopoietin receptor (TSLPR), IL-31R, IL-33R, IGF-1R, TNFR, FcRn large subunit p51, integrin alpha-D (ITGAD), Toll-like receptors (TLRs) including TLR3, TLR4, TLR5, and TLR7; Complement, such as complement C1q, complement C3, complement C5, etc.; Chemokines, such as CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, etc.; Cytokines, such as IL-1, TNF-alpha (TNFA), IL-6, IL-17, IL-4, IL-5, IL-13, IFN-gamma, IL-12, IL-21, IL-22, TGF-beta, IL-23, thymic stromal lymphopoietin (TSLP), IL-31, IL-33, etc.; It may be one or more selected from cell adhesion molecules, such as ICAM1, VCAM1, MADCAM1, integrin alpha 4, integrin beta 7, LFA-1 (or MAC-1), VLA-4, etc.

[0413] Diseases or conditions characterized by or associated with the accumulation of target substances in vivo may include, but are not limited to, the diseases or conditions listed in Table 3. In one embodiment, the diseases or conditions are:

[0414] Alzheimer's disease, hereditary intracerebral hemorrhage with amyloidosis, Parkinson's disease, Parkinsonian dementia, Lewy body dementia, multiple system atrophy, transmissible spongiform encephalopathy, fatal familial insomnia, Gerstmann-Sträussler-Scheinker disease, Creutzfeldt-Jakob disease, new variant Creutzfeldt-Jakob disease, tauopathy, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain disease, frontotemporal dementia, amyotrophic lateral sclerosis, familial British dementia, familial Danish dementia, light chain amyloidosis, heavy chain amyloidosis, AA amyloidosis, senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy. Leptomeningeal amyloidosis, dialysis-related amyloidosis, hereditary visceral amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, ApoCII amyloidosis, ApoCIII amyloidosis, familial amyloidosis (Finnish type), hereditary gelsolin amyloidosis, hereditary non-neuropathic systemic amyloidosis, fibrinogen amyloidosis, hereditary intracranial hemorrhage with amyloidosis (Icelandic type), type 2 diabetes, insulinoma, medullary thyroid cancer, cardiac arrhythmia, solitary atrial amyloidosis, pituitary prolactinoma, focal amyloidosis at the site of insulin injection, aortic valve medial amyloidosis, gelatinous drop-like corneal dystrophy, calcifying epithelial odontogenic tumor, Alveolar proteinosis, renal LECT2 amyloidosis, lichen amyloidosis, macular amyloidosis, hypotrichosis simplex of the scalp, lattice corneal dystrophy (type I,Type 3A or Avellino type), seminal vesicle amyloidosis, prostate cancer, injection-localized amyloidosis, amyloid cerebroangiopathy, cardiovascular disease, spinocerebellar ataxia, frontotemporal lobar degeneration, mild cognitive impairment, Parkinson's plus syndrome, progressive isolated aphasia, gray matter degeneration (Alpers) or subacute necrotizing encephalomyelopathy.

[0415] In one embodiment, the disease may be Alzheimer's disease or hereditary cerebral hemorrhage with amyloidosis.

[0416] Symptoms characterized by or associated with the accumulation of target substances within the body can include a variety of physical and functional changes, depending on the type of target substance and the site of accumulation. For example, accumulation of amyloid beta or amyloid beta derived from amyloid precursor protein in the brain can cause symptoms such as cognitive decline, memory loss, behavioral changes, language impairment, and impaired spatial cognition, which are commonly observed in Alzheimer's disease or hereditary intracerebral hemorrhage associated with amyloidosis.

[0417] Furthermore, accumulation of alpha-synuclein in the brain can lead to symptoms such as movement disorders, tremors, muscle stiffness, gait abnormalities, and postural instability, which are associated with diseases such as Parkinson's disease, Parkinson's dementia, and dementia with Lewy bodies. Accumulation of the microtubule-associated protein tau can be accompanied by symptoms such as memory loss, impaired judgment, personality changes, and motor dysfunction, which are characteristic of tauopathies, Pick's disease, and progressive supranuclear palsy.

[0418] Accumulation of target substances, such as transthyretin or immunoglobulin light chain fragments, in the heart can cause symptoms such as heart failure, arrhythmias, cardiac hypertrophy, and fatigue, as observed in familial amyloid cardiomyopathy or light chain amyloidosis. Accumulation of beta-2 microglobulin or immunoglobulin light chain fragments in the kidney can cause symptoms such as renal failure, proteinuria, hematuria, and edema, as associated with dialysis-associated amyloidosis.

[0419] Accumulation of N-terminal fragments of serum amyloid A protein or fibrinogen alpha chain fragments in the liver can cause symptoms such as liver dysfunction, jaundice, hepatomegaly, and ascites, as seen in AA amyloidosis or hereditary visceral amyloidosis. Accumulation of amyloid proteins (e.g., gelsolin fragments) in the skin can cause skin lesions, itching, pigmentation, and thickening, as seen in lichen amyloidosis and macular amyloidosis.

[0420] In the peripheral nervous system, accumulation of transthyretin or gelsolin fragments can cause symptoms such as paresthesias, muscle weakness, and autonomic dysfunction (e.g., orthostatic hypotension and gastrointestinal dysfunction), which are associated with familial amyloid polyneuropathy or hereditary gelsolin amyloidosis. In the eye, accumulation of the C-terminal fragment of TGFBI (keratoepithelin) or other amyloid proteins can cause symptoms such as decreased vision, corneal opacity, and tearing, which are observed in lattice corneal dystrophy or gelatinous droplet corneal dystrophy.

[0421] In one embodiment, the disease or condition may be an immune disease. In another embodiment, the immune disease is an autoimmune disease or an inflammatory disease. In another embodiment, the autoimmune disease or inflammatory disease may be multiple sclerosis (MS), rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, an antibody-mediated inflammatory or autoimmune disease, graft-versus-host disease, sepsis, type 1 diabetes, type 2 diabetes, psoriasis, atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, or inflammatory autoimmune myositis. Specifically, the spondyloarthropathies may be selected from ankylosing spondylitis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel disease, psoriatic arthritis, isolated acute anterior uveitis, undifferentiated spondyloarthropathy, Behcet's syndrome, and juvenile idiopathic arthritis. In another embodiment, the immune disease may be caused or worsened by excessive binding of an antigenic substance to immunoglobulins or immune cells, or by an increase in the amount of an antigenic substance or an increase in the expression of an antigenic substance. For example, the immune cell may be a dendritic cell. In another embodiment, the immune disease may be, but is not limited to, any of the diseases or conditions listed in Table 3.

[0422] The term "effective amount" means an amount of a variant, polypeptide, fusion molecule, nucleic acid molecule or expression vector sufficient to achieve the desired efficacy or activity.

[0423] The pharmaceutical composition may be administered orally or parenterally, preferably parenterally, and more preferably locally to the tissue where the target substance to be removed has accumulated.

[0424] “Parenteral administration” includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, intraperitoneal administration, vaginal administration, intrathecal administration, intrathecal administration, intracerebroventricular administration or infusion techniques.

[0425] When formulating a pharmaceutical composition into an injection, it can be prepared according to conventional injection manufacturing methods known in the art. The injection may be dispersed in a sterile medium so that it can be administered directly to a patient, or it may be dispersed in distilled water for injection to an appropriate concentration before administration.

[0426] When formulating a pharmaceutical composition into an oral dosage form, one or more of a diluent, a lubricant, a binder, a disintegrant, a sweetener, a stabilizer, and a preservative can be selected and used as a carrier, and one or more of a flavoring agent, a vitamin, and an antioxidant can be selected and used as an additive.

[0427] Techniques required for formulating pharmaceutical compositions and pharmaceutically suitable carriers, excipients, etc. are widely known to those skilled in the art of pharmaceutical science, and reference can be made to, for example, the Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000); and Remington's Pharmaceutical Sciences (19th ed., 1995).

[0428] The appropriate dosage of a pharmaceutical composition can vary depending on factors such as the formulation method, administration method, patient age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. An exemplary dosage of a pharmaceutical composition for adults is 0.0001 to 1000 μg / kg (body weight).

[0429] VII. Treatment Methods

[0430] In another aspect of the present invention, a method is provided for preventing or treating a disease comprising administering to a subject in need thereof an effective amount of a laminin G-like domain variant, engineered TAM receptor ligand polypeptide, fusion molecule, nucleic acid molecule or expression vector described herein, a disease or condition characterized by or associated with accumulation of a target substance.

[0431] In another aspect, a method is provided for removing a target agent from a subject, comprising administering to the subject in need thereof a laminin G-like domain variant, engineered TAM receptor ligand polypeptide, fusion molecule, nucleic acid molecule or expression vector described herein.

[0432] In another aspect, a method is provided for preventing or treating an immune disorder in a subject, delaying the progression of a symptom associated with an immune disorder, or alleviating a symptom of an immune disorder, comprising administering to the subject in need thereof a laminin G-like domain variant, engineered TAM receptor ligand polypeptide, fusion molecule, nucleic acid molecule, or expression vector described herein.

[0433] The target substances that can be removed by administering the laminin G-like domain variants, engineered TAM receptor ligand polypeptides, fusion molecules, nucleic acid molecules or expression vectors described herein and the diseases or conditions associated with or characterized by abnormal accumulation of such target substances, or immune diseases and symptoms associated therewith, are as described above.

[0434] For example, the disease could be Alzheimer's disease or hereditary cerebral hemorrhage with amyloidosis.

[0435] For example, the disease may be an autoimmune disease or an inflammatory disease.

[0436] All patents and references cited herein are incorporated herein by reference in their entirety.

[0437] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0438] Example

[0439] Example 1. Engineering of Gas6 (LG-like domain)

[0440] To engineer the Gas6 LG-like domain (SEQ ID NO: 1), we used theories related to existing known protein structures, Inpainting, a deep learning model for protein structure design, and Rosetta (Rosetta Commons) software to perform calculations on the design and stability of the protein structure. In addition, filtering was performed based on the designed structure prediction and stability using AlphaFold (Google DeepMind). The modules used to create the protein structure within the Rosetta software are the Remodel module and the Fast Design module. The Remodel module is a program that customizes loops according to the design, and the Fast Design module is a module that designs the protein structure while performing FastRelax, which optimizes and stabilizes the protein structure.

[0441] To improve structural stability, two crystal structures of Gas6 (PDB codes: 1H30, 4ra0) were analyzed using computational proteomic techniques, and nine mutation categories were classified, including regions with no or low electron density in the LG-like domain, the loop region, the N terminus, and the C terminus, and the following modifications were made. All sequences were designed using Rosetta software.

[0442] (1) Region 1 (residues 1 to 3 of the LG-like domain of SEQ ID NO: 1) - N-terminal stabilization: Deletion of one or two amino acids at the N-terminus of the Gas6 LG-like domain was thought to increase structural stability. Residues 1 and 2 at the N-terminus of the Gas6 LG-like domain were deleted, and the resulting leucine (Leu) at position 3 was substituted with aspartic acid (Asp) (this substitution replaces a hydrophobic amino acid with a hydrophilic amino acid; Asn, Glu, or Gln could also be used instead of Asp). The non-limiting rationale for this mutation is as follows: Removal of a flexible or non-contributing portion of the N-terminus increases structural stability.

[0443] (2) Region 2 (residues 21 to 27 of the LG-like domain of SEQ ID NO: 1) - Loop stabilization: This region is a loop region adjacent to the secondary receptor binding site. Sequence design was performed based on protein structure analysis. As a result, methionine (Met) at position 22 and phenylalanine (Phe) at position 23 were substituted with leucine (Leu) and tyrosine (Tyr), respectively. The non-limiting rationale for this mutation is as follows: Based on the predicted structure, Met22 faces inward and Phe23 is exposed on the surface. By replacing the inward-facing residue with a hydrophobic residue, the hydrophobic binding is strengthened (Met22Ile or Met22Val can also be used instead of Met22Leu), and the surface-exposed Phe is replaced with Tyr with an OH group to increase hydrophilicity.

[0444] (3) Region 3 (residues 173 to 183 of the LG-like domain of SEQ ID NO: 1) - Stabilization of the alpha helix structure: This region is a loop region corresponding to the main receptor binding site, and stability was improved by transforming this region into an alpha helix form. The length and sequence of amino acids within the alpha helix that can stabilize the alpha helix were designed using the inpainting model and Rosetta software. As a result, glycine (Gly) at position 175 and aspartic acid (Asp) at position 177 were replaced with aspartic acid (Asp) (Gly175Asn, Gly175Glu, or Gly175Gln can be used instead of Gly175Asp) and glutamic acid (Glu) (Asp177Gln, or Asp177Asn can be used instead of Asp177Glu), respectively, and two amino acids, asparagine (Asn) and threonine (Thr), were added between threonine (Thr) at position 178 and threonine (Thr) at position 179. Instead of the two amino acids asparagine (Asn) and threonine (Thr), two amino acids, glutamic acid (Glu) and valine (Val), can be added between positions 178 and 179. The non-limiting rationale for this mutation is as follows: it stabilizes the structure by rebuilding the alpha helix.

[0445] (4) Region 4 (residues 216 to 226 based on the LG-like domain of SEQ ID NO: 1) - Stabilization of the loop structure: Threonine (Thr) at position 217 to Glutamic acid (Glu) at position 224 were removed, residues 216 and 225 were connected, and glycine (Gly) was added between them (at position 217). Removal of one or more amino acids from Threonine (Thr) at position 217 to Glutamic acid (Glu) at position 224 may also contribute to the stabilization of the structure. In addition, instead of glycine (Gly), two amino acids, glycine (Gly) and serine (Ser), or glycine (Gly) and any other two amino acids, can be added (more residues can be added because it is a loop, but two is optimal). Sequence design was performed using Rosetta software. The non-limiting rationale for this mutation is as follows: it increases structural stability by removing a region predicted to be highly flexible due to low or absent electron density in the crystal structure. More specifically, the region from Thr217 to Ser225 (TPLDVGTES) behind Arg216 is removed, and glycine (Gly) and serine (Ser) are inserted to provide a more stable link at the cleavage site.

[0446] (5) Region 5 (residues 262 to 274 based on the LG-like domain of SEQ ID NO: 1) - Stabilization of the ring structure: Valine (Val) at position 262 to leucine (Leu) at position 270 were removed, and lysine (Lys) at position 272 and glutamine (Gln) at position 273 were replaced with aspartic acid (Asp) (Lys272Asn, Lys272Glu or Lys272Gln may be used instead of Lys272Asp) and arginine (Arg) (Gln273Lys or Gln273His may be used instead of Gln273Arg), respectively, and linked. Removal of one or more amino acids from Valine (Val) at position 262 to Leucine (Leu) at position 270 may contribute to structural stabilization. Sequence design was performed using Rosetta software. The non-limiting rationale for this mutation is as follows: Structural stability is increased by removing regions predicted to be highly flexible due to low or no electron density in the crystal structure. Lys272, which is left after removal, can be replaced with a negatively charged amino acid such as Asp to prevent charge repulsion with Lys271, or other amino acids such as Glu. Gln273 can also be replaced with a positively charged amino acid such as Arg to stabilize the charge with the substituted Asp272, or other amino acids such as Lys and His.

[0447] (6) Region 6 (residues 398 to 400 of the LG-like domain of SEQ ID NO: 1) - C-terminal stabilization: Amino acid residues 398 to 400 were deleted. The non-limiting rationale for this mutation is as follows: increasing structural stability by removing flexible or non-contributing portions of the C-terminus.

[0448] (7) Considering the three-dimensional structure, six hydrophobic amino acid residues exposed to the outside were replaced with hydrophilic amino acids (based on the LG-like domain of SEQ ID NO: 1, Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp, Phe361Tyr). The hydrophilic amino acids can be selected from the group consisting of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro, and Tyr.

[0449] (8) Based on the LG-like domain of SEQ ID NO: 1, a Thr90Tyr substitution was introduced for hydrophobic interactions (Thr90Ile, Thr90Leu, or Thr90Phe may also be used instead of Thr90Tyr). The non-limiting rationale for this mutation is as follows: replacing Thr90 with a hydrophobic amino acid is to stabilize the structure by strengthening the hydrophobic interaction with the structurally close Ile125 (hydrophobic and bulky amino acids were selected considering the structural space).

[0450] (9) In addition, eight hydrophobic amino acid residues exposed on the surface were additionally substituted with hydrophilic residues (based on the LG-like domain of SEQ ID NO: 1, Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu, Ala389Ser) (“secondary mutations.” In this specification, the term “primary or secondary mutation” is used for convenience in distinguishing mutations and should not be interpreted in any other way), and interactions with adjacent amino acid residues were also considered.

[0451] Gas6 candidates (LG-like domain variants) engineered according to the above design were derived. Since FastRelax, which is performed during the FastDesign process, calculates amino acid combinations, a large number of design results were generated. Among these, 30 candidates containing mutation(s) referred to as primary mutation or primary and secondary mutations for convenience were selected through visual inspection using PYMOL graphics software and used in the experiments (SEQ ID NOs: 2 to 31).

[0452] The primary mutation stabilizes the alpha-helix and beta-sheet structures, optimizing the ring structure, N-terminus, and C-terminus. Furthermore, mutant models of Gas6 with key mutations were designed using Rosetta software. The designed mutant models were used to measure changes in protein stability using the AlphaFold program. The key mutations are presented in Table 4.

[0453]

[0454] The secondary mutations serve to stabilize the structure and include mutations of the eight amino acid residues listed in Table 5.

[0455] Exemplary variants of the present invention include all primary mutations (mutants indicated in bold in Table 4) and optionally secondary mutations (Table 5). Variants 1 to 30 (SEQ ID NOs: 2 to 31) of Table 5 correspond to Gas6 LG1-LG2 V1 to Gas6 LG1-LG2 V30 of Table 6. Gas6 LG1-LG2 V1 to Gas6 LG1-LG2 V30 include all primary mutations (mutants indicated in bold in Table 4) and the inclusion or absence of secondary mutation(s) and the mutation details are presented in Table 5.

[0456]

[0457] The amino acid sequences for the wild-type LG-like domain and the mutants in Table 5 are presented in Table 6.

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464] In addition to SEQ ID NOS: 2 to 31, a total of 158 exemplary laminin G-like domain variants (SEQ ID NOS: 115 to 272) comprising variant(s) belonging to any one of the eight major variant categories described above and a total of 111 exemplary laminin G-like domain variants (SEQ ID NOS: 273 to 383) comprising combination(s) of variants belonging to two or more of the eight major variant categories were additionally designed. A description of the additionally designed variants is provided in Table 7.

[0465]

[0466]

[0467] The amino acid sequences of exemplary variants among the laminin G-like domain variants comprising a variant(s) belonging to any one of the eight major variant categories described above are presented in Table 8.

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474] Additionally, we designed laminin G-like domain variants (SEQ ID NOs: 385 to 397) containing only the secondary mutation(s) listed in Table 9. Using Rosetta software, we designed variant models of Gas6 containing only the secondary mutations. The designed variant models were then analyzed for changes in protein stability using the AlphaFold program.

[0475]

[0476] Examples of laminin G-like domain variants containing only secondary mutations and their amino acid sequences are presented in Table 10.

[0477]

[0478]

[0479]

[0480] Example 2. Preparation of engineered Gas6 (LG-like domain variant) and fusion molecules.

[0481] Example 2.1. Preparation of Engineered Gas6 (LG-Like Domain Variant)

[0482] Based on the LG-like domain variant sequence provided in Example 1, a DNA sequence was designed and codon-optimized to match the human (Homo sapiens) codon. To facilitate protein purification, a 6xhistidine-tag (6xHis-Tag) was added to the C-terminus, and a gene was synthesized based on the optimized DNA sequence, and then cloned into the pcDNA3.4 vector using restriction enzymes to construct an expression plasmid. Transient expression was performed by transfecting HEK293F cells with the constructed plasmid using the Lipofectamine method. After culturing HEK293F cells with the plasmid introduced at 37°C for 4 to 5 days, cells were removed by centrifugation, and the culture supernatant was collected. Thus, LG-like domain mutants (Gas6 LG1-LG2 V1 to Gas6 LG1-LG2 V30; Gas6 LG1-LG2 V31 to Gas6 LG1-LG2 V300; Gas6 LG1-LG2 V301 to Gas6 LG1-LG2 V305; and Gas6 LG1-LG2 V306 to Gas6 LG1-LG2 V313) were obtained. As controls, wild-type Gas6 or LG-like domains were prepared in the same manner.

[0483] Example 2.2. Preparation of fusion molecules

[0484] (1) Aducanumab-fused eGas6 (Adu-eGas6)

[0485] To enable Gas6 to specifically bind to a desired target substance, the Gla domain, which is a site that recognizes PS (phosphatidylserine), was removed from Gas6, and a single-chain variable fragment (scFv) of aducanumab, an amyloid beta (Aβ)-specific antibody, was introduced as a targeting substance in that position. In addition, the EGF-like domain present within the Gas6 protein was also removed. Adu-Gas6 (WT) manufactured in this way has the following structure from the N-terminus to the C-terminus: [signal peptide]-[Adu scFv]-[linker]-[Gas6 LG-like domain]-[His tag]. The specific sequence is as follows.

[0486] [Signal peptide]: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 418)

[0487] [Adu scFv]:

[0488] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQ LVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 464)

[0489] [Linker]: GGGGSGGGGS (SEQ ID NO: 423)

[0490] [Gas6 LG-like domain]: amino acid sequence of SEQ ID NO: 1

[0491] [His tag]: HHHHHH (SEQ ID NO: 424)

[0492] Adu-Gas6(WT), denoted as mIgK-Adu(VL-VH)-(G4S)2-GAS6-6xHis:

[0493]

[0494] Similarly, the scFV fragment of aducanumab was linked to the sequence of the LG-like domain presented in Example 2.1 as a linker to prepare Adu-eGas6 V1 to V30 and V31 to V300.

[0495] The sequences of Adu-eGas6 V1 to V30, V31 to V299 and V300 are identical except that the LG-like domain variant sequences presented in Example 1 (Gas6 LG1-LG2 V1 to Gas6 LG1-LG2 V30; Gas6 LG1-LG2 V31 to Gas6 LG1-LG2 V299; and Gas6 LG1-LG2 V300) are used instead of the LG-like domain sequence of Adu-Gas6 (WT).

[0496] Proteins were produced using the method described in Example 2.1 using plasmids capable of producing each protein.

[0497] (2) eGas6 fused with gantenerumab (scFv) (Gan-eGas6)

[0498] The scFv fragment of gantenerumab (Gan), an amyloid beta (Aβ)-specific antibody, was introduced as a targeting substance at the position of the aducanumab scFv of Adu-Gas6 (WT) or Adu-eGas6 V2 produced in (1) above. The Gan-Gas6 (WT) or Gan-eGas6 V2 produced in this way has the following structure from the N-terminus to the C-terminus: [signal peptide]-[Gan scFv]-[linker]-[Gas6 LG-like domain]-[His tag]. The specific sequence is as follows.

[0499] [Signal peptide]: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 419)

[0500] [Gan scFv]:

[0501] DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKGGGGSGGGGSGGGGSQVEL VESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS (SEQ ID NO: 465)

[0502] [Linker]: GGGGSGGGGS (SEQ ID NO: 423)

[0503] [Gas6 LG-like domain]: amino acid sequence of SEQ ID NO: 1 or amino acid sequence of SEQ ID NO: 3

[0504] [His tag]: HHHHHH (SEQ ID NO: 424)

[0505] Gan-Gas6, denoted as mIgK-Gan(VL-VH)-(G4S)2-GAS6-6xHis:

[0506]

[0507] or

[0508]

[0509] Here, the sequences of Gantenerumab_hVH and Gantenerumab_hVL are presented in SEQ ID NOs: 425 and 426, respectively.

[0510] Proteins were produced using the method described in Example 2.1 using plasmids capable of producing each protein.

[0511] (3) eGas6 (bivalent construct) fused with aducanumab (scFv) and Fc

[0512] Adu(scFv)-Fc-Gas6(WT) was prepared by linking the scFv fragment and Fc of the wild-type Gas6 described in (1) above and the aducanumab antibody. The Adu(scFv)-Fc-Gas6(WT) thus prepared has the following structure from the N-terminus to the C-terminus: [signal peptide]-[Adu scFv]-[Fc]-[linker]-[Gas6 LG-like domain]-[His tag]. In the CH2 portion of the Fc portion, asparagine at position 297 was mutated to alanine. The specific sequence is as follows.

[0513] [Signal peptide]: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 418)

[0514] [Adu scFv]:

[0515] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQ LVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 464)

[0516] [hinge]: EPKSCDKTHTCPPCP (SEQ ID NO: 427)

[0517] [Fc]:

[0518] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 466)

[0519] [Linker]: GGGGSGGGGS (SEQ ID NO: 423)

[0520] [Gas6 LG-like domain]: amino acid sequence of SEQ ID NO: 1

[0521] [His tag]: HHHHHH (SEQ ID NO: 424)

[0522]

[0523] Here, the sequences of Aducanumab_hVH and Aducanumab_hVL are presented in SEQ ID NOs: 420 and 421, respectively, CH2_N297A and CH3 are presented in SEQ ID NOs: 432 and 433, respectively, and the linker GGGGSGGGGSGGGGS is presented in SEQ ID NO: 422.

[0524] And in the same manner as described above, the scFv fragment and Fc of the gantenerumab antibody were linked to eGas6 V28 to produce Gan(scFv)-Fc-eGas6 V28. The Gan(scFv)-Fc-eGas6 V28 produced in this way has the following structure from the N-terminus to the C-terminus: [signal peptide]-[Gan scFv]-[Fc]-[linker]-[Gas6 LG-like domain]-[His tag]. Here, CH2 of the Fc portion had the 297th asparagine mutated to alanine. The specific sequence is as follows.

[0525] [Signal peptide]: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 418)

[0526] [Gan scFv]:

[0527] DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKGGGGSGGGGSGGGGSQVEL VESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS (SEQ ID NO: 465)

[0528] [Hinge]: DKTHTCPPCP (SEQ ID NO: 428)

[0529] [Fc]:

[0530] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 466)

[0531] [Linker]: GGGGSGGGGS (SEQ ID NO: 423)

[0532] [Gas6 LG-like domain]: amino acid sequence of SEQ ID NO: 29

[0533] [His tag]: HHHHHH (SEQ ID NO: 424)

[0534]

[0535] Here, the sequences of Gantenerumab_hVH and Gantenerumab_hVL are presented in SEQ ID NOs: 425 and 426, respectively, the sequences of CH2_YTE,N297A and CH3 are presented in SEQ ID NOs: 429 and 433, respectively, and the linker GGGGSGGGGSGGGGS is presented in SEQ ID NO: 422.

[0536] Proteins were produced using the method described in Example 2.1 using plasmids capable of producing each protein.

[0537] (4) Gas6 (bivalent structure) fused with adalimumab (IgG)

[0538] (1) Ada(IgG)-eGas6 V2 or Ada(IgG)-eGas6 V28 was produced by linking the heavy chain portion of adalimumab (Ada), a tumor necrosis factor alpha (TNF-α)-specific bivalent antibody, to eGas6 V2 or eGas6 V28. The Ada(IgG)-eGas6 V2 or Ada(IgG)-eGas6 V28 produced in this manner has the following structure from the N-terminus to the C-terminus: [signal peptide]-[Ada]-[linker]-[Gas6 LG-like domain]-[His tag]. Here, CH2 of the Fc portion in the adalimumab sequence had the 297th asparagine mutated to alanine. The specific sequence is as follows.

[0539] [Signal peptide]: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 418)

[0540] [Adalimumab heavy chain]:

[0541] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 467)

[0542] [Linker]: GGGGSGGGGS (SEQ ID NO: 423)

[0543] [Gas6 LG-like domain]: amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 29

[0544] [His tag]: HHHHHH (SEQ ID NO: 424)

[0545]

[0546] or

[0547]

[0548] Here, the sequence of Adalimumab_hVH is presented in SEQ ID NO: 430, the sequences of CH1, CH2_YTE, N297A and CH3 are presented in SEQ ID NOs: 431, 432 and 433, respectively, and the linker GGGGSGGGGSGGGGS is presented in SEQ ID NO: 422.

[0549] The amino acid sequence encoded by the plasmid capable of producing the light chain portion of adalimumab is as follows.

[0550] [Signal peptide]: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 418)

[0551] [Adalimumab light chain]:

[0552] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 468)

[0553]

[0554] Here, the sequences of Adalimumab_hVL and Adalimumab_hCL are presented in SEQ ID NOs: 434 and 435, respectively.

[0555] Proteins were produced using the method described in Example 2.1 using plasmids capable of producing each protein.

[0556] Example 3. Productivity Evaluation of Engineered Gas6 (LG-Like Domain Variant) and Fusion Molecules

[0557] Example 3.1. Expression / productivity of Adu-eGas6 (1)

[0558] Adu-Gas6 (WT) and Adu-eGas6 V1 to V30 were transfected into HEK293F cells using a plasmid that introduced a 6xHis-Tag at the C-terminus using the Lipofectamine method to perform transient expression. On the fifth day of culture, the culture medium was harvested, and the expression level was measured using an enzyme-linked immunosorbent assay (ELISA). First, the amount of eGas6 was measured using an anti-hGas6 antibody (R&D system) (Fig. 4a), and then the productivity was compared with that of the wild type using an anti-histidine antibody (Abcam) (Fig. 4b). Adu-eGas6s showed an approximately 2- to 10-fold increase in productivity compared to Adu-Gas6 (WT) (Figs. 4a, 4b, and Table 11).

[0559] Example 3.2. Purity / Productivity of Adu-eGas6

[0560] The Gas6 fusions produced in Example 2.2 were purified using two-step chromatography. First, for the first-step purification, the culture medium was loaded onto nickel (Ni)-affinity chromatography, and then eluted with a concentration gradient using a buffer containing imidazole to obtain an eluate containing Gas6 fusions. After analyzing the purity of the Gas6 fusions contained in the obtained eluate, the second-step purification was performed. The second-step purification was performed by diluting the eluate obtained in the first-step purification, injecting it into ion exchange resin chromatography, and then recovering the Gas6 fusions using a salt (NaCl) concentration gradient. The final purity of the recovered fusion was confirmed by size-exclusion chromatography for analysis, and the productivity was calculated by measuring the protein concentration of the fusion using a UV spectrometer, and is expressed as mg / L value converted to 1 L of culture medium scale (Table 11).

[0561]

[0562] All Gas6 fusion constructs containing these mutants were purified to a purity similar to or better than that of the wild-type construct. Furthermore, the productivity of the Gas6 fusion constructs containing the mutants was approximately threefold higher than that of the wild-type Gas6 fusion construct. We confirmed that the fusion constructs containing the mutants had higher productivity with a similar purity compared to the wild-type fusion construct.

[0563] Example 3.3. Expression level / productivity of Adu-eGas6 (2)

[0564] As demonstrated in Example 3.1, Adu-eGas6 V1 to V30 (containing either the primary mutation or the primary mutation plus the secondary mutation) exhibited increased productivity compared to the wild type. To determine whether mutants containing only the primary mutation(s) also exhibited increased productivity compared to the wild type, the laminin G-like domain mutants listed in Table 7 were used to evaluate productivity.

[0565] Among the Adu-eGas6 V31 to V299 (also referred to as “V31” to “V299”) mutants, representative mutants V57, V189, V193, V195, V202, V205, V208, V229, V255, V264, V273, V278, and V289 were compared in productivity compared to the wild type. The mutation positions and mutation categories of each mutant are shown in Table 12.

[0566]

[0567] To evaluate the productivity of the produced Adu-eGas6 mutants, sandwich ELISA was performed to measure the titer in the culture medium. Anti-G4S (E702V) rabbit antibody (Cell Signaling) was diluted to a concentration of 0.2 μg / mL in 50 mM carbonate buffer (pH 9.6) and dispensed into a 96-well plate at 100 μL per well. The wells were incubated overnight at 4°C to coat the antibody, and each well of the 96-well plate was washed five times with 0.05% Tween-20 / PBS (PBST). Subsequently, 200 μL of 1% BSA / PBST was added to each well, incubated at 37°C for 1 hour, and then washed five times with PBST. After washing, 100 μL of Adu-eGas6 culture medium diluted in DPBS was added to each well, incubated at 37°C for 1 hour, and washed five times with PBST. Subsequently, anti-histidine (AD1.1.10) HRP antibody (Invitrogen) was diluted to a concentration of 0.3 μg / mL in 1% BSA / PBST, dispensed 100 μL per well into a 96-well plate, incubated at 37°C for 1 hour, and washed five times with PBST. After washing, 100 μL of tetramethylbenzidine (TMB) solution was added to each well, and the reaction was carried out for 20 minutes at room temperature in a light-protected state. The reaction was stopped by adding 100 μL of stop solution to each well, and the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. The experiment was repeated three times.

[0568] All of these mutants exhibited at least a 1.5-fold increase in productivity compared to the wild type (Figure 5a). As can be seen from Figure 5a, all mutants that applied any one or a combination of two or more of the four mutation categories presented in Table 12 exhibited increased productivity compared to the wild type.

[0569] Additionally, to determine whether the productivity increases compared to the wild type even when applying a minimal mutation in any of the four mutation categories, the productivity of Adu(scFv)-V300 (hereinafter referred to as “V300”), which applied only two of the three mutations belonging to the loop optimization, was compared to the wild type, compared to Adu(scFv)-V273, which applied three mutations. The experiment was repeated three times. A detailed description of V300 is shown in Table 13.

[0570]

[0571] As a result, V300 was confirmed to exhibit nearly threefold increased productivity compared to the wild type (Fig. 5b). This indicates that not only mutants that applied only one of the four mutation categories exhibited increased productivity, but also mutants that applied only a portion of a single mutation category, but not all, of the mutations.

[0572] Example 3.4. Expression / Productivity of Engineered Gas6

[0573] (1) LG-like domain variants containing all primary mutations

[0574] The expression levels of Gas6 (WT) and eGas6 (eGas6 V2, V3, V18, V20, V21, and V28) recovered in Example 2.1 were evaluated by SDS-PAGE experiment. The experiment was repeated three times. Specifically, 10 μL of culture medium of Gas6 (WT) and eGas6 was loaded per well, electrophoresed on a 4-12% Bis-Tris gel (Thermo Fisher) at 150 V for 80 minutes, and then stained with Coomassie blue solution to confirm protein expression. After destaining, the stained gel was laser scanned at a wavelength of 700 nm with an ODYSSEY M (LI-COR) instrument, and the measurement results were analyzed and quantified with Empiria Studio 3.1 (LI-COR). The results are shown in Figs. 6a, 6b, and 6c. Referring to Figure 6c, which quantifies the results of Figure 6b as signal intensity, the eGas6s of the present invention showed expression enhancement of more than fourfold compared to the wild type. These results indicate that the expression level of the LG-like domain variants themselves exhibits a similar tendency to that of the form bound to the antigen-binding molecule (Adu). This suggests that the protein mutations resulting from the engineering of the present invention increase the expression level of LG-like domain-containing proteins, regardless of the presence or absence of antigen-binding molecule fusion.

[0575] (2) Variants containing only secondary mutation(s)

[0576] In addition, to confirm whether the LG-like domain variants that applied only the secondary mutations presented in Table 5 showed improved productivity compared to Gas6 (WT), eGas6 V2, eGas6 V3, eGas6 V18, eGas6 V20, and eGas6 V21 were selected from the variants with improved expression levels, and variants (eGas6 V301 to eGas6 V305) that applied only the secondary mutations were produced using the method described in Example 2.1. Subsequently, a comparison of the productivity between the variants was performed using the SDS-PAGE method in the same manner as described above. A description of the produced variants is shown in Table 10.

[0577] As a result, all mutants with only secondary mutations applied showed productivity 1.6 times higher than that of the wild type, and in particular, the productivity of eGas6 V303 increased approximately 3.19 times compared to the wild type (Fig. 7a).

[0578] Additionally, eight mutants (eGas6 V306 to eGas6 V313) containing one of the eight secondary mutations were produced. The descriptions of the produced mutants are shown in Table 10. As a result of performing a productivity comparison, all of these mutants showed improved productivity compared to the wild type, and in particular, the L314Q mutant (V306), in which leucine at position 314 was substituted with glutamic acid, showed an approximately 2.79-fold increase in productivity compared to the wild type (Fig. 7b).

[0579] Example 3.5. Productivity of Gas6 (Gan-Gas6) fused to gantenerumab (scFv)

[0580] The Gas6 protein fused with gantenerumab prepared in Example 2.2 was purified in the same manner as in Example 3.2, and its productivity was measured. It was confirmed that the mutant Gas6 protein fused with gantenerumab also exhibited superior productivity than the wild type (Table 14).

[0581]

[0582] Example 3.6. Gas6 (bivalent construct) fused to aducanumab (scFv) and Fc

[0583] The Gas6 protein fused with the bivalent antibody manufactured in Example 2.2 was purified by the method of Example 3.2, and the productivity was measured. Although the sequences of scFv in the bivalent antibodies Adu(scFv)-Fc-Gas6 WT and Gan(scFv)-Fc-eGas6 V28 used for comparison in the experiment were different, the productivities of Adu(scFv)-Gas6 WT and Gan(scFv)-Gas6 WT in Examples 3.2 and 3.5 purified by the same method (Tables 11 and 14) were similar, so it was determined that the difference in the sequence of scFv would not have a significant effect on the productivity. It was also confirmed that the mutant showed a superior productivity than the wild type in the Gas6 protein fused with the bivalent antibody (Table 15).

[0584]

[0585] Example 3.7. Gas6 (bivalent construct) fused to adalimumab (IgG)

[0586] The Gas6 protein fused with the adalimumab antibody prepared in Example 2.2 was purified using the same method as in Example 3.2, and its productivity was measured. It was confirmed that the variant of the Gas6 protein fused with the IgG antibody exhibited excellent productivity (Table 16).

[0587]

[0588] Example 4. Evaluation of Adu-eGas6 Productivity According to Production Cell Line

[0589] The productivity of Adu-Gas6 (WT) and Adu-eGas6 was evaluated using different production cell lines than in Example 2.1. Adu-Gas6 (WT) and Adu-eGas6 were produced and purified using Expi293F and ExpiCHO cell lines using the same process as in Example 2.2 and Example 3.2. As a result, the Expi239F cell line showed a 3 to 6-fold or greater improvement in productivity compared to Adu-Gas6 (WT), and the ExpiCHO cell line showed a 5 to 20-fold or greater improvement in productivity compared to Adu-Gas6 (WT) (Figs. 8a and 8b).

[0590] Example 5. Axl binding confirmation test of Adu-eGas6

[0591] Example 5.1. Enzyme-linked immunosorbent assay

[0592] To evaluate the Axl binding activity of Adu-eGas6, ELISA was performed. Human Axl protein (Sino Biological) was diluted to a concentration of 0.5 μg / mL in DPBS (Dulbecco's Phosphate-Buffered Saline) and added 100 μL per well to 96-well plates. Incubated overnight at 4°C to coat, followed by washing four times with 0.05% Tween-20 / PBS (PBST). After washing, 200 μL per well of 3% BSA / PBST was added for blocking, and then washed four times with PBST. 100 μL per well of Adu-eGas6 diluted to various concentrations was added and incubated for 2 hours at room temperature. The plates were washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature to induce a reaction. After washing four times with PBST, 100 μL of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well as a secondary antibody and incubated at room temperature for 1 hour. After washing, 100 μL of tetramethylbenzidine (TMB) solution was added per well and the color was developed for 10 minutes. After stopping the reaction with stop solution, the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer.

[0593] As a result, all tested Adu-eGas6s showed binding activity toward Axl protein (Fig. 9).

[0594] Example 5.2. Fluorescence-activated cell sorting (FACS)

[0595] To confirm the binding activity of Adu-eGas6 to Axl expressed on the cell surface, human Axl overexpressing THP-1 cell line (hereinafter referred to as THP-1Axl After treating THP-1 cells with Adu-eGas6, Adu-eGas6 bound to Axl on the cell surface was detected using a flow cytometer (Becton Dickinson). First, THP-1 cells were resuspended in FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen / Strep + 20 mM HEPES). Axl The cell lines were incubated with Adu-eGas6 at 4°C for 1 hour. To remove Adu-eGas6 that did not bind to cell surface Axl and remained in the supernatant, FACS solution was added to each well, centrifuged at 2,000 rpm for 3 minutes, and the supernatant was removed. This washing process was repeated twice. To detect Adu-eGas6 bound to cell surface Axl, G4S Linker (E7O2V) Rabbit mAb (Cell Signaling Technology) was diluted 1:50 in FACS solution, added 50 μL to each well, and incubated at 4°C for 30 minutes. After washing twice, the mean fluorescence intensity (MFI) was analyzed using a flow cytometer.

[0596] As a result, it was confirmed that all tested Adu-eGas6s could bind to human Axl protein expressed on the cell surface (Fig. 10).

[0597] Example 5.3. Bio-layer interferometry (BLI)

[0598] To measure the dissociation constant of Adu-eGas6, biolayer interferometry (BLI) experiments were performed using Octet R8 (Sartorius). Biotinylated AXL (Acro Biosystems) was attached to a streptavidin biosensor tip (Sartorius) for 120 s. Then, the sensor tip was immersed in kinetic buffer (Kinetics Buffer, Sartorius) for 60 s to determine the baseline. Antibody samples of different concentrations underwent a 480 s binding phase and a 480 s dissociation phase. All reactions were performed in kinetic buffer. The binding reactions were analyzed using Octet Data Analysis 10.0 software (Sartorius) to determine the dissociation constant (K D ) were calculated (Table 17). As a result of the calculation, the Adu-eGas6 used in the experiment showed higher binding affinity than Adu-Gas6 (WT).

[0599]

[0600] Example 5.4. Confirmation of Axl signal activation by Adu-eGas6

[0601] U2OS was used to evaluate the Axl receptor activating ability of Adu-eGas6. Axl The analysis system was used. The system stably overexpresses the human osteosarcoma cell line U2OS with ProLink-attached Axl and SH2 domain proteins with enzyme acceptor (EA) attached. Axl(Eurofins DiscoverX) is used. When the Axl receptor reacts to the Sh2 domain, Prolink and EA attached to Axl generate chemiluminescence, which can be used to measure receptor activity. Since this chemiluminescence is sensitive to changes in Axl receptor activity by Gas6, the dose-dependence of receptor activity was confirmed by treating Adu-Gas6 (WT) and Adu-eGas6 at various concentrations (Fig. 11a). In addition, amyloid beta aggregates (oligomeric Aβ) were additionally treated to evaluate amyloid beta aggregate-specific receptor activity and analyze the Alzheimer's disease-specific efficacy of Adu-eGas6 (Fig. 11b).

[0602] As a result, Adu-eGas6 dose-dependent receptor activity was observed in both the groups treated with amyloid beta aggregates and those not treated, and it was confirmed that the activity was enhanced in the group treated with amyloid beta aggregates.

[0603] Example 6. Evaluation of Adu-eGas6-based amyloid beta (Aβ)-specific phagocytosis activity.

[0604] THP-1 overexpressing the Axl receptor Axl Adu-Gas6 (WT), Adu-eGas6, and amyloid-beta aggregate-specific phagocytosis by Adu-eGas6 were analyzed in cells and human microglia cell line HMC3 using an Incucyte Live-Cell Analysis System device (Satorius##). This assay was performed by conjugating pHrodo, a pH-sensitive fluorescent indicator, to amyloid-beta, and then using THP-1 Axl When cells and HMC3 engulf amyloid beta aggregates and transport the amyloid proteins to intracellular lysosomes, the fluorescent indicator glows, allowing for assessment of phagocytic activity.

[0605] As a result, all tested Adu-eGas6s were confirmed to increase cellular phagocytosis (Figs. 12a and 12b). Phagocytosis activity induced by Adu-eGas6s (V2, V13, V21, V23, V28, and V29) was comparable to that of Adu-Gas6 (WT) (Fig. 12b).

[0606] Example 7. Stability test of Adu-eGas6

[0607] The liquid stability of Adu-eGas6 was evaluated by measuring thermal stability. Accelerated stability tests were performed at room temperature (25°C) on purified Adu-Gas6 (WT) and six types of Adu-eGas6. The stability test was performed by aliquoting Adu-eGas6 at a concentration of 4.5 mg / mL in a PBS liquid condition containing 0.05% polysorbate 20. Protein samples were collected at weeks 0, 1, 2, and 4, and the purity and protein concentration were analyzed by SE-HPLC and Micro-CE-SDS.

[0608] As a result of the stability evaluation, differences in purity and protein reduction were observed depending on the Adu-eGas6, but it was confirmed that stability was improved in all Adu-eGas6 compared to Adu-Gas6 (WT) (Figs. 13a and 13b).

[0609] statistical processing

[0610] Each experiment was repeated three times, and all values ​​are expressed as the mean ± standard deviation (SD). Statistical significance was assessed at the p<0.05 level. Error bars in the graphs represent standard deviation. Statistical analysis was performed using GraphPad Prism software, using one-way ANOVA followed by Dunnett's multiple comparison test.

Claims

1. A laminin G-like domain variant comprising one or more amino acid modifications compared to the amino acid sequence of the wild-type laminin G-like domain, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise at least one selected from the group consisting of (i) to (x): (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1; (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272, and Gln273 based on sequence number 1; (iii) addition of one, two or three amino acids between residues corresponding to Thr178 and Thr179 based on sequence number 1; (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1; (v) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on SEQ ID NO: 1; (vi) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO: 1; (vii) Deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO: 1; (viii) Substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 based on sequence number 1 with a hydrophilic amino acid; (ix) The amino acid of the residue corresponding to Thr90 in sequence number 1 is replaced with Tyr, Ile, Leu or Phe; and (x) An amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on sequence number 1.

2. In paragraph 1, One or more of the above amino acid mutations (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Met22, Phe23, Lys272, and Gln273 based on sequence number 1; (iv) addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on sequence number 1; (v) deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on sequence number 1; and (vi) comprising at least one selected from the group consisting of deletions of amino acids at residues corresponding to at least one continuous or discontinuous residue selected from Val262 to Leu270 (including both terminal residues) based on sequence number 1; Laminin G-like domain variants.

3. In paragraph 1, One or more of the above amino acid mutations (ii) an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Gly175 and Asp177 based on sequence number 1; and (iii) comprising at least one selected from the group consisting of one, two or three additional amino acids between residues corresponding to Thr178 and Thr179 based on sequence number 1; Laminin G-like domain variants.

4. In paragraph 1, One or more of the above amino acid mutations (i) Deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2 based on sequence number 1; (ii) amino acid substitution at the residue corresponding to the Leu3 residue based on sequence number 1; and (vii) comprising at least one selected from the group consisting of deletions of amino acids at residues corresponding to at least one continuous or discontinuous residue selected from Ala398 to Ala400 (including both terminal residues) based on sequence number 1; Laminin G-like domain variants.

5. In paragraph 1, A laminin G-like domain variant wherein said one or more amino acid mutations comprise (i) a deletion of an amino acid at a residue corresponding to Asp1, Ile2, or Asp1 and Ile2 based on SEQ ID NO:

1.

6. In paragraph 5, A laminin G-like domain variant wherein said one or more amino acid mutations comprise (i) a deletion of amino acids at residues corresponding to Asp1 and Ile2 based on SEQ ID NO:

1.

7. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise an amino acid substitution at a residue corresponding to one or more amino acid residues selected from the group consisting of Leu3, Met22, Phe23, Gly175, Asp177, Lys272 and Gln273 based on SEQ ID NO:

1.

8. In paragraph 7, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise (ii) one or more amino acid substitutions selected from the group consisting of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His, based on SEQ ID NO:

1.

9. In paragraph 1, A laminin G-like domain variant wherein said one or more amino acid mutations comprise (iii) an addition of one or two amino acids between residues corresponding to Thr178 and Thr179 based on SEQ ID NO:

1.

10. In paragraph 9, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179 based on SEQ ID NO:

1.

11. In paragraph 1, A laminin G-like domain variant, wherein said one or more mutations comprise (iv) an addition of one, two or three amino acids between residues corresponding to Arg216 and Thr217 based on SEQ ID NO:

1.

12. In paragraph 11, A laminin G-like domain variant, wherein said one or more mutations comprise (iv) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217 of SEQ ID NO:

1.

13. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Thr217 to Glu224 (including both terminal residues) based on SEQ ID NO:

1.

14. In paragraph 13, A laminin G-like domain variant, wherein said one or more mutations comprise a deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224 based on SEQ ID NO:

1.

15. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Val262 to Leu270 (including both terminal residues) based on SEQ ID NO:

1.

16. In paragraph 15, A laminin G-like domain variant wherein said one or more amino acid mutations comprise a deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270 based on SEQ ID NO:

1.

17. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise a deletion of an amino acid at a residue corresponding to one or more consecutive or discontinuous residues selected from Ala398 to Ala400 (including both terminal residues) based on SEQ ID NO:

1.

18. In paragraph 17, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise a deletion of amino acids at three consecutive residues corresponding to Ala398 to Ala400 based on SEQ ID NO:

1.

19. In paragraph 1, A laminin G-like domain variant, wherein the at least one amino acid mutation comprises a substitution of an amino acid corresponding to at least one residue selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353, and Phe361 based on SEQ ID NO: 1 with a hydrophilic amino acid.

20. In paragraph 19, A laminin G-like domain variant, wherein in the above (viii), the hydrophilic amino acid is selected from the group consisting of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Pro and Tyr.

21. In paragraph 19, A laminin G-like domain variant, wherein at least one amino acid substitution of (viii) above comprises at least one amino acid substitution selected from the group consisting of Gly85Lys, Val86Arg, Ala239Gln, Ala283Pro, Val353Asp and Phe361Tyr.

22. In paragraph 1, A laminin G-like domain variant wherein at least one amino acid mutation comprises a substitution of the amino acid of a residue corresponding to Thr90 in SEQ ID NO: 1 with Tyr, Ile, Leu or Phe.

23. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise an amino acid substitution at a residue corresponding to one or more residues selected from the group consisting of Leu36, Ala110, Leu138, Phe193, Ala249, Ala254, Ala381, and Ala389 based on SEQ ID NO:

1.

24. In paragraph 23, The one or more amino acid mutations are (x) Leu36Gln or Leu36Gly; Ala110Asp or Ala110Trp; Leu138Gln, Leu138Gly, Leu138 Ala or Leu138 Ser; Phe193Tyr, Phe193Gly, Phe193Ser, Phe193Ala, Phe193Thr, Phe193Asp, Phe193Glu, Phe193Cys, Phe193Gln, Phe193Asn, Phe193Pro, Phe193Val, Phe193Lys, Phe193His, Phe193Ile, Phe193Lys, Phe193Met or Phe193Arg based on SEQ ID NO: 1; A laminin G-like domain variant comprising at least one amino acid substitution selected from the group consisting of Ala249Asn, Ala249Asp, Ala249Glu, Ala249His, Ala249Ser or Ala249Thr; Ala254Thr, Ala254Asp, or Ala254His; Ala381Glu, Ala381Trp, Ala381Ser, Ala381Gly, Ala381His, Ala381Thr, Ala381Phe, Ala381Asn, Ala381Tyr, Ala381Arg or Ala381Lys; and Ala389Ser, Ala389Asp or Ala389Glu.

25. In paragraph 23, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise one or more amino acid substitutions selected from the group consisting of (x) Leu36Gln, Ala110Asp, Leu138Gln, Phe193Tyr, Ala249Asn, Ala254Thr, Ala381Glu and Ala389Ser.

26. In paragraph 1, A laminin G-like domain variant, wherein said one or more amino acid mutations comprise amino acid mutations of (i) to (ix).

27. In paragraph 26, A laminin G-like domain variant wherein said one or more amino acid mutations further comprise an amino acid mutation of said (x).

28. In paragraph 1, The laminin G-like domain variant comprises (1) an amino acid substitution of Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln and an amino acid substitution of Asp177Glu, Asp177Gln or Asp177Asn and (2) an addition of Asn and Thr or an addition of Glu and Val between residues corresponding to Thr178 and Thr179, based on SEQ ID NO:

1.

29. In paragraph 1, The laminin G-like domain variant comprises (1) amino acid substitutions of Met22Leu, Met22Ile or Met22Val; and Phe23Tyr, based on SEQ ID NO: 1, (2) deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224, (3) addition of Gly, addition of Gly and Ser, or addition of Gly and any other amino acid between residues corresponding to Arg216 and Thr217, (4) deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270, and (5) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His, or (1) amino acid substitutions of Met22Leu and Phe23Tyr; (2) amino acid deletions at residues corresponding to Val262 to Leu270; and (3) amino acid substitutions of Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln and Gln273Arg, Gln273Lys or Gln273His. Laminin G-like domain variants.

30. In paragraph 1, The laminin G-like domain variant comprises (1) a deletion of an amino acid at a residue corresponding to Asp1, Ile2 or and Ile2, (2) an amino acid substitution of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln and (3) a deletion of an amino acid at one, two or three consecutive residues corresponding to Ala398 to Ala400 based on SEQ ID NO:

1.

31. In paragraph 1, The above laminin G-like domain variant is based on SEQ ID NO:

1. (i) deletion of amino acids at residues corresponding to Asp1, Ile2, or Asp1 and Ile2; (ii) amino acid substitutions of Leu3Asp, Leu3Asn, Leu3Glu or Leu3Gln; Met22Leu, Met22Ile or Met22Val; Phe23Tyr; Gly175Asp, Gly175Asn, Gly175Glu or Gly175Gln; Asp177Glu, Asp177Gln or Asp177Asn; Lys272Asp, Lys272Asn, Lys272Glu or Lys272Gln; and Gln273Arg, Gln273Lys or Gln273His; (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179; (iv) addition of Gly between residues corresponding to Arg216 and Thr217, addition of Gly and Ser, or addition of Gly and any other amino acid; (v) Deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224; (vi) Deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270; (vii) Deletion of amino acids in three consecutive residues corresponding to Ala398 to Ala400; (viii) a substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353 and Phe361 with a hydrophilic amino acid; and (ix) A laminin G-like domain variant comprising an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu and Thr90Phe.

32. In paragraph 1, The above laminin G-like domain variant is based on SEQ ID NO:

1. (i) Deletion of amino acids at residues corresponding to Asp1 and Ile2; (ii) amino acid substitutions of Leu3Asp; Met22Leu; Phe23Tyr; Gly175Asp; Asp177Glu; Lys272Asp; and Gln273Arg; (iii) addition of Asn and Thr, or addition of Glu and Val, between residues corresponding to Thr178 and Thr179; (iv) addition of Gly between residues corresponding to Arg216 and Thr217, addition of Gly and Ser, or addition of Gly and any other amino acid; (v) Deletion of amino acids in eight consecutive residues corresponding to Thr217 to Glu224; (vi) Deletion of amino acids in nine consecutive residues corresponding to Val262 to Leu270; (vii) Deletion of amino acids in three consecutive residues corresponding to Ala398 to Ala400; (viii) a substitution of an amino acid corresponding to one or more residues selected from the group consisting of Gly85, Val86, Ala239, Ala283, Val353 and Phe361 with a hydrophilic amino acid; and (ix) A laminin G-like domain variant comprising an amino acid substitution selected from the group consisting of Thr90Tyr, Thr90Ile, Thr90Leu and Thr90Phe.

33. In paragraph 1, A laminin G-like domain variant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 31, SEQ ID NOs: 115 to 384, and SEQ ID NOs: 385 to 397.

34. In paragraph 1, A laminin G-like domain variant wherein the laminin G-like domain is derived from Gas6 or ProS1.

35. In paragraph 1, The above laminin G-like domain is a laminin G-like domain variant that is part of a fusion protein.

36. In paragraph 1, The laminin G-like domain variant is a laminin G-like domain variant comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of a wild-type laminin G-like domain.

37. An engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant of any one of claims 1 to 36.

38. In paragraph 37, The engineered TAM receptor ligand polypeptide is an engineered Gas6 polypeptide or an engineered ProS1 polypeptide.

39. In paragraph 34, The engineered TAM receptor ligand polypeptide is an engineered TAM receptor ligand polypeptide in which the Gla domain is deleted.

40. In paragraph 39, An engineered TAM receptor ligand polypeptide, wherein the engineered TAM receptor ligand polypeptide further lacks at least one selected from the group consisting of a thrombin-sensitive loop domain and an EGF-like domain. 41.(i) A fusion molecule comprising an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant of any one of claims 1 to 36 and not comprising a Gla domain, and (ii) a binding molecule that specifically binds to a target substance. 42.(i) A fusion molecule comprising an engineered TAM receptor ligand polypeptide comprising a laminin G-like domain variant of any one of claims 1 to 36 and (ii) a binding molecule that specifically binds to a target substance.

43. In paragraph 41 or 42, The engineered TAM receptor ligand polypeptide is a fusion molecule, wherein the engineered Gas6 polypeptide or the engineered ProS1 polypeptide.

44. In paragraph 41 or 42, A fusion molecule wherein the engineered TAM receptor ligand polypeptide further lacks at least one selected from the group consisting of a thrombin-sensitive loop domain and an EGF-like domain.

45. In paragraph 41 or 42, The above binding molecule is a fusion molecule, which is an antibody, an antigen-binding fragment, an antibody-like protein, a peptide, an aptamer or a soluble receptor.

46. ​​In paragraph 41 or 42, A fusion molecule wherein the binding molecule is a Gla domain or an antibody, antigen-binding fragment, antibody-like protein, peptide, aptamer, or soluble receptor that binds to phosphatidylserine (PS).

47. In paragraph 41 or 42, The above binding molecule is a fusion molecule that is an antibody, antigen-binding fragment, antibody-like protein, peptide, aptamer, or soluble receptor that binds to amyloid beta or TNF alpha.

48. In paragraph 41 or 42, The above target substance is a fusion molecule that accumulates in biological tissue and causes disease.

49. In paragraph 41 or 42, The target substance is a fusion molecule that is a self-antigen, an auto-antibody, a complex of an auto-antigen and an auto-antibody, a cytokine, a chemokine, a complement, a receptor, an immune cell-specific marker, a cell adhesion molecule, or a combination thereof.

50. In paragraph 41 or 42, The above engineered TAM receptor ligand polypeptide is a fusion molecule having phagocytosis-inducing activity through interaction with the TAM receptor.

51. In paragraph 50, The above induction of phagocytosis is a fusion molecule that does not involve an inflammatory response.

52. A nucleic acid molecule encoding a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, or a fusion molecule of any one of claims 41 to 51.

53. An expression vector comprising the nucleic acid molecule of item 52.

54. A host cell producing a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, or a fusion molecule of any one of claims 41 to 51.

55. A pharmaceutical composition comprising an effective amount of a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, a fusion molecule of any one of claims 41 to 51, a nucleic acid molecule of claim 52, or an expression vector of claim 53.

56. In paragraph 55, A pharmaceutical composition for the prevention or treatment of a disease or disorder characterized by abnormal accumulation of a target substance in a living body.

57. In paragraph 55, A pharmaceutical composition for preventing or treating an immune disease in a subject.

58. A method for preventing or treating a disease or disorder caused by abnormal accumulation or aggregation of a target substance in a subject, comprising administering to the subject a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, a fusion molecule of any one of claims 41 to 51, a nucleic acid molecule of claim 52, or an expression vector of claim 53.

59. A method for reducing or eliminating a target substance to a normal level or inhibiting the production or formation of a target substance in a subject, comprising administering to the subject a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, a fusion molecule of any one of claims 41 to 51, a nucleic acid molecule of claim 52, or an expression vector of claim 53.

60. A method for preventing or treating an immune disease in a subject, delaying the progression of a symptom associated with an immune disease, or alleviating a symptom of an immune disease, comprising administering to the subject a laminin G-like domain variant of any one of claims 1 to 36, an engineered TAM receptor ligand polypeptide of any one of claims 37 to 40, a fusion molecule of any one of claims 41 to 51, a nucleic acid molecule of claim 52, or an expression vector of claim 53.

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