WO2024101382A1

WO2024101382A1 - Composition to be used in immune checkpoint regulation

Info

Publication number: WO2024101382A1
Application number: PCT/JP2023/040173
Authority: WO
Inventors: 哲治仲; 聡世良田; 穣藤本
Original assignee: 学校法人岩手医科大学
Priority date: 2022-11-09
Filing date: 2023-11-08
Publication date: 2024-05-16

Abstract

The present disclosure addresses the problem of providing a novel composition to be used in immune checkpoint regulation. One aspect of the present disclosure provides a composition which is to be used in immune checkpoint regulation and contains a lipolysis-stimulated lipoprotein receptor (LSR) regulator. Another aspect of the present disclosure provides a composition which contains the LSR regulator and activates and/or increases the number of tumor-infiltrating CD8+ T-cells. The LSR regulator in some of the embodiments is an anti-LSR antibody or an LSR inhibitor, which is an antigen-binding fragment thereof.

Description

Compositions for use in modulating immune checkpoints

The present disclosure relates to compositions for use in regulating immune checkpoints.

Cancers that are known to highly express LSR include ovarian cancer, gastric cancer, breast cancer, endometrial cancer, colon cancer, bladder cancer, lung cancer, head and neck tumors, and pancreatic cancer. For any of these cancers, no effective treatment has been established for advanced stages or recurrent cases, and the development of new treatments is needed.

Currently, antibody drugs targeting PD-1, PD-L1, CTLA-4, etc. are being used clinically as immune checkpoint inhibitors. However, these immune checkpoint inhibitors only have a significant therapeutic effect in around 10% to 20% of patients, and there is an urgent need to develop therapeutic drugs for patients in whom these immune checkpoint inhibitors are not effective.

　In our laboratory, the inventors were the first in the world to identify LSR as a novel target antigen highly expressed in ovarian cancer (Hiramatsu K, Naka T, et al. Cancer Res. (2018) 78 (2): 516-527, WO2015/098113). LSR is a receptor involved in the uptake of lipoproteins into cells in lipid metabolism (Bihain BE, et al. Curr Opin Lipidol. 1998 Jun;9(3):221-4).

International Publication No. 2015/098113

In the analysis by the present inventors, LSR has variable-type (V-type) immunoglobulin (Ig) in the extracellular domain and is classified as the Ig superfamily. Interestingly, the PD-L1 molecule belonging to the B7 family is also classified as the Ig superfamily. Therefore, the present inventors suspected that LSR suppresses tumor immunity as an immune checkpoint molecule. Therefore, an anti-LSR antibody developed independently was administered to an LSR-positive mouse breast cancer cell line (4T1) syngenic model mouse. As a result, it was confirmed that the anti-LSR antibody exerted an anti-tumor effect on the 4T1 syngenic model mouse, that this anti-tumor effect was lost by the removal of CD8+ T cells, and that the administration of the anti-LSR antibody increased the number and activation rate of CD8+ T cells infiltrating into the tumor. These results suggest that anti-LSR antibodies exert their antitumor effects through activation of CD8+ T cells by inhibiting the binding of LSR on tumor cells to an unknown receptor on CD8+ T cells.

The purpose of this disclosure is to provide a treatment using immune checkpoint inhibitors that target lipolysis-stimulated lipoprotein receptors (LSRs) as an effective treatment for intractable solid cancers such as breast cancer and ovarian cancer.

The present disclosure provides, for example, the following:
(Item 1)
A composition for modulating an immune checkpoint comprising a modulator of the lipolysis-stimulating lipoprotein receptor (LSR).
(Item 2)
The composition described in the preceding item, wherein the LSR regulator is an inhibitor of LSR.
(Item 3)
The composition according to any one of the preceding items, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 4)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 5)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition according to any one of the preceding items.
(Item 6)
A composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells comprising a modulator of an LSR.
(Item 7)
The composition of any one of the preceding items, wherein the tumor-infiltrating CD8+ T cells are CD69 positive.
(Item 8)
The composition according to any one of the preceding items, wherein the LSR modulator is an inhibitor of LSR.
(Item 9)
The composition of any one of the preceding items, wherein the inhibitor of LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 10)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 11)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition of any one of the preceding claims, selected from the group consisting of:
(Item 12)
The composition according to any one of the preceding items, wherein the composition is for treating or preventing malignant tumors.
(Item 13)
The composition of any one of the preceding items, wherein the malignant tumor is an LSR-positive malignant tumor.
(Item 14)
The composition of any one of the preceding items, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
(Item 15)
A method for regulating an immune checkpoint using a composition described in any one of the preceding items.
(Item 16)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 17)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 18)
The method of any one of the preceding items, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
(Item 19)
A method for increasing and/or activating tumor-infiltrating CD8+ T cells using the composition according to any one of the preceding items.
(Item 20)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 21)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 22)
A method for treating or preventing malignant tumors using the composition described in any one of the preceding items.
(Item 23)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 24)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 25)
A composition according to any one of the preceding items for use in regulating immune checkpoints.
(Item 26)
The composition according to any one of the preceding items for use in increasing the number and/or activating tumor-infiltrating CD8+ T cells.
(Item 27)
A composition according to any one of the preceding items for use in the treatment or prevention of malignant tumors.
(Item 28)
Use of a composition described in any one of the preceding items for the manufacture of a composition for modulating an immune checkpoint.
(Item 29)
Use of a composition according to any one of the preceding items for the manufacture of a composition for increasing and/or activating tumor-infiltrating CD8+ T cells.
(Item 30)
Use of a composition according to any one of the preceding items for the manufacture of a composition for the treatment or prevention of malignant tumors.
It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The antibody targeting the LSR disclosed herein can be used as an immune checkpoint inhibitor, providing a new treatment for refractory solid cancers that highly express LSR. As a result, this disclosure makes it possible to improve the prognosis of cancer patients.

Figure 1 shows the results of sequence analysis of the V-type Ig region of LSR and B7 family molecules. FIG. 2 shows the results of ELISA analysis demonstrating that anti-LSR monoclonal antibody #27-6 mF-18 cross-reacts with cynomolgus monkey LSR, rat LSR and mouse LSR with binding affinity equivalent to that of human LSR. FIG. 3 shows the results of FACS (FIG. 3, left) and Western blotting (FIG. 3, right) demonstrating the expression of mouse LSR in 4T1 and MC-38-mLSR-13. Figure 4 shows a schematic diagram of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous transplantation). Figure 5 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous transplantation). Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test. FIG. 6 shows a schematic diagram of an in vivo efficacy test verifying the influence of CD8 ⁺ T cells on the efficacy of an anti-LSR antibody (#27-6mF18). Figure 7 shows the results of a drug efficacy test of anti-LSR monoclonal antibody #27-6 mF-18 using a 4T1 syngenic mouse model (subcutaneous implantation) in which CD8+ T cells were deleted by administration of anti-CD8 antibody. Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test. Figure 8 shows the results of FACS analysis to confirm the depletion of CD8+ T cells from mice by administration of anti-CD8 antibodies. Data were analyzed using Student's t-test. Figure 9 shows the results of FACS analysis of CD8+ T cells infiltrating into the tumors of the anti-LSR monoclonal antibody #27-6 mF-18 treated group. Data were analyzed using Student's t-test. Figure 10 shows the results of measuring chemokine concentrations in tumor tissues of a 4T1 syngenic mouse model (subcutaneously implanted) administered with an anti-LSR antibody (#27-6mF18). * indicates P<0.05. Data were analyzed using Student's t-test. Figure 11 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous implantation) lacking T cell function. Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test. Figure 12 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using an MC-38-mLSR syngenic mouse model (subcutaneous implantation). Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test. Figure 13 shows the results of a drug efficacy test of anti-LSR monoclonal antibody #27-6 mF-18 using an MC-38-mLSR syngenic mouse model (subcutaneous implantation) in which CD8+ T cells were deleted by administration of an anti-CD8 antibody. Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test.

The present disclosure is described below. Throughout this specification, singular expressions should be understood to include the concept of the plural, unless otherwise specified. Thus, singular articles (e.g., in the case of English, "a," "an," "the," etc.) should be understood to include the concept of the plural, unless otherwise specified. In addition, terms used in this specification should be understood to be used in the sense commonly used in the field, unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.

(Definition)
First, terms and general techniques used in this disclosure are explained.

In this specification, "about" means ±10% of the indicated value.

In this specification, "LSR (Lipolysis stimulated lipoprotein receptor)" is generally known as a molecule involved in the metabolism of low density lipoprotein (LDL). Details of the amino acid sequence of LSR can be found on websites such as NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee). The accession numbers of LSR listed in NCBI are, for example, NP_991403 (amino acid) and /NM_205834.3 (mRNA). The amino acid sequence of LSR is, for example, SEQ ID NO: 7. The base sequence of LSR mRNA is, for example, SEQ ID NO: 8. The amino acid sequence of LSR is not limited as long as it has LSR activity. Therefore, as long as it meets the specific objectives of this disclosure, it is understood that not only a protein having an amino acid sequence set forth in a particular SEQ ID NO or accession number (or a nucleic acid encoding the same), but also a functionally active analog or derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a mutant encoded by a nucleic acid that hybridizes to the nucleic acid encoding the protein under high or low stringency conditions, can be used in this disclosure.

As used herein, "derivatives," "analogs," or "variants" preferably include, but are not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., LSR), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical over the same size amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or nucleic acids encoding such molecules are hybridizable to sequences encoding the component proteins under (highly) stringent, moderately stringent, or non-stringent conditions. This refers to proteins that are the product of modification of naturally occurring proteins by amino acid substitutions, deletions, and additions, respectively, and whose derivatives still exhibit the biological functions of the naturally occurring proteins, although not necessarily to the same degree. For example, the biological functions of such proteins can be examined by suitable available in vitro assays described herein or known in the art. As used herein, "functionally active" refers to a polypeptide, i.e., a fragment or derivative, that has a structural, regulatory, or biochemical function of a protein, such as biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative, of the present disclosure relates. In this disclosure, LSR is primarily discussed in humans, but since many animals other than humans are known to express LSR, it is understood that these animals, particularly mammals, are also within the scope of the present disclosure. Preferably, the functional domains of the LSR, such as the transmembrane domain (positions 260-280), phosphorylation sites (positions 309, 328, 406, 493, 528, 530, 535, 540, 551, 586, 615, 646), are conserved.

A representative nucleotide sequence of an LSR is:
(a) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof;
(b) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(c) a polynucleotide encoding a variant polypeptide or a fragment thereof having one or more or one or several amino acids in the amino acid sequence set forth in SEQ ID NO:8, the variant polypeptide having biological activity;
(d) a polynucleotide which is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7, or a fragment thereof;
(e) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8, or a fragment thereof;
(f) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (e) and encodes a polypeptide having biological activity; or (g) a polynucleotide that has a base sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotides (a) to (e) or their complementary sequences and encodes a polypeptide having biological activity. Here, biological activity typically refers to the activity of an LSR or the ability to be distinguished from other proteins present in the same organism as a marker.

The amino acid sequence of LSR is:
(a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(b) a polypeptide having one or more amino acid mutations selected from the group consisting of substitutions, additions and deletions in the amino acid sequence set forth in SEQ ID NO:8, and having biological activity;
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7;
(d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 8; or (e) a polypeptide that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polypeptides (a) to (d) and has biological activity. Here, biological activity typically refers to the ability to distinguish the LSR activity or marker from other proteins present in the same organism (e.g., the inclusion of a region that can function as a specific epitope when used as an antigen).

In this disclosure, "LSR regulator" refers to a substance that has the effect of regulating the function of LSR. In this disclosure, it has been suggested that LSR is an immune checkpoint molecule, and that LSR suppresses tumor immunity. By regulating the function of LSR, it is possible to regulate tumor immunity.

In this disclosure, "immune checkpoint molecule" refers to a molecule that is expressed on cells and transmits a signal that inhibits an immune response by binding to a ligand.

In this disclosure, "LSR inhibitor" refers to a substance or factor that inhibits the biological action of LSR. Such factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, antisense, and nucleic acid forms such as RNAi factors such as siRNA.

In this disclosure, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as "elimination." In this specification, "elimination" is encompassed by "reduction" or "suppression."

As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or downregulates gene expression in cells, mediated by factors that cause RNAi. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and synthesis of gene products is suppressed by introducing factors that cause RNAi, such as double-stranded RNA (also called dsRNA), into cells, and the technology used therefor. In this specification, "RNAi" may also be used synonymously with "factors that cause RNAi," "factors that cause RNAi," "RNAi factors," and the like, in some cases. In addition, in this disclosure, the term RNAi is understood to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition, transcription inhibition, and epigenetics. In this specification, "factors that cause RNAi" may be anything as long as they cause "RNAi."

As used herein, "agents that cause RNAi" include "small interfering nucleic acids," "siNA," "small interfering RNA," "siRNA," "small interfering nucleic acid molecules," "small interfering oligonucleotide molecules," or "chemically modified small interfering nucleic acid molecules," and these terms refer to any nucleic acid molecule that can inhibit or downregulate gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms can refer to individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. These molecules can be double-stranded nucleic acid molecules that contain self-complementary sense and antisense regions.

The "siRNA" typically used in this disclosure is a double-stranded RNA that is short, typically about 20 bases long (e.g., typically about 21-23 bases long) or shorter. Such siRNA suppresses gene expression when expressed in cells. The siRNA used in this disclosure may take any form as long as it can induce RNAi.

In the present disclosure, in agents that cause RNAi, such as siRNA, the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense region includes a nucleotide sequence or a portion thereof that corresponds to the target nucleic acid sequence. These molecules can be assembled from two separate oligonucleotides, one strand being the sense strand and the other being the antisense strand, where the antisense strand and the sense strand are self-complementary (i.e., each strand includes a nucleotide sequence that is complementary to a nucleotide sequence in the other strand, such that the antisense strand and the sense strand form a duplex or double-stranded structure. Here, for example, the double-stranded region can be about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer. The antisense strand includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule. The sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, these molecules are assembled from a single oligonucleotide, the self-complementary sense and antisense regions of which are linked by a nucleic acid or non-nucleic acid linker. These molecules are polynucleotides having double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structures that include self-complementary sense and antisense regions. The antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a separate target nucleic acid molecule, and a sense region having a nucleotide sequence or a portion thereof corresponding to the target nucleic acid sequence. The molecules may be circular single-stranded polynucleotides having two or more loop structures and a stem including self-complementary sense and antisense regions. The antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and the circular polynucleotide may be processed in vivo or in vitro to generate an active molecule capable of mediating RNAi. The agents may also include single-stranded polynucleotides having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule (e.g., the agents do not require that a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof be present within the agents). The single-stranded polynucleotide may be a 5' phosphate (e.g., Martinez et al., 2002, Cell. , 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10,537-568), and may further comprise a terminal phosphate group, such as 5',3'-diphosphate. In certain embodiments, the LSR inhibitors of the present disclosure comprise separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or alternately non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the LSR inhibitors of the present disclosure comprise a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, the LSR inhibitors of the present disclosure interact with the nucleotide sequence of the target gene to inhibit expression of the target gene. As used herein, the LSR inhibitors are not necessarily limited to molecules that comprise only RNA, but also encompass chemically modified nucleotides and non-nucleotides. In some embodiments, when the disclosure is a small interfering nucleic acid molecule, it may lack 2' hydroxy (2'-OH) containing nucleotides. In some embodiments, the disclosure may be a small interfering nucleic acid that does not require the presence of a nucleotide with a 2' hydroxyl group to mediate RNAi. Thus, when the disclosure is a small interfering nucleic acid molecule, it may not include ribonucleotides (e.g., nucleotides with a 2'-OH group). However, when the presence of ribonucleotides in the LSR inhibitor is not required to maintain RNAi, it may have an attached linker or other linked or associated group, moiety, or strand that includes one or more nucleotides with a 2'-OH group. In some cases, the LSR inhibitor of the disclosure may include ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. In the present disclosure, the inhibitor of the LSR may be a nucleic acid molecule capable of mediating sequence-specific RNAi, such as a small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, or post-transcriptional gene silencing RNA (ptgsRNA).

As used herein, factors that induce RNAi include, but are not limited to, RNA or modified forms thereof that contain a double-stranded portion of at least 10 nucleotides in length, including a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Here, the factor preferably contains a 3' overhanging end, and more preferably, the 3' overhanging end can be DNA that is 2 nucleotides or more in length (e.g., DNA that is 2 to 4 nucleotides in length.

Alternatively, the RNAi used in the present disclosure may include, but is not limited to, a pair of short inverted complementary sequences (e.g., 15 bp or more, e.g., 24 bp, etc.).

Without being bound by theory, one of the possible mechanisms by which RNAi works is that when a molecule that induces RNAi, such as dsRNA, is introduced into a cell, in the case of a relatively long RNA (e.g., 40 base pairs or more), an RNaseIII-like nuclease called Dicer, which has a helicase domain, cuts out the molecule from the 3' end in units of about 20 base pairs in the presence of ATP, generating short dsRNA (also called siRNA). In this specification, "siRNA" is an abbreviation for short interfering RNA, and refers to short double-stranded RNA of 10 base pairs or more that is either artificially chemically synthesized or biochemically synthesized, or synthesized in a living organism, or is generated by decomposing double-stranded RNA of about 40 bases or more in the body, and usually has a 5'-phosphate, 3'-OH structure, with the 3' end overhanging by about 2 bases. A specific protein binds to this siRNA to form an RNA-induced silencing complex (RISC). This complex recognizes and binds to mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNaseIII-like enzyme activity. It is preferable that the sequence of the siRNA and the sequence of the target mRNA to be cleaved are 100% identical. However, for base mutations at positions away from the center of the siRNA, the cleavage activity by RNAi is not completely eliminated, but partial activity remains. On the other hand, the effect of base mutations at the center of the siRNA is large, and the cleavage activity of the mRNA by RNAi is extremely reduced. By utilizing such properties, for mRNA having a mutation, an siRNA with the mutation located at the center can be synthesized and introduced into cells to specifically degrade only the mRNA containing the mutation. Therefore, in the present disclosure, the siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA of about 40 bases or more) can be used as such a factor.

Furthermore, without wishing to be bound by theory, it is also contemplated that, apart from the above-mentioned pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP), synthesizing dsRNA, which then serves as a substrate for Dicer again, generating new siRNA and amplifying the effect. Therefore, in the present disclosure, siRNA itself and factors that generate siRNA are also useful. In fact, in insects, for example, 35 dsRNA molecules almost completely degrade 1,000 or more copies of intracellular mRNA, and therefore it is understood that siRNA itself and factors that generate siRNA are useful.

In another embodiment, the factor that causes RNAi of the present disclosure may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3' end. In this specification, "shRNA" refers to a molecule of about 20 base pairs or more that is a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and forming a hairpin-like structure. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase from hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in reverse. Although not wishing to be bound by theory, it should be understood that such shRNA, after being introduced into a cell, is decomposed to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in the cell, and causes RNAi in the same way as siRNA, and has the treatment effect of the present disclosure. It should be understood that such an effect is exerted in a wide range of organisms, such as insects, plants, and animals (including mammals). Thus, shRNA can be used as an active ingredient of the present disclosure since it induces RNAi in the same manner as siRNA. shRNA may also preferably have a 3' overhang. The length of the double-stranded portion is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3' overhang may preferably be DNA, more preferably at least 2 nucleotides in length, and even more preferably 2 to 4 nucleotides in length. The factor that induces RNAi used in the present disclosure may be either artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and there is no essential difference in the effect of the present disclosure between the two. For chemically synthesized factors, it is preferable to purify them by liquid chromatography or the like.

The factor that induces RNAi used in the present disclosure can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and a T7 promoter. When these are annealed in vitro and then introduced into a cell, RNAi is induced through the mechanism described above, and the effect of the present disclosure is achieved. Here, such RNA can be introduced into a cell by any suitable method, such as the calcium phosphate method. The factor that induces RNAi of the present disclosure also includes factors such as single strands that can hybridize with mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present disclosure.

For example, siDirect2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30;10:392.) can be used to design RNAi molecules. This can also be outsourced to a contract company (such as Takara Bio Inc.). RNAi action can be confirmed by quantifying the amount of RNA strand expression using real-time RT-PCR. Alternatively, it can be confirmed by analyzing the amount of RNA strand expression using Northern blot, analyzing the amount of protein using Western blot, and observing the phenotype. Plasmids that generate siRNA or shRNA for a specific gene can also be purchased from a contract company (such as Takara Bio Inc.).

In one embodiment of the present disclosure, "siRNA" includes an RNA strand capable of inducing RNAi. In general, the two strands of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC. The guide strand incorporated into RISC is used to recognize the target RNA. In RNAi research, artificially created ones are mainly used, but some are known to exist endogenously in living organisms. The guide strand may be composed of RNA of 15 or more bases. If it is 15 or more bases, the possibility of binding to the target polynucleotide with high accuracy increases. The guide strand may also be composed of RNA of 40 or less bases. If it is 40 or less bases, the risk of adverse phenomena such as interferon response is lower.

In one embodiment of the present disclosure, "shRNA" includes a single-stranded RNA strand capable of inducing RNAi and forming a structure folded into a hairpin (hairpin-like structure). Typically, shRNA is cleaved by Dicer in cells, and siRNA is excised. It is known that this siRNA causes cleavage of the target RNA. The above shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility of accurately forming the hairpin-like structure specific to shRNA increases. In addition, the above shRNA may be composed of RNA of 100 bases or less. If it is 100 bases or less, the risk of adverse phenomena such as interferon response is reduced. However, since many pre-miRNAs, which are generally similar in structure and function to shRNA, are about 100 nucleotides or more in length, it is considered that shRNA can function as an shRNA even if it is not necessarily 100 bases or less in length.

In one embodiment of the present disclosure, "miRNA" includes an RNA strand that has a function similar to that of siRNA, and is known to suppress the translation and degrade the target RNA strand. The difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.

In one embodiment of the present disclosure, "small RNA" refers to a relatively small RNA strand, such as siRNA, shRNA, miRNA, antisense RNA, and single- or double-stranded small RNA.

The RNAi molecule may contain an overhang of 1 to 5 bases at the 5' or 3' end. In this case, it is believed that the efficiency of RNAi increases. This number can be, for example, 5, 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 140, 145, 150, 165, 170, 186, 197, 198, 199
, 2, or 1 base, or within any two of these values. When the RNAi molecule is double-stranded, mismatched RNA may exist between each RNA strand. The number of bases may be, for example, 1, 2, 3, 4, 5, or 10 or less, or within any two of these values. The RNAi molecule may also include a hairpin loop, and the number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, or within any two of these values. The base sequence may have one or more bases deleted, substituted, inserted, or added, as long as it has the desired effect. In addition, the left side of each base sequence is the 5' end, and the right side is the 3' end.

The length of the RNAi molecule may be, for example, 15, 18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases, or may be within a range of any two of those values.

From the viewpoint of stably exerting the RNAi effect, it is preferable that the above RNAi molecule contains a base sequence complementary to a portion of the base sequence of the LSR mRNA. The above "portion" may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 50 or more bases, or may be within a range between any two of these values.

The siRNA contains the base sequence of SEQ ID NO: 9 or 10. These base sequences are complementary to a portion of the LSR mRNA and are considered to function as a guide strand. One embodiment of the present disclosure includes an RNAi molecule containing such a base sequence of SEQ ID NO: 9 or 10. This RNAi molecule may further contain a base sequence complementary to the base sequence shown in SEQ ID NO: 9 or 10 (e.g., SEQ ID NO: 11 or 12, respectively). In one embodiment of the present disclosure, a "complementary base sequence" is a base sequence possessed by another polynucleotide that is highly complementary to one polynucleotide and can hybridize with it. The full length of the sense strand of the siRNA is the base sequence of SEQ ID NO: 13 or 14, and the full length of the antisense strand is the base sequence of SEQ ID NO: 15 or 16.

The base sequences listed above may be (i) amino acid sequences in which one or more base sequences are deleted, substituted, inserted, or added in the above base sequences, or (ii) base sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of base sequences complementary to the above base sequences, so long as the LSR siRNA has the desired effect.

In the present disclosure, an "LSR-binding substance", "LSR binding agent" or "LSR-interacting molecule" or "binding factor for LSR" is a molecule or substance that binds to LSR at least temporarily. For detection purposes, it is preferably advantageous to be able to indicate the binding (e.g., be labeled or be capable of being labeled), and for therapeutic purposes, it is advantageous to further bind a therapeutic agent. These can be, for example, antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules (LMWs), binding peptides, aptamers, ribozymes, and peptidomimetics. The LSR-binding substance or LSR-interacting molecule can be an inhibitor of LSR, and also includes, for example, binding proteins or binding peptides directed against LSR, in particular against the active site of LSR, as well as nucleic acids directed against the LSR gene. Nucleic acids against LSR refer to, for example, double-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, that inhibit the expression of the LSR gene or the activity of the LSR, and include, but are not limited to, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs), and ribozymes. As used herein, "binding protein" or "binding peptide" with respect to an LSR refers to any protein or peptide that binds to an LSR, and includes, but is not limited to, antibodies (e.g., polyclonal or monoclonal antibodies), antibody fragments, and functional equivalents directed against an LSR.

Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly recognized one-letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.13.0 (published 5/17/2022). The identity value in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them shall be regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

In one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values or less. It is known that a polypeptide that has one or several amino acid residues deleted, added, inserted, or substituted with other amino acids maintains its biological activity (Market et al., Proc Natl Acad Sci USA. 1984 Sep; 81(18): 5662-5666.; Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10(20): 6487-6500.; Wang et al., Science. 1984 Jun 29; 224(4656): 1431-1433.). Antibodies with deletions or the like can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. For example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used for site-specific mutagenesis. It is possible to select antibodies with the same activity as the wild type from mutant antibodies with deletions, etc., by performing various characterizations such as FACS analysis and ELISA.

As used herein, a "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment can be changed as appropriate depending on the purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In addition, for a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. As used herein, such fragments are understood to be within the scope of the present disclosure, for example, if the full-length fragment functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule.

In accordance with the present disclosure, the term "activity" as used herein refers to the function of a molecule in its broadest sense. Activity generally includes, but is not intended to be limiting, the biological, biochemical, physical, or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, ability to localize to a particular subcellular location. Where applicable, the term also relates to the function of protein complexes in their broadest sense.

In this specification, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, and includes, but is not limited to, the production of specific antibodies, enzyme activity, and the conferring of resistance. In the present disclosure, the term "biological function" refers to, but is not limited to, the function of LSR in inhibiting the uptake of VLDL. In this specification, the biological function may be exerted by "biological activity." In this specification, "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes the activity of exerting various functions (e.g., transcription promoting activity), and also includes, for example, the activity of activating or inactivating another molecule by interacting with another molecule. When two factors interact with each other, the biological activity may be the binding between the two molecules and the biological change that occurs as a result of this. For example, when one molecule is precipitated using an antibody and the other molecule is also co-precipitated, the two molecules are considered to be bound. Therefore, observing such co-precipitation is one method of judgment. For example, if a factor is an enzyme, its biological activity includes its enzymatic activity. In another example, if a factor is a ligand, its biological activity includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.

As used herein, the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form. Preferably, the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but transcription to produce an mRNA is also an aspect of expression. Thus, as used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing. For example, the expression level of an LSR can be determined by any method. Specifically, the expression level of an LSR can be known by evaluating the amount of LSR mRNA, the amount of LSR protein, and the biological activity of the LSR protein. Such measurements can be used in companion diagnostics. The amount of LSR mRNA or protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.

In this specification, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Therefore, it is understood that the functional equivalent of an "LSR" or its antibody is not the LSR or its antibody itself, but a mutant or modified version of the LSR or its antibody (e.g., an amino acid sequence variant, etc.) that has the biological action of the LSR, as well as an entity that can change into the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody at the time of acting (e.g., a nucleic acid that encodes the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody, and a vector, cell, etc. that contains the nucleic acid). In this disclosure, it is understood that the functional equivalent of an LSR or its antibody can be used in the same way as the LSR or its antibody, even if not specifically mentioned. The functional equivalent can be found by searching a database, etc. In this specification, "search" refers to using a certain nucleic acid base sequence to find other nucleic acid base sequences that have a specific function and/or property, electronically, biologically, or by other methods. Electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444-2448(1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195-197(1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970)). Biological searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assays), PCR, and in situ hybridization. In this specification, it is intended that the gene used in this disclosure should also include corresponding genes identified by such electronic searches and biological searches.

As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini, can be used. In this specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that may occur naturally. The modified amino acid sequence may be, for example, an amino acid sequence in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or added to one or both termini. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of the LSR or its antibody. Here, "conservative substitution" means replacing one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally similar amino acids that can be used for such substitutions are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In the present specification, the term "antibody" broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab') ₂ and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc(Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc). Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes, such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, and the like. The anti-LSR antibody used in the present disclosure may be of any origin, type, shape, etc., as long as it binds to an LSR protein. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present disclosure, monoclonal or polyclonal antibodies can be used as the antibodies, but monoclonal antibodies are preferred. The antibody preferably binds specifically to the LSR protein. The antibody also includes modified and unmodified antibodies. The modified antibody may be bound to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method.

In this specification, "anti-LSR antibody" includes an antibody that has binding ability to LSR. There are no particular limitations on the method for producing this anti-LSR antibody, but it may be produced, for example, by immunizing a mammal or bird with LSR.

It is further understood that the "functional equivalent" of "an antibody against LSR (anti-LSR antibody) or a fragment thereof" includes, for example, in the case of antibodies, not only the antibody itself and its fragment having LSR binding activity and, if necessary, inhibitory activity, but also chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-chain antibodies, scFVs, diabodies, sc(Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc, and the like.

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an anti-LSR antibody that specifically binds to a specific epitope of LSR, from the viewpoint of particularly strong inhibition of malignant tumor growth.

The anti-LSR antibody according to one embodiment of the present disclosure may be a monoclonal antibody. A monoclonal antibody can act on LSR more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-LSR monoclonal antibody, it is preferable to immunize chickens with LSR.

The antibody class of the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is not particularly limited, but may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.

The anti-LSR antibody according to one embodiment of the present disclosure may be an antibody fragment having antigen-binding activity. In this case, there are effects such as increased stability or antibody production efficiency.

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be a fusion protein. This fusion protein may be an anti-LSR antibody with a polypeptide or oligopeptide bound to the N- or C-terminus. Here, the oligopeptide may be a His tag. The fusion protein may also be a fusion of a mouse, human, or chicken antibody partial sequence. Such fusion proteins are also included in one form of the anti-LSR antibody according to this embodiment.

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be, for example, an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization.

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody having a CDR set of an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cell cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization. The CDR set is a set of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3.

In one embodiment of the present disclosure, the "LSR-expressing cells" may be obtained, for example, by introducing a polynucleotide encoding LSR into a cell and then expressing LSR. Here, the LSR includes an LSR fragment. In another embodiment of the present disclosure, the "LSR-containing lipid membrane" may be obtained, for example, by mixing LSR with a lipid bilayer membrane. Here, the LSR includes an LSR fragment. In addition, from the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an antibody obtained through a process of immunizing chickens with an antigen, or an antibody having a CDR set of that antibody.

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may have any binding strength as long as the intended purpose is achieved, and examples thereof include, but are not limited to, at least 1.0×10 or ^more , 2.0× ¹⁰ or more, 5.0×10 ^or more, or 1.0×10 ^{or more} , and typically has a _K value (kd/ka) of 1.0× ¹⁰ or less, and may be 1.0× ^{10 or less} ⁽ M) or less (M).

The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody that binds to a wild-type or mutant form of LSR. Mutant forms include those resulting from differences in DNA sequences between individuals. The amino acid sequence of the wild-type or mutant form of LSR has a homology of preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO:8.

As used herein, a "polyclonal antibody" can be produced, for example, by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), bird, etc., in order to induce the production of polyclonal antibodies specific to the antigen. The administration of the immunogen may be by injection of one or more immunizing agents and, if desired, an adjuvant. Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin), etc. Immunization protocols are known in the art and may be performed by any method that induces an immune response, depending on the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-91.).

As used herein, the term "monoclonal antibody" includes antibodies in which the individual antibodies constituting the population are substantially identical to one another, except for antibodies with small amounts of naturally occurring mutations. Alternatively, the individual antibodies constituting the population may be substantially identical, except for antibodies with small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies that correspond to different epitopes. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins. The term "monoclonal" may indicate the characteristic of being obtained from a substantially homogeneous antibody population, but does not imply that the antibody must be produced by any particular method. For example, monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug7;256(5517):495-497." Alternatively, monoclonal antibodies may be produced by recombinant methods similar to those described in U.S. Pat. No. 4,816,567. Alternatively, monoclonal antibodies may be isolated from phage antibody libraries using techniques similar to those described in Clackson et al., Nature. 1991 Aug 15;352(6336):624-628, or Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597. Alternatively, monoclonal antibodies may be produced by methods described in Protein Experiment Handbook, Yodosha (2003):92-96.

Any method known in the art can be used for the mass production of antibodies, but the following is an example of a typical method for constructing a mass production system for antibodies and producing antibodies. That is, CHO cells are transfected with an H-chain antibody expression vector and an L-chain antibody expression vector, cultured using the selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and the culture supernatant containing the antibody is collected. The antibody can be purified from the collected culture supernatant by Protein A or Protein G purification.

As used herein, an "Fv antibody" is an antibody that contains an antigen recognition site. This region comprises a non-covalent dimer of one heavy chain variable domain and one light chain variable domain. In this configuration, the three CDRs of each variable domain can interact to form an antigen-binding site on the surface of the VH-VL dimer.

In this specification, a "Fab antibody" is, for example, an antibody fragment obtained by treating an antibody containing a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are bound via some disulfide bonds. Fab can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure containing a Fab region and an Fc region with the protease papain.

As used herein, the term "F(ab') ₂ antibody" refers to an antibody that contains two sites corresponding to Fab, among fragments obtained by treating an antibody containing a Fab region and an Fc region with the protease pepsin. F(ab') ₂ can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure that contains a Fab region and an Fc region with the protease pepsin. In addition, for example, it can be prepared by forming a thioether bond or disulfide bond with the following Fab'.

As used herein, a "Fab'antibody" refers to an antibody obtained, for example, by cleaving the disulfide bond in the hinge region of F(ab') _2. For example, it can be obtained by treating F(ab') ₂ with a reducing agent, dithiothreitol.

As used herein, an "scFv antibody" is an antibody in which VH and VL are linked via a suitable peptide linker. For example, an scFv antibody can be produced by obtaining cDNA encoding the VH and VL of an anti-LSR antibody used in a complex according to an embodiment of the present disclosure, constructing a polynucleotide encoding VH-peptide linker-VL, incorporating the polynucleotide into a vector, and using an expression cell.

As used herein, a "diabody" is an antibody that has bivalent antigen-binding activity. The bivalent antigen-binding activities can be the same, or one of the two can be a different antigen-binding activity. A diabody can be produced, for example, by constructing a polynucleotide encoding an scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length, incorporating the resulting polynucleotide into a vector, and using an expression cell.

As used herein, "dsFv" refers to an antibody in which polypeptides with cysteine residues introduced into VH and VL are linked via disulfide bonds between the cysteine residues. The position at which the cysteine residue is introduced can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Reiteret et al., Protein Eng. 1994 May;7(5):697-704.)

As used herein, a "peptide or polypeptide having antigen-binding activity" refers to an antibody that is composed of an antibody VH, VL, or CDR1, 2, or 3 thereof. A peptide containing multiple CDRs can be linked directly or via a suitable peptide linker.

The method of producing the Fv antibody, Fab antibody, F(ab') ₂ antibody, Fab' antibody, scFv antibody, diabody, dsFv antibody, antigen-binding peptide or polypeptide (hereinafter sometimes referred to as "Fv antibody, etc.") used in the present disclosure is not particularly limited. For example, DNA encoding a region of the Fv antibody, etc. in the anti-LSR antibody used in the complex according to an embodiment of the present disclosure can be inserted into an expression vector and produced using an expression cell. Alternatively, it may be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBOC method (t-butyloxycarbonyl method). The antigen-binding fragment according to an embodiment of the present disclosure may be one or more of the above Fv antibodies, etc.

As used herein, a "chimeric antibody" refers to, for example, an antibody variable region and an antibody constant region linked between different organisms, and can be constructed by recombinant gene technology. Mouse-human chimeric antibodies can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 1;91(3):969-973." The basic method for producing mouse-human chimeric antibodies is, for example, to link mouse leader sequences and variable region sequences present in cloned cDNA to sequences encoding human antibody constant regions already present in a mammalian cell expression vector. Alternatively, mouse leader sequences and variable region sequences present in cloned cDNA may be linked to sequences encoding human antibody constant regions, and then linked to a mammalian cell expression vector. The fragment of the human antibody constant region can be any human antibody H chain constant region or human antibody L chain constant region, for example, Cγ1, Cγ2, Cγ3, or Cγ4 for the human H chain, and Cλ or Cκ for the L chain.

As used herein, a "humanized antibody" is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and that binds to a desired antigen. Antibody humanization can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan 1;13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) can be used. To alter (preferably improve) antigen binding, amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.). For example, FR residues important for antigen binding can be identified by modeling the interactions of CDR and FR residues. Alternatively, unusual FR residues at specific positions can be identified by sequence comparison.

In this specification, a "human antibody" refers to an antibody whose heavy chain variable and constant regions and light chain variable and constant regions are derived from genes encoding human immunoglobulin. The main methods for producing human antibodies include the transgenic mouse method for producing human antibodies and the phage display method. In the transgenic mouse method for producing human antibodies, if a functional human Ig gene is introduced into a mouse whose endogenous Ig has been knocked out, human antibodies with diverse antigen-binding abilities are produced instead of mouse antibodies. Furthermore, if this mouse is immunized, human monoclonal antibodies can be obtained by the conventional hybridoma method. For example, they can be produced by the method described in "Lonberg et al., Int Rev Immunol. 1995;13(1):65-93." The phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminus of the coat protein (g3p, g10p, etc.) of a filamentous phage, such as M13 or T7, which is typically an E. coli virus, so as not to lose the infectivity of the phage. For example, it can be produced by the method described in "Vaughan et al., Nat Biotechnol. 1996 Mar;14(3):309-314."

The antibody may also be prepared by grafting the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure onto any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.). Alternatively, the antibody may be obtained by linking DNA encoding the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure and DNA encoding a region excluding the heavy chain CDR or light chain CDR of a known antibody derived from a human or non-human organism to a vector according to a method known in the art, and then expressing the vector using a known cell. In this case, in order to increase the efficiency of the action of the anti-LSR antibody on the target antigen, the region excluding the heavy chain CDR or light chain CDR may be optimized using a method known in the art (e.g., a method of randomly mutating amino acid residues of an antibody and screening for one with high reactivity, or a phage display method, etc.). In addition, the FR region may be optimized, for example, by using FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) or a method of substituting amino acid residues or packaging residues in the vernier zone (JP Patent Publication 2006-241026, or Footeet et al., J Mol Biol. 1992 Mar 20;224(2):487-499.).

Heavy chains, as used herein, are typically the main components of full-length antibodies. Heavy chains are usually bound to light chains by disulfide bonds and non-covalent bonds. The N-terminal domain of heavy chains contains a region called a variable region (VH), whose amino acid sequence is not constant even in antibodies of the same species and class, and it is generally known that VH contributes greatly to specificity and affinity for antigens. For example, "Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98." describes that when a molecule consisting of only VH was produced, it bound specifically to antigens with high affinity. Furthermore, "Wolfson W, Chem Biol. 2006 Dec;13(12):1243-1244." describes that camel antibodies contain only heavy chains and no light chains.

In this specification, "CDR (complementarity determining region)" refers to the region of an antibody that actually contacts an antigen and forms a binding site. Generally, CDR is located on the Fv (variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)) of an antibody. Generally, CDR consists of CDR1, CDR2, and CDR3, each consisting of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain in particular contributes to the binding of the antibody to the antigen. It is also known that among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of an antibody was increased by modifying the heavy chain CDR3. It is known that one or several (e.g., two, three, etc.) CDRs can be modified (e.g., conservatively modified) while retaining binding ability, and in this disclosure, these modifications are also understood to be within the scope of a particular CDR. The Fv region other than the CDRs is called the framework region (FR), which consists of FR1, FR2, FR3, and FR4, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest," US Dept. Health and Human Services, 1983.). In other words, it can be said that the factors that characterize the reactivity of an antibody are the CDRs, and in particular the heavy chain CDRs.

Several methods for determining the definition and location of CDRs have been reported. For example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987;196:901-917) may be adopted. In one embodiment of the present disclosure, the Kabat definition is adopted as a preferred example, but is not necessarily limited to this. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition. For example, the overlapping portion of the CDR according to each definition, or the portion including both the CDRs according to each definition, may be the CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989;86:9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions. Such CDR information can be used to produce variants that can be used in the present disclosure. Such antibody variants can be produced that contain one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, additions, or deletions in the framework of the original antibody, but do not contain mutations in the CDRs.

As used herein, "antigen" refers to any substance capable of being specifically bound by an antibody molecule. As used herein, "immunogen" refers to an antigen capable of initiating lymphocyte activation resulting in an antigen-specific immune response. As used herein, "epitope" or "antigenic determinant" refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by one of skill in the art using such well-known and conventional techniques when provided with the primary sequence of nucleic acid or amino acid. It is understood that the antibodies of the present disclosure can be similarly utilized with antibodies having other sequences, so long as the epitopes are the same.

It is understood that the antibodies used in this specification may have any specificity as long as false positives are reduced. Thus, the antibodies used in this disclosure may be polyclonal or monoclonal antibodies.

In this specification, the term "means" refers to any tool that can achieve a certain purpose (e.g., detection, diagnosis, treatment), and in particular, in this specification, "selective recognition means" refers to a means that can recognize one object differently from others.

As used herein, the term "malignant tumor" includes, for example, tumors that develop as a result of mutation of normal cells. Malignant tumors can arise from any organ or tissue in the body. Examples of malignant tumors include lung cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, ureter cancer, renal pelvis cancer, ureter cancer, penile cancer, testicular cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, and hematological malignant tumors. Here, ovarian cancer includes, for example, ovarian serous, liquid adenocarcinoma, or ovarian clear cell adenocarcinoma. Uterine cancer includes, for example, endometrial cancer or cervical cancer. Head and neck cancers include, for example, oral cancer, pharyngeal cancer, laryngeal cancer, nasal cancer, paranasal sinus cancer, salivary gland cancer, or thyroid cancer. Lung cancers include, for example, non-small cell lung cancer or small cell lung cancer. The malignant tumor may also be LSR positive.

Among malignant tumors, serous adenocarcinoma progresses very quickly, and it is difficult to completely eliminate the cancer even with commercially available anticancer drugs. Furthermore, in cases of recurrence, commercially available anticancer drugs are almost ineffective. Furthermore, for clear cell adenocarcinoma, commercially available anticancer drugs are unlikely to have any therapeutic effect. On the other hand, the anti-LSR antibody according to the embodiment of the present disclosure could become a new therapeutic agent for serous adenocarcinoma and clear cell adenocarcinoma.

In one embodiment of the present disclosure, "LSR-positive malignant tumor" includes malignant tumors that significantly or excessively express LSR. Whether a malignant tumor is LSR-positive may be evaluated, for example, by RT-PCR, Western blot, or immunohistochemical staining. In addition, when total protein of malignant tumor cells is subjected to Western blot and a band corresponding to LSR (e.g., a band around 649aa) can be visually confirmed, it may be determined to be LSR-positive. Alternatively, it may be determined to be LSR-positive when the amount of LSR expression in malignant tumor cells derived from a patient is significantly greater than that in normal cells. From the viewpoint of achieving more optimal medication by accurately diagnosing LSR positivity, it is preferable to test the expression of LSR using an anti-LSR antibody.

As used herein, the term "subject" refers to an object that is the subject of diagnosis, detection, or treatment according to the present disclosure (e.g., an organism such as a human, or cells, blood, serum, etc. extracted from an organism).

As used herein, the term "sample" refers to any substance obtained from a subject, etc., including, for example, blood, serum, plasma, saliva, urine, tears, cerebrospinal fluid, etc. Those skilled in the art will be able to select an appropriate and preferred sample based on the description in this specification.

In this specification, the terms "drug", "agent" or "factor" (all of which correspond to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof. Factors specific to a polynucleotide typically include, but are not limited to, polynucleotides that have a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions. Representative factors specific to a polypeptide include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.

In this specification, "treatment" refers to preventing the worsening of a certain disease or disorder (e.g., malignant tumor) when that condition occurs, preferably maintaining the status quo, more preferably alleviating, and even more preferably causing the disease or disorder to recede, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents used for this purpose are sometimes called "companion diagnostic agents."

In this specification, the term "therapeutic agent" refers broadly to any drug capable of treating a target condition (e.g., a disease such as a malignant tumor). In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be produced, for example, by mixing the active ingredient with the carrier and using any method known in the technical field of pharmaceutical formulations. The therapeutic agent may be in any form as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and any other ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The therapeutic agent for malignant tumors includes drugs used for preventing malignant tumors (prophylactic drugs) or agents for suppressing the proliferation of malignant tumor cells.

As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., malignant tumor) from occurring before that state occurs. A diagnosis can be made using the drug disclosed herein, and if necessary, the drug disclosed herein can be used to prevent, for example, malignant tumors, or preventive measures can be taken.

In this specification, the term "prophylactic drug" refers broadly to any drug that can prevent a target condition (e.g., a disease such as a malignant tumor).

As used herein, "interaction" refers to the mutual exertion of force (e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.) between one substance and the other. Usually, two substances that have interacted are in an associated or bonded state. The detection, testing, and diagnosis disclosed herein can be achieved by utilizing such interactions.

As used herein, the term "binding" refers to a physical or chemical interaction between two substances or a combination thereof. Binding includes ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. Physical interactions (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effect of another protein or compound and does not involve other substantial chemical intermediates.

Thus, as used herein, an "agent" (or drug, detection agent, etc.) that "specifically" interacts with (or binds to) a biological agent such as a polynucleotide or polypeptide includes one whose affinity for the biological agent, such as the polynucleotide or polypeptide, is typically equal to or higher than, and preferably significantly (e.g., statistically significantly) higher than, its affinity for other unrelated polynucleotides or polypeptides (e.g., those with less than 30% identity). Such affinity can be measured, for example, by hybridization assays, binding assays, etc.

As used herein, a first substance or factor "specifically" interacts with (or binds to) a second substance or factor means that the first substance or factor interacts with (or binds to) the second substance or factor with a higher affinity than it does with substances or factors other than the second substance or factor (particularly other substances or factors present in a sample containing the second substance or factor). Examples of specific interactions (or binding) for a substance or factor include, but are not limited to, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, and other reactions between nucleic acids and proteins, protein-lipid interactions, and nucleic acid-lipid interactions. Thus, when both substances or factors are nucleic acids, "specifically interacting" with a first substance or factor includes the first substance or factor having at least partial complementarity to the second substance or factor. For example, when both substances or factors are proteins, examples of "specific" interaction (or binding) of a first substance or factor with a second substance or factor include, but are not limited to, interactions due to antigen-antibody reactions, interactions due to receptor-ligand reactions, enzyme-substrate interactions, etc. When two types of substances or factors include proteins and nucleic acids, "specific" interaction (or binding) of a first substance or factor with a second substance or factor includes interactions (or binding) between an antibody and its antigen. By utilizing such specific interactions or binding reactions, it is possible to detect or quantify a target substance in a sample.

As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression may be accomplished using any suitable method, including, for example, measurement of mRNA and immunological measurement methods, including binding or interaction with a detection agent, test agent or diagnostic agent. Examples of molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR. Examples of immunological measurement methods include, for example, ELISA, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining using microtiter plates. Examples of quantification methods include ELISA or RIA. Genetic analysis may also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are broadly reviewed in "DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha. Protein arrays are reviewed in NatGenet. 2002　Dec;32　Suppl:526-532. In addition to the above, gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods: Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all descriptions therein are incorporated by reference.

As used herein, "expression level" refers to the amount of a polypeptide or mRNA, etc., expressed in a cell, tissue, etc. of interest. Examples of such expression level include the expression level at the protein level of the polypeptide of the present disclosure, evaluated by any appropriate method, including immunological measurement methods such as ELISA, RIA, fluorescent antibody technique, Western blotting, and immunohistochemical staining, using an antibody of the present disclosure, or the expression level at the mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. "Change in expression level" refers to an increase or decrease in the expression level at the protein level or mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including the immunological measurement method or molecular biological measurement method. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.

As used herein, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as "elimination." As used herein, "elimination" is encompassed within "reduction" or "suppression."

As used herein, "increase" or "activation" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to an increase or increasing activity in the amount, quality or effect of a particular activity, transcript or protein.

In this specification, the term "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When a plurality of markers of the present disclosure or factors or means for capturing them are labeled by a fluorescence method, the labels are labeled with fluorescent substances having mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but Alexa ^TM Fluor is preferable as the fluorescent substance. Alexa ^TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa ^TM 555 and Alexa ^TM 633, and a combination of Alexa ^TM 488 and Alexa ^TM 555. When labeling a nucleic acid, any dye that can bind to the base moiety can be used, but it is preferable to use cyanine dyes (e.g., Cy3 and Cy5 of the CyDye ^TM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), and the like. Examples of fluorescent substances having a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present disclosure, such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.

As used herein, a "tag" refers to a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a substance that acts as a binding partner for binding a specific substance (e.g., having a relationship such as biotin-avidin or biotin-streptavidin), and may be included in the category of a "label." Thus, for example, a specific substance bound to a tag can be selected by contacting the specific substance with a substrate bound to a binding partner of the tag sequence. Such tags or labels are well known in the art. Representative tag sequences include, but are not limited to, myc tags, His tags, HA, and Avi tags.

As used herein, "in vivo" refers to the inside of a living organism. In certain contexts, "within the organism" refers to the location where a substance of interest is to be placed.

In this specification, "in vitro" refers to a state in which a part of a living organism is removed or isolated "outside of a living organism" (e.g., in a test tube) for various research purposes. This term contrasts with in vivo.

As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit is advantageously provided with instructions or instructions describing how to use the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, etc.) or how to handle the reagents. When the kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.

In this specification, "instructions" refers to instructions for a physician or other user on how to use the present disclosure. The instructions include instructions on how to use the present disclosure's detection method, how to use a diagnostic agent, or how to administer a medicine. The instructions may also include instructions for administration via the mouth or esophagus (e.g., by injection, etc.) as the site of administration. The instructions are prepared in accordance with a format stipulated by the supervisory authority of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is clearly stated that the instructions have been approved by the supervisory authority. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

As used herein, "internalization" refers to a cell taking up an antigen-bound substance on the cell surface via endocytosis or phagocytosis, with the substance being mediated by the antigen. A substance that binds to an LSR having such activity (e.g., an anti-LSR antibody) can cause the intracellular internalization of a target active ingredient in a cell expressing LSR on the cell surface, thereby producing the desired effect of the target active ingredient in the LSR-expressing cell. Examples of target active ingredients include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, etc.

As used herein, "antibody drug conjugate (ADC)" refers to an antibody or antigen-binding fragment thereof chemically linked to one or more active ingredients of interest. In a preferred embodiment, the ADC is operably linked via a linker. As used herein, "operably linked" refers to a relationship in which the linked substances are capable of operating in a predicted manner. Active ingredients of interest that may be included in an ADC include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, and the like. The linker may be a cleavable linker or a non-cleavable linker. Examples of cleavable linkers include, but are not limited to, linkers having a sequence cleaved by a protease, acid-labile linkers, disulfide linkers, and the like. Examples of non-cleavable linkers include, but are not limited to, MCC linkers, and the like.

In this specification, "cytotoxic activity" refers to, for example, causing pathological changes in cells, not only direct trauma, but also any damage to the structure or function of cells, such as DNA breaks, formation of base dimers, chromosome breaks, damage to the cell division system, and reduced activity of various enzymes, thereby directly or indirectly blocking cell function and causing cell death. Therefore, examples of "drugs having cytotoxic activity" include, but are not limited to, alkylating agents, tumor necrosis factor inhibitors, intercalators, microtubule inhibitors, kinase inhibitors, proteasome inhibitors, and topoisomerase inhibitors.

As used herein, the term "50% inhibitory concentration ( _IC50 )" refers to the concentration of a compound required to kill 50% of cells. _IC50 is measured herein using the method described in Example 7.

Preferred Embodiments
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

The present inventors confirmed that anti-LSR antibodies exert an anti-tumor effect in 4T1 syngenic model mice, that this anti-tumor effect is lost by the removal of CD8+ T cells, and that administration of anti-LSR antibodies increases the number and activation rate of CD8+ T cells infiltrating into the tumor. These results suggest that anti-LSR antibodies exert their anti-tumor effect through activation of CD8+ T cells by inhibiting the binding of LSR on tumor cells to an unknown receptor on CD8+ T cells. Thus, in one aspect, the present disclosure provides a composition for regulating immune checkpoints, comprising a regulator of lipolysis-stimulating lipoprotein receptor (LSR).

The inventors have demonstrated in this disclosure that immune checkpoints can be regulated by modulating the lipolysis-stimulating lipoprotein receptor (LSR), and this can be explained by the following mechanism.

The inventors have confirmed in an animal model that anti-LSR antibodies exert an antitumor effect in a CD8+ T cell-dependent manner. Here, without wishing to be bound by theory, the fact that immune checkpoints can be regulated by regulating LSR can be explained as follows. That is, LSR expressed on tumor cells interacts with receptors present on CD8+ T cells. As a result, inhibitory stimuli are transmitted to CD8+ T cells intracellularly, and LSR has the function of suppressing tumor immunity. In this specification, it has actually been confirmed that ovarian cancer, gastric cancer, and endometrial cancer with high LSR expression have a significantly poorer prognosis compared to those with low LSR expression, and this can be reasonably explained as being appropriate. One of the reasons for this is thought to be that LSR suppresses tumor immunity, thereby causing tumor growth to progress (Hiramatsu, Naka et al., Cancer Res. 2018 Jan 15;78(2):516-527, Sugase, Naka et al., Oncotarget. 2018 Aug 31;9(68):32917-32928, Nagase, Naka et al., BMC Cancer. 2022 Jun 21;22(1):679.).

In some embodiments, the modulator of LSR may be an inhibitor of LSR. Examples of inhibitors of LSR include, but are not limited to, anti-LSR antibodies or antigen-binding fragments thereof, nucleic acids such as antisense nucleic acids or siRNAs, and small molecule compounds. siRNAs may include, for example, SEQ ID NOs: 9 to 14.

In some embodiments, the binding agent for LSR or a fragment thereof can be an anti-LSR antibody or an antigen-binding fragment thereof or a functional equivalent.

The antibody of the present disclosure may be a specific sequence described elsewhere in this disclosure. The antibody may be an antibody or antigen-binding fragment thereof that includes any sequence including the CDRs of the full-length sequence, or an antibody or antigen-binding fragment thereof that includes a variable region of the following sequence, and that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 or more substitutions, additions, or deletions in the framework region. The antibody may be produced, etc., using the embodiments described elsewhere in this disclosure and/or techniques known in the art. For the therapeutic or preventive purposes of the present disclosure, such an antibody or fragment or functional equivalent preferably has an inhibitory activity downstream of the LSR or its signal transduction pathway. Such activity may be confirmed by observing the expression level or activity of the LSR, or by directly using a malignant tumor cell line such as ovarian leiomyoma cells to inhibit cell growth, or by transplanting the cell line into a model animal to observe tumor regression. These techniques are well known in the art and may be used in the present disclosure.

The administration route of the composition of the present disclosure is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, or granule. When administering an antibody or polynucleotide, it is effective to use it as an injection. The aqueous solution for injection may be stored, for example, in a vial or stainless steel container. The aqueous solution for injection may also be mixed with, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may also be mixed with, for example, a buffer (for example, a phosphate buffer), a stabilizer, etc.

Generally, the compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present disclosure include a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharma- ceutically acceptable carrier or excipient. As used herein, "pharma-ceutically acceptable" means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. As used in this disclosure, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the medicament is administered orally, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose are the preferred carriers. Preferably, saline solutions, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The composition can also contain a small amount of a wetting or emulsifying agent, or a pH buffer, if desired. These compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. The composition can also be formulated as a suppository, using traditional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide a form that is properly administered to the patient. The formulation should be suitable for the mode of administration. In addition to these, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavorings, etc. may be included.

When the antibodies, conjugates, compositions, etc. of the present disclosure are administered as pharmaceuticals, various delivery systems are known and such systems can be used to administer the therapeutic agents of the present disclosure to the appropriate site (e.g., esophagus), such as encapsulation in liposomes, microparticles, and microcapsules; using recombinant cells capable of expressing the therapeutic agent (e.g., polypeptides); using receptor-mediated endocytosis; constructing the therapeutic nucleic acid as part of a retroviral or other vector. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical agent can be administered by any suitable route, such as by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), using an inhaler or nebulizer with an aerosolizing agent if necessary, and can be administered together with other biologically active agents. Administration can be systemic or local. When the present disclosure is used in the ovarian region, it may be administered by any suitable route, such as by direct injection into the affected area, such as the ovary.

In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans, according to known methods. Such a composition can be administered by injection. Typically, a composition for administration by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachette indicating the quantity of active agent. If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

The compositions, pharmaceuticals, therapeutic agents, and prophylactic agents of the present disclosure may be formulated in neutral or salt form or as other prodrugs (e.g., esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxide, etc.

The amount of the therapeutic agent of the present disclosure effective in treating a particular disorder or condition may vary depending on the nature of the disorder or condition, but can be determined by one of skill in the art using standard clinical techniques based on the description herein. In addition, in vitro assays may be used in some cases to help identify optimal dosage ranges. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dose, or within any two of these values. The administration interval is not particularly limited, and may be, for example, 1 or 2 doses per 1, 7, 14, 21, or 28 days, or 1 or 2 doses per two of these values. The dosage, administration interval, and administration method may be selected appropriately depending on the age, weight, symptoms, target organ, etc. of the patient. In addition, the therapeutic agent preferably contains an active ingredient in a therapeutically effective amount, or in an effective amount that exerts a desired effect. If the malignant tumor marker is significantly reduced after administration, it may be determined that the therapeutic effect has been achieved.

In one embodiment of the present disclosure, a "patient" includes a human or a non-human mammal (e.g., one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee). The patient may also be a patient who has been determined or diagnosed as having an LSR-positive malignant tumor. In this case, the determination or diagnosis is preferably made by detecting the protein level of LSR.

The pharmaceutical composition or therapeutic or prophylactic agent disclosed herein can be provided as a kit.

In certain embodiments, the disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of an antibody, conjugate, composition, or medicament of the disclosure. Optionally, such containers may bear information indicating approval by a government agency of the manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products.

In another embodiment, the anti-LSR antibody may be an anti-LSR antibody that specifically binds to an epitope of LSR. More specifically, the antibody may have an epitope at positions 116-135 and/or 216-230 of SEQ ID NO:7.

One embodiment of the present disclosure relates to an antibody comprising: (a) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 1; and (b) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 103, 152 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 2. (c) an antibody in which heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO: 3, respectively; (d) an antibody in which heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO: 4, respectively. 5, (e) an antibody in which heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences represented by positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; and (f) an antibody in which heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and The anti-LSR antibody may be an anti-LSR antibody, in which the amino acid sequences of heavy chain CDR1, 2, and 3 are at least one antibody selected from the group consisting of antibodies comprising the amino acid sequences of positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 6, respectively, or a mutant of the antibody, in which the mutant comprises one or several substitutions, additions, or deletions in the framework of the antibody, but does not comprise a mutation in the CDR. The use of this anti-LSR antibody can be particularly effective in suppressing the proliferation of LSR-positive malignant tumor cells. In addition, LSR-positive malignant tumors can be efficiently diagnosed. Another embodiment of the present disclosure is an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain CDR1, 2, and 3 listed above. These antibodies may be antibodies selected from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFVs, diabodies, sc(Fv) ₂ (single chain (Fv) ₂ ), and scFv-Fc.

In another embodiment, the antibody may contain one or several substitutions, additions or deletions in one, two, three, four, five or six of the six CDRs while maintaining binding to the LSR. In another embodiment, the antibody may contain up to three, preferably up to two, more preferably up to two, preferably one substitution, addition or deletion in one CDR. In a preferred embodiment, the substitutions may be conservative substitutions.

An anti-LSR antibody according to one embodiment of the present disclosure comprises a set of amino acid sequences of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3, and further, at least one, preferably two, three, four, five, six, seven, or all of the frameworks of heavy chain FR1, 2, 3, and 4, and light chain FR1, 2, 3, and 4 may be identical or substantially identical to any of SEQ ID NOs: 1 to 6, or identical except for conservative substitutions. There may be one or more types of antibodies. Another embodiment of the present disclosure is an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain FR1, 2, 3, and 4 listed above.

The anti-LSR antibody according to one embodiment of the present disclosure may be in the form of an scFv, in which case the linker between the heavy and light chains may have the amino acid sequence shown in positions 116 to 132 of SEQ ID NO:1, positions 116 to 132 of SEQ ID NO:2, positions 116 to 132 of SEQ ID NO:3, positions 116 to 132 of SEQ ID NO:4, positions 116 to 132 of SEQ ID NO:5, or positions 116 to 132 of SEQ ID NO:6.

　The VHs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 1 to 115 of SEQ ID NO:1, positions 1 to 115 of SEQ ID NO:2, positions 1 to 115 of SEQ ID NO:3, positions 1 to 115 of SEQ ID NO:4, positions 1 to 115 of SEQ ID NO:5, and positions 1 to 115 of SEQ ID NO:6, respectively. The VLs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 133 to 238 of SEQ ID NO:1, positions 133 to 239 of SEQ ID NO:2, positions 133 to 238 of SEQ ID NO:3, positions 133 to 238 of SEQ ID NO:4, positions 133 to 238 of SEQ ID NO:5, and positions 133 to 238 of SEQ ID NO:6, respectively.

The amino acid sequences listed above may be one or more amino acid sequences selected from the group consisting of: (i) the above amino acid sequences in which one or more base sequences have been deleted, substituted, inserted, or added; (ii) amino acid sequences having 90% or more identity (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the above amino acid sequences; and (iii) amino acid sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of a base sequence complementary to the base sequence encoding the above amino acid sequences, so long as the anti-LSR antibody has the desired effect.

A transformant can be created by introducing a polynucleotide or vector encoding an anti-LSR antibody according to an embodiment of the present disclosure into a cell. The transformant can be used to produce an anti-LSR antibody according to an embodiment of the present disclosure. The transformant may be a cell of a human or a mammal other than a human (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells) and monkey cells COS-7. Alternatively, the transformant may be Escherichia bacteria, yeast, etc.

The above vectors may be, for example, plasmids derived from E. coli (e.g., pET-Blue), plasmids derived from Bacillus subtilis (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5/His-TOPO), bacteriophages such as λ phage, and vectors derived from viruses. These vectors may contain components necessary for protein expression, such as a promoter, an origin of replication, or an antibiotic resistance gene. The vector may be an expression vector.

　The above-mentioned polynucleotides or vectors can be introduced into cells by, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, or microinjection (Revised 4th Edition New Genetic Engineering Handbook, Yodosha (2003): 152-179.). For the production of antibodies using cells, for example, the method described in "Protein Experiment Handbook, Yodosha (2003): 128-142." can be used. For the purification of antibodies, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography can be used (Protein Experiment Handbook, Yodosha (2003): 27-52.).

In another aspect, the present disclosure provides a composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells, comprising a modulator of an LSR. In some embodiments, the CD8+ T cells can be CD69+. CD69 is known as an activation marker for CD8+ T cells.

In some embodiments, the compositions and the like of the present disclosure may be for treating or preventing malignant tumors. The malignant tumor may be LSR positive. The malignant tumor may be breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer, or colon cancer, which may be LSR positive.

In a further aspect, a companion reagent for determining whether or not treatment with the complex, composition, pharmaceutical, etc. of the present disclosure is required, the companion reagent including a detection reagent for LSR.

In a further aspect, a kit for treating or preventing malignant tumors is provided, comprising an LSR detection reagent, a complex, composition, medicine, etc. of the present disclosure, and instructions.

　All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

The present disclosure will be further explained below with reference to examples, but the present disclosure is not limited to these.

Example 1: Sequence analysis of LSR and V-type Ig region of B7 family molecules Sequence analysis of LSR (Figure 1, left)
The identity of the amino acid sequences of human LSR (Q86X29) and mouse LSR (Q99KG5) was analyzed using the Aliment tool of Uniprot.

Sequence analysis of the V-type Ig region of LSR and B7 family molecules (Figure 1, right)
The amino acid sequence identity of the V-type Ig region of human LSR (Q86X29), human CD80 (P33681), human CD86 (P42081), human ICOSL (O75144), human PDL1 (Q9NZQ7), human PDL2 (Q9BQ51), human CD276 (Q5ZPR3), human VTCN1 (B7-H4) (Q7Z7D3), and human VISTA (Q9H7M9) was analyzed using the Aliment tool of Uniprot.

(result)
The amino acid sequences of human LSR (UniProtKB accession: Q86X29-1) and mouse (LSR UniProtKB accession: Q99KG5-1) were compared. The signal peptide of human LSR could not be determined by prediction software, but mouse LSR had a signal peptide sequence of 35 amino acids. Human LSR had a long V-type Ig domain, a stalk region, and a transmembrane domain. The amino acid sequence identity of human LSR and mouse LSR over their entire length was 73.2%, but the V-type Ig domain showed 91.3% amino acid identity (Figure 1).

The B7 family belongs to the Ig superfamily and is a single-pass transmembrane protein with V-type and constant (C)-type Ig domains. Comparison of amino acid sequences between human LSR and mouse LSR and other B7-like proteins revealed that the DxGxYxC motif in the V-type Ig domain is conserved (Figure 1). The amino acid sequence identity between human LSR and other B7-like proteins in the V-type Ig domain ranged from 6.77% to 16.9% (CD80: 8.87%, CD86: 6.77%, ICOSL: 10.24%, PD-L1: 11.2%, PD-L2: 8.94%, B7-H3: 7.67%, B7-H4: 8.43%, VISTA: 16.89%). Human LSR showed the highest amino acid sequence identity with VISTA, and both proteins lacked the C-type Ig domain. The DxGxYxC motif is a typical sequence conserved in Ig domains. Some proteins with Ig domains may have immune checkpoint activity. From the above, it is suggested that LSR is also a B7-like protein, and in the Examples below, we will confirm whether LSR has immune checkpoint activity.

Example 2: Analysis of cross-reactivity of chicken mouse chimeric anti-LSR antibody (#27-6mF18) with human, cynomolgus monkey, rat, and mouse LSR by ELISA (Materials and Methods)
Buffer Preparation
The buffers used in this example were prepared as follows.
- Coating buffer: 50 mM carbonate-bicarbonate buffer, pH 9.6.
Dilution buffer: The dilution solution used was 10% Block Ace, which was prepared by dissolving 4 g of Block Ace powder in 100 mL of MiliQ and diluting the solution 10-fold with PBS(-).
Blocking buffer: 4 g of Block Ace powder was dissolved in 100 mL of MiliQ, and then diluted 4-fold with PBS (-) to prepare a blocking buffer. The solution obtained by dissolving 4 g of Block Ace powder in 100 mL of MiliQ was used as the stock solution (100% solution).
Washing buffer: One packet of PBS-T (Sigma, P3563) was dissolved in 1 L of ultrapure water (0.05% Tween 20/PBS).

(Primary antibody preparation)
The primary antibody used in this example was prepared. Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was serially diluted with dilution buffer to 1000, 100, 10, 1, 0.1, 0.01, and 0.001 nM. The antigens used were recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc.

(Method)
Recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc were diluted to 2.5 μg/mL with 50 mM carbonate-bicarbonate buffer, pH 9.6, and added to an Immuno 96-well MicroWell Solid Plate (Maxisorp, Nunc) at 50 μL/well, and the plate was sealed. The plate was rocked to ensure that the liquid was distributed throughout. The following procedure was then followed.
- Leave it still (4℃, overnight)
Washing (200 μL/well x 3)
Add 200 μL of blocking buffer per well to a 96-well microplate. Incubate at room temperature for 1 hour. Wash (200 μL per well, 3 times).
・Dilute chicken mouse chimeric anti-LSR antibody (#27-6mF18) in dilution buffer in a 10-fold serial dilution starting from 100 nM, add 100 μL/well to a 96-well microplate, react at room temperature for 1 hour, and wash (200 μL/well, 3 times).
・Goat Anti-Chicken IgY H&L (HRP) (Abcam, cat. No.: ab6877) was diluted to 2 μg/ml with dilution buffer (10% BlockAce) (prepared 5000-fold dilution just before use), and added to a 96-well microplate at 100 μL/well. Incubated at room temperature for 1 hour and washed (200 μL/well, 3 times).
・Bring TMB (SurModics, TMBW-1000-01) to room temperature before use, add 100 μL/well to a 96-well microplate, and mix (in the dark, at room temperature).
- Stop with 50 μL/well of 1N sulfuric acid - Measure absorbance at 450 nm - Data analysis with GraphPad Prism 6.04.

(result)
The anti-LSR monoclonal antibody #27-6 created by the inventors also cross-reacts with mouse LSR (WO/2015/098113). By substituting the amino acid sequence of the Fc region of the anti-LSR monoclonal antibody #27-6, the anti-LSR monoclonal antibody #27-6 mF-18, which does not exhibit ADCC activity or CDC activity, was created. This is to demonstrate that the antitumor effect shown in the following examples is not due to ADCC activity or CDC activity. ELISA analysis revealed that the anti-LSR monoclonal antibody #27-6 mF-18 reacts with human LSR and mouse LSR (Figure 2).

Example 3: LSR expression analysis by FACS and Western blot LSR expression analysis by FACS (Figure 3, left)
The expression of LSR was analyzed by FACS analysis for 4T1 (mouse breast cancer cell line), HM-1 (OV2944-HM-1, mouse ovarian cancer cell line), B16F10 (mouse malignant melanoma cell line), CT26 (mouse colon cancer cell line), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mLSR-expressing mouse colon cancer). Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was used as the primary antibody, and FITC-labeled Goat Anti-Mouse IgG (H+L chain specific) (southern biotech) was used as the secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using FlowJo (trademark) software (Tree star).

Expression analysis of LSR by Western blot (Figure 3, right)
After the cell lines seeded on a 10 cm plate reached approximately 90% confluence, the culture supernatant was discarded, the cells were washed with ice-cold PBS, and then detached and collected with a cell scraper. Proteins were extracted using RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1× phosphatase inhibitor cocktail (Nacalai Tesque) and 1× protease inhibitor cocktail (Nacalai Tesque)), and differential protein expression was analyzed by Western blotting. The primary antibodies used were anti-LSR antibody (CST #14804) and anti-beta-actin antibody (sc-69879) (Santa Cruz Biotechnology, Santa Cruz, CA).

(result)
Expression of mouse LSR was confirmed using 4T1 (mouse breast cancer), HM-1 (OV2944-HM-1, mouse ovarian cancer), B16F10 (mouse melanoma), CT-26 (mouse colon cancer), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mouse colon cancer) cell lines. Expression of mouse LSR was confirmed in 4T1 and MC-38-mLSR-13 by both FACS and Western blotting (Figure 3).

Example 4: In vivo efficacy test of anti-LSR antibody (#27-6mF18) using 4T1 syngenic mouse model (subcutaneous transplantation) Paraffin-embedded tissue was sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for LSR was performed using anti-LSR antibody (CST #14804) and ChemMate Envision kit HRP 500T (Dako: K5007).

A schematic diagram of the in vivo efficacy test of anti-LSR antibody (#27-6mF18) using the 4T1 syngenic mouse model (subcutaneous transplantation) in this example is shown in Figure 4. Specifically, 2.0 x 10 ⁵ 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups, and administration of PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) was started. Intraperitoneal administration of drugs, measurement of tumor volume, and body weight were performed at the time points described in the experimental outline in Figure 4.

(result)
A 4T1 syngenic mouse model was created, and the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was evaluated according to the experimental schedule in Figure 4. Compared to the PBS-administered group and the control antibody-administered group (20 mg/kg), the anti-LSR monoclonal antibody #27-6 mF-18-administered group showed significant antitumor effects at both doses of 5 mg/kg and 20 mg/kg (Figure 5).

Example 5: Effect of CD8 ⁺ T cells on efficacy of anti-LSR antibody (#27-6mF18) A schematic diagram of the in vivo efficacy test in this example to verify the effect of CD8 ⁺ T cells on the efficacy of anti-LSR antibody (#27-6mF18) is shown in FIG.

Specifically, ^2.0x105 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups and administered isotype control antibody (5mg/kg), anti-LSR antibody (#27-6mF18) (5mg/kg), anti-CD8 antibody (0.2mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5mg/kg) + anti-CD8 antibody (0.2mg/body). Drugs were intraperitoneally administered, tumor volume was measured, and body weight was measured at the time points described in the experimental outline in Figure 6.

(result)
To verify whether tumor immunity is involved in the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 on the 4T1 syngenic mouse model, a drug efficacy test using anti-LSR monoclonal antibody #27-6 mF-18 under conditions in which CD8+ T cells were deleted by administration of anti-CD8 antibody was performed according to the experimental schedule shown in Figure 6. It was confirmed that the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was negated under conditions in which anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 were combined (Figure 7). This indicates that the antitumor effect of anti-LSR antibody is dependent on CD8+ T cells.

Example 6: Experiment to confirm CD8+ T cell depletion by administration of anti-CD8 antibody An experiment was conducted to confirm that CD8+ T cells in mice were removed by administration of anti-CD8 antibody. 2.0×10 ⁵ 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and anti-CD8 antibody (0.2 mg/body) was intraperitoneally administered twice, on the day of transplantation and two days later. On the fourth day, cells were isolated from the spleen, and surface antigens were stained with anti-CD45 (30-F11; catalog No. 103116), anti-CD8 (53-6.7; catalog No. 100734), and anti-CD4 (RM4-5; catalog No. 100516) antibodies at 4°C for 30 minutes. After washing the stained cells, they were measured using FACS CantoII (BD), and the data was analyzed using BD FACS Diva software (BD Biosciences).

(result)
Regarding the removal of CD8+ T cells from mice by administration of anti-CD8 antibodies, we administered anti-CD8 antibodies to a 4T1 syngenic mouse model, and then performed FACS analysis of T cells in the spleen to confirm the disappearance of CD8+ T cells (Figure 8).

Example 7: Analysis of tumor-infiltrating CD8+ T cells To evaluate the number of tumor-infiltrating CD8+ T cells and the rate of activation by anti-LSR antibody administration, tumors were excised on the final day (day 19) for the two groups of isotype control antibody (5 mg/kg) and anti-LSR antibody (#27-6mF18) (5 mg/kg) in Example 5. Single cell suspensions were prepared from the excised tumor tissues using gentleMACS Octo Dissociator (Miltenyi Biotec) and mouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730). The cell suspension was passed through 100 μm pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458), and the cell suspension that passed through the filters was collected. The cell suspension was reacted with mouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575), and then surface antigens were stained with anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), and anti-CD8 (53-6.7; catalog No.100734, BioLegend) at 4°C for 30 minutes. After washing, the stained cells were measured using a FACS CantoII (BD) and the data were analyzed using FlowJo software (Tree Star).

(result)
CD8+ T cells infiltrating into the tumors of the anti-LSR monoclonal antibody #27-6 mF-18 treated group were analyzed by FACS. As a result, the number of CD8+ T cells infiltrating into the tumors and the proportion of activated (CD69) cells were significantly increased in the anti-LSR monoclonal antibody #27-6 mF-18 treated group compared to the control antibody treated group (Figure 9). Therefore, it is believed that inhibition of LSR inhibits immune checkpoint stimulation and promotes migration and activation of CD8+ T cells into the tumors.

Example 8: Analysis of chemokine expression in 4T1 tumor tissue 2.0 x 10 ⁵ cells of the 4T1 cell line were subcutaneously transplanted into 8-week-old Balb/c female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. On the 19th day after cell transplantation, tumors were excised from the mice in each group. 2 μl of PBS was added per 1 mg of the tumor tissue to the excised 4T1 tumor tissue, and then the tumor tissue was incubated at 37° C. for 4 hours. The tumor tissue was then centrifuged (14,000 rpm, 4° C., 10 min), and the supernatant was then collected. The concentrations of chemokines (CXCL9, CXCL10) in the supernatants were quantified using BioLegend Legendplex™, a mouse proinflammatory chemokine panel.

(result)
CXCL9 and CXCL10 are known to be chemokines that attract CD8+ T cells, so the chemokines (CXCL9, CXCL10) in the tumor tissue were quantified. As a result, it was confirmed that the concentrations of CXCL9 and CXCL10 in the tumor tissue were significantly higher in the anti-LSR monoclonal antibody #27-6 mF-18 administration group than in the control antibody administration group (Figure 10). Therefore, it is considered that the increase in the concentrations of CXCL9 and CXCL10 in the tumor promotes the infiltration of CD8+ T cells into the tumor in the anti-LSR monoclonal antibody #27-6 mF-18 administration group.

Example 9: Drug efficacy analysis of anti-LSR antibody (#27-6mF18) using immunodeficient mice 2.0 x 10 ⁵ cells of the 4T1 cell line were subcutaneously transplanted into 7-week-old Balb/c nu/nu female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. At the time points shown in FIG. 11, tumor volume, body weight, and tumor weight were measured.

(result)
Balb/c nu/nu mice lacking T cell function were used to create tumor-bearing mice subcutaneously implanted with 4T1, and the in vivo efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was examined. As a result, no antitumor effect was observed. Therefore, it was confirmed that the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 in 4T1 tumor-bearing mice is T cell-dependent (Figure 11).

Example 10: Analysis of the efficacy of anti-LSR antibody (#27-6mF18) using syngenic model mice created by transplanting MC38-mLSR cells into C57BL/6 mice 5.0 x 10 ⁵ MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. Measurements of tumor volume, body weight, and tumor weight were performed at the time points shown in FIG. 12.

(result)
In Example 3, since the expression of LSR is negative in the mouse colon cancer cell line MC-38, MC38-mLSR-13, which stably expresses the mouse LSR gene, was established (Figure 3). By examining the in vivo antitumor effect of the anti-LSR monoclonal antibody #27-6 mF-18 using MC38-mLSR-13, it was examined whether the antitumor effect was observed as in the case of using 4T1 (Figure 5). As a result, the anti-LSR monoclonal antibody #27-6 mF-18 administration group showed a significant antitumor effect at both doses of 5 mg/kg and 20 mg/kg, compared with the PBS administration group and the control antibody administration group (20 mg/kg) (Figure 12).

Example 11: In vivo efficacy test to verify the influence of CD8 ⁺ T cells on the efficacy of anti-LSR antibody (#27-6mF18) in MC38-mLSR syngenic model mice 5.0 × 10 ⁵ MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and each group was administered isotype control antibody (5 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), anti-CD8 antibody (0.2 mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5 mg/kg) + anti-CD8 antibody (0.2 mg/body). Tumor volume, body weight, and tumor weight were measured at the time points shown in Figure 13.

(result)
The antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was examined when CD8+ T cells were removed by administering anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 to MC38-mLSR-13-implanted mice. As a result, it was confirmed that the antitumor effect exerted by anti-LSR monoclonal antibody #27-6 mF-18 was negated (Figure 13). It was confirmed that LSR monoclonal antibody #27-6 mF-18 exerts an antitumor effect by immune checkpoint inhibitory activity not only in 4T1 but also in MC38-mLSR-13.

These results suggest that anti-LSR antibodies activate tumor immunity by inhibiting the interaction between LSR on tumor cells and an unknown receptor on T cells, and exert an antitumor effect through activation of CD8+ T cells.

Example 12: Other LSR inhibitors In this example, tests similar to those in Examples 4 to 11 are performed using anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, and #1-43, siRNA (SEQ ID NO: 15 or 16), or small molecule compounds as LSR inhibitors.

Specifically, as in Example 4, anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm in vivo efficacy tests in a 4T1 syngenic mouse model (subcutaneous implantation). Also, as in Example 7, anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm the increase and activation of tumor-infiltrating CD8+ T cells.

These results suggest that regulating LSR can regulate immune checkpoints, regardless of the type of regulator.

The present disclosure has been described above based on examples. These examples are merely illustrative, and those skilled in the art will understand that various modifications are possible and that such modifications are also within the scope of the present disclosure.

As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted only in terms of the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth in this specification.

This application claims priority based on Japanese Patent Application No. 2022-179712, filed on November 9, 2022, the disclosure of which is incorporated herein in its entirety.

Malignant tumor control technology is provided, and technology that can be used in industries involved in technologies related to the treatment and prevention of malignant tumors (reagents, pharmaceuticals, etc.) is provided.

SEQ ID NO:1: Anti-LSR antibody 9-7 sequence SEQ ID NO:2: Anti-LSR antibody 16-6 sequence SEQ ID NO:3: Anti-LSR antibody 26-2 sequence SEQ ID NO:4: Anti-LSR antibody 27-6 sequence SEQ ID NO:5: Anti-LSR antibody 1-25 sequence SEQ ID NO:6: Anti-LSR antibody 1-43 sequence SEQ ID NO:7: Human LSR protein sequence (NP_991403.1)
SEQ ID NO: 8: Human LSR nucleic acid sequence (NM_205834.3)
SEQ ID NO: 9: Core sequence (guide sequence) of LSR siRNA 1
SEQ ID NO: 10: LSR siRNA 2 core sequence (guide sequence)
SEQ ID NO: 11: Antisense sequence of LSR siRNA 1 core sequence (guide sequence) SEQ ID NO: 12: Antisense sequence of LSR siRNA 2 core sequence (guide sequence) SEQ ID NO: 13: Full-length sense sequence of LSR siRNA 1 SEQ ID NO: 14: Full-length sense sequence of LSR siRNA 2 SEQ ID NO: 15: Full-length antisense sequence of LSR siRNA 1 SEQ ID NO: 16: Full-length antisense sequence of LSR siRNA 2

Claims

A composition for regulating immune checkpoints, comprising a regulator of the lipolysis-stimulating lipoprotein receptor (LSR).
The composition according to claim 1, wherein the LSR regulator is an inhibitor of LSR.
The composition according to claim 2, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
The composition according to claim 3, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
5. The composition according to claim 3 or 4.
A composition for increasing and/or activating tumor-infiltrating CD8+ T cells, comprising an LSR regulator.
The composition of claim 6, wherein the tumor-infiltrating CD8+ T cells are CD69 positive.
The composition according to claim 6 or 7, wherein the LSR regulator is an inhibitor of LSR.
The composition of claim 8, wherein the LSR inhibitor is an anti-LSR antibody or an antigen-binding fragment thereof.
The composition according to claim 9, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition according to claim 9 or 10, selected from the group consisting of:
The composition according to any one of claims 1 to 11, wherein the composition is for treating or preventing a malignant tumor.
The composition according to claim 12, wherein the malignant tumor is an LSR-positive malignant tumor.
The composition according to claim 12 or 13, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
A method for regulating immune checkpoints using a composition according to any one of claims 1 to 5.
The method of claim 15, for use in vitro or in vivo.
The method of claim 15 or 16, comprising administering to a patient a therapeutically effective amount of the composition.
The method according to any one of claims 15 to 17, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
A method for increasing and/or activating tumor-infiltrating CD8+ T cells using a composition according to any one of claims 6 to 11.
The method of claim 19, for use in vitro or in vivo.
The method of claim 19 or 20, comprising administering to a patient a therapeutically effective amount of the composition.
A method for treating or preventing malignant tumors using a composition according to any one of claims 1 to 15.
The method of claim 22, for use in vitro or in vivo.
The method of claim 22 or 23, comprising administering to a patient a therapeutically effective amount of the composition.
A composition according to any one of claims 1 to 5 for use in regulating immune checkpoints.
The composition according to any one of claims 6 to 11, for use in increasing the number and/or activating tumor-infiltrating CD8+ T cells.
A composition according to any one of claims 1 to 15 for use in the treatment or prevention of malignant tumors.