Antibody targeting canine CD20, chimeric antigen receptor, and use thereof

Abstract

The present invention discloses antibodies specifically binding to canine CD20 and uses thereof. The antibody according to the present invention can be used for early diagnosis, disease progression monitoring, and treatment of B-cell lymphoma in dogs.

Description

Antibodies targeting canine CD20, chimeric antigen receptors, and uses thereof

[0001] The present invention relates to antibodies, chimeric antigen receptors and uses thereof, and more particularly to antibodies targeting canine CD20, chimeric antigen receptors and uses thereof.

[0002]

[0003] Chimeric antigen receptor (CAR) T cells are molecules that combine antibody-based specificity for a target antigen with a T cell receptor-activating intracellular domain to produce chimeric proteins that exhibit specific anti-cancer immune activity. Typically, a chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain may comprise a single-chain variable fragment (scFv) that targets a identified tumor antigen.

[0004] CARs can be expressed on the surface of T cells, immune effector cells, using genetic transfection technology. When the CAR expressed on the T cell surface binds to a target tumor antigen, the CAR can activate the T cell to initiate a specific anti-tumor response in an antigen-dependent manner.

[0005] Lymphoma is a type of blood cancer whose cause is unclear. It is difficult to detect with routine blood tests or X-rays, making early diagnosis and treatment challenging. Furthermore, it progresses rapidly, gradually spreading throughout the body over time. Even if lymphoma initially develops in a single lymph node, it can gradually spread to multiple lymph nodes, eventually progressing to lymph nodes throughout the body and eventually metastasizing to the liver, spleen, and eventually to other organs and bone marrow. Furthermore, because lymphoma can develop in multiple locations throughout the body, chemotherapy is often chosen over surgery for a complete cure, offering life-prolonging benefits. For a good prognosis, prompt and aggressive chemotherapy is recommended.

[0006] In the case of lymphoma arising from B cells, various target antigens such as CD20, CD19, CD22, and CD79B exist on the cell surface. In particular, CD20 plays an important role in the maturation and activation process of B cells, and is highly expressed on the cell surface of B-cell-derived cancers, so CD20 is an important target for diagnosing and treating B-cell lymphoma.

[0007] Canine lymphomas are classified into B-cell and T-cell types based on their cytological phenotypes, depending on their origin. Most canine lymphomas are B-cell malignancies, accounting for 65–75% of canine lymphomas, while T-cell malignancies account for 25–35% (Ponce et al., 2010; Vezzali et al., 2010). The standard treatment for canine lymphoma is the widely used cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy protocol. However, most cases of canine lymphoma treated with the standard treatment experience relapse and chemotherapy refractoriness, necessitating the use of separate chemotherapy agents or salvage therapy (Sorenmo et al., 2010; Zandvliet, 2016).

[0008] Accordingly, the inventors of the present invention have conducted extensive research efforts to develop a new treatment method applicable to canine lymphoma in addition to the standard treatment, and as a result, have developed an antibody that can diagnose canine B-cell lymphoma by targeting canine CD20 and completed the present invention so that the antibody can be applied as a CAR (chimeric antigen receptor)-NK cell treatment.

[0009]

[0010] The present invention provides an amino acid sequence of a scFv domain capable of specifically recognizing a canine CD20 antigen for diagnosing companion dog B-cell lymphoma, thereby enabling the identification of differences in expression in the blood of individuals with B-cell lymphoma and non-lymphoma individuals, and thereby enabling the diagnosis of B-cell lymphoma with a small amount of blood in a short period of time.

[0011] Another object of the present invention is to provide a composition for treating canine B-cell lymphoma, which comprises, as an active ingredient, an antigen-specific antibody-presenting cell (CAR-NK cell) transformed with a chimeric antigen receptor (CAR) comprising an antigen-binding domain that specifically binds to the canine CD20 antigen, thereby providing therapeutically effective anticancer or anticancer immune enhancement efficacy against malignant tumors while minimizing harmful or side effects.

[0012]

[0013] The purpose of the present invention is not limited to what has been mentioned above, and other purposes not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the description below.

[0014]

[0015] According to one aspect of the present invention for solving the above technical problem, the present invention provides an antibody comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0016] Here, the antibody can be produced in the form of scFv, Fab, F(ab')2 or minibody, which can have high specificity and binding affinity to dog CD20.

[0017] Here, the present invention can provide a pharmaceutical composition for treating canine B-cell lymphoma comprising the antibody.

[0018] Here, the present invention can enable mass production of antibodies by providing a nucleic acid encoding the antibody and an expression vector containing the same.

[0019] Here, the present invention provides a chimeric antigen receptor (CAR) including the antibody, thereby laying the foundation for the development of CAR-T cell and CAR-NK cell therapy.

[0020] Here, the present invention can provide an immune cell for treating canine B-cell lymphoma comprising the CAR.

[0021] Here, the present invention can provide a composition for diagnosing canine B-cell lymphoma comprising the antibody.

[0022] Here, the present invention can provide a composition for detecting canine CD20 expression comprising the antibody.

[0023] Here, the present invention can provide a composition for monitoring treatment of B-cell lymphoma comprising the antibody.

[0024] The terms used in this specification may be defined as follows.

[0025] As used herein, numerical terms such as Kd are calculated based on scientific measurements and are therefore subject to reasonable measurement errors. In some cases, numerical terms may include values ​​rounded to the nearest significant digit.

[0026] As used herein, "a" or "an" means "at least one" or "one or more," unless otherwise stated. As used herein, the term "or" means "and / or," unless otherwise stated. In the context of multiple dependent claims, the use of "or" when referring back to another claim refers only to the alternate claim.

[0027] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. The term also includes chimeric antibodies (e.g., humanized murine antibodies) and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies. A whole 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 whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes such as IgG1, IgG2, IgG3, and IgG4. The above antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, Fab', F(ab')2, and Fv, etc. The Fab has a structure having variable regions of the light chain and heavy chain, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site.

[0028] The term “CAR-NK” in the present invention may be defined as a genetically engineered immune cell produced by introducing a chimeric antigen receptor (CAR) into a natural killer cell. CAR-NK cells are composed of an extracellular binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain, and through this structure, can recognize tumor-specific antigens and eliminate target cells. CAR-NK cells can eliminate tumor cells through two mechanisms: a CAR-dependent manner and a CAR-independent manner. It can be understood that CAR-NK cells are capable of not only recognizing and eliminating specific tumor cells through the CAR, but also eliminating tumor cells through activating and inhibitory receptors unique to NK cells. CAR-NK cells can be transplanted allogeneically regardless of HLA match, have a low risk of graft-versus-host disease, a low risk of cytokine release syndrome, and almost no neurotoxicity. In addition, CAR-NK cells can eliminate tumor cells through various mechanisms, including direct secretion of perforin and granzyme, induction of apoptosis through ADCC effect, and induction of apoptosis through Fas ligand or TRAIL.

[0029] 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 by disulfide bonds between the cysteine ​​residues in the hinge region of Fab'.

[0030] Fv(variable fragment) refers to the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. Double-chain Fv (dsFv) has the heavy chain variable region and the light chain variable region linked by a disulfide bond, and single-chain Fv (scFv) has the heavy chain variable region and the light chain variable region covalently linked, generally through a peptide linker (SEQ ID NO: 9). These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin. Specifically, the enzyme papain can cleave an antibody monomer into two Fab (fragment antigen binding) fragments and one Fc (fragment crystallizable) fragment, and pepsin can cleave one f(ab')2 fragment and one Fc fragment.

[0031] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

[0032] Typically, immunoglobulins have heavy chains and light chains, each of which comprises a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains comprise three variable regions called complementarity-determining regions (CDRs) and four framework regions. The CDRs are primarily responsible for binding to epitopes on an antigen. The CDRs of each chain are typically named CDR1, CDR2, and CDR3, starting from the N-terminus, and are also identified by the chain on which the particular CDR is located.

[0033] The term "epitope" of the present invention refers to a site on the canine CD20 protein to which the canine CD20 antibody of the present invention binds. In the present invention, the first part (144-167) of the 144-188 amino acid sequence (SHFFKMENLN LIKAPMPYVD IHNCDPANP S EKNSLSIQYC GSIRS) located in the extra domain of canine CD20 corresponds to the major epitope. An epitope includes a chemically active surface group of an amino acid and has specific three-dimensional structural characteristics and specific charge characteristics. The epitope recognized by the canine CD20 antibody of the present invention is composed of adjacent amino acid residues of the canine CD20 protein, which is an important factor in determining the specificity of antigen-antibody binding. In the present invention, the epitope of the canine CD20 antibody is located within the extra domain of canine CD20, consisting of approximately 24 amino acid residues (positions 144-167), plays a crucial role in B cell-specific binding, and has the characteristic of directly interacting with the CDR region of the antibody. This epitope recognition may play a key role in enabling the canine CD20 antibody of the present invention to effectively detect and treat B cell lymphoma.

[0034] The term "canine antibody" in the present invention refers to a molecule derived from canine immunoglobulin, wherein the entire amino acid sequence constituting the antibody, including the complementarity determining region and the structural region, is comprised of the amino acid sequence of canine immunoglobulin. Canine antibodies are commonly used to treat diseases in companion dogs, and may have at least three potential advantages. First, they interact better with the canine immune system, allowing for more efficient destruction of target cells, for example, through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, there is the advantage that the canine immune system does not recognize the antibody as foreign. Third, there is the advantage that the half-life in the canine circulation is similar to that of naturally occurring antibodies, even when administered in smaller doses or less frequently. Therefore, the canine monoclonal antibody according to the present invention exhibits strong affinity for CD20 and exhibits low immunogenicity because both the heavy and light chain domains are of canine origin, and thus can be usefully used in the treatment of diseases related to CD20 expression, such as B-cell lymphoma.

[0035] The monoclonal antibody specifically binding to the above-mentioned canine CD20 may be, but is not limited to, a canine monoclonal antibody specifically binding to canine CD20.

[0036] The term "CDR" refers to a complementarity determining region, as defined by at least one identification scheme, to those skilled in the art. In some embodiments, a CDR may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, the contact definition, or a combination of the Kabat, Chothia, AbM, or contact definitions. The various CDRs within an antibody may be designated by any suitable number and chain type, including but not limited to CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR" may also be understood to include a "hypervariable region" or HVR, which comprises a hypervariable loop.

[0037] The canine monoclonal antibody specifically binding to the above-mentioned canine CD20 may be a canine monoclonal antibody or fragment thereof comprising a heavy chain variable region comprising a heavy chain CDR1 as set forth in SEQ ID NO: 1; a heavy chain CDR2 as set forth in SEQ ID NO: 2; and a heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 as set forth in SEQ ID NO: 4; a light chain CDR2 as set forth in SEQ ID NO: 5; and a light chain CDR3 as set forth in SEQ ID NO: 6.

[0038] When the canine monoclonal antibody of the present invention includes a constant region, it may include a constant region derived from canine IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof.

[0039] In the present invention, the term "combination" means that a polypeptide encoding a canine immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of different origin when forming a dimer or multimer.

[0040] In the present invention, the term "hybrid" means that a sequence corresponding to two or more different origin canine immunoglobulin heavy chain constant regions exists within a single-chain immunoglobulin heavy chain constant region.

[0041] In addition, when the canine monoclonal antibody specific for the canine CD20 of the present invention includes a light chain constant region, the light chain constant region may be derived from a canine lambda (λ) or kappa (κ) light chain.

[0042] The term "CD20" in the present invention refers to B lymphocyte differentiation antigen, also known as MS4A1 (Membrane Spanning 4-Domains A1), a membrane protein expressed on the surface of B cells. CD20 is known to play an important role in the development, activation, and differentiation of B cells. The CD20 may include any mammalian CD20 without limitation, but preferably refers to canine CD20.

[0043] For the purpose of the present invention, the above CD20 may refer to a protein that binds to CD20 expressed in cells of a malignant tumor, specifically a blood cancer, more specifically a lymphoma, and more specifically a B-cell lymphoma, and can inhibit the growth, onset, or progression of B-cell lymphoma, but is not limited thereto.

[0044] In addition, the CD20 protein includes, but is not limited to, both native and mutant CD20 proteins. The native CD20 protein generally refers to a polypeptide comprising the amino acid sequence of the native CD20 protein, and the amino acid sequence of the native CD20 protein generally refers to an amino acid sequence found in naturally occurring CD20. In particular, the present invention includes a sequence of amino acids 144-188 located in the extra domain of canine CD20 (SHFFKMENLN LIKAPMPYVD IHNCDPANP S EKNSLSIQYC GSIRS). Information on the CD20 can be obtained from known databases such as GenBank of the National Institutes of Health (accession number: NM_001048028).

[0045] It should be understood that the protein, polypeptide and / or amino acid sequence of the CD20 antibody and CAR included in the present invention includes at least functional variants or homologs having the same or similar function as the original protein or polypeptide.

[0046] In the present invention, functional variants may be proteins or polypeptides obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the canine CD20 antibody and / or CAR. For example, functional variants may include proteins or polypeptides having a different amino acid sequence due to substitution, deletion, and / or insertion of one or more amino acids, such as 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5. Such functional variants may substantially maintain the biological properties of the unmodified canine CD20 antibody or CAR. For example, a functional variant may retain at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the biological activity (such as canine CD20 antigen binding ability or T cell activation ability) of the original canine CD20 antibody or CAR.

[0047] In the present invention, the homologue may be a protein or polypeptide (e.g., an antibody capable of specifically binding to canine CD20 or a fragment thereof) having an amino acid sequence homology of about 85% or more (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with the canine CD20 antibody and / or CAR.

[0048] In the present invention, homology generally refers to similarity or correlation between two or more sequences, and is particularly characterized by maintaining binding specificity for the 144-188 amino acid sequence located in the extra domain of canine CD20.

[0049] "Constant region" refers to a region comprising at least three constant domains. "Heavy chain constant region" or "constant heavy chain" refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. There are IgG antibody classes in dogs including IgG-A, IgG-B, IgG-C, and IgG-D. "Affinity" refers to the strength of the sum of noncovalent interactions between the canine CD20 antibody of the present invention and the canine CD20 antigen. Affinity can be measured by methods such as ELISA, biolayer interferometry (BLI), or surface plasmon resonance. "Variant" refers to a polypeptide that has at least about 50% amino acid sequence identity to a native sequence polypeptide after aligning the sequences and introducing gaps to achieve the maximum percent sequence identity, and that retains binding activity to canine CD20. "Pharmaceutical formulation" and "pharmaceutical composition" refer to a form that allows the biological activity of the canine CD20 antibody or CAR-NK cell of the present invention to be effective. Pharmaceutically acceptable carriers may include phosphate buffered saline (pH 7.2) or 50 mM Na citrate (pH 7, 150 mM NaCl). "Treatment" means administration of a therapeutic agent for B-cell leukemia, including B-cell lymphoma, and may include effects such as inhibition or delay of progression of lymphoma, reduction of lymphoma cells, improvement of symptoms associated with B-cell lymphoma, and improvement of disease state.

[0050] In the present invention, the Minibody is a modified antibody structure of 75-80 kDa in size, in which a single-chain variable region (scFv) and the CH3 domain of an antibody are combined, and has a scFv-(hinge)-CH3 configuration. The Minibody has excellent tissue penetration due to its smaller size than a whole antibody, has enhanced binding affinity through dimer formation due to the CH3 domain, and exhibits the characteristics of increased drug persistence due to a longer blood half-life than the scFv.

[0051] In the present invention, FSC-A (Forward Scatter-Area) is a parameter that measures the area of ​​forward scattered light of a laser beam passing through a cell in flow cytometry, which reflects the size of the cell and is used to classify a specific cell group such as lymphocytes, distinguish cell aggregates, and distinguish between living and dead cells.

[0052] In the present invention, PARR (PCR for Antigen Receptor Rearrangement) is a molecular diagnostic method that analyzes the rearrangement pattern of B cell or T cell receptor genes. By extracting DNA from lymphocytes, performing PCR with antigen receptor gene-specific primers, and then analyzing the size of the amplified product, it is possible to determine whether there is clonal proliferation of lymphoma.

[0053] The Dog Lymphocyte Activation Cocktail used in the present invention is a mixture of stimulants for lymphocyte activation consisting of PMA, Ionomycin, and Protein Transport Inhibitor. PMA activates protein kinase C, and Ionomycin increases the intracellular calcium concentration to activate lymphocytes and induce cytokine production.

[0054] In the present invention, MFI (Mean Fluorescence Intensity) is a quantitative indicator representing the average value of the fluorescence signal of a specific cell group in flow cytometry, reflecting the expression level of cell surface proteins and evaluated as a relative increase rate compared to the negative control group.

[0055] In the present invention, the framework region is a structural skeleton region that supports the CDR within the antibody variable region, and there are four of them for each variable region. They have a relatively conserved amino acid sequence and a β-sheet structure, and thus perform the functions of maintaining the three-dimensional structure of the CDR, providing stability of the antibody, and maintaining the appropriate orientation of the antigen binding site.

[0056] Various immunoassay methods can be used to detect the specific binding of the canine CD20 antibody. Exemplary immunoassays performed in the present invention include flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blot. Fluorescent substances (e.g., FITC) or enzymes (e.g., horseradish peroxidase) can be used as markers for detection. In the present invention, a c-myc tag was introduced to detect the canine CD20 antibody, which enables secondary detection using an anti-c-myc antibody. Specifically, the present invention used detection methods such as flow cytometry (cell surface expression analysis using the canine CD20 minibody and a fluorescently labeled secondary antibody, lymphocyte population selection through FSC-A gating, and CD14-negative cell population analysis), ELISA (clonal selection for canine CD20-specific antibodies, antigen-binding ability confirmation of antibodies), and Western blot (determination of CAR protein expression using an anti-c-myc antibody, confirmation of canine CD20 protein expression). The specificity and functionality of the developed antibodies were evaluated through these analysis methods, and their potential use as diagnostic tools for B-cell lymphoma was confirmed.

[0057] The present invention relates to an antibody that specifically binds to canine CD20 and applications thereof, and provides an antibody comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6. The antibody of the present invention can be produced in the form of scFv, Fab, F(ab')2, or minibody, and all of these various forms of antibodies exhibit high specificity and binding affinity for canine CD20. In particular, the antibody of the present invention specifically binds to the first part (144-167) of the 144-188 amino acid sequence located in the extra domain of canine CD20. The present invention also provides a pharmaceutical composition for treating canine B-cell lymphoma, comprising the antibody. The composition can be formulated with a pharmaceutically acceptable carrier, and can be effectively used for the treatment of canine B-cell lymphoma. The present invention provides a nucleic acid encoding the antibody and an expression vector comprising the same. These nucleic acid constructs enable the mass production of antibodies, which is essential for the commercial production of therapeutic agents. Furthermore, the present invention provides a chimeric antigen receptor (CAR) comprising the antibody. This provides a foundation for the development of CAR-NK cell therapy, and the present invention also provides immune cells comprising such CARs for treating canine B-cell lymphoma. In the field of diagnostics, a composition for diagnosing canine B-cell lymphoma, a composition for detecting canine CD20 expression, and a composition for monitoring B-cell lymphoma treatment can be provided. These diagnostic compositions can be useful for the early diagnosis of canine B-cell lymphoma, disease progression monitoring, and treatment efficacy evaluation.

[0058] According to another aspect of the present invention, the present invention provides a method for treating B-cell lymphoma by administering to a dog an antibody comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0059] Here, the antibody can be produced in the form of scFv, Fab, F(ab')2 or minibody and used for treatment, and it exhibits a therapeutic effect through high specificity and binding affinity to canine CD20.

[0060] Here, the present invention provides a method for diagnosing B-cell lymphoma by detecting CD20 expression in dogs using the antibody. In particular, CD20 expression can be quantitatively analyzed using flow cytometry.

[0061] Here, the present invention provides an immunocytotherapy method that administers NK cells expressing a chimeric antigen receptor (CAR) comprising the antibody to a dog. The NK cells used may be autologous NK cells of the dog, thereby minimizing immune rejection.

[0062]

[0063] The present invention provides an amino acid sequence of a scFv domain capable of specifically recognizing a canine CD20 antigen for diagnosing companion dog B-cell lymphoma, thereby confirming the difference in expression in the blood of individuals suffering from B-cell lymphoma and individuals without lymphoma, and using this, it is possible to diagnose B-cell lymphoma with a small amount of blood in a short period of time.

[0064] The present invention provides a composition for treating canine B-cell lymphoma, which comprises, as an active ingredient, an antigen-specific antibody-presenting cell (CAR-NK cell) transformed with a chimeric antigen receptor (CAR) including an antigen-binding domain that specifically binds to the canine CD20 antigen, thereby providing a therapeutically effective anticancer or anticancer immune enhancement effect against malignant tumors while minimizing harmful effects or side effects.

[0065]

[0066] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the description below.

[0067]

[0068] Figure 1 shows the cloning results for establishing a recombinant canine CD20 antigen overexpressing cell line.

[0069] Figure 2 shows the expression pattern of a recombinant canine CD20 antigen overexpressing cell line.

[0070] Figure 3 illustrates a process for discovering and producing antibodies using the phage display of the present invention.

[0071] Figure 4 shows the results of flow cytometry analysis for the diagnosis of B-cell lymphoma and non-B-cell lymphoma.

[0072] Figure 5 shows the results of confirming the expression of B cell markers CD20 and CD21 after isolating PBMC from the peripheral blood of a non-B cell lymphoma patient (Dongja) and a B cell lymphoma patient (Byeol-i) using the antibody of the present invention (A) and (B), respectively.

[0073]

[0074] The purposes and effects of the present invention, as well as the technical configurations for achieving them, will become clearer with reference to the embodiments described in detail below, along with the attached drawings. In describing the present invention, detailed descriptions of known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the invention. Furthermore, the terms described below are defined in consideration of the contributions made to the present invention, and may vary depending on the intentions or practices of the user or operator.

[0075] However, the present invention is not limited to the embodiments disclosed below and can be implemented in various different forms. These embodiments are provided solely to ensure complete disclosure of the present invention and to fully inform those skilled in the art of the invention of the scope of the invention. The present invention is defined solely by the scope of the claims. Therefore, such definitions should be based on the contents of this specification.

[0076]

[0077] The present invention relates to the development and application of an antibody that specifically binds to canine CD20 and a chimeric antigen receptor (CAR) using the same. The present invention involves the production of a recombinant antigen based on the extra-domain sequence of canine CD20, the establishment of a cell line overexpressing the same, the production and characterization of an antibody that specifically binds to canine CD20, and the design and production of a canine CD20-specific CAR construct using the developed antibody. In addition, the diagnostic and therapeutic potential of the developed antibody and CAR-NK cells for B-cell lymphoma were evaluated.

[0078] The present invention aims to provide an anti-CD20 antibody therapeutic for the treatment of canine B-cell lymphoma. The developed antibody exhibits high specificity and affinity for canine CD20 and was confirmed to bind to both normal and lymphoma B cells. CAR-NK cells developed based on this antibody were confirmed to exhibit effective cytotoxicity against B-cell lymphoma both in vitro and in vivo.

[0079] In particular, the present invention comparatively analyzed the expression patterns of canine CD20 in normal individuals and dogs with B-cell lymphoma, confirming the potential of the developed antibody as a diagnostic biomarker. This finding is expected to be utilized in the early diagnosis and treatment monitoring of canine B-cell lymphoma in the future.

[0080] Furthermore, the present invention proposes novel antibody and / or CAR-NK cell therapies targeting canine CD20, which are expected to bring about groundbreaking advancements in the diagnosis and treatment of canine B-cell lymphoma. The results of the present invention represent a significant advance that could contribute to the health and longevity of companion animals, and suggest potential future applications in the treatment of human B-cell lymphoma.

[0081]

[0082] Hereinafter, embodiments of the present invention will be described in detail.

[0083]

[0084] Example 1: Establishment of a recombinant canine CD20 antigen-overexpressing cell line

[0085]

[0086] In this example, a recombinant canine CD20 antigen was produced based on the amino acid sequence 144-188 located in the extra domain of canine CD20, and a cell line overexpressing it was established. The specific method is as follows:

[0087]

[0088] 1. Synthesis of the CD20 sequence:

[0089] - Synthesize 894 base pairs of the coding sequence of the CD20 mRNA base sequence (Genebank accession number, NM_001048028)

[0090] - This sequence includes the sequence of amino acids 144-188 (SHFFKMENLN LIKAPMPYVD IHNCDPANP S EKNSLSIQYC GSIRS) located in the extra domain of the full-length amino acid sequence of dog CD20.

[0091]

[0092] 2. PCR amplification:

[0093] - To amplify 135 base pairs located in the extracellular domain, use the following primers:

[0094] - Forward primer: 5'-CCT CTC TCC CCA GGG GGA TCC ATG TCT CTG TTT GCC GCA ATC

[0095] - Reverse primer: 5'-CTC ACC ATG GTG GCG ACC GGT AGC GGT CAC AAG CTT TTG AAA

[0096] - PCR conditions: Initial denaturation 94°C for 5 minutes; 35 cycles (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds); Final elongation 72°C for 7 minutes.

[0097]

[0098] 3. Cloning:

[0099] - Cloning the amplified sequence into an expression vector (e.g. pFUGW) using the In-Fusion method

[0100] - Cloning results were confirmed through restriction enzyme analysis and DNA sequencing.

[0101] - The structure of the cloned plasmid is shown in Figure 1.

[0102]

[0103] 4. Virus production:

[0104] - Transfecting 293T cells with cloned plasmids and helper plasmids (e.g., psPAX2, pMD2.G)

[0105] - Harvest the supernatant containing the virus 48 hours after transfection.

[0106] - The harvested virus was filtered through a 0.45 μm filter and concentrated by ultracentrifugation (25,000 rpm, 4°C, 2 hours).

[0107]

[0108] 5. Cell line establishment:

[0109] - The produced virus was treated with Jurkat cells (1 × 10^6 cells / ml) at MOI 30.

[0110] - Increased transduction efficiency by adding 8 μg / ml of polybrene

[0111] - 72 hours after transduction, GFP-positive cells were isolated using a flow cytometer.

[0112]

[0113] 6. Confirmation of expression:

[0114] - Confirmation of CD20 expression pattern of established cell lines by flow cytometry (FACS)

[0115] - Staining using anti-dog CD20 antibody (e.g. NCD1.2 or self-made antibody)

[0116] - Non-transduced Jurkat cells were used as a negative control.

[0117] - The expression results are shown in Figure 2.

[0118]

[0119] 7. Functional Verification:

[0120] - Confirmation of expression of canine CD20 protein through Western blot

[0121] - Quantitative analysis of canine CD20 overexpression at the mRNA level using RT-qPCR

[0122]

[0123]

[0124] Example 2: Production of recombinant canine CD20 antibody

[0125]

[0126] In this example, a recombinant antibody was produced using the extra domain sequence of canine CD20, and the specific method is as follows:

[0127]

[0128] 1. Antigen preparation:

[0129] i. Synthesize a peptide by dividing the extra domain sequence of CD20 (144-188 amino acids) into two parts:

[0130] - Front: 144-168 amino acids (SHFFKMENLN LIKAPMPYVD IHNCD)

[0131] - Back: 169-188 amino acids (PANPS EKNSLSIQYC GSIRS)

[0132] ii. To increase antigen exposure, each peptide was conjugated with BSA (bovine serum albumin) and KLH (keyhole limpet hemocyanin) using the EDC / NHS coupling method.

[0133]

[0134] 2. Antibody Library Creation:

[0135] i. Immunization was administered to three 8-week-old normal beagles at two-week intervals.

[0136] ii. 3 days after the final immunization, 100 ml of blood was collected and canine PBMC was isolated.

[0137] iii. Total RNA extraction from isolated PBMCs

[0138] iv. cDNA synthesis through reverse transcription reaction

[0139] v. Performing first and second PCR for antibody gene amplification

[0140] vi. Insertion of PCR products into phagemid vector (Phagemid ligation)

[0141] vii. Production of antibody library through rescue and PEG down process after transformation

[0142]

[0143] 3. Antibody discovery (repeated 3 times):

[0144] i. Antigen coating (Ag coating): immobilizing the synthesized CD20 peptide on a microplate

[0145] ii. Library binding: Binding the manufactured phage library to the antigen-coated plate.

[0146] iii. Wash: Removal of non-specific binding

[0147] iv. Elution: Elution of specifically bound phages

[0148] v. TG1 infection: Infection of E. coli with released phage

[0149] vi. Agar plating: Spreading infected E. coli on agar plates

[0150] vii. Rescue and PEG down: Amplification of selected phages

[0151] viii. 96-well seeding: isolation of individual clones

[0152] ix. Induction: Induction of antibody expression

[0153] x. ELISA: Confirmation of antibody activity

[0154] xi. Wash: Final Selection

[0155]

[0156] 4. Antibody production:

[0157] i. Phagemid prep: Extraction of phagemid DNA from selected clones

[0158] ii. Subcloning: Recloning into an expression vector

[0159] iii. Transfection: Transfection into mammalian cell lines

[0160] iv. Purification: Antibody purification

[0161] v. ELISA: Final activity confirmation

[0162]

[0163] 5. Antibody Characterization:

[0164] - As a result of analyzing the amino acid sequence of the selected antibody, a positive clone with an OD value of 0.997 was detected in the part corresponding to the front part (144-167) of the extra domain of canine CD20.

[0165] - Analysis of the heavy chain sequence of this clone showed 64.57% similarity to the heavy chain of Rituximab, a human CD20 target antigen treatment, and 61.87% similarity to the heavy chain of Ofatumumab.

[0166]

[0167] Table 1 shows the selected antibody sequences.

[0168]

[0169] Sequence number Description Sequence 1 Heavy chain CDR 1 SSYRV 2 Heavy chain CDR 2 AISSGGS AYYAS 3 Heavy chain CDR 3 GVLVRGSYYFNI 4 Light chain CDR 1 YDS CDR 5 Light chain CDR 2 SSFNSL 6 Light chain CDR 3 YPAALPNHK 7 scFv Full sequence ELSLTQPASPVLFLPPPLPPGKLGGQDSFESIESYDSCDRLTQSWSSQSSFNSLQRVPSYDSFDSEDYPAALPNHKPKGTFKDYVRNPAHRPQVVVPLRSSSSGGGGSGGGGEVQLVKSGGSLKLSCKASGFALSSYRVSWVRQAPGNGLEWIGAISSGGSAYYASWAKSRSTITRNTNLNTVTLKMTSLTPADTATYFCARGVLVRGSYYFNIWGPGTLVTVSSGQPKLPPRPPRSS 8 scFv coding서열GAGCTCTCGTTGACGCAACCTGCCTCCCCTGTCCTCTTCCTACCTCCTCCTTTACCTCCAGGTAAACTCGGGGGGCCAGGACTCTTTTGAAAGCATAGAGAGCTACGATAGTGTGATCGCCTCACCCAGTCCTGGAGCAGCCAGTCATCTTTCAACAGCCTGCAGCGTGTTCCCTCCTATGACAGCTTCGACTCAGAGGACTATCCGGCTGCCCTGCCCAACCACAAGCCCAAGGGCACCTTCAAGGACTATGTGCGGAACCCAGCTCACCGTCCCCAGGTGGTGGTTCCTCTTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGAGGTGCAACTGGTGAAGTCTGGGGGATCCCTGAAACTCTCCTGTAAAGCTTCTGGATTCGCCCTCAGTAGCTACAGAGTGAGCTGGGTCCGCCAGGCCCCGGGGAATGGGCTGGAATGGATCGGAGCCATTAGTAGTGGTGGTAGCGCATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACACCTGCGGACACGGCCACCTATTTCTGTGCGAGGGGGAGTACTGGTTCGGGGGGTCATACTACTTCAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGCTTCCACCACGGCCCCCCTCGTTCTAGT9링커 서열RSSSSGGGGSGGGG10경쇄 가변영역(VL)ELSLTQPASPVLFLPPPLPPGKLGGQDSFESIESYDSCDRLTQSWSSQSSFNSLQRVPSYDSFDSEDYPAALPNHKPKGTFKDYVRNPAHRPQVVVPLRS11중쇄 가변영역(VH)EVQLVKSGGSLKLSCKASGFALSSYRVSWVRQAPGNGLEWIGAISSGGSAYYASWAKSRSTITRNTNLNTVTLKMTSLTPADTATYFCARGVLVRGSYYFNIWGPGTLVTVSSGQPKLPPRPPRSS

[0170] Example 3: Design and fabrication of a CD20 CAR construct

[0171]

[0172] In this example, a second-generation CAR construct was designed and manufactured based on the canine CD20 scFv DNA sequence. The specific methods and details are as follows:

[0173]

[0174] 1. CAR construct design:

[0175] The CAR construct is designed with the following components:

[0176] a) Extracellular domain

[0177] - Leader sequence: CD8α derived (MALPVTALLLPLALLLHAARP)

[0178] - Antigen binding site: canine CD20-specific scFv (VH-linker-VL format)

[0179] - Hinge region: derived from canine CD8α (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD)

[0180] - c-myc tag: EQKLISEEDL (for expression verification)

[0181] - Transmembrane domain: derived from canine CD28 (IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP)

[0182]

[0183] b) Intracellular domain

[0184] - Costimulatory region: derived from canine CD28 (RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS)

[0185] - Signal transduction domain: derived from canine CD3ζ (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR)

[0186]

[0187] 2. DNA sequence optimization:

[0188] - Optimizing the DNA sequence of CAR components by considering the frequency of dog codon usage

[0189] - Adjust the sequence considering GC content and possibility of secondary structure formation

[0190]

[0191] 3. CAR gene synthesis and cloning:

[0192] - Synthesize the optimized CAR DNA sequence through a commercial gene synthesis service.

[0193] - Designed with restriction enzyme sites (5' NheI, 3' NotI)

[0194] - Cloning the synthesized CAR gene into the lentiviral vector pCDH-EF1-MCS-T2A-Puro

[0195]

[0196] 4. Production of CAR expression vector:

[0197] - Link the restriction enzyme-treated CAR insert and vector using T4 DNA ligase.

[0198] - Extract plasmid DNA from transformed E. coli colonies and select the correct clone through restriction enzyme analysis and DNA sequencing.

[0199]

[0200] 5. Confirm CAR expression:

[0201] a) Transient expression in 293T cells

[0202] - The CAR expression vector was transfected into 293T cells using lipofection.

[0203] - After 48 hours, cells were harvested and CAR expression was confirmed using Western blot and flow cytometry analysis.

[0204] b) Western blot analysis

[0205] - Confirmation of CAR protein expression using anti-c-myc antibody

[0206] - Expected molecular weight: approximately 75 kDa

[0207] c) Flow cytometry

[0208] - Confirmation of CAR expression on the cell surface using anti-c-myc antibody and fluorescently labeled secondary antibody

[0209]

[0210] 6. CAR Function Verification:

[0211] a) Confirmation of CD20 antigen binding ability

[0212] - Co-culture of CAR-NK cells with cell lines expressing canine CD20 to evaluate binding capacity

[0213] b) Cell activation analysis

[0214] - After co-culturing CAR-NK cells and target cells, cytokine secretion such as IL-2 and IFN-γ was measured using ELISA.

[0215] c) Cytotoxicity analysis

[0216] - Assessing the cytotoxicity of CAR-NK cells against target cells using the LDH release assay.

[0217]

[0218] 7. Production of CAR-NK cell lines:

[0219] After transducing the CAR gene into canine NK cells using a viral vector, a population of NK cells stably expressing the CAR was obtained through antibiotic selection and magnetic separation.

[0220]

[0221] Example 4: Analysis of expression of canine CD20 in canine B-cell lymphoma and non-lymphoma individuals.

[0222] In this example, flow cytometry analysis was performed on dogs with blood cancer to confirm the efficacy of the manufactured canine CD20 antibody and to evaluate the effectiveness of canine CD20 as a biomarker.

[0223] For the diagnosis of B-cell lymphoma, an analysis was performed using the B-cell surface marker CD21 and the Dog lymphocyte activation cocktail. The dog with B-cell lymphoma (Byeoli) was CD21 positive (+) and showed a positive rate of 85.3% in the Dog lymphocyte activation cocktail, while the dog with non-B-cell lymphoma (Dongja) was CD21 negative (-) and showed a positive rate of 2.15% in the Dog lymphocyte activation cocktail.

[0224] During flow cytometry, CD14-negative cell populations were selected for analysis. This was done to specifically analyze lymphocyte populations excluding monocytes / macrophages.

[0225] The specific methods and results are as follows:

[0226]

[0227] 1. Antibody preparation:

[0228] - Minibody type antibody was produced using the dog CD20 scFv produced in Example 2.

[0229] - Minibody is produced in the form of scFv-CH3 using an E. coli expression system.

[0230] - Purification using IMAC (Immobilized Metal Affinity Chromatography) and size exclusion chromatography

[0231] - Confirm the purity and specificity of the purified minibody through SDS-PAGE and Western blot.

[0232]

[0233] 2. Target selection:

[0234] a) Case 1 (circular): non-B cell lymphoma (confirmed as T-cell lymphoma)

[0235] - Age: 8 years old, Breed: Golden Retriever, Gender: Female

[0236] - Clinical symptoms: Systemic lymphadenopathy, loss of appetite, weight loss

[0237] b) Vision 2 (Star): B cell lymphoma

[0238] - Age: 10 years old, Breed: Shih Tzu, Gender: Male

[0239] - Clinical symptoms: Systemic lymphadenopathy, splenomegaly, hepatomegaly

[0240]

[0241] 3. Sample preparation:

[0242] - Collect 3 ml of peripheral blood from each patient into an EDTA tube.

[0243] - Isolation of peripheral blood mononuclear cells (PBMC) using density gradient centrifugation using Ficoll-Paque PLUS

[0244] - Wash the separated PBMC twice with PBS and resuspend at a concentration of 1 × 10^6 cells / ml.

[0245]

[0246] 4. Flow cytometry:

[0247] During flow cytometry, lymphocyte populations were selected using Forward Scatter-Area (FSC-A) gating, which allowed for specific analysis of lymphocytes based on cell size and granularity.

[0248] a) Antibody staining

[0249] - Add 1 μg of CD20 minibody to 100 μl of PBMC (1 × 10^5 cells) and incubate at 4°C for 30 minutes.

[0250] - After washing twice with PBS, FITC-conjugated anti-dog IgG secondary antibody was added and reacted at 4°C for 30 minutes.

[0251] - After washing twice with PBS, 7-AAD was added to label dead cells.

[0252] b) Setting up a control group

[0253] - Negative control: Sample treated with secondary antibody only

[0254] - Positive control: Sample using commercially available anti-canine CD20 antibody (clone NCD1.2)

[0255] c) Flow cytometer setup

[0256] - Using a BD FACSCanto II flow cytometer

[0257] - Selection of lymphocyte populations through FSC / SSC gating

[0258] - Only 7-AAD negative cells were selected for analysis.

[0259]

[0260] 5. Data Analysis:

[0261] - Analyze data using FlowJo software (version 10.7.1)

[0262] - Calculate the percentage of CD20 positive cells and mean fluorescence intensity (MFI)

[0263]

[0264] 6. Results:

[0265] - In this example, flow cytometry was performed to confirm the B cell specificity of the canine CD20 minibody. Figure 4 shows the results of flow cytometry analysis of a non-B cell lymphoma patient (Dongja) and a B cell lymphoma patient (Byeol-i), and Figure 5 shows the results of confirming the expression of B cell markers CD20 and CD21 after isolating PBMC from the peripheral blood of a non-B cell lymphoma patient (Dongja) and a B cell lymphoma patient (Byeol-i) using the antibody of the present invention (A) and (B), respectively.

[0266] - As a result of analyzing the expression of CD20 in the B-cell lymphoma patient (Byeoli), the mean fluorescence intensity (MFI) was measured to be 15,624, which is a 78.5-fold increase compared to the negative control group. This high MFI value quantitatively shows that CD20 is overexpressed in B-cell lymphoma. Referring to Figures 4 and 5, after isolating PBMC from the peripheral blood of the non-B cell lymphoma patient (Dongja) and the B cell lymphoma patient (Byeoli) using the developed canine CD20 antibody, the expression of B cell markers CD20 and CD21 was confirmed, and as a result, CD20-positive and CD21-positive cells were confirmed in the B cell lymphoma patient (Byeoli).

[0267] a) Analysis results of non-B cell lymphoma patients (swarms):

[0268] - Selection of lymphocyte population through FSC-A gating

[0269] - Analysis of CD14 negative cell population

[0270] - Flow cytometry analysis revealed no significant positive response to the CD20 minibody.

[0271] - CD20 positivity rate: 1.2% (no significant difference from the negative control group)

[0272] - CD20 MFI: 243 (1.2-fold increase compared to the negative control group)

[0273] b) Analysis results of B cell lymphoma patient (Star):

[0274] - Selection of lymphocyte population through FSC-A gating

[0275] - Analysis of CD14 negative cell population

[0276] - Flow cytometry analysis results showed a high positivity rate of 92.3% for the canine CD20 minibody.

[0277] - CD20 MFI: 15,624 (78.5-fold increase compared to the negative control group)

[0278]

[0279] 7. Additional Analysis:

[0280] - A significant increase was observed in B-cell lymphoma patients compared to the proportion of CD20-positive B cells (5-10%) in the peripheral blood of normal individuals.

[0281] - Overexpression of CD20 protein was confirmed in lymph node tissue of B-cell lymphoma patients using Western blot.

[0282]

[0283] 8. Conclusion:

[0284] - The manufactured CD20 minibody was confirmed to bind specifically to B-cell lymphoma.

[0285] - Expression analysis of canine CD20 can be used as a useful biomarker for the diagnosis and monitoring of B-cell lymphoma.

[0286]

[0287] This specification and drawings disclose preferred embodiments of the present invention. Although specific terms have been used, they are used in a general sense only to easily explain the technical content of the present invention and to aid understanding of the invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that other modifications based on the technical concept of the present invention are possible in addition to the embodiments disclosed herein.

Claims

1. An antibody comprising a heavy chain CDR1 of sequence number 1, a heavy chain CDR2 of sequence number 2, a heavy chain CDR3 of sequence number 3, a light chain CDR1 of sequence number 4, a light chain CDR2 of sequence number 5, and a light chain CDR3 of sequence number 6.

2. In paragraph 1, An antibody characterized in that the antibody is scFv, Fab, F(ab')2 or minibody.

3. A pharmaceutical composition for treating canine B-cell lymphoma comprising the antibody of clause 1.

4. A nucleic acid encoding the antibody of paragraph 1.

5. An expression vector comprising the nucleic acid of clause 4.

6. A chimeric antigen receptor (CAR) comprising the antibody of claim 1.

7. Immune cells for treating canine B-cell lymphoma, comprising the CAR of clause 6.

8. A composition for diagnosing canine B-cell lymphoma comprising the antibody of clause 1.

9. A composition for detecting expression of canine CD20 comprising the antibody of clause 1.

10. A composition for monitoring treatment of B-cell lymphoma comprising the antibody of clause 1.

11. A method for treating canine B-cell lymphoma, comprising administering to the dog the antibody of claim 1.

12. In paragraph 11, A method characterized in that the antibody is scFv, Fab, F(ab')2 or minibody.

13. A method for diagnosing canine B-cell lymphoma, comprising the step of detecting CD20 expression in the canine using the antibody of claim 1.

14. In paragraph 13, A method characterized in that the above detection step uses flow cytometry.

15. A method for immunocytotoxic treatment of canine B-cell lymphoma, comprising the step of administering to the dog NK cells expressing a chimeric antigen receptor (CAR) comprising the antibody of claim 1.

16. In paragraph 15, A method characterized in that the above NK cells are autologous NK cells of the dog.

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