Composition for preventing or treating lymphoma, comprising Anti-CD20 antibody and pikfyve inhibitor as active ingredients

Abstract

The present invention relates to a composition comprising an anti-CD20 antibody and a PIKfyve inhibitor for preventing or treating CD20 positive cancer such as B cell lymphoma. The present invention can maximize the anticancer effect of an anti-CD20 antibody, specifically, obinutuzumab, by co-administering an anti-CD20 antibody and a PIKfyve inhibitor, which is a negative regulator of a tumor cell killing mechanism thereof. In addition, by using PIKfyve as a screening target, the present invention can rapidly identify, with high reliability, combination therapy candidate agents that can synergistically enhance the direct cell death (DCD), lysosomal membrane permeabilization (LMP), and subsequent cancer cell-killing activity of anti-CD20 antibodies.

Description

Composition for preventing or treating lymphoma comprising anti-CD20 antibody and PIKFYVE inhibitor as active ingredients

[0001] The present invention relates to an inhibitor of PIKfyve that synergistically enhances the tumor cell killing mechanism of an anti-CD20 antibody and a method for treating CD20-expressing tumors using the same.

[0002]

[0003] Non-Hodgkin lymphoma (NHL) refers to a group of diverse, heterogeneous hematologic malignancies. NHL originates from B cells and is clinicopathologically characterized by more than 20 different subtypes. Standard treatment for NHL patients includes chemotherapy and CD20-directed immunotherapy, such as rituximab and obinutuzumab. Rituximab was approved by the US Food and Drug Administration (FDA) in 1997 for the treatment of relapsed and refractory NHL. Rituximab, a type 1 anti-CD20 antibody, eliminates malignant B cells through complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and apoptosis. While rituximab was successful in early clinical trials, a significant number of patients have relapsed or become resistant to the drug.

[0004] Obinutuzumab is FDA-approved for the treatment of untreated follicular lymphoma (FL), relapsed or refractory FL, and untreated chronic lymphocytic leukemia (CLL). Obinutuzumab is a second-generation Fc-engineered type II antibody with modified sugar chains to enhance ADCC by enhancing interaction with FcγRIIIa expressed on immune effector cells. Obinutuzumab is notable for promoting both antigen-dependent cell cytotoxicity and direct cell death (DCD).

[0005] Herter et al. generated effector-silent Fc variants of obinutuzumab and rituximab harboring a PGLALA mutation, which fail to induce ADCC, ADCP, and CDC. When these variants were administered to a lymphoma transplantation mammary gland model, obinutuzumab-PGLALA exhibited cytotoxicity comparable to that of rituximab-WT and obinutuzumab-WT, while inducing near-complete tumor remission. This study demonstrated the importance of obinutuzumab-induced DCD and demonstrated that DCD alone is sufficient for cytotoxicity in lymphoma.

[0006] The key phenotypes of obinutuzumab-induced DCD include homotypic adhesion, lysosomal membrane permeabilization (LMP), and membrane damage. LMP in cells leads to the influx of lysosomal material into the cytoplasm, triggering lysosome-dependent apoptosis. Because cancer cells exhibit altered lysosomal composition and activity to meet their high energy demands and weaken their lysosomal membranes, targeting lysosomes could be an effective strategy for eliminating malignant tumor cells. Although the precise mechanism by which obinutuzumab induces DCD remains unclear, several studies have reported that LMP is essential for DCD. For example, co-administration of obinutuzumab with the anti-lysosomal agent LLOME enhances DCD in CLL-derived cells. Elucidating the molecular mechanisms underlying this effect could potentially improve NHL treatment.

[0007] Meanwhile, CRISPR (clustered regularly interspaced short palindromic repeats)-based screening platforms are used to predict gene function by analyzing large-scale genetic changes. CRISPRi (CRISPR interference) uses a catalytically inactive Cas9 (dCas9, a mutant RuvC1 and HNH nuclease domain) fused to a RAB repression domain. The dCas9-KRAB unit is guided to the target gene by a single guide RNA (sgRNA). The sgRNA is designed to target the promoter region, spatially disrupting the interaction between cis-acting DNA motifs and trans-acting transcription factors, thereby blocking transcription initiation and repressing gene expression.

[0008] Lysosomes are membrane-bound organelles that contain enzymes involved in the breakdown and regeneration of cellular components, making lysosomal homeostasis crucial for cellular maintenance. Lysosomal homeostasis is maintained through fusion and fission, and inhibition of lysosomal fission significantly impacts cellular stability and therapeutic responsiveness. Inhibition of lysosomal fission leads to lysosomal fusion, forming larger vacuoles, which adversely affects cellular homeostasis and leads to cellular instability.

[0009] We aimed to explore optimal combination therapies for the treatment of B-cell malignancies by identifying molecules that regulate obinutuzumab-induced DCD through an iCas-mediated knockdown system using an sgRNA library composed of drug target genes.

[0010]

[0011] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0012]

[0013] The present inventors have conducted extensive research to explore effective combinational pharmacological agents that can maximize the therapeutic efficacy of anti-CD20 antibodies in various CD20-positive tumors, including B-cell lymphoma. As a result, using a CRISPR-based screening platform, we have for the first time elucidated the involvement of the PIKfyve protein in the direct apoptosis (DCD) and lysosomal membrane permeabilization (LMP) mechanisms of anti-CD20 antibody-induced B-cell lymphoma. Furthermore, we have discovered that co-administration of a PIKfyve inhibitor with anti-CD20 antibodies can be utilized as an effective pharmaceutical combination with significantly enhanced B-cell killing activity.

[0014] Therefore, the purpose of the present invention is to provide a composition for preventing or treating CD20-expressing cancer.

[0015] Another object of the present invention is to provide screening of a pharmaceutical composition for co-administration with an anti-CD20 antibody.

[0016] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0017]

[0018] According to one aspect of the present invention, the present invention provides a composition for preventing or treating a CD20-expressing cancer, comprising as active ingredients an antibody or an antigen-binding fragment thereof that specifically binds to CD20; and an inhibitor of PIKfyve (FYVE finger-containing phosphoinositide kinase).

[0019] According to another aspect of the present invention, the present invention provides a method for preventing or treating a CD20-expressing cancer, comprising administering to a subject an antibody or an antigen-binding fragment thereof that specifically binds to CD20; and an inhibitor of PIKfyve (FYVE finger-containing phosphoinositide kinase).

[0020] The present inventors have conducted extensive research to explore effective combinational pharmacological agents that can maximize the therapeutic efficacy of anti-CD20 antibodies in various CD20-positive tumors, including B-cell lymphoma. As a result, using a CRISPR-based screening platform, we have for the first time elucidated the involvement of the PIKfyve protein in the direct cell death (DCD) and lysosomal membrane permeabilization (LMP) mechanisms of anti-CD20 antibody-induced B-cell lymphoma. We have also discovered that co-administration of a PIKfyve inhibitor with anti-CD20 antibodies significantly increases anticancer activity, thereby completing the present invention.

[0021] The term “antibody” as used herein refers to a peptide that recognizes and specifically binds to a specific epitope of CD20, and includes not only a complete antibody form but also an antigen-binding fragment (antibody fragment) of a full-length antibody molecule. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant region is of the kappa (κ) and lambda (λ) types.

[0022] The term “antigen-binding fragment of an antibody” as used herein means a fragment having significant antigen-antibody binding function within a full-length antibody molecule, and includes Fab, F(ab'), F(ab')2, Fv, and nanobody (or sybody).

[0023] Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, constant regions of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site.

[0024] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv has a heavy chain variable region and a single chain variable region linked covalently, usually through a peptide linker, or directly at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv.

[0025]

[0026] The term “inhibitor” as used herein means a substance that causes a decrease in the activity or expression of the PIKfyve protein, not only such that the activity or expression of PIKfyve becomes undetectable or present at an insignificant level, but also such that the inhibition of anti-CD20 antibody-induced apoptosis by PIKfyve can be significantly restored.

[0027] The term “reduction in expression” in this specification may mean a state in which the expression level of PIKfyve is reduced by, for example, 20% or more compared to the control group, more specifically, 30% or more, or even more specifically, 40% or more.

[0028] As used herein, the term “reduction in activity” refers to a measurable and significant decrease in the in vivo intrinsic biological function of PIKfyve compared to a control, and specifically refers to a decrease in the activity of PIKfyve to the extent that inhibition of apoptosis in a subject administered an anti-CD20 antibody can be improved or restored. The decrease in activity includes not only a simple decrease in function but also ultimate inhibition of activity due to a decrease in stability.

[0029] Inhibitors of PIKfyve include, but are not limited to, shRNA, siRNA, miRNA, ribozyme, peptide nucleic acids (PNA), antisense oligonucleotides, CRISPR systems that suppress the expression of PIKfyve proteins, the amino acid sequences and encoding nucleotide sequences of which are already known in the art, at the gene level, guide RNAs that recognize target genes, antibodies or aptamers that suppress at the protein level, as well as small molecule compounds, peptides, and natural products that suppress their activity, and all possible suppression means at the gene and protein levels can be used.

[0030] According to a specific embodiment of the present invention, the inhibitor of PIKfyve is at least one inhibitor selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the PIKfyve protein; an aptamer that specifically binds to the PIKfyve protein; and a nucleic acid molecule that inhibits the expression of a nucleotide encoding the PIKfyve protein.

[0031] According to the present invention, the PIKfyve inhibitor of the present invention may be a PIKfyve-specific antibody that inhibits the activity of PIKfyve at the protein level. The antibody that specifically recognizes PIKfyve is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody. The antibody of the present invention can be produced by methods commonly practiced in the art, for example, a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), a recombinant DNA method (U.S. Patent No. 4,816,567), or a phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). The general procedure for antibody preparation is described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999.

[0032] The present invention can also inhibit the activity of PIKfyve by using an aptamer that specifically binds to it instead of an antibody. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. General information on aptamers is described in detail in Hoppe-Seyler F, Butz K “Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R . “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24):14272-7(1998).

[0033] According to the present invention, the PIKfyve inhibitor of the present invention may be a nucleic acid molecule that inhibits the expression of a nucleotide encoding a PIKfyve protein.

[0034] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0035] The term “nucleic acid molecule that suppresses expression” as used herein means a nucleic acid molecule that can specifically recognize a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and induce a modification in the nucleotide structure that causes a decrease in its function, and includes, for example, the shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and gRNA included in the CRISPR system described above.

[0036] As used herein, the term “complementary” means that a nucleic acid molecule for suppressing expression is sufficiently complementary to a target nucleic acid sequence to selectively hybridize under certain annealing or hybridization conditions, and has a meaning that includes both substantially complementary and perfectly complementary, and preferably means perfectly complementary. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared, within the range where sequence-specific hybridization can occur by annealing to a specific sequence.

[0037] The term “shRNA (small hairpin RNA)” as used herein refers to an RNA sequence that forms a tight hairpin structure to suppress the expression of a target gene through RNA interference, which is a single-stranded structure consisting of 50-70 nucleotides that forms a stem-loop structure in vivo. Typically, a long RNA of 19-29 nucleotides complementarily forms a double-stranded stem by base pairing on both sides of a loop region of 5-10 nucleotides, and is transduced into cells through a vector containing a U6 promoter to ensure constant expression, and is usually passed on to daughter cells to ensure heritable suppression of the expression of the target gene.

[0038] The term “siRNA” in this specification refers to a short double-stranded RNA that can induce RNAi (RNA interference) by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and either blunt-ended or cohesive-ended is possible as long as it can suppress the expression of the target gene through the RNAi effect. The cohesive-ended structure can be either a 3-terminal protruding structure or a 5-terminal protruding structure.

[0039] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0040] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme, recognizing a specific base sequence in RNA and cleaving it. A ribozyme consists of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0041] As used herein, the term "PNA (Peptide Nucleic Acid)" refers to a molecule that possesses properties of both nucleic acids and proteins and can complementarily bind to DNA or RNA. PNA is not found in nature and is artificially synthesized through chemical methods. It forms a double strand through hybridization with a natural nucleic acid of complementary base sequence, thereby regulating the expression of target genes.

[0042] As used herein, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to a sequence of a specific mRNA and binds to the complementary sequence in the target mRNA, thereby inhibiting its translation into protein, translocation into the cytoplasm, maturation, or any other essential activity for its overall biological function. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance their potency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatoms, heterocyclic sugar sulphonates, etc.

[0043] As used herein, the term "gRNA (guideRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized region. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0044] The nucleic acid molecule of the present invention described above can suppress the expression of PIKfyve at the genetic level by expressing it in a subject who is or is scheduled to be administered an anti-CD20 antibody.

[0045]

[0046] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD20 comprises a heavy chain variable region comprising an HCDR1 region of sequence listing 1; an HCDR2 region of sequence listing 2; and an HCDR3 region of sequence listing 3.

[0047] The term “heavy chain” as used herein refers to a full-length heavy chain and fragments thereof comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen and three constant region domains CH1, CH2 and CH3.

[0048] As used herein, the term “CDR (complementarity determining region)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0049] The scope of the antibodies or antigen-binding fragments of the present invention includes variants having conservative amino acid substitutions in the CDR regions. In addition, the antibodies or antigen-binding fragments of the present invention may include variants of the amino acid sequences set forth in the attached sequence listing, as long as they can specifically recognize CD20. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody, and are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0050] Furthermore, amino acid substitutions in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath et al., The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0051] Considering the mutations having the above-described biological equivalent activity, the amino acid sequence constituting the antibody of the present invention is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence listing. The substantial identity means a sequence showing at least 61% homology, in one specific example 70% homology, in another specific example 80% homology, and in yet another specific example 90% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods and algorithms for sequence comparison are disclosed in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.

[0052] According to a more specific embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD20 additionally comprises a light chain variable region comprising an LCDR1 region of sequence number 4; an LCDR2 region of sequence number 5; and an LCDR3 region of sequence number 6.

[0053] The term “light chain” as used herein refers to both full-length light chains and fragments thereof comprising a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen.

[0054] More specifically, the antibody or antigen-binding fragment thereof may include a heavy chain variable region of sequence number 7 and / or a light chain variable region of sequence number 8.

[0055] Most specifically, the antibody or antigen-binding fragment thereof that specifically binds to CD20 is obinutuzumab.

[0056]

[0057] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD20 comprises a heavy chain variable region comprising an HCDR1 region of sequence number 9; an HCDR2 region of sequence number 10; and an HCDR3 region of sequence number 11.

[0058] According to a more specific embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD20 additionally comprises a light chain variable region comprising an LCDR1 region of sequence number 12; an LCDR2 region of sequence number 13; and an LCDR3 region of sequence number 14.

[0059] More specifically, the antibody or antigen-binding fragment thereof may include a heavy chain variable region of sequence number 15 and / or a light chain variable region of sequence number 16.

[0060] Most specifically, the antibody or antigen-binding fragment thereof that specifically binds to CD20 is rituximab.

[0061]

[0062] According to a specific embodiment of the present invention, the inhibitor of PIKfyve is at least one small molecule inhibitor selected from the group consisting of YM201636, APY0201, MOMIPP, Vacuolin-1, Umbralisib, a compound represented by the following chemical formula 1, and a pharmaceutically acceptable salt thereof:

[0063] Chemical Formula 1

[0064]

[0065] In the above chemical formula, R1 and R2 are each independently N or CH, R3 is C1-C3 alkyl, and n is an integer from 1 to 3.

[0066] The term “alkyl” as used herein means a straight-chain or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl means an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the carbon number of the substituent is not included.

[0067] As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium.

[0068] More specifically, in the chemical formula, R1 is N, R2 is CH, R3 is C1 alkyl, and n is 2. The compound of chemical formula 1, wherein R1, R2, R3, and n are N, CH, C1 alkyl(methyl), and 2, respectively, is apilimod (3-methyl-benzaldehyde-2-[6-(4-morpholinyl)-2-[2-(2-pyridinyl)ethoxy]-4-pyrimidinyl]hydrazone). Apilimod is an anti-inflammatory agent that inhibits the pro-inflammatory cytokines IL-12 and IL-23, and was developed as a treatment for Crohn's disease or rheumatoid arthritis. It is a small molecule drug that is currently widely used as an inhibitor of the lipid kinase enzyme PIKfyve.

[0069]

[0070] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0071] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating a disease, condition, or symptom; or (c) eliminating a disease, condition, or symptom. When the composition of the present invention is administered to a subject, it blocks the proliferation of cancer cells expressing CD20 on the surface and synergistically induces direct apoptosis, thereby suppressing the progression of symptoms caused by CD20-expressing malignant tumors such as B-cell lymphoma, eliminating them, or alleviating them. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and applied as an adjuvant treatment for B-cell lymphoma, etc. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0072] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0073] The term “therapeutically effective amount” as used herein means the content of a composition containing a pharmacological ingredient in the composition sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0074] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0075]

[0076] According to a specific embodiment of the present invention, the CD20 expressing cancer is a B cell origin malignant tumor, and more specifically, non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL).

[0077] More specifically, the non-Hodgkin's lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and mantle cell lymphoma.

[0078]

[0079] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of CD20-expressing cancer, which is a pharmaceutical composition for co-administration with an antibody or an antigen-binding fragment thereof that specifically binds to CD20, comprising an inhibitor of PIKfyve (FYVE finger-containing phosphoinositide kinase) as an active ingredient.

[0080] The PIKfyve inhibitor used in the present invention, the anti-CD20 antibody, and the CD20-expressing cancer that can be prevented or treated by the same have already been described above, so their description is omitted to avoid excessive duplication.

[0081] As described above, the PIKfyve inhibitor of the present invention and the anti-CD20 antibody showed a remarkable synergistic effect when administered together, and therefore, a PIKfyve inhibitor including apilimod can be administered together with a CD20 antibody including obinutuzumab to maximize the anticancer effect and improve the survival rate of patients.

[0082] Co-administration may be accomplished by administering a single formulation containing both the PIKfyve inhibitor of the present invention and the anti-CD20 antibody, or by administering separate formulations containing each active ingredient individually, either simultaneously or sequentially with an appropriate time difference in any order.

[0083]

[0084] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

[0085] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0086] The pharmaceutical composition of the present invention can be administered orally or parenterally, specifically parenterally, and more specifically intravenously, intraperitoneally, intramuscularly, or subcutaneously.

[0087] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0088] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0089] According to another aspect of the present invention, the present invention provides a method for screening a pharmaceutical composition for co-administration with an antibody or antigen-binding fragment thereof that specifically binds to CD20, comprising the following steps:

[0090] (a) contacting a candidate substance with a biological sample containing cells expressing PIKfyve (FYVE finger-containing phosphoinositide kinase); and

[0091] (b) a step of measuring the activity or expression level of PIKfyve in the sample;

[0092] If the activity or expression level of the above PIKfyve is reduced, the candidate substance is determined to be a pharmaceutical composition for co-administration with an antibody or antigen-binding fragment thereof that specifically binds to CD20.

[0093] As used herein, the term “biological sample” refers to any sample obtained from a mammal, including a human, that contains cells expressing PIKfyve, including, but not limited to, tissues, organs, cells, or cell cultures.

[0094] The term “candidate substance” used in referring to the screening method of the present invention refers to an unknown substance used in the screening to examine whether it affects the activity or expression level of PIKfyve when added to a sample containing cells expressing PIKfyve. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of PIKfyve in a biological sample treated with the test substance can be performed by various expression level and activity measurement methods known in the art. If the measurement result shows that the expression level or activity of PIKfyve is decreased, the test substance can be determined to be a pharmacological ingredient for combination administration that exhibits a synergistic anticancer effect when administered together with an anti-CD20 antibody in the prevention or treatment of CD20-expressing cancer.

[0095] According to the present invention, the expression level of the PIKfyve protein, which is the screening target of the present invention, can be measured by an immunoassay method using an antigen-antibody reaction. This immunoassay can be performed according to various immunoassay or immunostaining protocols developed in the past. For example, when the method of the present invention is performed according to a radioimmunoassay method, a radioisotope (C) 14 , I 125 , P 32 and S 35 ) can be used.

[0096] According to another embodiment of the present invention, the expression level of PIKfyve can be measured at the gene level using an agent that measures the expression level of a gene encoding the PIKfyve protein. The agent that measures the expression level of the gene is, for example, a primer or probe that specifically binds to a nucleic acid sequence of the gene encoding the PIKfyve protein.

[0097] As used herein, the term “primer” refers to an oligonucleotide that acts as an initiation point for synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerization agent such as DNA polymerase, and conditions of suitable temperature and pH. Specifically, the primer is a single chain of deoxyribonucleotides. The primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

[0098] The extension primer used in the present invention includes a hybridizing nucleotide sequence complementary to a specific base sequence of a target nucleic acid, for example, a target gene. The term “complementary” means that the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under predetermined annealing or hybridization conditions, and includes both substantially complementary and perfectly complementary sequences, and specifically means perfectly complementary. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared, within a range that can anneal to a specific sequence and act as a primer.

[0099] As used herein, the term “probe” refers to a linear oligomer having a natural or modified monomer or linkage comprising deoxyribonucleotides and ribonucleotides that can hybridize to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As the probe used in the present invention, a sequence that is perfectly complementary to a specific base sequence of a target gene can be used, but a sequence that is substantially complementary can also be used as long as it does not interfere with specific hybridization. Conditions suitable for hybridization can be determined with reference to the matters disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0100] According to a specific embodiment of the present invention, the cell used in the screening method of the present invention is a B cell, and more specifically, a Raji B cell.

[0101]

[0102] The features and advantages of the present invention are summarized as follows:

[0103] (a) The present invention provides a composition for the prevention or treatment of CD20-positive cancer such as B-cell lymphoma, comprising an anti-CD20 antibody and an inhibitor of PIKfyve.

[0104] (b) The present invention can maximize the anticancer effect of an anti-CD20 antibody, specifically, obinutuzumab, by co-administering an inhibitor of PIKfyve, a negative regulator of the tumor cell death mechanism thereof, together with an anti-CD20 antibody.

[0105] (c) In addition, the present invention can rapidly and reliably search for an efficient combination administration candidate substance that can synergistically increase direct cell death (DCD), lysosomal membrane permeabilization (LMP) and cancer cell killing activity through this of an anti-CD20 antibody using PIKfyve as a screening target.

[0106]

[0107] Figure 1 illustrates the CRISPRi screening process to identify regulators of the DCD-induced pathway by obinutuzumab. Figure 1a is a schematic diagram of the CRISPRi screening process. dCas9-Raji cells were transduced via lentivirus with a sgRNA sub-library targeting kinases, phosphatases, and drug targets. dCas9-library-Raji cells were treated with 10 μg / ml OBI-WT for 4 hours and stained with 50 nM Lysotracker (LT) for 30 minutes. LT-positive cells were isolated on a FACS Aria III, expanded in vitro for NGS analysis, and re-sorted. Figure 1b shows the results of fluorescence microscopy of sgRNA-BFP transduced into HEK293T cells. Figure 1c shows the flow cytometry results for dCas9-Raji cells transduced with sgRNA vectors via lentivirus before and after selection with 0.75 μg / mL puromycin. Figures 1d and 1e are diagrams showing the results of flow cytometry (Figure 1d) and Western blot (Figure 1e) verification of the dCas9-expressing Raji cell line, respectively. Figure 1f shows the results of filtering the 2,318 genes of Jonathan Weissman's kinase, phosphatase, and drug target CRISPRi sgRNA sub-library using Metascape (Uniprot: kinase class, DrugBank: drug target) and the HGNC phosphatase set to identify genes belonging to each functional class.

[0108] Figure 2 shows the results of selecting hit genes based on fluorescence-activated cell sorting (FACS). Figure 2a shows the results of re-treating FACS-isolated lysotracker-positive dCas9-Raji cells with OBI-WT and lysotracker. The LMP (Lysotracker-negative) rate was measured by flow cytometry and a representative histogram is shown. Figure 2b shows the results of analyzing the proportion of dCas9-CD20 sgRNA-BFP-Raji cells (positive control) within the entire subpopulation. Control cells were passed through FACS to measure FACS-induced cell damage and a representative histogram is shown. Figure 2c shows the results of the LMP assay for NGS analysis of the isolated cell population. The % LMP (Lysotracker-negative) decreased with repeated cell sorting. Figure 2d shows the results of the DCD assay for NGS analysis of the isolated cell population. % DCD (calcein-negative) decreased with repeated cell separation.

[0109] Figure 3 shows the results of a CRISPRi screening to identify regulators of the obinutuzumab-induced apoptosis pathway. Figure 3a is a volcano plot showing genes that were upregulated or depleted within the screen. The data were analyzed using a model-based approach using the genome-wide CRISPR / Cas9 knockout (MAGeCK) algorithm, and each candidate gene is indicated. Figure 3b is a schematic diagram illustrating the criteria for selecting hit genes during the negative selection process. Figure 3c is an sgRNA enrichment plot showing the relative positions of five sgRNAs / gene. Depleted genes are indicated in blue, and positive control genes (MS4A1, CD20) are indicated in pink. Figure 4d is a box plot showing LFC per gene, with the numbers at the top indicating the median LFC for each gene.

[0110] Figure 4 shows the results of CRISPRi screening, which identified PIKfyve as a combination target for obinutuzumab-induced apoptosis. Figure 4a shows the results of quantitative PCR (qPCR) to verify the knockdown of candidate genes. Figures 4b and 4c show the results of LMP analysis (Figure 4b) and cell morphology-based DCD assay (Figure 4c) for a single knockdown cell line. Figure 4d shows the correlation between the LMP and DCD pathways. Figures 4e and 4f show the results of cytotoxicity evaluations of expression inhibitors used in the present invention. Cells were pretreated with inhibitors for 24 hours, and then 0.3 μg / mL of obinutuzumab was administered. Apilimod exhibited superior cytotoxicity even at low concentrations compared to other superior inhibitors. Figure 4g shows the results of comparing the Raji B cell killing activity by flow cytometry in combination with ubralisib, another PIKfyve inhibitor, and acalabrutinib, a BTK inhibitor, in obinutuzumab to further verify that PIKfyve is a valid combination target for obinutuzumab-induced apoptosis.

[0111] Figure 5 shows that apilimod and obinutuzumab synergistically induce B cell apoptosis. Figure 5a shows the results of the Raji DCD assay in which Raji cells were pretreated with apilimod for 24 hours and then treated with rituximab (left) and obinutuzumab (right) for 4 hours. Figure 5b shows the results of the Raji CDC assay in which Raji cells were pretreated with apilimod for 24 hours and then treated with complement and 0.3 μg / mL rituximab or obinutuzumab for 1 hour.

[0112] Figure 6 illustrates the lysosomal fusion and fission mechanisms in relation to apilimod, PIKfvye, and obinutuzumab activity. Figure 6a is a schematic diagram of the core lysosomal and fission mechanisms. Fusion inhibition reduces obinutuzumab-mediated cell death, and division inhibition enhances obinutuzumab-mediated cell death. Figure 6b shows that co-treatment with OSI-027 and obinutuzumab does not induce apoptosis. Figure 6c shows that OSI-027 induces vacuolization. Scale bar 10 μM. Figure 6d shows that WIPI2 mutations do not increase obinutuzumab-induced cell death. Figure 6e shows that a WIPI2 dominant-negative hypermutant exhibits vacuolization. Scale bar 10 μM. Figure 6f shows that co-treatment with 10 μM MLSI-3 and 0.3 μg / mL obinutuzumab for 4 hours reduced apoptosis. Figure 6g shows that co-treatment with BAY-1797 and obinutuzumab reduced obinutuzumab-induced apoptosis. Figure 6h shows that apilimod treatment induces vacuolization, and it can be seen that apilimod-induced vacuolization is reversibly restored by BAY-1797 treatment. Scale bar 10 μM.

[0113] Figure 7 is a schematic diagram showing how PIKfyve inhibition by apilimod enhances bOBI-induced LMP. Apilimod treatment inhibits lysosomal fission, leading to lysosome expansion, and obinutuzumab treatment further inhibits further lysosomal fission by blocking TRPML2 activity. Thus, apilimod and obinutuzumab can synergistically induce LMP.

[0114]

[0115] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0116]

[0117] Example

[0118] Experimental method

[0119] cell culture

[0120] Raji B cells (provided by Professor Seung-Hwan Kim of Chungnam National University) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (26140-079) and 1% penicillin-streptomycin (15140-122, Gibco, Life technologies, Carlsbad, CA, USA). Cells were cultured in a humidified incubator (37°C, 5% CO2), and 1.0 x 10 Raji cells were plated. 5 Cells were seeded at 10 cells / mL and subcultured every 2-3 days.

[0121]

[0122] Production and purification of obinutuzumab-WT (OBI-WT)

[0123] 1.0 x 10 for 10 x 100 mm plates 6 Cells were seeded and cultured at 37°C for 1 day. The following day, the culture medium was gently aspirated and replaced with EX-CELL CD CHO medium (10743011, Thermofisher) and 1 mM sodium butyrate (LS 033-01, WELGENE, Daegu, Korea). Cells were cultured at 30°C for 2 weeks, and the culture medium containing secreted antibodies was collected, centrifuged at 1,000 rpm for 3 minutes, and filtered through a 0.45 μM syringe filter to remove cell debris.

[0124] Antibody-containing medium was mixed with agarose A beads (Pierce Protein A Agarose, Thermofisher, QE218104), placed on a rotator, and incubated overnight at 4°C. The solution was loaded onto a gravity filtration column, and the beads were washed three times with cold PBS. The antibodies were eluted with 0.1 M citric acid and 1 M Tris, loaded onto a dialysis cassette, and dialyzed overnight. The dialyzed antibodies were concentrated in Amicon® Ultra-15 Centrifugal Filter Units (UFC900324, Merck, 3K). Antibody concentrations were measured using a NanoDrop TM Quantification was performed using a Lite spectrophotometer, and purity was analyzed using SDS-PAGE.

[0125]

[0126] Lysosomal membrane permeabilization (LMP) assay

[0127] 1.0×10 suspended in 100μL complete RPMI 5 Raji cells were treated with 10 μg / mL antibodies (IgG, rituximab, obinutuzumab) at 37°C for 4 h. Cells were stained with 50 nM Lysotracker Deep Red for 30 min at 37°C and subjected to flow cytometry. Cells were gated as whole cells, single cells, and Lysotracker-positive cells.

[0128]

[0129] Direct apoptosis assay

[0130] 1.0 x 10 5 Raji cells were resuspended in 100 μL of complete RPMI and treated with 10 μg / mL antibodies (IgG, rituximab, and obinutuzumab) at 37°C for 4 h. Cells were stained with 0.25 μM calcein-AM for 30 min at 37°C, and flow cytometric analysis was performed on 10,000 samples. Cells were gated on total cells, single cells, and calcein-AM-positive cells.

[0131]

[0132] CD20 quantitation

[0133] 2.0 x 10 5 Raji cells were resuspended in 100 μL PBS and treated with 10 μg / mL rituximab for 30 min at 4°C. After washing the cells once with PBS, they were resuspended in 100 μL PBS and incubated with 1:500 anti-human Ig Fc-specific FITC-conjugated secondary antibody (109-095-008, 1:200 dilution; Jackson Laboratories) at 4°C for 30 min. The cells were washed once with PBS and resuspended in fresh PBS. Flow cytometry was performed on 10,000 samples to measure changes in CD20 content.

[0134]

[0135] Generation of CRISPRi Raji B cell lines

[0136] Raji B cells were transduced with SSFV-dCas9-mCherry-KRAB (Addgene #180264) via lentivirus. Polyclonal dCas9-Raji B cell lines were obtained by FACS for mCherry-positive cells. Cells were sorted into mCherry-low, -intermediate, and -high cells based on mCherry fluorescence intensity using a BD FACSAriaⅢ Cell Sorter.

[0137] The dCas9-Raji B cell line was validated using sgRNA targeting MS4A1, a B cell surface marker. sgMS4A1 or a non-targeting control (sgERBB2) was lentivirally transduced into the dCas9-Raji B cell line, and knockdown cells were selected by treatment with 1 μg / mL puromycin (ant-pr-1, Invivogen). After binding to rituximab, cells were stained with goat anti-human Fc-FITC and analyzed by flow cytometry using LSRⅡ (BD Biosciences) to measure the intramembrane MS4A1 content.

[0138]

[0139] sgRNA packaging and transformation

[0140] sgRNA sub-pooled libraries targeting kinases, phosphatases, and drug targets, respectively, were generously provided by Jonathan Weissman (Addgene #83971). The sgRNA sub-libraries were initially amplified by E. coli transformation. Briefly, 50 ng of the sgRNA sub-library was transformed into SS320 electrocompetent E. coli by electroporation at 2.4 kV, 25 μF, and 300 Ω using a Bio-Rad Gene Pulser II Electroporator. A portion of the transformed cells was recovered in 25 mL of SOC medium at 37°C and 160 rpm for 30 min, serially diluted, and plated onto LB medium and ampicillin-containing agar plates, and the transformation efficiency was calculated. The remaining transformed cells were cultured overnight in 200 mL of LB broth, and plasmids were extracted by mini-prep.

[0141] The sgRNA sublibrary was packaged into a lentiviral vector and introduced into dCas9-Raji cells. Briefly, 3.0 x 10 6 HEK293T cells were seeded, and when cells reached 80% confluency the following day, 8 μg of the sgRNA-sublibrary was transfected with 6 μg psPA X 2, 2 μg pMD2.G, and 48 μL polyethylenimine (PEI) for 16 h. The PEI-containing medium was gently aspirated, and fresh DMEM supplemented with 10% FBS and 1% penicillin-streptomycin was added. The culture supernatant was collected 3 days after transfection, filtered through a 0.22 μM syringe filter, and the virus was precipitated.

[0142] To establish sgRNA-dCas9-Raji knockdown cell lines, 1.3 x 10 7dCas9-Raji cells were transduced via lentivirus with an initial target transfection rate of 20–50%. Initial transfection efficiency was measured by flow cytometry. Stably transduced cells were enriched by treating the knockdown pool with 0.75 μg / mL puromycin.

[0143]

[0144] CRISPRi screening

[0145] 1.3 x 10 7 sgRNA-dCas9-Raji cells (corresponding to 1,000 x library coverage) were resuspended in 20 mL complete RPMI and seeded in 6-well plates (1.3 x 10 6 Cells were divided equally into 10 × 10 cells / well. Cells were treated with 10 μg / mL obinutuzumab for 4 h at 37°C and then stained with 50 nM LysoTracker Deep Red for 30 min at 37°C. Cells were collected, washed once, resuspended in 1 mL complete RPMI, and filtered with a Falcon 40 μM cell stainer. Lysotracker-positive cells were isolated by BD FACS Aria III and cultured in complete RPMI for 1 week. 1.0 × 10 cells were recovered. 5 Lysosomal content was measured by performing the LMP assay on cells. FACS was repeated until the majority of cells became Lysotracker-positive.

[0146]

[0147] CRISPRi genomic DNA (gDNA) isolation

[0148] DNA extraction was performed for control cells and obinutuzumab-treated cells using the MCherry-Nagel's Nucleospin Blood Midi kit according to the manufacturer's instructions. Briefly, 1.3 x 10 7Cells were washed with PBS and resuspended in 2 ml of PBS. The cells were then mixed with proteinase K and lysis buffer and incubated at 56°C for 15 minutes. The sample was cooled to room temperature, pure ethanol was added, and the lysate was loaded onto a DNA binding column, centrifuged, and washed. Finally, the DNA was eluted with ultrapure distilled water, and the extracted gDNA was loaded onto a 1% (w / v) agarose gel for electrophoresis.

[0149]

[0150] Amplification of sgRNA fragments

[0151] The sgRNA-containing fragment in gDNA was amplified by PCR using the following PCR reaction mixture: 10 μg template, 1 μM forward primer (5'-CAGCACAAAAG GAAACTCACCCTAACTG-3'), 1 μM reverse primer (5'-CGACTCGGTGCCACTTTT-3'), 50 μL NEB Ultra Ⅱ Q5 2 x master mix, and 100 μL distilled water. Fifteen to twenty tubes, each containing 100 μL of PCR reaction, were amplified by 23 cycles of three-step PCR using an Applied Biosystems 2720 thermal cycler. All reactions were combined and a portion was loaded onto a 1.5% (w / v) agarose gel.

[0152] The PCR product (280 bp) was purified with SPRIselect beads (Beckman and Coulter B23317), and a second SPRI purification was performed using 300 μL of the PCR product. In the first purification, 300 μL of the PCR product was mixed with 195 μL of SPRI beads (0.65 x SPRI beads) and incubated at room temperature for 10 minutes. During this step, fragments >300 bp bind to the SPRI beads, while fragments <300 bp remain in the supernatant. The tube was then placed on a magnetic stand for 5 minutes to separate the supernatant containing the PCR product. The supernatant was then transferred to a new Eppendorf tube to prepare for the second purification. During this process, 300 μL of SPRI beads (1 x SPRI beads) were added and mixed with the sample. Here, fragments >150 bp bind to the magnetic beads. After incubation at room temperature for 10 minutes, the tube was placed back on the magnetic stand for 5 minutes. The supernatant was carefully removed, and the beads were washed twice with fresh 80% ethanol. The beads were completely air-dried and eluted with 20 μL of ultrapure deionized water.

[0153] Purified PCR products were applied to the Agilent 4200 TapeStation using the D1000 kit according to the manufacturer's instructions. Briefly, 3 μL of sample buffer was added to 1 μL of DNA ladder or sample in an optical tube strip. The sample was vortexed at 2,000 rpm for 1 minute and gently spun down. Each tube was analyzed using TapeStation software.

[0154]

[0155] PCR amplification of sgRNA fragments (second PCR round)

[0156] Macrogen, a next-generation sequencing (NGS) service provider, performed secondary PCR and NGS on primary PCR products using the TruSeq Nano DNA (LMW) library kit according to the manufacturer's instructions. The Index 7 sequence is 5'-TGGCCGGT-3' and 5'-CAATTAAC-3' for the control and obinutuzumab-treated samples, respectively. The Index 5 sequence is 5'-TAGAGCGC-3' and 5'-CGAGATAT-3' for the control and obinutuzumab-treated samples, respectively. The secondary PCR products were analyzed using the Agilent 4200 TapeStation and D1000 kit according to the previously described method.

[0157]

[0158] Next-generation sequencing (NGS)

[0159] The secondary PCR products were loaded onto the Illumina NovaSeq platform and hybridized to surface-bound oligonucleotides complementary to the Index 7 and 5 sequences described above. In situ amplification for clustering was then performed by adding polymerase, dNTPs, and buffer. The clusters were sequenced using SBS (sequencing by synthesis) chemistry.

[0160]

[0161] CRISPRi-sgRNA screening data analysis

[0162] The read sequences from the fastq files were first mapped to kinase, phosphatase, and drug target sublibraries after removing adapter sequences. sgRNA read counts per guide were generated for both control and obinutuzumab-treated samples. Genes were ranked using the model-based analysis algorithm of Whole Genome CRISPR / Cas9 Knockout (MAGeCK). Rankings were determined based on robust ranking aggregation (RRA) scores, log2 fold change (LFC), and false discovery rate (FDR). Hit genes with FDR <0.25 were further selected based on DepMap gene variability scores, gene expression profiles, and the presence of available inhibitors.

[0163]

[0164] Validation of CRISPRi hit genes using individual gene knockdown cell lines.

[0165] Individual sgRNAs targeting the hit genes were cloned into the CRISPRi / a V2 library parental plasmid (AddGene #84832). The parental vector was digested with BstXI and BlpI for 2 h at 37°C and purified by gel extraction. The sense and antisense strands of sgRNA oligonucleotides were synthesized to span BstXI and BlpI. Each oligo pair was hybridized by mixing the two strands with 10 x T4 ligation buffer (NEB) and T4 PNK (NEB). Samples were incubated at 37°C for 30 min, then at 95°C for 5 min, and then cooled to 25°C. The annealed oligos were diluted 10-fold, and the digested vector, 1 μl of the diluted oligo duplexes, 10 x T4 NEB, and T4 ligase (NEB) were added to a PCR tube and incubated at room temperature for 2 hours. The ligation products were introduced into DH5a cells by heat shock transformation. The transformed DH5a cells were cultured overnight on LB agar plates with ampicillin, and a single colony was inoculated into LB broth the following day and mini-prepped. The extracted plasmids were sequenced using the U6 primer.

[0166] sgRNAs targeting each gene were introduced into dCas9-Raji B cells via lentivirus. To measure transfection efficiency, cell images were obtained using a fluorescence microscope for BFP 2 days after transfection. Two days after transfection, cells were treated with 1 g / mL puromycin, and complete RPMI was added to dilute the virus. Transfection efficiency was measured by flow cytometry. Cell lines with each gene knockdown were treated with obinutuzumab and then treated with Lysotracker Deep Red for the LMP assay.

[0167]

[0168] Obinutuzumab-drug combination treatment (cytotoxicity assay)

[0169] 5.0 x 10 4 Raji cells were seeded in 96-well plates and pretreated with the indicated concentrations of drugs for 24 hours in a humidified incubator (37°C, 5% CO2). Cells were then treated with the indicated concentrations of obinutuzumab for 4 hours at 37°C, 5% CO2, and stained with propidium iodide (PI) for 30 minutes. Cell morphology changes in 10,000 cells were analyzed by flow cytometry using a BD FACSLyric as previously reported (Iulianna, T. et al., Cell Death & Disease, 13:509 (2022)). Briefly, cells were first gated for singlets (based on FSC-A vs. FSC-H scatter) and then gated by lymphocytes (based on FSC-A and SSC-A scatter). Cell viability was quantified as the percentage of cells within the lymphocyte gate. Cytotoxicity profiles were analyzed by quantifying PI, a fluorescent dye that binds to nucleic acids in apoptotic cells. Cytotoxicity was defined as “% PI positivity.”

[0170]

[0171] Complement-dependent cytotoxicity (CDC) assay

[0172] 5.0 x 10 4 Raij was pretreated with 50 nM apilimod for 24 h in 96-well plates in a humidified incubator (37°C, 5% CO2). Cells were treated with increasing concentrations of rabbit complement-MA and 0.3 μg / mL obinutuzumab-GE or RTX for 1 h. Cells were stained with PI for 30 min before flow cytometry, and 2,000 samples were analyzed using a BD FACS. % CDC was defined as “% PI-positive.”

[0173]

[0174] RNA extraction and quantitative PCR (qPCR)

[0175] RNA was extracted from Raji cells using the TRIzol protocol. 3.0 x 106 Raji cells were washed once with PBS and spun down. Cells were resuspended in 500 μl of TRIzol and homogenized by pipetting and vortexing. 100 μl of chloroform was added, and the two layers were vortexed. The tubes were centrifuged to separate the layers, and the liquid layer was carefully transferred to a new tube. RNA was washed, dried, and eluted in distilled water. The concentration and purity of the RNA sample were confirmed using a NanoDrop, and cDNA was synthesized using an RNA template, an oligo dT primer, and reverse transcriptase. qPCR primers were designed using PrimerBank, and qPCR was performed according to the standard protocol for SYBR green.

[0176]

[0177] vacuolization imaging

[0178] 5.0 x 10 4 Raji cells were seeded in 96-well plates and treated with 50 nM apilimod, 50 μM OSI-027, or 50 μM BAY-1797 for 24 h. Cells were then stained with 100 nM Lysotracker Red for 30 min. Cells were centrifuged, resuspended in 4 μl RPMI, and observed under a fluorescence microscope.

[0179]

[0180] Statistical analysis

[0181] Data were expressed as mean ± standard deviation, and statistical significance was assessed by Student's t-test as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0182]

[0183] Amino acid sequence of antibody used in the present invention Sequence number name Sequence 1 Obi-HCDR 1 YSWIN 2 Obi-HCDR 2 RIFPGDGDTDYNGKFK 3 Obi-HCDR 3 NVFDGYWLVY 4 Obi-LCDR 1 RSSKSLLHSNGITYLY 5 Obi-LCDR 1 QMSNLVS 6 Obi-LCDR 1 AQNLELPYT 7 Obi-heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLE WMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS8Obi-Light Chain Variable RegionDIVMTQTPLSL PVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGT KVEIK9Rit-HCDR1SYNMH10Rit-HCDR1AIYPGNGDTSYNQKFKG11Rit-HCDR1STYYGGDWYFNV12Rit-LCDR1RASSSVSYIH13R it-LCDR1ATSNLAS14Rit-LCDR1QQWTSNPPT15Rit-Heavy chain variable regionQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIG AIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVASA16Rit-Light Chain Variable RegionQIVLSQSPAI LSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0184] 본 발명에서 사용된 sgRNA 올리고뉴클레오타이드 서열유전자방향서열 (서열벴눥서열) GGAGGGAGCGCAGTAACGGG G TTT AAG AGC (17)RTTA GCT CTT AAAC CCCGTTACTGCGCTCCCTCC CAA CAA G (18)CAPN1FTTG GCACCGGGAAGCCAGCCTCA G TTT AAG AGC (19)RTTA GCT CTT AAAC TGAGGCTGGCCGTT GGCCGTT CACHGTT2 GCGGGGCGGCCGCAGCGCGAG G TTT AAG AGC (21)RTTA GCT CTT AAAC CTCGCGCTGCGGCCGCCCGC CAA CAA G (22)DCTDFTTG GGCGCGGAGCCGGCACCGGA G TTT AAG AGC (23)RTTA GCT CTT AAAC TCCGGTCGCCGCCGAGCTCAGCT424 GAGGGCCGCCGAACTACCCC G TTT AAG AGC (25)RTTA GCT CTT AAAC GGGGTAGTTCGGCGGCCCTC CAA CAA G (26)ODC1FTTG GTAGGGAGCGGCGTGCCGTG G TTT AAG AGC (27)RTTA GCT CTT AAAC CACGGCCACCCGCACTCACTGCACT28) GCGGAGTCCTGCGCACGCCA G TTT AAG AGC (29)RTTA GCT CTT AAAC TGGCGTGCGCAGGACTCCGC CAA CAA G (30)PIK3R4FTTG GCC AGC AGC TGG AGC GGA GT G TTT AAG AGC (31)RTTA GCT CTT AAA CAC TCC GCT GCT GCT GCT CA GCT CA (32)PIKFYVEFTTG GAGTCGGCCCCCGAGAGCGG G TTT AAG AGC (33)RTTA GCT CTT AAAC CCGCTCTCGGGGGCCGACTC CAA CAA G (34)SLC16A1FTTGGTGGCTAGCTGCGTGGGTAC G TTT AAG AGC (35)RTTA GCT CTT AAAC GTACCCACGCAGCTAGCCAC CAA CAA G (36)SMSFTTG GCTGGGAGTGTGCTGCGCCC G TTT AAG AGC (37)RTTA GCT CTT AAAC GGGGCAGGCCACTCA GCTCA GCTCA GTCCAQCA ​​GTT38 GCACCAGCATCCCTCGCGGG G TTT AAG AGC (39)RTTA GCT CTT AAAC CCCGCGAGGGATGCTGGTGC CAA CAA G (40)TRPM7FTTG GCGGCCTGTAGCCATCTATC G TTT AAG AGC (41)RTTA GCT CTT AAAC GATAGATGGGCCAGCCAGCCAGCA CAGCA (42)VDAC1FTTG GCCCGCCGCCACATCCTCTG G TTT AAG AGC (43)RTTA GCT CTT AAAC CAGAGGATGTGGCGGCGGGC CAA CAA G (44)

[0185] Download 사용된 qPCR용 Primer sequenceGenePrimer sequence (SEQ ID)Tm(℃)Amplicon size(bp)PrimerBank IDATP1A1FACAGACTTGAGCCGGGGATTA (45)62.7100237681108c1RTCCATTCAGGAGTAGTGGGAG (46)60CAPN1FGCCAAGCAGGTGAACTACC (47)60.4125311893361c3RTATGGGTCCACGTTGTTCCAC (48)62CHKAFATTACAGGGGATTCGACATTGGA (49)61.111747078275c3RGCTGTTGTTTCTTGGTGGGAT (50)60.8DCTDFCCAAATGGGTGCAGTGATGAC (51)61.513861742818c2RACTACAGCCTTTCACATCGGT (52)61.1MST4FATCTTGTGCAAACCCTGAGTTG (53)61104109633024c2RTTTCAATCGCCTGATTCCTGCT (54)61.8ODC1FTTTACTGCCAAGGACATTCTGG (55)60.21274505488c1RGGAGAGCTTTTAACCACCTCAG (56)60.3PAK2FCACCCGCAGTAGTGACAGAG (57)61.9112191250770c3RGGGTCAATTACAGACCGTGTG (58)60.6PIK3R4FGCTCTTTAGGCAGTATGTGCG (59)61.1148116812580c3RGATGTCCCCATGACGAACTCC (60)62.2PIKFYVEFACCTCCGAGCTTGCACATATT (61)61.595295789161c2RTGAAAGAGCATTCAAGTCTTCCC (62)60.5SLC16A1FGGTGGAGGTCCTATCAGT (63)62.7107115583684c2RCAGAAAGAAGCTGCAATCAAGC (64)60.4SMSFTGGGCGGGTGAAACGATTAC (65)62.5162311083638c1RCCAAACTGCTTCGAGTGTAGAA (66)60.2SQLEFGATGATGCAGCTATTTTCGAGGC (67)60.717962865634c2RCCTGAGCAAGGATTTCACGACA (68)60TRPM7FGTTGGAAAGTATGGGCGGAA (69)62.4190296080776c2RCACACACAACTACTGGAACAGG (70)60.8VDAC1FCTGACCTTCGATTCATCCTTCTC (71)62.475307133764c1RCTCCCGCTTGTACCCTGTC (72)63.

[0186] Experimental results

[0187] Identification of modulators of obinutuzumab-induced LMP

[0188] To identify key regulators of obinutuzumab-induced LMP, we utilized a CRISPR-dCas9 screening platform. A drug-targeting sgRNA sublibrary (provided by Dr. Weissman) was delivered to dCas9-Raji cells to generate knockdown pools. Lysotracker-positive cells were then selected by repeated treatment with obinutuzumab and lysotracker followed by FACS separation. Lysotracker, an intracellular dye that accumulates in acidic organelles, is a marker of LMP and subsequent apoptosis. After amplification of viable cells, genomic DNA (gDNA) was extracted and subjected to next-generation sequencing (NGS). The amplicons were analyzed using an Illumina sequencer and the MAGeCK algorithm. Synergistic B cell death can be induced by depleting or increasing sgRNAs, respectively, when combined with their inhibitors or agonists (Figure 1a).

[0189] First, dCas9-Raji-expressing Raji cells were genetically engineered via lentiviral packaging and transfection. The SSFV-dCas9-mCherry-KRAB vector (provided by Dr. Tippale) was introduced into Raji cells, and mCherry-expressing cells were isolated using a FACSAria III. MS4A1 (CD20)-targeting sgRNA was introduced into dCas9-expressing Raji cells. Knockdown efficiency was assessed by quantifying surface expression of CD20 using flow cytometry (Fig. 1b).

[0190] Subsequently, known drug targets were selectively targeted using kinase, phosphatase, and drug target sgRNA sublibraries (provided by Dr. Weissman) (Fig. 1c). The sublibrary targets 2,318 genes, each targeted by five sgRNAs, for a total of 12,775 sgRNAs. To generate knockdown pools, the sgRNA sublibrary was amplified by electroporation using SS320 electrocompetent E. coli cells and packaged into lentiviral vectors. These lentiviruses were transduced into dCas9-Raji cells. The initial transfection rate was set to 20% to 50% to ensure that a single sgRNA could be introduced into a single cell (single knockdown). Transformed cells were selected with low-dose puromycin (0.75 μg / mL), and the selection efficiency was quantified by flow cytometry. 1.3 x 10 7 Cells (maintaining 1000 x coverage) were treated with 10 μg / mL obinutuzumab for 4 h and then stained with 50 nM Lysotracker deep red. Lysotracker-positive cells were isolated and amplified until most cells became unresponsive to obinutuzumab.

[0191]

[0192] Candidate genes are enriched through repeated FACS-based screening.

[0193] To validate the screening process using repeated FACS-based sorting, a pilot study was conducted in which Raji cells were treated and isolated twice. Results showed that the isolated cells remained responsive to obinutuzumab (Fig. 2a). However, in the second FACS round, the lysotracker histogram shifted slightly to the right, indicating that the cells began to become less responsive (Fig. 2a).

[0194] Subsequently, two rounds of FACS-based sorting were performed using a mixture of Raji cells and dCas9-sgMS4A1 cells (CD20 knockdown cells), confirming that CD20 knockdown cells were not responsive to obinutuzumab. The observed increase in the content of CD20 knockdown cells with each repeated FACS run confirmed the proper functioning of this system (Fig. 2b).

[0195] In large-scale experiments, knockdown cells were treated with obinutuzumab and Lysotracker deep red and repeated FACS analyses were performed to identify synergistic targets that enhance B cell death as modulators of obinutuzumab-induced LMP. After five rounds of FACS, LMP and DCD rates were measured by flow cytometry. Cell responsiveness to obinutuzumab was reduced, with LMP decreasing from 56.1% to 16.0% and DCD decreasing from 42.6% to 16.8% (Figs. 2c and 2d).

[0196]

[0197] PIKfyve is a negative regulator of obinutuzumab-induced apoptosis

[0198] CRISPRi screening identified positive regulators of obinutuzumab-induced apoptosis. After five rounds of obinutuzumab treatment and FACS-based selection, sequence analysis was performed via next-generation sequencing (NGS) using an Illumina sequencer. Each read was further analyzed using the MAGeCK algorithm to rank genes. The CRISPRi screening results were visualized using a volcano plot (Fig. 3a), revealing that most genes were depleted, as most known drug targets and kinases are negative regulators of apoptosis.

[0199] We employed a prioritization strategy for gene selection (Fig. 3b). First, we performed a primary selection using a 25% FDR cutoff. Next, we determined the control sgRNA read count threshold using the Cancer Dependency Map (DepMap) database. Third, Wang et al. 20 After excluding the Raji cell essential genes reported in 2015, genes with finally available small molecule inhibitors were selected, and a total of 15 candidate genes were selected for validation.

[0200] We further validated the 15 candidate genes using single knockdown cell lines. sgRNAs targeting the candidate genes were introduced into dCas9-Raji cells, and knockdown efficiency was assessed by qPCR (Figure 4a). LMP and DCD assays were performed on the single knockdown cell lines (Figures 4b and 4c), and each gene showed a high correlation between LMP and DCD results (Figure 4d). Six of the 15 initial candidate genes were selected for additional inhibitor combination experiments. Raji cells were pretreated with a small molecule inhibitor for 24 hours, followed by obinutuzumab treatment for 4 hours. Combination treatment of the small molecule inhibitor and obinutuzumab synergistically killed Raji cells (Figures 4e and 4f). Among the six inhibitors, PIKfyve-targeting apilimod showed the most enhanced apoptotic effect when combined with obinutuzumab (Fig. 4f).

[0201] Co-treatment with apilimod and rituximab or obinutuzumab synergistically induced direct cell death (Fig. 5a). CDC assay results showed that apilimod treatment did not affect the CDC activity of either rituximab or obinutuzumab (Fig. 5b). Varying the exposure time to apilimod revealed that pretreatment with apilimod for 24 h synergistically induced cell death compared to 4 h (Fig. 5b), which is thought to be due to vacuolization observed only after 24 h of treatment. PIKfyve is a lysosomal lipid kinase that catalyzes the conversion of PIP2 to PI(3,5)P2. PIKfyve is known to regulate lysosomal fission, and inhibition of PIKfyve with apilimod blocks lysosomal fission without interfering with fusion, leading to continued lysosomal coalescence and expansion. We aimed to investigate how apilimod initiates obinutuzumab-induced apoptosis in Raji cells by blocking key regulators of lysosomal fission.

[0202]

[0203] Co-treatment with apilimod and obinutuzumab induces enhanced cytotoxicity through modulation of lysosomal fission.

[0204] We hypothesized that co-treatment with apilimod, which inhibits PIKfyve, and obinutuzumab would result in greater cytotoxicity due to lysosomal vacuolization following inhibition of lysosomal fission. Therefore, we aimed to investigate factors affecting lysosomal homeostasis, including the P2X4 receptor, TRPML2, mTOR, and WIPI (Fig. 6a). First, we focused on factors involved in lysosomal fission, such as TRPML2, mTOR, and WIPI. We expected that simultaneous inhibition of CD20 and the factors listed above would increase apoptosis, but a significant increase was observed only when MLSI-3 and obinutuzumab were combined (Figs. 6b-6h). Simultaneous inhibition of TRPML2 and CD20 with 10 μM MLSI-3 and 0.3 μg / mL obinutuzumab, respectively, slightly increased apoptosis, but not significantly (Fig. 6f). TRPML2 is Ca 2+ Ca transport from lysosomes to the cytoplasm 2+ It is a permeable channel. Previous studies using cell lines expressing TRPML2 fused to the GCaMP calcium reporter have reported that obinutuzumab blocks TRPML2. Addition of the TRPML2 agonist MLSA-1 did not alter TRPML2 activity in obinutuzumab-treated cells. Although it was expected that MLSA-3-induced apoptosis would only slightly increase because obinutuzumab strongly blocks TRPML2 activity, TRPML2 inhibition enhanced obinutuzumab-induced apoptosis, suggesting that lysosomal fragmentation enhances obinutuzumab-induced apoptosis.

[0205] mTOR participates in lysosomal fission by phosphorylating dynamin, a motor protein involved in membrane fission. Treatment of cells with OSI-027 resulted in mTOR inhibition and vacuolization, confirming the drug's effectiveness (Fig. 6c). However, OSI-027 did not significantly increase cell death (Fig. 6b).

[0206] WIPI2 is a member of the PROPPIN protein family and is a Ca2+ receptor secreted by TRPML2. 2+ and is regulated by PIP2 produced by PIKfyve. WIPI2 regulates lysosomal fission by interacting with dynamin and induces autophagy by binding to ATG16L1. The present inventors have generated a WIPI2 dominant-negative mutant cell line-WIPI2 H85E and WIPI R108E We attempted to verify whether WIPI2 is involved in the induction of DCD by OBI by producing a dominant-negative mutant, and confirmed that the dominant-negative mutant was functional by observing that these mutants exhibited vacuolization (Fig. 6e). However, when obinutuzumab was administered to the mutant cell line, no significant increase in apoptosis was observed (Fig. 6d).

[0207] The P2X4 receptor is a lysosomal membrane protein involved in lysosomal fusion. The P2X4 receptor is an ATP-gated Ca receptor that is regulated by lysosomal pH and ATP concentration. 2+ Once activated as a channel, Ca 2+ By secreting , it makes lysosomes alkaline and induces lysosomal fusion. When cells were administered with BAY-1797, a P2X4 receptor inhibitor, and obinutuzumab, a significant decrease in apoptosis was confirmed (Fig. 6g). When cells were treated with apilimod and BAY-1797, it was confirmed that BAY-1797 treatment reduced apilimod-induced vacuolization (Fig. 6h).

[0208]

[0209] Cross-validation with additional PIKfyve inhibitors

[0210] Raji B cells (donated by Professor Seonghwan Kim of Chungnam National University) were cultured using RPMI-1640 medium (Welgene) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Welgene).

[0211] 5.0 x 10 4 Raji cells were cultured in 96-well plates in a humidified incubator for 24 h with the PIKfyve inhibitor umbralisib (MedChemExpress) or the BTK inhibitor acalabrutinib (MedChemExpress) at specific concentrations, respectively, followed by treatment with obinutuzumab (0.3 μg / ml) for 4 h at 37°C. Cells were then stained with 50 nM propidium iodide (PI) for 30 min at 37°C to assess cytotoxicity. Samples were analyzed by flow cytometry, and 10,000 events were recorded per sample. Analysis was gated on whole cells, single cells, and PI-positive cells depending on the experiment.

[0212] As a result, as shown in Fig. 4g, the PIKfyve inhibitor umbralisib most strongly increased DCD when co-administered with obinutuzumab, whereas the BTK inhibitor acalabrutinib did not increase DCD at all at any concentration when co-administered with obinutuzumab. Through this, not only was it additionally confirmed that the PIKfyve discovered in the present invention is an effective target for maximizing the anticancer effect of anti-CD20 antibodies, but also a significant synergistic effect was observed with umbralisib in addition to apilimod, suggesting that various PIKfyve inhibitors can be used as effective combination agents for anti-CD20 antibodies.

[0213]

[0214] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing or treating a CD20-expressing cancer, comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD20; and an inhibitor of PIKfyve (FYVE finger-containing phosphoinositide kinase) as active ingredients.

2. A composition according to claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 comprises a heavy chain variable region comprising an HCDR1 region of the first sequence in the sequence listing; an HCDR2 region of the second sequence in the sequence listing; and an HCDR3 region of the third sequence in the sequence listing.

3. A composition according to claim 2, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 additionally comprises a light chain variable region comprising an LCDR1 region of sequence number 4; an LCDR2 region of sequence number 5; and an LCDR3 region of sequence number 6.

4. A composition according to claim 3, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 is obinutuzumab.

5. A composition according to claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 comprises a heavy chain variable region comprising an HCDR1 region of sequence number 9; an HCDR2 region of sequence number 10; and an HCDR3 region of sequence number 11.

6. A composition according to claim 5, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 additionally comprises a light chain variable region comprising an LCDR1 region of sequence number 12; an LCDR2 region of sequence number 13; and an LCDR3 region of sequence number 14.

7. A composition according to claim 6, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD20 is rituximab.

8. A composition according to claim 1, wherein the PIKfyve inhibitor is at least one inhibitor selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the PIKfyve protein; an aptamer that specifically binds to the PIKfyve protein; and a nucleic acid molecule that inhibits the expression of a nucleotide encoding the PIKfyve protein.

9. A composition according to claim 1, wherein the PIKfyve inhibitor is at least one small molecule inhibitor selected from the group consisting of YM201636, APY0201, MOMIPP, Vacuolin-1, Umbralisib, a compound represented by the following chemical formula 1, and a pharmaceutically acceptable salt thereof: Chemical Formula 1 In the above chemical formula, R1 and R2 are each independently N or CH, R3 is C1-C3 alkyl, and n is an integer from 1 to 3.

10. A composition characterized in that in the chemical formula of claim 9, R1 is N, R2 is CH, R3 is C1 alkyl, and n is 2.

11. A composition according to claim 1, characterized in that the CD20-expressing cancer is a B-cell-origin malignant tumor.

12. A composition according to claim 11, wherein the B cell-derived malignant tumor is selected from the group consisting of non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL).

13. A pharmaceutical composition for combination administration with an antibody or an antigen-binding fragment thereof that specifically binds to CD20, comprising an inhibitor of PIKfyve (FYVE finger-containing phosphoinositide kinase) as an active ingredient, for the prevention or treatment of CD20-expressing cancer.

14. A method for screening a pharmaceutical composition for co-administration with an antibody or an antigen-binding fragment thereof that specifically binds to CD20, comprising the following steps: (a) contacting a candidate substance with a biological sample containing cells expressing PIKfyve (FYVE finger-containing phosphoinositide kinase); and (b) a step of measuring the activity or expression level of PIKfyve in the sample; If the activity or expression level of the above PIKfyve is reduced, the candidate substance is determined to be a pharmaceutical composition for co-administration with an antibody or antigen-binding fragment thereof that specifically binds to CD20.

15. A method according to claim 14, characterized in that the cell is a B cell.

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