CN117751141A

CN117751141A - anti-CD 300C monoclonal antibodies and biomarkers thereof for preventing or treating cancer

Info

Publication number: CN117751141A
Application number: CN202280049724.8A
Authority: CN
Inventors: 全在原; 李秀仁; H·金; 林昌基
Original assignee: Shancuikesi Biotechnology Co ltd
Current assignee: Shancuikesi Biotechnology Co ltd
Priority date: 2021-05-13
Filing date: 2022-05-13
Publication date: 2024-03-22

Abstract

The present invention relates to anti-CD 300c monoclonal antibodies and their use for the prevention or treatment of cancer. The anti-CD 300c monoclonal antibody according to the present invention binds to CD300c antigen while having high specificity and promoting anti-cancer immune activity, and thus, is expected to be effective for combating the growth, development and metastasis of various cancers.

Description

anti-CD 300C monoclonal antibodies and biomarkers thereof for preventing or treating cancer

Technical Field

The present disclosure relates to anti-CD 300c antibodies or antigen-binding fragments thereof, and biomarkers, compositions, methods, and kits for preventing or treating cancer, each comprising an anti-CD 300c antibody or antigen-binding fragment thereof.

Background

Cancer is one of the most common diseases among the causes of death in modern people. Such diseases are caused by changes in normal cells due to genetic mutations caused by various causes, and refer to malignant tumors that do not follow the differentiation, proliferation, growth patterns, etc. of normal cells. Cancer is characterized by "uncontrolled cell growth". This abnormal cell growth results in the formation of a cell mass called a tumor, which infiltrates the surrounding tissue and, in severe cases, may metastasize to other organs of the body. Cancer is a refractory chronic disease that is treated by surgery, radiation therapy, chemotherapy, etc., and in many cases cannot be fundamentally cured, causing pain to the patient and ultimately leading to death. In particular, in recent years, the global cancer incidence has increased by 5% or more annually due to an increase in the aged population, environmental deterioration, and the like. According to WHO reports, it is estimated that the number of cancer patients will increase to 3 tens of millions over the next 25 years, with 2 tens of millions dying from cancer.

Cancer drug therapy, cancer chemotherapy, is generally a cytotoxic compound, and treats cancer by attacking and killing cancer cells. However, these chemotherapies show higher side effects, as they damage not only cancer cells but also normal cells. Thus, targeted cancer chemotherapy has been developed to reduce side effects. These targeted cancer chemotherapies can reduce side effects, but are limited in the likelihood of developing resistance. Accordingly, in recent years, interest in cancer immunotherapy has rapidly increased, which utilizes the body's immune system to reduce problems due to toxicity and drug resistance. As an example of such cancer immunotherapy, immune checkpoint inhibitors have been developed that specifically bind to PD-L1 on the surface of cancer cells and inhibit binding to PD-1 on T cells, such that T cells are activated and attack cancer cells. However, even these immune checkpoint inhibitors are not effective in treating various types of cancer. Therefore, there is a need to develop novel cancer immunotherapies that exhibit equivalent therapeutic effects in various cancers.

Prior art literature

Patent literature

(patent document 1) Korean patent laid-open No. 10-2018-0099557

Disclosure of Invention

Technical problem

The present disclosure aims to solve all the problems described above.

It is an object of the present disclosure to provide anti-CD 300c antibodies for use in the prevention or treatment of cancer.

It is another object of the present disclosure to provide an anti-cancer therapy using an anti-CD 300c antibody.

It is yet another object of the present disclosure to provide a pharmaceutical composition for preventing or treating cancer using anti-CD 300c antibody therapy.

It is yet another object of the present disclosure to provide a method of preventing or treating cancer using an anti-CD 300c antibody.

It is yet another object of the present disclosure to provide a therapeutic kit for preventing or treating cancer using an anti-CD 300c antibody.

The objects of the present disclosure are not limited to the above objects. The objects of the present disclosure will become more apparent from the following description and will be attained by means of the means as described in the claims and combinations thereof.

Technical proposal

Representative configurations of the present disclosure for achieving the above objects are as follows.

According to one aspect of the disclosure, there is provided an antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof that specifically binds CD300 c.

According to another aspect of the present disclosure, there is provided an anti-CD 300c antibody or antigen-binding fragment thereof and use thereof for preventing or treating cancer.

According to yet another aspect of the present disclosure, there is provided the use of an anti-CD 300c antibody or antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of cancer.

According to yet another aspect of the present disclosure, there is provided an anticancer therapy comprising an anti-CD 300c antibody or antigen-binding fragment thereof as an active ingredient.

According to still another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises an anti-CD 300 antibody or antigen-binding fragment thereof as an active ingredient.

According to yet another aspect of the present disclosure, there is provided a method of preventing or treating cancer comprising administering to an individual in need thereof an anti-CD 300 antibody or antigen-binding fragment thereof.

According to yet another aspect of the present disclosure, there is provided a kit for preventing or treating cancer, the kit comprising a composition comprising an effective amount of an anti-CD 300c antibody or antigen-binding fragment thereof and instructions for how to use the antibody or antigen-binding fragment thereof.

Advantageous effects

The anti-CD 300c monoclonal antibody according to the present disclosure specifically binds to CD300c expressed on the surface of various cancers with high binding affinity, which activates T cells and simultaneously promotes differentiation into M1 macrophages, thereby effectively inhibiting proliferation of cancer cells. Thus, the anti-CD 300c monoclonal antibody can be effectively used as an immunotherapy for various cancers. In addition, the anti-CD 300c monoclonal antibodies of the present disclosure may exhibit further increased therapeutic effects by co-administration with conventional cancer immunotherapy, and also have inter-species cross-reactivity, which makes the antibodies widely applicable to a variety of mammals. Furthermore, it is expected that in the case of treating a resistant cancer cell exhibiting anti-apoptotic ability with the anti-CD 300c monoclonal antibody of the present disclosure, the antibody can significantly attenuate the resistance of the cancer cell, thereby exhibiting excellent efficacy in preventing cancer recurrence. In addition, cancer cells often inhibit the production of the proinflammatory cytokine IL-2 to evade the immune system. anti-CD 300c monoclonal antibodies have been identified to activate the immune system by restoring production of IL-2 blocked by these cancer cells, inducing cancer cell death. Thus, anti-CD 300c monoclonal antibodies are expected to be useful as a more basic cancer immunotherapy.

Drawings

FIGS. 1a-1y show the heavy and light chain variable region sequences (nucleotide and amino acid sequences), respectively, of 25 anti-CD 300c monoclonal antibodies of the present disclosure. In each figure, the CDR regions (CDR 1, CDR2, and CDR 3) are indicated sequentially.

Figure 2 shows a schematic diagram briefly showing the mechanism by which anti-CD 300c monoclonal antibodies and/or CD300c siRNA of the present disclosure exhibit anticancer effects.

Fig. 3 shows a schematic diagram briefly showing the mechanism by which the anti-CD 300c monoclonal antibodies of the present disclosure act on monocytes, T cells and cancer cells, respectively.

FIG. 4 shows the results obtained by SDS-PAGE of anti-CD 300c monoclonal antibodies under non-reducing conditions according to example 1.4.

FIG. 5 shows the results obtained by SDS-PAGE of anti-CD 300c monoclonal antibodies under reducing conditions according to example 1.4.

Fig. 6 shows the results obtained by comparing CD300c expression in normal cells, immune cells and cancer cell lines according to experimental example 1.1.

Fig. 7a and 7b show the results obtained by identifying CD300c expression in cancer tissue (fig. 7 a) and immune cells (fig. 7 b) according to experimental example 1.2.

Fig. 8a and 8b show the results obtained by identifying CD300c expression in tonsil tissue (fig. 8 a) and cancer tissue (fig. 8 b) according to experimental example 1.3.

Fig. 9 shows the results obtained by identifying the binding affinity of an anti-CD 300c monoclonal antibody to the CD300c antigen according to experimental example 2.1.

Figure 10 shows an S-shaped curve according to the FACS binding results in experimental example 2.2.

Fig. 11 shows the results of the binding ELISA according to experimental example 2.3.

Fig. 12 shows the result of surface plasmon resonance according to experimental example 2.4.

Fig. 13 shows the results of the binding ELISA according to experimental example 2.5.

Fig. 14 shows the results of the binding ELISA according to experimental example 2.6.

FIG. 15 shows the results obtained by comparing the total survival dependent on CD300c expression levels in different cancer patients in combination with experimental example 2.7.

FIG. 16 shows the results obtained by identifying the anti-cancer effect of anti-CD 300c monoclonal antibodies by T cell activation according to experimental example 3.1.

FIGS. 17 and 18 show the results obtained by identifying the effect of anti-CD 300c monoclonal antibodies on the differentiation into M1 macrophages according to experimental example 3.2.

FIGS. 19 and 20 show the results obtained by identifying the concentration-dependent effect of anti-CD 300c monoclonal antibodies in differentiating into M1 macrophages according to experimental example 3.3.

FIG. 21 shows the results obtained by identifying the effect of anti-CD 300c monoclonal antibodies on differentiation into M1 macrophages according to experimental example 3.4.

FIG. 22 shows the results obtained by again identifying whether the anti-CD 300c monoclonal antibody promotes differentiation of human monocytes into M1 macrophages according to experimental example 3.5.

FIGS. 23-25 show the results obtained by identifying whether anti-CD 300c monoclonal antibodies can induce the differentiation of M2 macrophages into M1 macrophages according to experimental example 3.6.

FIG. 26 shows the results obtained by identifying the ability of an anti-CD 300c monoclonal antibody to cause differentiation and re-differentiation (repolarization) into M1 macrophages according to experimental example 3.7.

FIGS. 27 and 28 show the results obtained by identifying the effect of a monoclonal antibody targeting CD300c on cancer cell growth according to experimental example 4.1.

Fig. 29 shows the results obtained by identifying the inhibitory effect of an anti-CD 300c monoclonal antibody on the growth of cancer cells according to its concentration, according to experimental example 4.2.

FIG. 30 shows the results obtained by identifying whether an anti-CD 300c monoclonal antibody can promote differentiation of mouse macrophages into M1 macrophages according to experimental example 4.3.

FIG. 31 shows the results obtained by identifying whether an anti-CD 300c monoclonal antibody exhibits an anticancer effect according to experimental example 4.4.

FIGS. 32-35 show the results obtained by comparing the ability of anti-CD 300c monoclonal antibody to elicit differentiation into M1 macrophages by conventional cancer immunotherapy, according to experimental example 5.1.

Fig. 36 shows the results obtained by comparing the ability of M0 macrophages to differentiate into M1 macrophages between an anti-CD 300c monoclonal antibody and conventional cancer immunotherapy according to experimental example 5.2.

Fig. 37 shows the results obtained by comparing the ability to cause differentiation into M1 macrophages between anti-CD 300c monoclonal antibodies and conventional cancer immunotherapy according to experimental example 5.3.

Fig. 38 and 39 show the results obtained by comparing the cancer cell growth inhibition between the anti-CD 300c monoclonal antibody and the conventional cancer immunotherapy according to experimental example 5.4.

FIG. 40 shows the results obtained by identifying the in vivo effects of anti-CD 300c monoclonal antibodies on tumor-associated macrophages according to experimental example 6.1.

FIG. 41 shows the results obtained by identifying the in vivo effects of an anti-CD 300c monoclonal antibody on CD8+ T cells according to experimental example 6.2.

FIG. 42 shows the results obtained by identifying whether anti-CD 300c monoclonal antibodies increased the number of CD8+ T cells in a tumor specific manner according to experimental example 6.3.

FIG. 43 shows the results obtained by identifying the in vivo effects of an anti-CD 300c monoclonal antibody on the increase in CD8+ T cell activity according to experimental example 6.4.

FIG. 44 shows the results obtained by identifying the increased in vivo effects of anti-CD 300c monoclonal antibodies on cytotoxic T cells relative to regulatory T cells according to experimental example 6.5.

FIG. 45 shows the results obtained by identifying the effect of anti-CD 300c monoclonal antibodies on cytotoxic T cells, regulatory T cells and tumor-associated macrophages according to experimental example 6.6.

FIG. 46 shows the results obtained by identifying anti-CD 300c monoclonal antibodies against cancer in vivo according to experimental example 6.7.

Fig. 47 shows Nanostring immune profile results obtained in the case of treatment of a solid cancer model with CL7 according to example 2.1.

Fig. 48 shows the variation in expression of various immune cells and tumor microenvironment related markers obtained in the case of treatment of a solid cancer model with CL7 according to example 2.1. * A marker showing a statistically significant change in the expression level compared to that before CL7 treatment.

Fig. 49 shows the variation of immune checkpoint marker expression identified based on the Nanostring immune profile results obtained in example 2.1 according to example 2.2. * A marker showing a statistically significant change in the expression level compared to that before CL7 treatment.

Fig. 50 shows the results of monocytes differentiated into M1 macrophages (whether or not M1 macrophages increased) by treatment with anti-CD 300c monoclonal antibodies and cancer immunotherapy alone or in combination according to experimental example 7.1.

Fig. 51 shows the results of differentiation of monocytes into M1 macrophages (which indicate whether the M1 macrophage marker increased) by treatment with anti-CD 300c monoclonal antibody according to experimental example 7.2.

Fig. 52 shows the results of monocytes differentiated into M1 macrophages (which indicate whether the M1 macrophage marker increased) by co-treatment with anti-CD 300c monoclonal antibody and cancer immunotherapy according to experimental example 7.2.

FIGS. 53-55 show the results obtained by identifying signal transduction of MAPK (FIG. 53), NF- κB (FIG. 54) and IκB (FIG. 55), which are signals for M1 macrophage differentiation, caused by co-treatment of anti-CD 300c monoclonal antibody and cancer immunotherapy, according to experimental example 7.4.

Fig. 56 shows the results obtained by identifying changes in apoptosis signals caused by co-treatment of anti-CD 300c monoclonal antibodies and cancer immunotherapy according to experimental example 8.1.

FIGS. 57 and 58 show the results obtained by identifying the growth inhibitory effect of cancer cells caused by co-treatment with an anti-CD 300c monoclonal antibody and cancer immunotherapy, according to experimental example 8.2.

Fig. 59 schematically shows an experimental method used in experimental example 9.1.

Figure 60 shows the in vivo cancer growth inhibition observed with anti-CD 300c monoclonal antibodies and anti-PD-1 antibodies administered alone or in combination to mice transplanted with colorectal cancer cell lines according to experimental example 9.1.

FIG. 61 shows the results obtained by identifying whether an anti-CD 300c monoclonal antibody increases M1 macrophages in cancer tissue of a mouse model according to experimental example 9.3.

Fig. 62 shows the results obtained by identifying whether anti-CD 300c monoclonal antibodies promote cd8+ T cell immunity in a mouse tumor model according to experimental example 9.4.

Fig. 63 schematically shows the experimental method used in experimental example 10.1.

FIG. 64 shows the results obtained by identifying whether an anti-CD 300c monoclonal antibody is effective in cancers other than the CT26 colorectal cancer mouse model, according to experimental example 10.1.

Fig. 65 shows the results obtained by identifying in vivo effects of anti-CD 300c monoclonal antibodies and cancer immunotherapy on cd8+ T cells in a B16F10 melanoma model administered alone or in combination (including duplex and triplex co-administration) according to experimental example 10.2.

FIG. 66 shows the results obtained by identifying in vivo effects of anti-CD 300c monoclonal antibodies and cancer immunotherapy on regulatory T cells in B16F10 melanoma models, alone or in combination (including duplex and triplex co-administration), according to experimental example 10.3.

Fig. 67 shows the results obtained by identifying in vivo effects of anti-CD 300c monoclonal antibodies and cancer immunotherapy on macrophages in a B16F10 melanoma model, alone or in combination (including duplex and triplex co-administration), according to experimental example 10.4.

FIGS. 68a and 68b show the results obtained by identifying in vivo anticancer effects caused by co-administration of an anti-CD 300c monoclonal antibody and cancer immunotherapy according to experimental example 11. Fig. 68a shows the tumor volume reduction rate, and fig. 68b shows the complete remission rate.

Fig. 69 shows the results obtained by identifying the effect of improving long-term survival caused by co-administration of an anti-CD 300c monoclonal antibody and cancer immunotherapy according to experimental example 12.

Fig. 70 shows the results obtained by identifying in vivo effects of preventing cancer recurrence caused by co-administration of an anti-CD 300c monoclonal antibody and cancer immunotherapy according to experimental example 13.

Fig. 71 shows the results obtained by identifying the immunological memory effect caused by co-administration of an anti-CD 300c monoclonal antibody and cancer immunotherapy according to experimental example 14.

Figures 72a and 72b show the results obtained by identifying whether anti-CD 300c monoclonal antibodies and cancer immunotherapy administered alone or in combination (including duplex and triplex co-administration) can promote differentiation of monocytes into M1 macrophages according to experimental example 15.

Fig. 73a and 73b show the results obtained by identifying whether an anti-CD 300c monoclonal antibody can inhibit the growth of cancer cells by co-administration with cancer immunotherapy according to experimental example 16.

Best Mode for Carrying Out The Invention

The following detailed description of the present disclosure will be described with reference to specific drawings for specific embodiments in which the disclosure may be practiced. However, the present disclosure is not limited thereto, and the scope of the present disclosure is limited only by the appended claims, appropriately interpreted, along with the full range of equivalents to which the claims are entitled. It is to be understood that the various embodiments of the disclosure, although different from each other, are not necessarily mutually exclusive. For example, the particular features, structures, or characteristics described herein may be varied from one embodiment to another, or implemented as a combination of embodiments, without departing from the spirit and scope of the present disclosure. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this disclosure belongs. For the purposes of explaining the present specification, the following definitions will be applied, and the singular forms "a," "an," and "the" include plural referents and vice versa, unless the context clearly dictates otherwise.

Definition of the definition

As used herein, the term "about" means within an acceptable error range for each value known to one of ordinary skill in the art.

The term "antibody" is used broadly and includes monoclonal antibodies (including full length antibodies) of any isotype (e.g., igG, igM, igA, igD and IgE), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fusions (e.g., fusions of antibodies with (poly) peptides or fusions of antibodies with compounds), and antibody fragments (including antigen-binding fragments). As used herein, the prefix "anti" when bound to an antigen indicates that a given antibody reacts with a given antigen. Antibodies reactive with a particular antigen may be produced by synthetic and/or recombinant methods without limitation, such as selecting a library of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or nucleic acid encoding the antigen. A typical IgG antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. The heavy chain variable region (HVR) and the light chain variable region (LVR) contain three segments, called "complementarity determining regions" ("CDRs") or "hypervariable regions", respectively, which are primarily responsible for binding epitopes of an antigen. They are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions outside the CDRs are called "framework regions" ("FR"). The antibody herein may be, for example, an animal antibody, a chimeric antibody, a humanized antibody or a human antibody.

The term "humanization" (also known as remodeling or CDR grafting) includes a mature technique for reducing the immunogenicity of monoclonal antibodies from heterologous sources (usually rodents) and for improving their affinity or effector functions (ADCC, complement activation, C1q binding).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, exhibit the properties of antibodies, and should not be construed as requiring production of antibodies by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

The term "antigen binding fragment" refers to a portion of an antibody that has the ability to specifically bind to an antigen or a polypeptide comprising the same. The terms "antibody" and "antigen-binding fragment" may be used interchangeably, except in the context that "antibody" is understood to specifically exclude "antigen-binding fragment" and "antibody" may be interpreted to include the case of "antigen-binding fragment". Examples of antigen binding fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2, diabodies, triabodies, tetrabodies, cross Fab fragments, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments and single domain antibodies.

The term "cancer therapy" refers generally to known agents used in conventional cancer treatment that act on various metabolic pathways of cells and exhibit cytotoxic or cytostatic effects on cancer cells. Cancer therapies include chemotherapy, targeted chemotherapy, and immunotherapy.

The term "immunotherapy" (also referred to as "cancer immunotherapy") is a cancer therapy or anticancer agent that activates immune cells to kill cancer cells.

The term "individual" is used interchangeably with "patient" and may be a mammal in need of prevention or treatment of cancer, such as primates (e.g., humans), companion animals (e.g., dogs and cats), livestock (e.g., cows, pigs, horses, sheep and goats), and laboratory animals (e.g., rats, mice and guinea pigs). In one embodiment of the present disclosure, the individual is a human.

The term "treatment" generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be therapeutic for partially or completely curing the disease and/or side effects due to the disease. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or slowing the disease state, and alleviating or improving prognosis. Preferably, "treatment" may refer to a medical intervention of a disease or disorder that has progressed.

The term "prevention" relates to prophylactic treatment, i.e. to measures or procedures which aim at preventing, rather than curing, a disease. "preventing" means obtaining a desired pharmacological and/or physiological effect, which is prophylactic in terms of completely or partially preventing a disease or symptom thereof.

The term "administering" refers to providing a substance (e.g., an anti-CD 300c antibody or antigen-binding fragment thereof and another cancer therapy) to an individual for prophylactic or therapeutic purposes (e.g., prevention or treatment of cancer).

The term "biological sample" includes a variety of sample types obtained from an individual and may be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples and solid tissue samples of biological origin, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. Thus, biological samples include clinical samples, and also include cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples, particularly tumor samples. The term "biological data" refers to any analytical data obtained using a biological sample.

The term "expression level" may be determined by measuring the expression level of at least one of mRNA and protein of the corresponding marker. For the method of measuring the expression level of mRNA or protein, any method known in the art may be used. For example, the reagent for measuring the expression level of mRNA may be a pair of primers or probes that specifically bind to the corresponding marker gene, and the reagent for measuring the expression level of protein may be an antibody, substrate, ligand or cofactor that specifically binds to the corresponding marker. Assays for measuring mRNA levels include, but are not limited to, reverse transcription polymerase chain reaction, competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase protection assay, northern blotting, or DNA chip assay. Assays to measure protein levels include, but are not limited to, western blotting, ELISA, radioimmunoassay, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip assay.

The term "therapeutic responsiveness" refers to whether an individual having or suspected of having cancer is beneficial or detrimental to a response to treatment with a therapeutically active ingredient (e.g., a CD300c antibody or antigen binding fragment thereof). Therapeutic responsiveness can be assessed by changes in the immune system associated with tumor therapy observed following administration of CD300c antibodies or antigen binding fragments thereof.

anti-CD 300c antibodies

According to one aspect of the present disclosure, an anti-CD 300c antibody or antigen-binding fragment thereof is provided. An anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure is an antigen-binding molecule that specifically binds to CD300c protein. Preferably, the anti-CD 300c antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD300c protein.

The term "CD300c protein" is used interchangeably with "CD300c" or "CD300c antigen", and is a protein encoded by the CD300c gene. CD300c proteins are known to exhibit significant sequence identity with B7 family proteins and to be expressed on antigen presenting cell membranes. Inhibition of CD300c protein expression or activity may induce T cell activation and/or promote differentiation into M1 macrophages.

Furthermore, the term "anti-CD 300c antibody" may be used interchangeably with polypeptides that bind to CD300c protein. The term "polypeptide" refers to any polymer of amino acids linked to each other by peptide bonds, irrespective of their length. That is, polypeptides as used herein also include peptides and proteins.

In one embodiment, the anti-CD 300c antibody or antigen-binding fragment thereof is capable of specifically binding to the extracellular domain (ECD) of the CD300c protein. The extracellular domain of CD300c may be the extracellular domain of human CD300c protein. Furthermore, the extracellular domain of CD300c may comprise SEQ ID NO: 402.

In one embodiment, it was identified that the expression level of CD300c protein exhibited a very high correlation with the survival of various cancer patients. In particular, cancer patients with high CD300c expression levels are identified as having a shorter survival period than cancer patients with low CD300c expression levels relative to the average CD300c expression level of the cancer patients. This means that inhibiting expression or activity of CD300c using an anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure may produce a cancer therapeutic effect or an effect of increasing the survival of a cancer patient.

The anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure may specifically bind to CD300c expressed on the surface of various cancer cells, and thus exhibit anticancer effects. Binding of anti-CD 300c antibodies to CD300c can activate T cells while promoting differentiation into M1 macrophages to effectively inhibit proliferation of cancer cells, which allows anti-CD 300c antibodies to be effectively used as immunotherapies for a variety of cancers. In addition, anti-CD 300c antibodies according to the present disclosure may exhibit further improved therapeutic effects by co-administration with conventional cancer immunotherapy, and also have inter-species cross-reactivity (e.g., between human and mouse antigens), which allows the antibodies to be widely used in a variety of mammals. Furthermore, it is expected that in the case of treating a resistant cancer cell exhibiting anti-apoptotic ability with the anti-CD 300c antibody of the present disclosure, the antibody can significantly attenuate the resistance of the cancer cell, thereby exhibiting excellent efficacy of preventing cancer recurrence. In addition, cancer cells often inhibit the production of the proinflammatory cytokine IL-2 to evade the immune system. anti-CD 300c antibodies have been identified to activate the immune system by restoring production of IL-2 blocked by these cancer cells, which induces cancer cell death. Thus, anti-CD 300c antibodies are expected to be useful as a more basic cancer immunotherapy. For details on the CD300c protein or anti-CD 300c antibody, reference may also be made to Korean patent publication No. 10-2019-0136949, the entire contents of which are incorporated herein by reference.

In one embodiment, the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof may comprise:

(i) A heavy chain variable region comprising: a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 7. SEQ ID NO: 19. SEQ ID NO: 31. SEQ ID NO: 43. SEQ ID NO: 55. SEQ ID NO: 67. SEQ ID NO: 79. SEQ ID NO: 91. SEQ ID NO: 103. SEQ ID NO: 115. SEQ ID NO: 127. SEQ ID NO: 139. SEQ ID NO: 151. SEQ ID NO: 163. SEQ ID NO: 175. SEQ ID NO: 187. SEQ ID NO: 199. SEQ ID NO: 211. SEQ ID NO: 223. SEQ ID NO: 235. SEQ ID NO: 247. SEQ ID NO: 259. SEQ ID NO: 271. SEQ ID NO:283 and SEQ ID NO:295;

a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 32. SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO: 92. SEQ ID NO: 104. SEQ ID NO: 116. SEQ ID NO: 128. SEQ ID NO: 140. SEQ ID NO: 152. SEQ ID NO: 164. SEQ ID NO: 176. SEQ ID NO: 188. SEQ ID NO: 200. SEQ ID NO: 212. SEQ ID NO: 224. SEQ ID NO: 236. SEQ ID NO: 248. SEQ ID NO: 260. SEQ ID NO: 272. SEQ ID NO:284 and SEQ ID NO:296; and

A CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 9. SEQ ID NO: 21. SEQ ID NO: 33. SEQ ID NO: 45. SEQ ID NO: 57. SEQ ID NO: 69. SEQ ID NO: 81. SEQ ID NO: 93. SEQ ID NO: 105. SEQ ID NO: 117. SEQ ID NO: 129. SEQ ID NO: 141. SEQ ID NO: 153. SEQ ID NO: 165. SEQ ID NO: 177. SEQ ID NO: 189. SEQ ID NO: 201. SEQ ID NO: 213. SEQ ID NO: 225. SEQ ID NO: 237. SEQ ID NO: 249. SEQ ID NO: 261. SEQ ID NO: 273. SEQ ID NO:285 and SEQ ID NO:297; and

(ii) A light chain variable region comprising: a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. SEQ ID NO: 22. SEQ ID NO: 34. SEQ ID NO: 46. SEQ ID NO: 58. SEQ ID NO: 70. SEQ ID NO: 82. SEQ ID NO: 94. SEQ ID NO: 106. SEQ ID NO: 118. SEQ ID NO: 130. SEQ ID NO: 142. SEQ ID NO: 154. SEQ ID NO: 166. SEQ ID NO: 178. SEQ ID NO: 190. SEQ ID NO: 202. SEQ ID NO: 214. SEQ ID NO: 226. SEQ ID NO: 238. SEQ ID NO: 250. SEQ ID NO: 262. SEQ ID NO: 274. SEQ ID NO:286 and SEQ ID NO:298;

A CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 11. SEQ ID NO: 23. SEQ ID NO: 35. SEQ ID NO: 47. SEQ ID NO: 59. SEQ ID NO: 71. SEQ ID NO: 83. SEQ ID NO: 95. SEQ ID NO: 107. SEQ ID NO: 119. SEQ ID NO: 131. SEQ ID NO: 143. SEQ ID NO: 155. SEQ ID NO: 167. SEQ ID NO: 179. SEQ ID NO: 191. SEQ ID NO: 203. SEQ ID NO: 215. SEQ ID NO: 227. SEQ ID NO: 239. SEQ ID NO: 251. SEQ ID NO: 263. SEQ ID NO: 275. SEQ ID NO:287 and SEQ ID NO:299; and

a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 12. SEQ ID NO: 24. SEQ ID NO: 36. SEQ ID NO: 48. SEQ ID NO: 60. SEQ ID NO: 72. SEQ ID NO: 84. SEQ ID NO: 96. SEQ ID NO: 108. SEQ ID NO: 120. SEQ ID NO: 132. SEQ ID NO: 144. SEQ ID NO: 156. SEQ ID NO: 168. SEQ ID NO: 180. SEQ ID NO: 192. SEQ ID NO: 204. SEQ ID NO: 216. SEQ ID NO: 228. SEQ ID NO: 240. SEQ ID NO: 252. SEQ ID NO: 264. SEQ ID NO: 276. SEQ ID NO:288 and SEQ ID NO:300.

in another embodiment, (i) the heavy chain variable region may comprise:

CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 7. SEQ ID NO: 19. SEQ ID NO: 43. SEQ ID NO: 55. SEQ ID NO: 67. SEQ ID NO: 79. SEQ ID NO: 103. SEQ ID NO: 115. SEQ ID NO: 127. SEQ ID NO: 139. SEQ ID NO: 151. SEQ ID NO: 163. SEQ ID NO:199 and SEQ ID NO:211;

CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO: 104. SEQ ID NO: 116. SEQ ID NO: 128. SEQ ID NO: 140. SEQ ID NO: 152. SEQ ID NO: 164. SEQ ID NO:200 and SEQ ID NO:212; and

CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 9. SEQ ID NO: 21. SEQ ID NO: 45. SEQ ID NO: 57. SEQ ID NO: 69. SEQ ID NO: 81. SEQ ID NO: 105. SEQ ID NO: 117. SEQ ID NO: 129. SEQ ID NO: 141. SEQ ID NO: 153. SEQ ID NO: 165. SEQ ID NO:201 and SEQ ID NO:213; and

(ii) The light chain variable region may comprise:

CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. SEQ ID NO: 22. SEQ ID NO: 46. SEQ ID NO: 58. SEQ ID NO: 70. SEQ ID NO: 82. SEQ ID NO: 106. SEQ ID NO: 118. SEQ ID NO: 130. SEQ ID NO: 142. SEQ ID NO: 154. SEQ ID NO: 166. SEQ ID NO:202 and SEQ ID NO:214;

CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 11. SEQ ID NO: 23. SEQ ID NO: 47. SEQ ID NO: 59. SEQ ID NO: 71. SEQ ID NO: 83. SEQ ID NO: 107. SEQ ID NO: 119. SEQ ID NO: 131. SEQ ID NO: 143. SEQ ID NO: 155. SEQ ID NO: 167. SEQ ID NO:203 and SEQ ID NO:215, respectively; and

CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 12. SEQ ID NO: 24. SEQ ID NO: 48. SEQ ID NO: 60. SEQ ID NO: 72. SEQ ID NO: 84. SEQ ID NO: 108. SEQ ID NO: 120. SEQ ID NO: 132. SEQ ID NO: 144. SEQ ID NO: 156. SEQ ID NO: 168. SEQ ID NO:204 and SEQ ID NO:216.

in another embodiment, (i) the heavy chain variable region may comprise:

CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 43. SEQ ID NO: 79. SEQ ID NO:115 and SEQ ID NO:211;

CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 44. SEQ ID NO: 80. SEQ ID NO:116 and SEQ ID NO:212; and

CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 45. SEQ ID NO: 81. SEQ ID NO:117 and SEQ ID NO:213; and

(ii) The light chain variable region may comprise:

CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 46. SEQ ID NO: 82. SEQ ID NO:118 and SEQ ID NO:214;

CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 47. SEQ ID NO: 83. SEQ ID NO:119 and SEQ ID NO:215, respectively; and

CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 48. SEQ ID NO: 84. SEQ ID NO:120 and SEQ ID NO:216.

in another embodiment, the heavy chain variable region may comprise a heavy chain variable region comprising SEQ ID NO:43 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:44 and CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:45 or CDR3 consisting of the amino acid sequence shown in seq id no; and the light chain variable region may comprise a light chain variable region comprising SEQ ID NO:46 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:47 and CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:48 or CDR3 consisting of the same.

In another embodiment, the heavy chain variable region may comprise a heavy chain variable region comprising SEQ ID NO:79 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:80 and CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:81 or CDR3 consisting of the amino acid sequence depicted; and the light chain variable region may comprise a light chain variable region comprising SEQ ID NO:82 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:83 and CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:84 or CDR3 consisting of the same.

In another embodiment, the heavy chain variable region may comprise a heavy chain variable region comprising SEQ ID NO:115 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:116 or CDR2 consisting of the amino acid sequence shown in SEQ ID NO:117 or CDR3 consisting of the amino acid sequence shown; and the light chain variable region may comprise a light chain variable region comprising SEQ ID NO:118 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:119 or CDR2 consisting of the amino acid sequence shown in SEQ ID NO:120 or CDR3 consisting of the amino acid sequence depicted therein.

In another embodiment, the heavy chain variable region may comprise a heavy chain variable region comprising SEQ ID NO:211 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:212 or CDR2 consisting of the amino acid sequence shown in SEQ ID NO:213 or CDR3 consisting of the amino acid sequence shown in seq id no; and the light chain variable region may comprise a light chain variable region comprising SEQ ID NO:214 or CDR1 consisting of the amino acid sequence shown in SEQ ID NO:215 and CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:216 or CDR3 consisting of the same.

In another embodiment, the heavy chain variable region may comprise an amino acid sequence selected from the group consisting of: SEQ ID No: 303. 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395 and 399; and the light chain variable region may comprise an amino acid sequence selected from the group consisting of: SEQ ID No: 304. 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, and 400.

In another embodiment, the heavy chain variable region may comprise an amino acid sequence selected from the group consisting of: SEQ ID No: 315. 327, 339 and 371; and the light chain variable region may comprise an amino acid sequence selected from the group consisting of: SEQ ID No: 316. 328, 340 and 372. Preferably, the heavy chain variable region may comprise SEQ ID No:315, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID No: 316; or the heavy chain variable region may comprise SEQ ID No:327, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID No:328, an amino acid sequence shown in seq id no; or the heavy chain variable region may comprise SEQ ID No:339, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID No:340, an amino acid sequence shown in seq id no; or the heavy chain variable region may comprise SEQ ID No:371, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID No: 372.

In another aspect, there is provided an anti-CD 300C monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1 to CDR3 comprising or consisting of the amino acid sequences represented by formulae (1) to (3), respectively, and a light chain variable region comprising CDR1 to CDR3 comprising or consisting of the amino acid sequences represented by formulae (4) to (6), respectively (each amino acid sequence is shown in the n→c direction):

FTFX1X2X3X4MX5WVR(1)(SEQ ID NO：403)

In the above-mentioned formula (i), the water,

x1=g or S

X2= S, R or D

X3=n or Y

X4= Y, A, G or H

X5=s or H

X1ISX2SGX3X4TYYAX5(2)(SEQ ID NO：404)

In the above-mentioned formula (i), the water,

x1=t or a

X2=g or S

X3=t or G

X4=s or Y

X5=d or E

YCAX1X2X3X4X5X6X7X8X9W(3)(SEQ ID NO：405)

In the above-mentioned formula (i), the water,

x1=r or S

X2=g or S

X3= M, S, Y or I

X4= W, Q, G or R

X5=g or L

X6= M, I or P

X7= D, F or L

X8=v or D

X9= I, Y or absence of

CX1X2X3X4X5X6X7X8X9X10X11VX12W (4) (SEQ ID NO: 406) in the above formula,

x1=t or S

X2=g or R

X3= K, N or S

X4= H, N or S

X5= R, I or G

X6= H, G or I

X7= T, I or S X8= R, A, K or absence of

X9= R, S, G or absence of

X10=n or absence of

X11=y or absence of

X12= N, H or Q X1X2X3X4RPSGVX5 (5) (SEQ ID NO: 407)

In the above-mentioned formula (i), the water,

x1= L, S, R or E

X2= D, K or N

X3=s or N

X4= E, N, Q or K

In the above formula, x5=p or rycx 1X2X3X4X5X6X7X8X9X10VF (6) (SEQ ID NO: 408),

x1= Q, A or S

X2=s or a

X3=y or W

X4=d or a

X5= S, D or G

X6= S, N or T

X7= S, L, N or K

X8= V, S, N or G

X9= G, L, V or absence of

X10=p or absent.

In certain embodiments, an anti-CD 300c antibody or antigen binding fragment may comprise a sequence having 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the CDR sequences described above or the sequences set forth in tables 3, 4, and 5.

In certain embodiments, amino acid sequence variants of the antibodies of the present disclosure are contemplated. For example, it may be desirable to increase the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding. The sites of interest for substitution mutations include the heavy chain variable region (HVR) and the Framework Region (FR). Conservative substitutions are provided under the heading "preferred substitutions" in table 1, and are further described below with reference to amino acid side chain categories (1) through (6). Amino acid substitutions may be introduced into the molecule of interest and products may be screened for a desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 1

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will require the exchange of members of one of these classes for another class.

As used herein, the term "amino acid sequence variant" includes basic variants in which there are amino acid substitutions in one or more hypervariable region residues of a parent antigen binding molecule (e.g., a humanized or human antibody). Typically, the resulting variants selected for further investigation will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule and/or will substantially retain certain biological properties of the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated using, for example, phage display-based affinity maturation techniques known in the art. Briefly, one or more HVR residues are mutated, variant antigen binding molecules are displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not substantially reduce the ability of the antigen binding molecule to bind to an antigen. For example, conservative changes (e.g., conservative substitutions provided herein) may be made in the HVR that do not substantially reduce binding affinity.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the molecule include fusion to the N-or C-terminus of a polypeptide that increases the serum half-life of the antibody. In addition, other insertional variants of the molecule may include fusion to the N-terminus or C-terminus of the polypeptide to facilitate passage of the Blood Brain Barrier (BBB).

Furthermore, variants of the antibodies of the disclosure, or antigen binding fragments thereof, are provided that have increased affinity for the CD300c antigen. These variants can be obtained by a number of affinity maturation protocols, including CDR mutations (Yang et al, j.mol.biol.,254,392-403,1995); chain shuffling (Marks et al, bio/Technology,10,779-783,1992); mutant strains of E.coli were used (Low et al, J.mol.biol.,250,359-368,1996); DNA shuffling (Patten et al, curr. Opin. Biotechnol.,8,724-733, 1997); phage display (Thompson et al, j. Mol. Biol.,256,77-88,1996) and sexual PCR (sexual PCR) (Crameri et al, nature,391,288-291,1998). Methods of these affinity maturation are discussed by Vaughan et al (Science, 239,1534-1536,1988).

In one embodiment, the anti-CD 300c monoclonal antibody or antigen binding fragment thereof may have cross-reactivity between species. In particular, anti-CD 300c monoclonal antibodies or antigen binding fragments thereof may exhibit cross-reactivity to both human and mouse CD300c antigens. Such cross-reactivity was identified in experimental examples 4.1-4.4.

In another embodiment, the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof may comprise a form of antibody-conjugated drug, wherein the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof is conjugated to another drug, or may be provided in such a form.

As used herein, the term "antibody-conjugated drug" refers to a form in which the drug and antibody are chemically linked to each other without reducing the biological activity of the antibody and drug. In the present disclosure, an antibody-conjugated drug means a form in which the drug binds to an amino acid residue at the N-terminus of an antibody heavy chain and/or light chain, in particular, a form in which the drug binds to an α -amino group at the N-terminus of an antibody heavy chain and/or light chain.

"drug" may refer to any substance that has some biological activity on cells (e.g., cancer cells), which is a concept that includes DNA, RNA, or peptides. The drug may be in a form containing a reactive group capable of reacting and crosslinking with an alpha-amino group, and also includes a form containing a reactive group capable of reacting and crosslinking with an alpha-amino group and to which a linker is attached.

The type of reactive group capable of reacting and crosslinking with an α -amino group is not particularly limited as long as the reactive group is capable of reacting and crosslinking with an α -amino group at the N-terminus of an antibody heavy chain or light chain. Reactive groups include all types of groups known in the art to react with amino groups. The reactive group may be, for example, any one of isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imidoester, carbodiimide, anhydride and fluorophenyl ester, but is not limited thereto.

Regardless of the type of drug that is capable of treating a disease targeted by an anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure, the drug is contained, but may preferably be an anti-cancer agent.

Nucleic acids, vectors, host cells and methods of production

The anti-CD 300c monoclonal antibodies of the present disclosure, or antigen-binding fragments thereof, may be produced by any antibody production technique known in the art.

According to yet another aspect of the present disclosure, there is provided a nucleic acid molecule (e.g., a polynucleotide) encoding an anti-CD 300c monoclonal antibody or antigen-binding fragment thereof. Such nucleic acid molecules may encode an amino acid sequence comprising a heavy chain variable region or a heavy chain CDR region and/or an amino acid sequence comprising a light chain variable region or a light chain CDR region of an anti-CD 300c monoclonal antibody. For sequences of nucleic acid molecules encoding the heavy/light chain variable and CDR regions of the anti-CD 300c monoclonal antibodies of the present disclosure, see tables 3-5 and fig. 1.

The term "nucleic acid molecule" includes DNA (gDNA and cDNA) and RNA molecules. In nucleic acid molecules, nucleotides as basic units also include analogues in which the sugar or base moiety is modified, as well as natural nucleotides. The sequences of nucleic acid molecules encoding the heavy and light chain variable regions of the present disclosure may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides. The nucleic acid molecules of the present disclosure are to be construed as also including nucleotide sequences having substantial identity to the nucleotide sequences as described above. Substantial identity refers to nucleotide sequences of the present disclosure that exhibit at least 80% homology, in one embodiment at least 90% homology, in another embodiment at least 95% homology, or in yet another embodiment at least 98% homology when aligned to correspond as much as possible to any other sequence and the aligned sequences are analyzed using algorithms commonly used in the art.

According to yet another aspect of the present disclosure, there is provided at least one vector (e.g., an expression vector) comprising the nucleic acid.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments may be inserted. Another type of vector is a viral vector, wherein a DNA or RNA sequence of viral origin is present in the vector for packaging into the virus. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Expression vectors commonly used in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which provide equivalent functions.

According to yet another aspect of the present disclosure, there is provided a host cell comprising at least one nucleic acid molecule (e.g., polynucleotide) encoding a monoclonal antibody of the present disclosure.

The host cell may be a cell transformed with a recombinant vector of the present disclosure. Any host cell known in the art capable of stably and continuously cloning or expressing a recombinant vector may be used. Suitable prokaryotic host cells may include E.coli, strains of Bacillus species such as Bacillus subtilis (Bacillus subtillis) or Bacillus thuringiensis (Bacillus thuringiensis), enterobacteria and strains such as Salmonella typhymurum, serratia marcescens (Serratia marcescens), and various Pseudomonas species. Suitable eukaryotic host cells to be transformed may include yeasts such as Saccharomyces cerevisiae, insect cells, plant cells and animal cells, e.g., sp2/0, chinese Hamster Ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, hepG2, huh7, 3T3, RIN and MDCK cell lines. In addition, "host cell" is used to refer to a cell that has been transformed, or that is capable of being transformed with a nucleic acid sequence and then expressing a selected gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent, as long as the selected gene is present.

According to another aspect of the invention, there is provided a method of producing an anti-CD 300c monoclonal antibody or antigen-binding fragment thereof, comprising culturing a host cell.

The culturing of the host cell for producing the antibody or antigen-binding fragment thereof may be performed in a suitable medium and under culture conditions known in the art. The person skilled in the art can easily adapt the method of such cultivation according to the host cell chosen. According to the type of cell growth, the culture process is classified into suspension culture and adherent culture, and according to the type of culture, the culture process is classified into batch, fed-batch and continuous culture. Various methods of cultivation are disclosed, for example, in James M.Lee, "Biochemical Engineering", prentice-Hall International Editions, pages 138-176.

For collection of antibodies, any method known in the art for purification of immunoglobulins may be used, examples of which may include chromatography (ion exchange, affinity (e.g., protein a), size exclusion, etc.), centrifugation, differential solubility, or other standard techniques for purifying proteins.

Combination with cancer immunotherapy

The inventors have found that anti-CD 300c antibodies or antigen binding fragments thereof may exhibit enhanced anti-cancer effects when used in combination with one or more other cancer immunotherapies. Thus, the anti-CD 300c antibodies or antigen-binding fragments thereof of the present disclosure may be used in combination with one or more other cancer immunotherapies to prevent or treat cancer.

Cancer immunotherapy has a new mechanism by which immune cells in the body are activated to kill cancer cells, so that it has an advantage in that it can be widely used for most cancers without specific genetic mutation. In addition, immunotherapy has fewer side effects because they treat cancer by strengthening the patient's own immune system, and have the effect of improving the patient's quality of life and significantly extending survival. These immunotherapies include immune checkpoint inhibitors and may be manufactured by known methods or commercially available products. Examples of immunotherapies include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-CD 47, anti-KIR, anti-LAG 3, anti-CD 137, anti-OX 40, anti-CD 276, anti-CD 27, anti-GITR, anti-TIM 3, anti-41 BB, anti-CD 226, anti-CD 40, anti-CD 70, anti-ICOS, anti-CD 40L, anti-BTLA, anti-TCR, and anti-TIGIT antibodies. In addition, examples of immunotherapy include, but are not limited to, divarumab (durvalumab) Abilizumab (atezolizumab +)>) Avelumab (avelumab->) Parbolizumab (pembrolizumab +)>) Nawuzumab (nivolumab->) Alpha CD47, cimip Li Shan anti (cemiplimab +) >) Mo Luoli mab (magrolimab (Hu 5F 9-G4)) and ipilimumab (ipilimumab>)。

In one embodiment, the immunotherapy may comprise at least any one selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-CD 47, anti-KIR, anti-LAG 3, anti-CD 137, anti-OX 40, anti-CD 276, anti-CD 27, anti-GITR, anti-TIM 3, anti-41 BB, anti-CD 226, anti-CD 40, anti-CD 70, anti-ICOS, anti-CD 40L, anti-BTLA, anti-TCR, and anti-TIGIT antibodies. In one example, the immunotherapy may include at least one selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4 and anti-CD 47 antibodies.

In another embodiment, the immunotherapy may comprise at least any one selected from the group consisting of: devaluzumab (durvalumab)) Abilizumab (atezolizumab +)>) Parbolizumab (pembrolizumab +)>) Nawuzumab (nivolumab->) And ipilimumab (ipilimumab->)。

Method for preventing or treating cancer

According to another aspect of the present disclosure, there is provided a method of preventing or treating cancer, ameliorating or reducing the severity of at least one symptom or sign of cancer, inhibiting metastasis or inhibiting cancer growth using an anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure. As used herein, "preventing or treating cancer" may include inhibiting proliferation, survival, metastasis, recurrence, or resistance to therapy of the cancer. Such methods may comprise administering an anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure to an individual in need of prevention or treatment of cancer.

As used herein, the term "cancer" refers to a physiological condition in a mammal that is generally characterized by uncontrolled cell growth. Depending on the site of occurrence, cancers to be prevented or treated in the present disclosure may include colorectal cancer, small intestine cancer, rectal cancer, colon cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, tongue cancer, pharynx cancer, larynx cancer, esophagus cancer, cervical cancer, uterine cancer, fallopian tube cancer, ovarian cancer, brain cancer, head and neck cancer, lung cancer, lymphoid cancer, gall bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer (or melanoma), breast cancer, stomach cancer, bone cancer, blood cancer, and the like. However, any cancer may be included as long as it expresses CD300c protein on the surface of cancer cells. In one embodiment, the cancer may include at least any one selected from the group consisting of: colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer. In another embodiment, the cancer may be a solid cancer.

In one embodiment, the method may further comprise determining the expression level of the CD300c protein based on a biological sample or data of the individual prior to administering the anti-CD 300c antibody or antigen-binding fragment thereof.

In addition, the method may include determining that the individual is suitable for treatment with the anti-CD 300c antibody or antigen-binding fragment thereof if the expression level of the CD300c protein determined with the biological sample or data of the individual is above a predetermined level. In particular, the method may comprise determining that the individual is suitable for treatment with an anti-CD 300c antibody or antigen-binding fragment thereof, if the expression level of CD300c protein determined using a biological sample or data of the individual is statistically significantly higher (e.g., at least 10% higher) than a control (e.g., the expression level of a normal person not suffering from cancer or the average expression level of a cancer patient). However, the differences in the expression levels of CD300c protein as described above are merely exemplary. The difference may be, but is not limited to, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. Preferably, the control may be the average expression level in an allogeneic cancer patient.

In one embodiment of the present disclosure, a comparison of total survival dependent on CD300c expression levels was made using renal cancer (530 patients), pancreatic cancer (177 patients), and liver cancer (370 patients) patient data obtained from a cancer genomic profile (TCGA) database. As a result, cancer patients with higher CD300c expression levels have a shorter overall survival relative to the average CD300c expression level for each cancer type compared to cancer patients with lower CD300c expression levels. Thus, it may be desirable to consider the expression level of CD300c protein in an individual to increase the therapeutic response rate of anti-CD 300c antibodies in the individual.

In another embodiment, the method may further comprise administering one or more immunotherapies. In the case where (i) an anti-CD 300c antibody or antigen-binding fragment thereof is used in combination with (ii) one or more immunotherapies, (i) and (ii) may be administered simultaneously or sequentially.

"sequential administration" refers to administration of one component first, and administration of the other component immediately after the first administration or at predetermined intervals, wherein the components may be administered in any order. That is, the anti-CD 300c antibody or antigen-binding fragment thereof may be administered first, and one or more immunotherapies may be administered immediately after the first administration or at predetermined intervals, or vice versa. Furthermore, one or more of the immunotherapies may be administered first, followed by administration of an anti-CD 300c antibody or antigen binding fragment thereof, followed by administration of another of the one or more immunotherapies.

In another embodiment, an anti-CD 300c antibody or antigen-binding fragment thereof may be administered in combination with two or more immunotherapies. In one example, the highest cancer cell proliferation inhibition is observed where an anti-CD 300c antibody or antigen-binding fragment thereof is used in combination with two immunotherapies (e.g., an anti-PD-L1 antibody and an anti-PD-1 antibody, or an anti-PD-1 antibody and an anti-CTLA-4 antibody).

Each of the antibodies or antigen binding fragments thereof according to the present disclosure and optionally one or more additional immunotherapies may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. The method of administering these ingredients to an individual may vary depending on the purpose of administration, the site of the disease, the condition of the individual, and the like. The route of administration may be oral, parenteral, inhalation, in situ, or topical (e.g., intralesional administration). For example, parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intraarterial, intramuscular, rectal, vaginal, intraarticular, intraprostatic, intranasal, intraocular, intravesical, intrathecal, or intraventricular administration (e.g., intraventricular administration). Furthermore, when used in combination, the anti-CD 300c antibody and the additional immunotherapy may be administered by the same route or may be administered by different routes.

In this method, the effective amount of each of the anti-CD 300c antibodies or antigen-binding fragments thereof and optionally one or more additional cancer therapies according to the present disclosure may vary depending on the age, sex, and weight of the individual (patient). Typically, administration may be in an amount of about 0.01mg to about 100mg or about 5mg to about 50mg per kg body weight. The amount may be administered in divided doses once a day or several times a day. However, depending on the route and time of administration, severity of the disease, sex, weight, age, etc., the effective amount may be increased or decreased. Accordingly, the scope of the present disclosure is not limited thereto.

Methods according to the present disclosure may include pre-identifying the expression level of CD300c protein from the individual. Based on the expression level, it can be determined whether an anti-CD 300c antibody or antigen-binding fragment thereof is administered.

In another embodiment, a method according to the present disclosure may comprise measuring a change in the expression level of a particular marker following administration of an individual anti-CD 300c antibody or antigen-binding fragment thereof to select for additional (at least one) immunotherapy suitable for use in combination with the anti-CD 300c antibody or antigen-binding fragment thereof.

In particular, the method may further comprise using biological samples or data from an individual who received an anti-CD 300c antibody or antigen binding fragment thereof to determine the expression level of one or more markers selected from the group consisting of:

bst2, cd40, cd70, cd86, ccl8, xcl, ccr7, cd80, cd206, msr1, arg1, vegfa, pdgfrb, col a1, hif1a, vcam1, icam1, gzma, gzmb, icos, cd69, ifng, tnf, cd d1, cd1d2, cd38, cxcr6, xcr1, tbx21, stat1, stat4, cxcr3, IL-12b, IL-4, IL-6, IL-13, PD-1, PD-L1, CTLA-4, lag3, tim3, ox40, gitr, hvem, CD27, CD28, cma1, timd4, bcl6, cxcl5, and Ccl21a.

See table 2 for description of markers.

TABLE 2

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In another embodiment, the method may further comprise selecting an additional immunotherapy based on the determined marker expression level. Herein, markers may include, but are not limited to, PD-1, PD-L1, CTLA-4, lag3, tim3, icos, ox40, gitr, hvem, CD27 and CD28. In another embodiment, the marker may comprise at least one selected from the group consisting of: PD-1, PD-L1, CTLA-4, lag3, tim3, icos, ox40, gitr, hvem, CD27 and CD28. Preferably, the marker may comprise at least one selected from the group consisting of: PD-1, PD-L1, CTLA-4, lag3 and Tim3. Further, the marker may include at least one selected from the group consisting of: icos, ox40, gitr, hvem, CD27 and CD28.

The changes in the expression levels of these markers refer to changes in tumor/immune-related markers that affect tumor suppression observed in the case of administration of an anti-CD 300c antibody or antigen binding fragment thereof of the present disclosure to an individual. For example, changes in marker expression levels may include changes in the expression patterns of protein markers or immune checkpoint protein markers associated with immune cell (e.g., dendritic cell, macrophage, T cell, NKT cell) activity, tumor Microenvironment (TME) protein markers that affect tumor proliferation, and markers associated with Th1 and Th2 responses. For specific examples of markers, reference is made to the description shown above. By these changes in expression patterns, the likelihood that the patient will respond to the drug can be predicted, or a cancer therapy can be selected that maximizes the anti-cancer effect, including another immune checkpoint inhibitor. Furthermore, the use of markers makes it possible to determine whether a patient can be treated with antibodies. In addition, the therapeutic effect of the drug may be monitored. In addition, information may be provided about the method of treatment, including dosage of the drug, use, combination therapy, and the like.

According to one embodiment of the present disclosure, an anti-CD 300c antibody or antigen binding fragment thereof of the present disclosure is identified as being conjugated to another immune checkpoint inhibitor (anti-PD-L1 antibody, cerulomumabanti-PD-1 antibody Nawuzumabanti-PD-1 antibody palbociclizumab +.>At least one of the anti-CTLA-4 antibody and the anti-CD 47 antibody (αcd 47) in combination exhibits an enhanced anti-tumor effect, and the other immune checkpoint inhibitor is selected by a change in the level of expression of the marker in the individual observed after administration of the anti-CD 300c antibody or antigen-binding fragment thereof.

In yet another embodiment, the method may further comprise determining the therapeutic responsiveness of the anti-CD 300c antibody or antigen binding fragment thereof based on the determined marker expression level. Markers may include, but are not limited to vegfa, pdgfrb, col a1, hif1a, bst2, CCL8, xcl1, CCR7, CD80, tbx21, stat1, stat4, ifng, cxcr3, IL-6, gzma, icos, cd69, cd1d1, cd38, cxcr6, ox40, gitr, CD27, and CD28. Preferably, the marker may comprise at least one selected from the group consisting of: vegfa, pdgfrb, col4a1, hif1a, bst2, CCL8, xcl1, CCR7, CD80, tbx21, stat1, stat4, ifng, cxcr3, IL-6, gzma, icos, cd69, cd1d1, cd38 and Cxcr6. In addition, the marker may include at least one selected from the group consisting of: vegfa, pdgfrb, col4a1 and Hif1a. In addition, the marker may include at least one selected from the group consisting of: bst2, CCL8 and Xcl1. In yet another embodiment, the marker may comprise CCR7, CD80, or a combination thereof. In addition, the marker may include at least one selected from the group consisting of: tbx21, stat1, stat4, ifng, cxcr3 and IL-6.

In yet another embodiment, the method may further comprise determining that the therapeutic responsiveness of the anti-CD 300c antibody or antigen-binding fragment thereof is good or excellent in the event that the expression level of at least one marker is reduced compared to an individual not receiving the anti-CD 300c antibody or antigen-binding fragment thereof. For example, according to the method, it can be determined that the therapeutic responsiveness of the anti-CD 300c antibody or antigen-binding fragment thereof is good or excellent in the case that the expression level of at least one marker selected from vegfa, pdgfrb, col a1, hif1a and IL-6 is reduced compared to an individual who does not receive the anti-CD 300c antibody or antigen-binding fragment thereof. Here, a decrease in expression level means a statistically significant decrease. The rate of decrease in expression level may include, but is not limited to, about 10% or greater, about 20% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, and about 100% or greater.

In addition, the method may further comprise determining that the therapeutic responsiveness of the anti-CD 300c antibody or antigen-binding fragment thereof is good or excellent in the event that the expression level of at least one marker is increased compared to an individual not receiving the anti-CD 300c antibody or antigen-binding fragment thereof. For example, according to the method, therapeutic responsiveness of an anti-CD 300c antibody or antigen-binding fragment thereof can be determined to be good or excellent in the event that the expression level of at least one marker selected from Bst2, CCL8, xcl1, CCR7, CD80, tbx21, stat1, stat4, ifng, cxcr3, gzma, icos, cd69, CD1d1, CD38, cxcr6, ox40, gitr, CD27, and CD28 is increased compared to an individual not receiving the anti-CD 300c antibody or antigen-binding fragment thereof. Here, an increase in expression level means a statistically significant increase. The rate of increase in expression level may include, but is not limited to, about 10% or greater, about 20% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, and about 100% or greater.

Pharmaceutical composition

According to yet another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating cancer, the composition comprising an anti-CD 300 antibody or antigen-binding fragment thereof as an active ingredient.

The anti-CD 300c antibody or antigen-binding fragment thereof may be included in the composition in a prophylactically or therapeutically effective amount. The pharmaceutical composition may be administered to an individual to inhibit proliferation, survival, metastasis, recurrence, or resistance to therapy of cancer.

In one embodiment, the pharmaceutical composition may further comprise at least one additional immunotherapy. In particular, the anti-CD 300c antibody or antigen-binding fragment thereof and optionally the additional immunotherapy may be included in the same composition or may be included in separate compositions. When included in separate compositions, the anti-CD 300c antibody or antigen-binding fragment thereof and the additional immunotherapy may be formulated separately, and may be administered simultaneously or sequentially.

For preparing the pharmaceutical compositions of the present disclosure, the antibodies or antigen-binding fragments thereof and optionally additional immunotherapy may be admixed with pharmaceutically acceptable carriers and/or excipients. The pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution. For example, please refer to Remington's Pharmaceutical Sciences and US Pharmacopeia: national Formulary, mack Publishing Company, easton, pa. (1984).

Acceptable carriers and/or excipients (including stabilizers) are non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers (e.g., phosphate, citrate, and other organic acids); antioxidants (e.g., ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol), alkyl parahydroxybenzoates (e.g., methyl parahydroxybenzoate and propyl parahydroxybenzoate; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (about less than about 10 residues) polypeptides, proteins (e.g., serum albumin, gelatin, and immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, and lysine), monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, and dextrins), chelators (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, and sorbitol), salt-forming counter ionsSub (e.g., sodium); metal complexes (e.g., zn-protein complexes); and/or a nonionic surfactant (e.g., TWEEN ^TM 、PLURONICS ^TM And polyethylene glycol (PEG)).

The pharmaceutical compositions of the present disclosure may be formulated into suitable forms known in the art according to the route of administration.

As used herein, the term "prophylactically or therapeutically effective amount" or "effective amount" refers to an amount of an active ingredient in a composition effective to prevent or treat cancer in an individual. The amount is also sufficient to prevent or treat cancer at a reasonable benefit/risk ratio applicable to medical treatment, and without causing side effects. The level of effective amount can be determined based on the patient's health, type of disease, severity of disease, activity of the drug, sensitivity to the drug, method of administration, frequency of administration, route of administration and rate of excretion, duration of treatment, drug combination therewith or concurrent therewith, and other factors well known in the medical arts. Here, taking all the above factors into consideration, it is important to administer the minimum amount that allows maximum effect to be achieved with minimal or no side effects, as can be readily determined by one skilled in the art.

In particular, the effective amount of each active ingredient in the pharmaceutical compositions of the present disclosure may vary depending on the age, sex and weight of the individual (patient). Typically, about 0.01mg to about 100mg or about 5mg to about 50mg per kg body weight may be administered once a day or several times a day in divided doses. However, the scope of the present disclosure is not limited thereto, as the effective amount may be increased or decreased according to the administration route and duration, severity of disease, sex, weight, age, etc.

Kit for preventing or treating cancer

According to another aspect of the present disclosure, there is provided a kit for preventing or treating cancer, the kit comprising a composition comprising an anti-CD 300c antibody or antigen-binding fragment thereof according to the present disclosure, and instructions for using the antibody or antigen-binding fragment thereof. Herein, the composition may contain a prophylactically or therapeutically effective amount of an anti-CD 300c antibody or antigen-binding fragment thereof.

In one embodiment, the instructions may include instructions directing the use of the antibody or antigen binding fragment thereof in combination with at least one additional cancer therapy.

In one embodiment, the instructions may include instructions containing instructions on how to take or administer the active ingredient. For example, the instructions may include instructions for measuring the level of CD300c protein expression using a biological sample or data obtained from an individual prior to administration of the antibody or antigen binding fragment thereof. Optionally, the apparatus or device required to administer the active ingredient may be included in a kit.

Dosage of

Each effective or effective non-toxic amount of an anti-CD 300c antibody or antigen binding fragment thereof of the present disclosure and optionally additional immunotherapy may be determined by routine experimentation. For example, the therapeutically active amount of an antibody or immunotherapy may vary depending on, for example, the following factors: the stage of the disease, the severity of the disease, the age, sex, medical complications and weight of the individual, and the ability of the component to elicit a desired response in the individual, as well as the dosage of the concurrent cancer therapy. The respective dosages and dosing regimen of the anti-CD 300c antibody or antigen-binding fragment thereof or the additional immunotherapy may be adjusted to provide an optimal therapeutic response. For example, several doses may be administered daily, weekly, biweekly, tricyclically, weekly, etc., and/or the dose may be proportionally reduced or increased depending on the urgency of the treatment.

Methods and kits for providing information for preventing or treating cancer

According to another aspect of the present disclosure, there is provided a method of providing information for preventing or treating cancer, comprising determining the expression level of CD300c protein using a biological sample or data obtained from an individual in need of prevention or treatment of cancer. The method may also be used for administration of a pre-screening anti-CD 300c antibody or antigen binding fragment thereof.

In one embodiment, determining the expression level of the CD300c protein (marker) may comprise using a molecule, such as an antibody, substrate, ligand or cofactor, that specifically binds to the CD300c protein, or a molecule, such as a primer pair or probe, that specifically binds to the mRNA of CD300 c. Methods for determining expression levels using these agents are well known in the art and include those described above. In one embodiment, the method may comprise determining that the individual is suitable for treatment with an anti-CD 300c antibody or antigen-binding fragment thereof, if the expression level of CD300c protein determined using a biological sample or data of the individual is above a predetermined level. In particular, the method may comprise determining that the individual is suitable for treatment with an anti-CD 300c antibody or antigen-binding fragment thereof, if the expression level of CD300c protein determined using a biological sample or data of the individual is statistically significantly higher (e.g., at least 10% higher) than a control (e.g., the expression level of a normal person not suffering from cancer or the average expression level of a cancer patient). However, the differences in the expression levels of CD300c protein as described above are merely exemplary. The difference may be, but is not limited to, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. Preferably, the control may be the average expression level in an allogeneic cancer patient.

In another embodiment, the information for preventing or treating cancer may include, but is not limited to, information about any one or more of therapeutic responsiveness of a therapeutic agent associated with CD300c protein (e.g., an anti-CD 300c antibody or antigen binding fragment thereof), selection of a therapeutic agent, selection of an individual to be treated, prognosis of an individual, and survival of an individual. Preferably, the information for preventing or treating cancer may include the cancer therapeutic responsiveness of the anti-CD 300c antibody or antigen binding fragment thereof, the survival of the individual, or both.

According to yet another aspect of the present disclosure, there is provided a kit for providing information for preventing or treating cancer, the kit comprising a substance for measuring the expression level of CD300c protein using biological samples or data obtained from an individual in need of prevention or treatment of cancer. Kits may include one or more other component compositions, solutions, or devices for performing the assay. The kit may be a kit for measuring the expression level of a protein marker, such as an enzyme-linked immunosorbent assay (ELISA) kit. The kit may contain other reagents necessary for the immunological detection of antibodies known in the art. The kit may further comprise a pharmaceutical composition comprising as an active ingredient an anti-CD 300c antibody or antigen-binding fragment thereof.

Detailed Description

Hereinafter, the present disclosure will be described in more detail by way of examples. However, the following examples are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited thereto.

Examples

I. Preparation of anti-CD 300c monoclonal antibody

Example 1 preparation of anti-CD 300c monoclonal antibody

EXAMPLE 1.1 construction of anti-CD 300c monoclonal antibody library

To select anti-CD 300c monoclonal antibodies, biopanning was performed using a lambda phage library, a kappa phage library, a VH3VL1 phage library, and an opat phage library. More specifically, CD300c antigen was added to the immune tube at a concentration of 5. Mu.g/mL, and the reaction was allowed to proceed for 1 hour to allow the antigen to adsorb on the surface of the immune tube. 3% skim milk was added to suppress non-specific reactions. Then, 10 dispersed in 3% skim milk ¹² PFU antibody phage libraries were added to each immune tube for antigen binding. Washing 3 times with Tris buffered saline-Tween 20 (TBST) solution to remove non-specifically bound phage, followed by elution of the specifically bound CD300c antigen with 100mM triethylamine solutionSingle chain variable fragment (scFv) phage antibodies. The eluted phage were neutralized with 1.0M Tris-HCl buffer (pH 7.8). The resulting product was then treated with E.coli ER2537 and the infection was allowed to proceed for 1 hour at 37 ℃. The infected E.coli was spread on LB agar medium containing carbenicillin and cultured at 37℃for 16 hours. The E.coli colonies formed were then suspended in 3mL of Super Broth (SB) -carbenicillin culture. Some suspensions were added to 15% glycerol, stored at-80℃for later use, the remainder was re-inoculated into SB-carbenicillin-2% glucose solution, cultured at 37℃and the resulting culture was centrifuged, and biopanning was repeated 3 times again with the supernatant containing phage particles to obtain and concentrate antigen-specific antibodies.

After repeating biopanning 3 times, E.coli containing the antibody gene was spread on LB agar medium containing carbenicillin and cultured at 37℃for 16 hours. The E.coli colonies formed were inoculated again into SB-carbenicillin-2% glucose solution and incubated at 37℃until the absorbance (OD 600 nm) reached 0.5. Then, IPTG was added and the culture was continued at 30℃for 16 hours. Periplasmic extraction is then performed. According to the results, a library of antibodies that specifically bind to the CD300c antigen was initially obtained.

Example 1.2 selection of anti-CD 300c monoclonal antibodies

To select an anti-CD 300c monoclonal antibody that specifically binds to CD300c antigen with high binding affinity, ELISA was performed using a library pool (library pool) obtained in the same manner as example 1.1. More specifically, each of the CD300c antigen and CD300a antigen in a coating buffer (0.1M sodium carbonate, pH 9.0) was dispensed onto an ELISA plate at a concentration of 5 μg/mL per well, and then the reaction was allowed to proceed at room temperature for 3 hours for antigen binding onto the plate. Wash 3 times with phosphate buffered saline-Tween 20 (PBST) to remove unbound antigen, then add 350 μl of PBST supplemented with 2% Bovine Serum Albumin (BSA) to each well. The reaction was allowed to proceed for 1 hour at room temperature and washed again with PBST. Then, 25. Mu.g of a scFv-containing periplasmic extract obtained in the same manner as in example 1.1 was added thereto, and the reaction was allowed to proceed at room temperature for 1 hour for antigen binding. After 1 hour, the unbound scFv was removed by washing 3 times with PBST, and then 4. Mu.g/mL of the antibody for detection was added. The reaction was allowed to proceed for an additional 1 hour at room temperature. Subsequently, the unbound detection antibody was removed using PBST. Then, HRP-conjugated anti-rabbit IgG was added and the reaction was allowed to proceed at room temperature for 1 hour. Unbound antibody was removed again using PBST. Subsequently, 3', 5' -Tetramethylbenzidine (TMB) solution was added, and the reaction was allowed to proceed for 10 minutes to perform color development. Then, the color development was stopped by adding 2N sulfuric acid solution, and absorbance was measured at 450nm to identify an antibody that specifically binds to CD300c antigen.

EXAMPLE 1.3 identification of anti-CD 300c monoclonal antibody sequences

The nucleotide sequence of the anti-CD 300c monoclonal antibody selected using the same method as example 1.2 was identified. More specifically, for each antibody clone selected, plasmid DNA was extracted therefrom using a plasmid miniprep kit (plasmid miniprep kit). Then, DNA sequencing was performed to analyze Complementarity Determining Region (CDR) sequences. As a result, 25 kinds of anti-CD 300c monoclonal antibodies having different amino acid sequences were obtained. The heavy and light chain variable regions of these 25 anti-CD 300c monoclonal antibodies are shown in tables 3 and 4 below.

TABLE 3 Table 3

TABLE 4 Table 4

In each of the figures mentioned in tables 3 and 4 above, the CDR regions (CDR 1, CDR2, and CDR 3) are underlined and appear sequentially (i.e., CDR1 appears, followed by CDR2, followed by CDR 3). In addition, CDR regions included in each figure are represented by SEQ ID nos:

TABLE 5

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As described above, 25 anti-CD 300c monoclonal antibodies were identified that specifically bind to CD300c antigen with high binding affinity and are useful for preventing or treating cancer.

EXAMPLE 1.4 preparation and purification of anti-CD 300c monoclonal antibodies

Using each nucleotide sequence of the anti-CD 300c monoclonal antibody identified in example 1.3, an expression vector capable of expressing each antibody was prepared in which a heavy chain and a light chain were inserted, respectively. More specifically, expression vectors were prepared by inserting genes into pciw3.3 vectors using the analyzed CDR sequences so that the vectors can express heavy and light chains, respectively. The prepared heavy and light chain expression vectors were mixed with Polyethylenimine (PEI) in a mass ratio of 1:1 and transfected into 293T cells to induce antibody expression. Then, on day 8, the culture was centrifuged to remove cells. The resulting culture was obtained. The resulting culture was filtered, then treated with 0.1M NaH ₂ PO ₄ And 0.1M Na ₂ HO ₄ Is resuspended in a mixed solution (pH 7.0). The resuspended solution was purified by affinity chromatography using protein a magnetic beads (GE Healthcare), and finally eluted using elution buffer (thermoshier).

To identify the antibodies produced, a reducing sample buffer and a non-reducing sample buffer were each added to 5 μg of purified antibody and run using a preformed SDS-PAGE (Invitrogen). The protein was then stained using coomassie blue. The results under non-reducing conditions are shown in fig. 4, and the results under reducing conditions are shown in fig. 5.

As shown in fig. 4 and 5, the generation and purification of high purity anti-CD 300c monoclonal antibodies were identified.

Expression of CD300c in cancer cells and binding of anti-CD 300c monoclonal antibodies to CD300c antigen

Experimental example 1 expression of CD300c in cancer cells and immune cells

Experimental example 1.1 identification of expression of CD300c in cancer cell lines

To evaluate whether CD300c is expressed in various cancer cells, various cancer cell lines such as MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colorectal cancer cell line), a549 (human lung cancer cell line), HCT116 (human colorectal cancer cell line), MDA-MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) were cultured, and expression of CD300c was evaluated at mRNA and protein levels. In addition, immune cells THP-1 cells (human monocyte cell line) were also evaluated. Here, HEK293T (normal cell line) was used as a control.

Simultaneously, protein expression was identified by Western blot and flow cytometry (FACS) of fluorescently labeled cells. Specifically, each cultured cell line was fixed with 4% formaldehyde and then blocked with 5% normal bovine serum albumin. Then, staining was performed with 0.5. Mu.g of eFluor 660-labeled anti-CD 300c antibody (Invitrogen). Subsequently, flow cytometry (FACS) was used to identify fluorescently labeled cells.

The results identify that CD300c antigen is expressed at mRNA and protein levels in various cancer cells such as colorectal cancer, lung cancer, breast cancer, and the like. In addition, as shown in fig. 6, according to the analysis using flow cytometry (FACS), it was identified that CD300c significantly highly expressed was observed in the human lung cancer cell line (a 549) and the human monocyte line (THP-1) compared to the normal cell line (HEK 293T).

Experimental example 1.2 identification of expression of CD300c in cancer tissues and immune cells (I)

To identify whether CD300c is expressed in cancer tissue of a patient, a tissue microarray was performed as follows. The tissues of colorectal cancer patients were fixed with formalin and embedded in paraffin blocks. The paraffin block was then cut into sections of 2.0mm diameter and 3 to 5 μm thickness using a microtome. The sections were then attached to slides in a direction and dried. Cancerous tissues were stained with H & E staining and then treated with anti-CD 300c antibody (Invitrogen) at 1:500 to stain CD300c. The results are shown in fig. 7a, identifying CD300c expression in colorectal cancer tissue of patients.

To identify whether CD300c is expressed not only in colorectal cancer tissue of a patient, but also in immune cells within cancer tissue, 2 x 10 is injected subcutaneously ⁵ CT26 cells were transplanted into 8-week-old BALB/c mice. On day 25 post tumor implantation (D25), mice were sacrificed and tumor tissue was collected from 6 control mice that did not receive anti-CD 300c antibody. The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, single cell suspensions were blocked with CD16/32 antibody (from Invitrogen) and cells were stained with staining solution (from Invitrogen) to check cell viability, and cells were stained with antibodies to the total macrophage markers F4/80 (from Abcam), CD11b (from Abcam), CD11c (from Abcam), CD3 (from Abcam), CD4 (from thermosusher) and CD8 (from thermosusher) and anti-CD 300c antibody (from yi, state). Then, using a CytoFLEX flow cytometerThe data was read and analyzed using FlowJo software.

As a result, as shown in FIG. 7b, it was identified that immune cells expressing CD300c were present in the tumor tissue of the mouse. These are immune cells that express both CD11b and CD11c markers, examples of which include dendritic cells and macrophages. From these results, CD300c expression in both cancer tissues and immune cells was identified.

Experimental example 1.3 identification of expression of CD300c in cancer tissues and immune cells (II)

To identify whether CD300c is expressed in human immune tissue and cancer cells, a tissue microarray was performed as follows. Each of normal tonsil tissue and colorectal cancer patient tissue was formalin fixed and embedded in paraffin blocks. Subsequently, in normal tonsil tissue and tissue of colorectal cancer patients, the locations where the tissue microarray was performed were determined, and then each paraffin block was cut into sections having a diameter of 2.0mm and a thickness of 3 to 5 μm using a microtome. The sections were then attached to slides in a direction and dried. Cancerous tissues were stained with H & E staining and then treated with anti-CD 300c antibody (Invitrogen) at 1:500 to stain CD300 c.

As a result, as shown in fig. 8a and 8b, CD300c was identified to be expressed in normal tonsil tissue (fig. 8 a), which is an immune tissue, and colon cancer tissue of the patient (fig. 8 b). Since a large number of immune cells such as T cells and monocytes are distributed in tonsils, the expression of CD300c in tonsil tissue means that CD300c is expressed in immune cells. CD300c expression in tissues of colorectal cancer patients was identified as in experimental example 1.2; however, this experimental example is interesting because expression of CD300c was observed in the tissues of all 4 colorectal cancer patients, indicating that CD300c is expressed in a large number of colon cancer tissues.

Experimental example 2 identification of the anti-CD 300c monoclonal antibody recognizing the CD300c antigen and binding to the CD300c antigen Experimental example 2.1 determination of the antigen binding affinity of the anti-CD 300c monoclonal antibody

To identify the antigen binding capacity of the anti-CD 300c monoclonal antibody produced in example 1, a binding ELISA was performed. Specifically, each of the CD300c antigen (11832-H08H, sense-of-makino) or CD300a antigen (12449-H08H, sense-of-makino) in a coating buffer (0.1M sodium carbonate, ph 9.0) was dispensed onto an ELISA plate at a concentration of 8 μg/mL per well, and then the reaction was allowed to proceed at room temperature for 3 hours so that the antigen could bind to the plate. Wash 3 times with phosphate buffered saline-Tween 20 (PBST) to remove unbound antigen, then add 300 μl of PBST supplemented with 5% Bovine Serum Albumin (BSA) to each well. The reaction was allowed to proceed for 1 hour at room temperature and washed again with PBST. anti-CD 300c monoclonal antibodies were then diluted in quadruplicate and added thereto. The reaction was allowed to proceed at room temperature for 1 hour for antigen binding. After 1 hour, the unbound anti-CD 300c monoclonal antibody was removed by washing 3 times with PBST, followed by the addition of 4. Mu.g/mL of detection antibody (HRP conjugated anti-Fc IgG). The reaction was allowed to proceed for an additional 1 hour at room temperature. Subsequently, unbound detection antibody was removed using PBST, and then TMB solution was added. The reaction was allowed to proceed for 10 minutes to develop color. Then, the color development was stopped by adding 2N sulfuric acid solution, and absorbance was measured at 450nm to identify an antibody that specifically binds to CD300c antigen. The results are shown in table 6 and fig. 9.

TABLE 6

CB301 antibody	EC50(μg/mL)
		CK1	0.056
CK2	0.033
		CK3	0.793
CL4	0.031
		CL5	0.032
CL6	0.148
		CL7	0.047
CL8	49.7
		CL9	0.094
CL10	0.039
		SK11	0.052
SK12	0.067
		SK13	0.044
SK14	0.065
		SK15	14.74
SK16	2.42
		SK17	0.054
SL18	0.17

As shown in table 6, as a result of measuring EC50 (the pharmaceutically effective concentration that causes 50% of the maximum reaction) values of the anti-CD 300c monoclonal antibody, it was identified that all of the remaining 14 clones except for 4 clones (CK 3, CL8, SK15, SK 16) exhibited high binding affinity of 0.2 μg/mL or less. Furthermore, as shown in fig. 9, the anti-CD 300c monoclonal antibodies of the present disclosure were found to bind to CD300c antigen with high binding affinity, even in the sigmoid curve of binding ELISA results.

Experimental example 2.2 identification of cell antigen recognized by anti-CD 300c monoclonal antibody

To identify whether the anti-CD 300c monoclonal antibody (CL 7) recognizes a cellular antigen, FACS binding was performed.

CD300c was overexpressed in 293T cells (ATCC) and THP-1 cells (ATCC) and each cell was then incubated at 2X 10 ⁵ Individual cells/tubes were dispensed into individual microcentrifuge tubes. Then, the cells were allowed to react with an anti-CD 300c monoclonal antibody in CO ₂ The monoclonal antibodies were serially diluted 3-fold from 10. Mu.g/mL in the incubator for 30 minutes. The FACS buffer was washed twice. Then, in CO ₂ Cells were reacted with FITC-conjugated anti-human IgG (h+l) in FACS buffer for 30 min, diluted 1:100. The FACS buffer was washed twice. Next, FITC signals were measured with a CytoFLEX instrument manufactured by Beckman Coulter, inc., and MFI values were obtained using the cytexert program. Using the MFI values obtained, an S-shaped curve was drawn by the Sigmaplot program to calculate EC50 (effective concentration of drug that elicited 50% of the maximum response) values. As a result, 293T cells were obtained with an EC50 of 2.7nM and THP-1 cells were obtained with an EC50 of 2.6nM.

As shown in fig. 10, in the S-shaped curve of FACS binding results, the anti-CD 300c monoclonal antibody generated in example 1 binds with strong binding affinity to THP-1 and CD300c overexpressed on the surface of 293T cells. Thus, anti-CD 300c monoclonal antibodies were identified that bind CD300c in an antigen-specific manner.

Experimental example 2.3. Determination of binding affinity of anti-CD 300c monoclonal antibodies to CD300c antigen (I): binding ELISA

CD300c antigen (250. Mu.g/mL) was diluted to a concentration of 800ng/mL in coating buffer (0.1M sodium carbonate, pH 9.0), placed in 96-well microplates at 100. Mu.L/well, and incubated overnight at 4 ℃. The next day, wash three times with 200 μl PBST. Then, 200. Mu.L of a blocking buffer (5% skim milk) was added thereto, and blocked at room temperature for 1 hour. anti-CD 300c monoclonal antibody CL7 was diluted to 200. Mu.g/mL in PBS by using Nanodrop (product name: nanodrop One/One) ^c Manufactured by thermosusher) to check its concentration. CL7 was then diluted in quadruplicate starting from 10 μg/mL with PBS, 100 μl each was added, and the reaction was allowed to proceed for 1 hour at room temperature. After the reaction, the reaction mixture was washed three times with 200. Mu.L of PBST. The secondary antibody (HRP conjugated anti-Fc IgG) was diluted 1:10,000 in blocking buffer, 100. Mu.L was added, and the reaction was allowed to proceed for 1 hour at room temperature. Then, the cells were washed three times with 200. Mu.L of PBST. Subsequently, TMB and hydrogen peroxide were mixed at 1:1 and added at 100. Mu.L/well, and then the reaction was allowed to proceed at room temperature for 7-9 minutes. Then, 50. Mu.L of 1N sulfuric acid was added to terminate the color development, and measurement was performed at 450nm using a microplate reader (product name: varioskan LUX) to obtain a binding affinity result.

As a result, as shown in fig. 11, it was identified that the anti-CD 300c monoclonal antibody bound to CD300c in a concentration-dependent manner, indicating that the anti-CD 300c monoclonal antibody has excellent binding affinity and specificity for the antigen CD300 c.

Experimental example 2.4. Determination of binding affinity of anti-CD 300c monoclonal antibody to CD300c antigen (II): surface plasmon resonance

To identify the binding affinity between antigen CD300c and anti-CD 300c monoclonal antibody CL7, a surface plasmon resonance experiment was performed.

To immobilize CD300c on CMS chip, 5 μg/mL of CD300c was diluted in 10mM acetate buffer (pH 5.5). Then, the flow rates were all set equal to 10mL/min, and the target RUs were set to 300 RUs, respectively. Activation was performed with a mixture of 0.2M EDC and 0.05M NHS and blocked with 1M ethanolamine. Immobilization was performed so that the final RU of CD300c could become 399.2RU. CL7 was then diluted in PBSP to concentrations of 0, 0.195, 0.39, 0.78, 1.56, 3.125 and 6.25 μg/mL, respectively. The kinetics/affinity test is performed with a binding time of 240 seconds, a dissociation time of 900 seconds and a flow rate of 30 μl/min. Subsequently, the surface was regenerated by flowing with 50mM NaOH at a rate of 30. Mu.L/min for 30 seconds.

The results are shown in FIG. 12, where the analytical KD value is 5.199E-10M, and the binding affinity of the anti-CD 300c monoclonal antibody is identified as 0.52nM, at a sub-nanomolar level. This means that the anti-CD 300c monoclonal antibodies exhibit high binding affinity for the antigen.

Experimental example 2.5 identification of binding specificity of anti-CD 300c monoclonal antibodies to CD300c antigen (I)

Binding ELISA was performed to identify that the anti-CD 300c monoclonal antibody CL7 specifically binds only CD300c and not other B7 family proteins. More specifically, each of the CD300c antigen, CD300a antigen, or seven B7 family protein antigens (PD-L1 [ B7-H1] (sense state), ICOS ligand [ B7-H2] (sense state), CD276[ B7-H3] (sense state), B7-H4 (sense state), CD80[ B7-1] (sense state), CD86[ B7-2] (sense state), CD273[ PD-L2] (sense state)) in a coating buffer (0.1M sodium carbonate, ph 9.0) was coated on an ELISA plate at a concentration of 8 μg/mL per well, followed by overnight incubation at 2 ℃ to 8 ℃ to enable antigen binding to the plate. Unbound antigen was removed by three washes with PBST, then 300mL of blocking buffer (5% skim milk in PBST) was added to each well. Subsequently, blocking was performed at room temperature for 1 hour, and then washing was performed again with PBST. CL7 was then diluted in quadruplicate with PBS and reacted with antigen at room temperature for 1 hour to bind antigen. After 1 hour, the cells were washed three times with PBST. Then, secondary antibody (HRP-conjugated anti-Fc IgG) diluted to 4. Mu.g/mL in blocking buffer was added thereto, and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, unbound detection antibody was removed with PBST, and then TMB solution was added thereto. The reaction was allowed to proceed for 10 minutes to develop color. Then, the color development was stopped by adding 2N sulfuric acid solution, and absorbance was measured at 450nm to identify an antibody that specifically binds to CD300c antigen.

As a result, as shown in fig. 13, it was identified that the anti-CD 300c monoclonal antibody specifically recognizes only CD300c, and does not bind other similar proteins.

Experimental example 2.6 identification of binding specificity of anti-CD 300c monoclonal antibodies to CD300c antigen (II)

To identify the specificity of the anti-CD 300c monoclonal antibody CL7 for the CD300c antigen, it was further examined whether CL7 exhibited cross-reactivity with CD300a antigen known to antagonize the CD300c antigen and having a similar protein sequence thereto. More specifically, CD300a antigen (from Yinqiao Shenzhou) was treated at concentrations of 0.039. Mu.g/mL, 0.63. Mu.g/mL and 10. Mu.g/mL, and binding ELISA was performed in the same manner as in experimental example 2.1.

As a result, as shown in fig. 14, it was found that the anti-CD 300c monoclonal antibody did not bind to an antigen other than CD300c, and exhibited high binding specificity only to CD300c antigen.

Experimental example 2.7 comparison of total survival of patients with various cancers depending on CD300c expression level

The comparison of total survival dependent on CD300c expression levels was performed using data obtained from the cancer genomic profile (TCGA) database for kidney cancer (530 patients), pancreatic cancer (177 patients) and liver cancer (370 patients). First, each cancer patient is classified according to the high or low degree of CD300c expression level. The extent to which the level is high or low is determined by comparison to the average CD300c expression level for each cancer type. For renal cancer patients, 394 patients had low CD300c expression levels and 136 patients had high CD300c expression levels. For pancreatic cancer patients, 57 patients had low CD300c expression levels and 120 patients had high CD300c expression levels. Meanwhile, for liver cancer patients, 192 patients had low CD300c expression levels, and 178 patients had high CD300c expression levels. Each cancer patient was analyzed for total survival dependent on CD300c expression levels by Kaplan-Meier method. Subsequently, the survival between the patient group with high CD300c expression level and the patient group with low CD300c expression level was compared by log rank test (log-rank test).

Results as shown in fig. 15, patients with high CD300c expression levels were identified with shorter survival than patients with low CD300c expression levels, indicating significant results with P values in mind. These results indicate that expression of CD300c is highly correlated with survival of cancer patients, and that in the case where expression or activity of CD300c is inhibited, a cancer therapeutic effect or survival-increasing effect can be expected.

Anti-cancer effect of anti-CD 300c monoclonal antibodies

Experimental example 3 identification of anticancer Effect by administration of anti-CD 300c monoclonal antibody

Experimental example 3.1 identification of T cell activation

To identify whether the anti-CD 300c monoclonal antibody produced in example 1 can exhibit an anticancer effect by activating T cells, the production level of interleukin-2 (IL-2) was identified in the case of treating human T cells with the anti-CD 300c monoclonal antibody. IL-2 is an immune factor that contributes to T cell growth, proliferation and differentiation, while an increase in IL-2 production levels means T cell activation due to an increase in stimulation that induces T cell differentiation, proliferation and growth. Specifically, each of the anti-CD 3 monoclonal antibody and the anti-CD 28 monoclonal antibody was added to a 96-well plate at a concentration of 2 μg/well and fixed for 24 hours. Then, with 1X 10 ⁵ Cell/well Jurkat T cells (human T lymphocyte cell line) were co-processed with 10. Mu.g/well of anti-CD 300c monoclonal antibody. Subsequently, ELISA kit (IL-2Quantikine Kit,R was used&D Systems) measures the level of IL-2 production and then compares it to a control group that was not treated with anti-CD 300c monoclonal antibody. The results are shown in fig. 16.

As shown in FIG. 16, it was identified that the IL-2 production level was increased in the case of treating Jurkat T cells with an anti-CD 300c monoclonal antibody, which were activated by treatment with an anti-CD 3 monoclonal antibody and an anti-CD 28 monoclonal antibody. Based on these results, it was found that the anti-CD 300c monoclonal antibody was able to activate T cells, indicating that the anti-CD 300c monoclonal antibody was able to induce anticancer immunity to inhibit cancer tissue growth.

Experimental example 3.2. Identification of macrophages differentiated into M1 (I): measurement of differentiation marker (TNF-. Alpha.) production level

To identify whether the anti-CD 300c monoclonal antibody selected in example 1 promotes differentiation of monocytes to M1 macrophages, THP-1 (human monocyte lineage) was used at 1.5X10 ⁴ Cells/well were distributed onto 96-well plates and treated with 10 μg/mL anti-CD 300c monoclonal antibody and/or 100ng/mL LPS. The reaction was allowed to proceed for 48 hours and then was performed using ELISA Kit (Human TNF-. Alpha.Quantikine Kit, R &D Systems) measures the level of production of tumor necrosis factor- α (TNF- α), a differentiation marker for M1 macrophages. The results are shown in fig. 17 and 18.

As shown in fig. 17, anti-CD 300c monoclonal antibodies CL4, CL7, CL10 and SL18 were identified, which increased TNF- α production levels by about 2-fold or more over the control group (Con) treated with LPS alone.

Furthermore, as shown in fig. 18, all experimental groups treated with anti-CD 300c monoclonal antibody alone without LPS showed an increase in TNF-a production level compared to the control group treated with LPS alone (Con).

Experimental example 3.3 identification of increased ability to differentiate into M1 macrophages depending on the concentration of anti-CD 300c monoclonal antibody

To identify whether the induction of differentiation into M1 macrophages by anti-CD 300c monoclonal antibodies increased with the concentration of anti-CD 300c monoclonal antibodies, the level of TNF- α production was identified in the same manner as in example 3.2. anti-CD 300c monoclonal antibody treatment was performed at concentrations of 10. Mu.g/mL, 1. Mu.g/mL, and 0.1. Mu.g/mL. The results are shown in FIG. 19. As shown in FIG. 19, it was identified that the production level of TNF- α increased with increasing treatment concentration of anti-CD 300c monoclonal antibodies (CL 7, CL10 or SL 18).

To identify the results of further sub-divided concentrations, treatment with anti-CD 300c monoclonal antibody (CL 7) at concentrations of 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.313. Mu.g/mL, 0.157. Mu.g/mL and 0.079. Mu.g/mL was performed to identify the level of TNF- α production. The results are shown in fig. 20. As shown in FIG. 20, it was identified that the level of TNF- α production was increased in a concentration-dependent manner relative to the anti-CD 300c monoclonal antibody.

Experimental example 3.4. Identification of macrophages differentiated into M1 (II): observation of cell morphology

To identify the differentiation pattern of M1 macrophages upon treatment of monocytes with anti-CD 300c monoclonal antibody by cell morphology, THP-1 was treated with 10. Mu.g/mL of anti-CD 300c monoclonal antibody, cultured for 48 hours, and then the cell shape was observed under a microscope. The results are shown in FIG. 21.

As shown in FIG. 21, round adherent cells were identified that changed the shape of THP-1 cells from suspension cells to M1 macrophage form for the experimental group (CL 7) treated with anti-CD 300c monoclonal antibody. From these results, treatment with anti-CD 300c monoclonal antibodies was identified to promote differentiation of monocytes into M1 macrophages.

Experimental example 3.5. Re-identification of differentiated M1 macrophages is facilitated

To again identify whether the anti-CD 300c monoclonal antibody CL7 promotes differentiation of human monocytes into M1 macrophages, secretion levels of TNF- α, interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) were measured using ELISA kits. More specifically, THP-1 was used at 1.5X10 ⁴ Cells/well were dispensed onto 96-well plates and treated with 10 μg/mL of anti-CD 300c monoclonal antibody. The reaction was allowed to proceed for 48 hours, then ELISA Kit (Human TNF-. Alpha.Quantikine Kit, R&D Systems) measures the levels of TNF- α, IL-1β and IL-8 production, which are markers of differentiation into M1 macrophages. The results are shown in fig. 22.

As shown in fig. 22, it was identified that all three types of markers differentiated into M1 macrophages were increased in the experimental group (CL 7) treated with the anti-CD 300c monoclonal antibody compared to the control group (Con) not treated with the anti-CD 300c monoclonal antibody.

Experimental example 3.6 identification of the ability to cause repolarization of M2 macrophages to M1 macrophages

To identify whether anti-CD 300c monoclonal antibodies can re-differentiate (repolarize) M2 macrophages to M1 macrophages, THP-1 was used at 1.5X10 ⁴ Cells/well were dispensed onto 96-well plates and pre-treated for 6 hours by treatment with 320nM PMA . Then, 20ng/mL of interleukin-4 (IL-4) and interleukin-13 (IL-13) as well as 10. Mu.g/mL of anti-CD 300c monoclonal antibody were used for treatment, and the reaction was allowed to proceed for 18 hours. Levels of TNF- α, IL-1β and IL-8 production were identified using ELISA kits. The results are shown in fig. 23 to 25.

As shown in fig. 23 to 25, it was identified that the experimental group co-treated with IL-4 and IL-13 and anti-CD 300c monoclonal antibody showed an increase in TNF- α, IL-1β and IL-8 production levels among the experimental groups not pre-treated with PMA; of the experimental groups pretreated with PMA, the experimental group co-treated with IL-4& IL-13 and anti-CD 300c monoclonal antibodies similarly showed increased levels of TNF- α, IL-1β and IL-8 production, and based on these results, it was found that the anti-CD 300c monoclonal antibodies were effective in re-differentiating (repolarizing) M2 macrophages into M1 macrophages.

Experimental example 3.7 identification of the ability to cause differentiation and repolarization to M1 macrophages

To identify the ability of anti-CD 300c monoclonal antibodies to cause differentiation and re-differentiation (repolarization) into M1 macrophages, THP-1 was expressed at 1.5X10 ⁴ Cells/well were distributed to 96-well plates, pre-treated with 10. Mu.g/mL of anti-CD 300c monoclonal antibody for 48 hours, and treated with 100ng/mL PMA, 100ng/mL LPS, and 20ng/mL IL-4 and IL-13, allowing the reaction to proceed for 24 hours. Levels of TNF- α production were identified using ELISA kits. The results are shown in fig. 26.

As shown in fig. 26, all experimental groups pretreated with anti-CD 300c monoclonal antibodies were identified to show a significant increase in TNF- α production levels compared to the M0 macrophage control group treated with PMA alone, the M1 macrophage control group treated with LPS alone, and the M2 macrophage control group treated with IL-4 and IL-13 alone. From these results, it was found that the anti-CD 300c monoclonal antibody has excellent ability to differentiate M0 macrophages into M1 macrophages, THP-1 into M1 macrophages, and M2 macrophages to re-differentiate (repolarize) into M1 macrophages.

Experimental example 4 identification of Cross-reactivity between species of anti-CD 300c monoclonal antibody by observing anticancer Effect

Experimental example 4.1 identification of human cancer cell growth inhibitory Effect

To identify the effect of CD300 c-targeted monoclonal antibodies on cancer cell growth, a cell proliferation assay was performed using a549 (human lung cancer cell line). More specifically, cells were treated with 2X 10 under 0% Fetal Bovine Serum (FBS) conditions ⁴ Cells/well were distributed into 96-well plates and the cells were plated at 6X 10 under 0.1% fetal bovine serum ³ Cells/well were distributed into 96-well plates. Then, the cells were treated with 10. Mu.g/mL of an anti-CD 300c monoclonal antibody and incubated for 5 days. The inhibition of cancer cell growth by anti-CD 300c monoclonal antibody was identified by treatment with CCK-8 (DOJINDO) and absorbance at OD450 nm. The results are shown in fig. 27 and 28.

As shown in fig. 27, all anti-CD 300c monoclonal antibodies except SK11 and SK17 were identified to have an effect of inhibiting proliferation of cancer cells under 0% fbs conditions.

As shown in fig. 28, all anti-CD 300c monoclonal antibodies used in the experiments were identified as having an effect of inhibiting proliferation of cancer cells under 0.1% fbs conditions.

Experimental example 4.2 identification of the growth inhibitory Effect of anti-CD 300c monoclonal antibody on cancer cell growth depending on its concentration

To identify the growth inhibitory effect of anti-CD 300c monoclonal antibodies on their concentration on cancer cells, A549 cells were treated at 2X 10 under 0% Fetal Bovine Serum (FBS) conditions ⁴ Cells/well were dispensed onto 96-well plates. Treatment with 10. Mu.g/mL anti-CD 300c monoclonal antibody was performed and incubated for 5 days. Subsequently, CCK-8 (DOJINDO) was used for treatment, and the reaction was allowed to proceed for 3 hours. The absorbance was then measured at OD450 nm to identify the cancer cell growth inhibition by the anti-CD 300c monoclonal antibody. The results are shown in fig. 29.

As shown in fig. 29, it was identified that the growth of cancer cells was inhibited with increasing concentration of anti-CD 300c monoclonal antibody.

Experimental example 4.3 identification of increased ability to differentiate into M1 macrophages in mice

To identify whether anti-CD 300c monoclonal antibodies could promote differentiation from mouse macrophages to M1 macrophages, mouse macrophages (Raw264.7) were incubated at 1X 10 ⁴ Cell/well concentration was distributed to 96-well plates using 10. Mu.g/mL of anti-CD 300c monoclonal antibodyBody treatment and incubation were performed. Levels of TNF- α production were detected using ELISA kits. The results are shown in fig. 30.

As shown in FIG. 30, an increase in TNF- α production levels was identified in the experimental group treated with anti-CD 300c monoclonal antibody. From these results, it can be seen that the anti-CD 300c monoclonal antibodies perform the same in humans and mice, indicating that cross-reactivity of the anti-CD 300c monoclonal antibodies promotes differentiation into M1 macrophages.

Experimental example 4.4 identification of cancer cell growth inhibition in mice

To identify whether anti-CD 300c monoclonal antibodies CL7, CL10 and SL18 exhibited anticancer effects, CT26 (mouse colorectal cancer cell line) was used at 1×10 ⁴ The cell/well concentration was distributed to 96-well plates, treated with 10. Mu.g/mL of each monoclonal antibody, and incubated for 5 days. Then, cell proliferation assay was performed by detecting CCK-8.

As shown in fig. 31, the anti-CD 300c monoclonal antibodies showed cancer cell proliferation inhibition 66% (CL 7), 15% (CL 10), 38% (SL 18) higher than the control group, respectively. From these results, it was identified that the anti-CD 300c monoclonal antibody exhibited a cancer therapeutic effect in mice. Thus, it can be seen that the anti-CD 300c monoclonal antibodies perform the same in mice as well as in humans, indicating that cross-reactivity of the anti-CD 300c monoclonal antibodies results in anti-cancer effects.

Experimental example 5 comparison of in vitro anticancer Effect between anti-CD 300c monoclonal antibody and conventional cancer immunotherapy

The manufacturers of each immunotherapy used in the following experimental examples were as follows:(AstraZeneca) and(Merck Sharp&Dohme)。

experimental example 5.1 comparison of the ability of anti-CD 300c monoclonal antibodies to cause differentiation into M1 macrophages with conventional cancer immunotherapy: measurement of production levels of three differentiation markers (TNF- α, IL-1β and IL-8)

To compare the ability to elicit differentiation into M1 macrophages between anti-CD 300c monoclonal antibodies and conventional cancer immunotherapy, the level of TNF- α production was identified using an ELISA kit in the same manner as example 3.2. As a conventional cancer treatment, a concentration of 10. Mu.g/mL was usedThe results are shown in fig. 32.

As shown in FIG. 32, the use of a single immune therapy called cancer was identifiedThe (Imf) treated control resulted in a significant increase in the level of TNF- α production by the anti-CD 300c monoclonal antibody. From these results, it was found that the anti-CD 300c monoclonal antibody resulted in a significant increase in the ability to cause differentiation into M1 macrophages compared to conventionally known cancer immunotherapy.

anti-PD-L1 immunotherapy at a concentration of 10 μg/mL each was used for comparison with other cancer immunotherapy anti-PD-1 immunotherapy->And isotype control (immunoglobulin G) antibodies, and the levels of TNF- α, IL-1β and IL-8 production were identified using ELISA kits. The results are shown in fig. 33 to 35.

As shown in fig. 33 to 35, the correlation was identifiedanti-CD 300c monoclonal antibodies resulted in significantly increased levels of TNF- α, IL-1β and IL-8 production compared to IgG antibodies. Based on these results, it was found that the anti-CD 300c monoclonal antibody can lead to a significant increase in facilitating differentiation into M1 macrophages compared to conventional cancer immunotherapy.

Experimental example 5.2 comparison of the ability to elicit differentiation from M0 macrophages to M1 macrophages between anti-CD 300c monoclonal antibody and conventional cancer immunotherapy

To compare the ability of anti-CD 300c monoclonal antibodies to elicit differentiation from M0 macrophages to M1 macrophages between cancer immunotherapy, 1.5X10 were used ⁴ Cell/well THP-1 was dispensed onto 96-well plates and used with 10. Mu.g/mL anti-CD 300c monoclonal antibody, 10. Mu.g/mLAnd/or 200nM phorbol-12-myristate-13-acetate (PMA). The reaction was allowed to proceed for 48 hours, and then the level of TNF- α production was measured using ELISA kit. The results are shown in fig. 36.

As shown in FIG. 36, immunotherapy alone with cancer was identified TNF- α was not produced in the treated comparative group, whereas TNF- α production levels were increased in the experimental group treated with anti-CD 300c monoclonal antibody alone. In addition, it was identified that even in the case of differentiation of THP-1 cells into M0 macrophages by PMA treatment, the same as that of +.>The treated groups also showed significantly increased levels of TNF- α production compared to the groups treated with the anti-CD 300c monoclonal antibody. Based on these results, it was found that the anti-CD 300c monoclonal antibody promoted differentiation from M0 macrophages to M1 macrophages compared to conventional cancer immunotherapy.

Experimental example 5.3 comparison of the ability of anti-CD 300c monoclonal antibodies to elicit differentiation into M1 macrophages with conventional cancer immunotherapy

To compare the ability of anti-CD 300c monoclonal antibodies to elicit differentiation into M1 macrophages with conventional cancer immunotherapy, TNF- α production levels were identified in the same manner as in example 3.2. The results are shown in fig. 37.

As shown in FIG. 37, it was identified that in the case of differentiation of monocytes into M1 macrophages by treatment with LPS, the following were usedThe group co-treated with LPS showed no significant difference in the level of TNF- α production, whereas the group co-treated with anti-CD 300c monoclonal antibody and LPS showed a significant increase in the level of TNF- α production compared to the group treated with anti-CD 300c monoclonal antibody alone.

Experimental example 5.4 comparison of cancer cell growth inhibition between anti-CD 300c monoclonal antibody and conventional cancer immunotherapy

To compare the cancer cell growth inhibition by anti-CD 300c monoclonal antibodies with conventional cancer immunotherapy, a549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line) were used to identify cell growth inhibition. More specifically, each cell was treated with 2X 10 under 0% Fetal Bovine Serum (FBS) conditions ⁴ Cells/well were distributed to 96-well plates and each cell was plated at 6X 10 under 0.1% fetal bovine serum conditions ³ Cells/well were distributed to 96-well plates. Subsequently, 10. Mu.g/mL of anti-CD 300c monoclonal antibody was used for treatment and incubation for 5 days. Then, observation was performed under an optical microscope. The results are shown in fig. 38 and 39.

As shown in FIG. 38, it was identified that in the A549 cell line, the anti-CD 300c monoclonal antibody was specific for cancer immunotherapyMore effectively inhibit the proliferation of cancer cells.

As shown in FIG. 39, it was identified that in the MDA-MB-231 cell line, the anti-CD 300c monoclonal antibody was specific as an immunotherapy for cancerMore effectively inhibit the proliferation of cancer cells.

Experimental example 6: identification of anti-CD 300c monoclonal antibodies for in vivo anti-cancer

Experimental example 6.1 identification of increased tumor-associated macrophages (TAMs)

To identify the in vivo effect of anti-CD 300c monoclonal antibody CL7 on tumor-associated macrophages, the antigen is administered subcutaneouslyInjection will be 2X 10 ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in Specific Pathogen Free (SPF) equipment. After 12 days of colorectal cancer cell line transplantation, tumors of 50mm in size were given to each ³ -100 mm ³ Is injected with an anti-CD 300c monoclonal antibody and an equal amount of Phosphate Buffered Saline (PBS) is injected as a control group. Mice were injected intraperitoneally with 25mg/kg of each material twice weekly for two weeks for a total of 4 times. On day 25 post injection, mice were sacrificed and tumor tissue was harvested from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution (from Invitrogen) to check cell viability, and with antibodies to the total macrophage marker F4/80 and the M1 macrophage marker iNOS (from Abcam). The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

The results are shown in fig. 40, which identifies that the expression level of M1-type tumor-associated macrophages increases in mouse cancer tissue when treated with anti-CD 300c monoclonal antibody alone. This means that administration of anti-CD 300c monoclonal antibodies causes an increase in tumor-associated macrophages in cancer tissue, thereby inhibiting cancer growth.

Experimental example 6.2 identification of cytotoxic T cell increase

To identify the in vivo effect of anti-CD 300c monoclonal antibody CL7 on cd8+ T cells, a syngeneic mouse tumor model was prepared as in experimental example 6.1, and anti-CD 300c monoclonal antibody was administered at the same concentration. On day 25 post injection, mice were sacrificed and tumor tissue was harvested from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution to check cell viability, and cells were stained with cd8+ antibody (from Abcam) and cd4+ antibody (from Abcam). The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

The results are shown in fig. 41, which identifies an increase in the number of cd8+ T cells in the tumor when treated with anti-CD 300C monoclonal antibody alone. This means that administration of an anti-CD 300c monoclonal antibody results in an increase in intratumoral cytotoxic T cells, thereby exhibiting a therapeutic effect on cancer.

Experimental example 6.3 identification of increased tumor specificity in cytotoxic T cells

To identify whether anti-CD 300c monoclonal antibody CL7 increased the number of cd8+ T cells in a tumor specific manner, a syngeneic mouse tumor model was prepared as in experimental example 6.1, and anti-CD 300c monoclonal antibody was administered at the same concentration. On day 25 post injection, mice were sacrificed and tumor tissue was harvested from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution to check cell viability, and with AH1 tetrameric antibody (from Abcam). The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in fig. 42, it was identified that the expression of AH1 tetramer as a CT26 tumor marker factor in cd8+ T cells was increased when treated with anti-CD 300c monoclonal antibody alone, indicating that the number of cd8+ T cells was increased in a tumor (CT 26) -specific manner. This means that administration of anti-CD 300c monoclonal antibodies increased cd8+ T cell targeting and inhibited CT26 cancer cells.

Experimental example 6.4 identification of increased cytotoxic T cell Activity

To identify the in vivo effect of anti-CD 300c monoclonal antibody CL7 on cd8+ T cells, a syngeneic mouse tumor model was prepared as in experimental example 6.1, and anti-CD 300c monoclonal antibody was administered at the same concentration. On day 25 post injection, mice were sacrificed and spleens were collected from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). Subsequently, IFN-g was measured by the ELISPOT assay to identify the results. Specifically, the mouse IFN-g ELISPot kit was purchased from R & D Systems (# EL 485), and IFN-g was measured according to the protocol of the kit.

The results are shown in FIG. 43, which identifies an increase in IFN-g expression when treated with anti-CD 300c monoclonal antibody alone. This means that administration of the anti-CD 300c monoclonal antibody alone not only results in an increase in the number of cd8+ T cells (see fig. 41), but also results in an increase in the activity of cd8+ T cells, thereby inhibiting cancer growth in various ways to show the effect of cancer treatment.

Experimental example 6.5 identification of an increase in cytotoxic T cells relative to regulatory T cells

To identify the effect of anti-CD 300c monoclonal antibody CL7 on the increase of cytotoxic T cells relative to regulatory T cells in vivo, a syngeneic mouse tumor model was prepared as described in experimental example 6.1, and anti-CD 300c monoclonal antibody was administered at the same concentration. On day 25 post injection, mice were sacrificed and tumor tissue was harvested from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution to check cell viability, and cells were stained with antibodies to Treg marker protein CD25 (from sengzhuzhou) and Foxp3 (from Abcam), cd3+ antibody and cd8+ antibody. The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

The results are shown in figure 44, identifying an increase in cd8+ T cells relative to Treg T cells with anti-CD 300C monoclonal antibody alone. This means that the increase in the number of cd8+ T cells caused by administration of the anti-CD 300C monoclonal antibody further inhibits cancer growth.

Experimental example 6.6 identification of the Effect on cytotoxic T cells, regulatory T cells and tumor-associated macrophages

To identify the effect of the anti-CD 300c monoclonal antibody CL7 on cytotoxic T cells, regulatory T cells and tumor-associated macrophages, the following experiments were performed. A syngeneic mouse tumor model was prepared as in Experimental example 6.1, and after 12 days of implantation of colorectal cancer cell lines, tumors of 50mm in size were given to each ³ -100 mm ³ Is injected with an anti-CD 300c monoclonal antibody and an equal amount of Phosphate Buffered Saline (PBS) is injected as a control group. Mice were injected intraperitoneally with 25mg/kg of each material twice weekly for two weeks for a total of 4 times. On day 25 post injection, mice were sacrificed and tumor tissue was harvested from each of 6 mice in the CL7 25mg/kg administration group (highest antitumor effect was observed compared to the control group). The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/mL) and DNase I (2 mg/mL) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 (Invitrogen) antibody and the cells were stained with staining solution to check cell viability, and cells were stained with antibody to cd8+ T cell marker CD8 and antibody to CD4, antibody to Treg cell marker Foxp3 and antibody to CD4, or antibody to total macrophage marker F4/80 and M1 macrophage marker iNOS (from Abcam). The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software. The results are shown in FIG. 45.

As shown in fig. 45, anti-CD 300c monoclonal antibody (CL 7) was identified that significantly increased activated cd8+ T cells, inhibited regulatory T cells, and repolarized tumor-associated macrophages toward the M1 phenotype.

Experimental example 6.7 identification of in vivo cancer growth inhibition

To identify the in vivo anticancer effect of the anti-CD 300c monoclonal antibody CL7, 2×10 was injected subcutaneously ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in an SPF facility. On day 11 after implantation of colon cancer cell lines (D11), tumors were 50mm in size, respectively ³ -100mm ³ 1mg/kg, 5mg/kg, 10mg/kg or 25mg/kg of anti-CD 300c monoclonal antibody, and an equivalent amount of Phosphate Buffered Saline (PBS) as a control group. Specifically, mice were given intraperitoneal injections of each dose twice a week for two weeks (4 total in D11, D14, D18 and D21). Tumor volumes were measured for 25 days. The results are shown in fig. 46.

As shown in fig. 46, the anti-CD 300c monoclonal antibody CL7 was identified to delay the growth of CT26 colorectal cancer in a dose-dependent manner.

Changes in biomarker expression caused by administration of anti-CD 300c monoclonal antibodies

Example 2 variation of immune cell-related marker and tumor microenvironment-related marker expression by administration of anti-CD 300c monoclonal antibody

EXAMPLE 2.1 nanostring immune profile

To identify changes in immune cell and tumor microenvironment related marker expression in the case of administration of the anti-CD 300c monoclonal antibody (CL 7) generated in example 1 to a solid cancer model, 2 x 10 was injected subcutaneously ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in an SPF facility. After 12 days of colorectal cancer cell line transplantation, tumors of 50mm in size were each transplanted ³ -100mm ³ anti-CD 300c monoclonal antibodies, and an equivalent amount of Phosphate Buffered Saline (PBS) was administered as a control group. Mice were injected intraperitoneally with 25mg/kg of each material twice weekly for two weeks for a total of 4 times. At the position ofOn day 25 post injection, mice were euthanized to prepare tumor tissue. Purified RNA was extracted therefrom and changes in dendritic cell markers, macrophage markers, tumor Microenvironment (TME) markers, th1 response markers, or Th2 response markers were identified by Nanostring immunology.

The Nanostring immune profile results are shown in fig. 47, from which the extensively reprogrammed tumor immune microenvironment with the administration of anti-CD 300c monoclonal antibody was identified.

In addition, fig. 48 shows the results obtained by observing changes in dendritic cell markers, macrophage markers, tumor microenvironment markers, th1 response markers, or Th2 response markers caused by administration of an anti-CD 300c monoclonal antibody relative to a control group. As shown in fig. 48, it was identified that administration of anti-CD 300c monoclonal antibodies resulted in a significant increase in expression of Bst2, CCL8, and Xcl1 of the dendritic cell markers; significantly increased expression of the M1 macrophage markers CCR7 and CD 80; reduced expression of vegfa, pdgfrb, col a1 and Hif1a which aid in cancer growth in the tumor microenvironment; and increased expression of markers Tbx21, stat1, stat4, ifn-g and Cxcr3 that identify Th1 responses.

EXAMPLE 2.2 variation in immune checkpoint marker expression

From the Nanostring immune profile results obtained in example 2.1, it was identified which immune checkpoint markers showed significant expression differences compared to the control group in case of administration of anti-CD 300c monoclonal antibodies to syngeneic mouse tumor models.

As a result, as shown in fig. 49, it was identified that administration of the anti-CD 300c monoclonal antibody resulted in increased expression of PD-1, CTLA-4, and lang 3 as inhibitory Immune Checkpoints (ICs), and increased expression of ICOS, OX40, gitr, CD27, and CD28 as agonistic ICs.

These results are of considerable importance as they can provide useful information about which immune checkpoint related immunotherapy should be selected in the case of combined administration of anti-CD 300c monoclonal antibodies and other immunotherapies to obtain further enhanced anti-cancer efficacy.

Combination of anti-CD 300c monoclonal antibodies and immunotherapy

EXAMPLE 3 Co-administration of anti-CD 300c monoclonal antibody (CL 7) and immunotherapy

The anti-CD 300c monoclonal antibody (CL 7) produced in example 1 was used in other cancer immunotherapies, such as anti-PD-L1 antibodiesAnd->anti-PD-1 antibody->anti-CD 47 antibodies (αcd 47) and anti-CTLA-4 antibodies are used in combination. Then, the result was obtained.

The manufacturer of the respective immunotherapy is as follows: IMFINZI (AstraZeneca); OPDIVO and anti-CTLA-4 antibodies (Bristol Myers Squibb company); keyruda (Merck Sharp & Dohme); and anti-CD 47 antibodies (Abcam).

Experimental example 7 identification of increased macrophage Activity (synergy) by combination

Experimental example 7.1 identification of an increase in M1 macrophages

To identify by cell morphology the pattern of differentiation into M1 macrophages when monocytes are co-treated with anti-CD 300c monoclonal antibody CL7 produced in example 1 and cancer immunotherapy such as anti-PD-L1 antibody immunotherapy, anti-PD-1 antibody immunotherapy and anti-CD 47 antibody (. Alpha.CD47), THP-1 (human monocyte line) was treated with anti-CD 300c monoclonal antibody and cancer immunotherapy, respectively, alone or in combination at 10. Mu.g/mL. After 48 hours of incubation, the cell shape was observed under a microscope.

As a result, as shown in fig. 50, in the case of co-treatment with the anti-CD 300c monoclonal antibody and the cancer immunotherapy, the shape of THP-1 cells was changed from the suspension cells to round adherent cells in the shape of M1 macrophages, compared with the case of the cancer immunotherapy alone. Based on these results, it was identified that co-treatment with anti-CD 300c monoclonal antibody and cancer immunotherapy further promoted differentiation of monocytes into M1 macrophages.

Experimental example 7.2 identification of increased M1 macrophage marker

To identify when immunotherapy such as anti-PD-L1 antibody was used with anti-CD 300c monoclonal antibody CL7 generated in example 1, respectively, cancer immunotherapyImmunotherapy with anti-PD-1 antibodies>Immunotherapy with anti-PD-1 antibodies>In the case of the co-treatment of monocytes with an anti-CD 47 antibody (. Alpha.CD 47) and an anti-CTLA-4 antibody, the induction of differentiation of monocytes into M1 macrophages was increased, and THP-1 was expressed as 1.5X10 ⁴ Cells/well were dispensed onto 96-well plates and treated with anti-CD 300c monoclonal antibody and cancer immunotherapy, respectively, at 10 μg/mL alone or in combination. The reaction was allowed to proceed for 48 hours and ELISA Kit (Human TNF-. Alpha.Quantikine Kit, R&D Systems) measures the production levels of tumor necrosis factor-alpha (TNF-alpha), IL-1b and IL-8, which are markers of differentiation of M1 macrophages.

As a result, as shown in fig. 51, it was identified that the production level of all three differentiation markers was increased in the case of treatment with the anti-CD 300c monoclonal antibody alone, wherein the production level of IL-8 was particularly significantly increased. In addition, as shown in FIG. 52, it was identified that the anti-CD 300c monoclonal antibody was used in combination with the anti-CD 300c monoclonal antibody alone, as compared with the case of treatment with the anti-CD 300c monoclonal antibody aloneAnd αcd47, the level of TNF- α production as a differentiation marker of M1 macrophages is further increased. From these results, it was identified that there was more single core fineness in the case of co-treatment with the anti-CD 300c monoclonal antibody and the anti-PD-1 antibody and/or the anti-CD 47 antibody than in the case of treatment with the anti-CD 300c monoclonal antibody aloneCells differentiate into M1 macrophages.

Experimental example 7.3 identification of M2 macrophage marker reduction

To identify anti-CD 300c monoclonal antibodies for use in monocytes and cancer immunotherapy such as In the case of co-treatment with anti-CTLA-4 or αCD47, the induction of monocyte differentiation into M2 macrophages was reduced, and THP-1 was expressed as 1.5X10 ⁴ Cells/well were dispensed onto 96-well plates and pretreated with 320nM PMA for 6 hours. Then, the treatment with the anti-CD 300c monoclonal antibody and immunotherapy, respectively, alone or in combination with 10. Mu.g/mL, was performed together with the treatment with 20ng/mL of interleukin-4 (IL-4) and interleukin-13 (IL-13). The reaction was allowed to proceed for 48 hours. Then, ELISA kit (R &D Systems) measures the production levels of the M2 macrophage differentiation markers IL-10 and IL-12.

The results identify that when treated with anti-CD 300c monoclonal antibody and compared to the anti-CD 300c monoclonal antibody aloneAnd αCD47, the production level of IL-10 and IL-12 is further reduced by 30% or more.

Experimental example 7.4 identification of increased ability to differentiate into M1 macrophages

To identify the increase in differentiation of monocytes into M1 macrophages in the case of co-treatment of monocytes with anti-CD 300c monoclonal antibody CL7 and cancer immunotherapy such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies and anti-CD 47 antibodies, signal transduction of mitogen-activated protein kinases (MAPKs), IκB and NF- κB, which are representative signals for M1 macrophage differentiation, were examined. Specifically, THP-1 was used at 8.8X10 ⁵ Cells/well were distributed on 6-well plates and used 10. Mu.g/mL of anti-CD 300c monoclonal antibody, 10. Mu.g/mLAnd/or 10. Mu.g/mL->And (5) processing. For the control group, the same amount of Phosphate Buffer (PBS) was used for treatment. Incubate for 48 hours. Western blotting was then performed to identify phosphorylated SAPK/JNK, phosphorylated ERK, phosphorylated p38 for MAPK signaling, phosphorylated NF- κB for NF- κB signaling, and phosphorylated IκB for IκB signaling. The results are shown in fig. 53 to 55.

FIGS. 53, 54 and 55 show the results obtained by identifying signal transduction of MAPK, NF- κB and IκB, respectively. It was identified that in the case of co-treatment of THP-1 with anti-CD 300c monoclonal antibodies and cancer immunotherapy such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies and anti-CD 47 antibodies, the levels of phosphorylated MAPK, IκB and NF- κB were increased compared to the case of treatment of THP-1 with anti-CD 300c monoclonal antibodies alone. From these results, it was identified that in the case of co-treatment of THP-1 with an anti-CD 300-300c monoclonal antibody and a cancer immunotherapy such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-CD 47 antibody, cell signals representing differentiation into M1 macrophages are increased compared with the case of treatment of THP-1 with an anti-CD 300-300c monoclonal antibody alone.

Experimental example 8 identification of (synergistic) increase in inhibition of cancer cells by combination (in vitro)

Experimental example 8.1 identification of apoptosis Signal

It was identified whether the apoptosis signal was increased in the case of co-treatment with anti-CD 300c monoclonal antibody CL7 and cancer immunotherapy such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody and anti-CD 47 antibody. Specifically, A549 was set at 8X 10 ⁵ Cells/well were dispensed onto 6-well plates and used 10. Mu.g/mL of anti-CD 300c monoclonal antibody and 10. Mu.g/mLAnd anti-CD 47 antibodies, alone or in combination. Incubation for 48 hoursWestern blotting was then performed to identify apoptotic signals or cell cycle signals. Cleaved caspase-9, caspase-3, caspase-2 and caspase-8 are identified as markers of apoptosis signals, cyclin D1, CDK2, P27kip1, CDK6, cyclin D3, P21 Waf1, cip1, etc. are identified as markers of cell cycle signals.

As shown in FIG. 56, when the anti-CD 300c monoclonal antibody was used and the anti-PD-1 antibody was used, compared with the case of treatment with the anti-CD 300c monoclonal antibody aloneIn the case of co-treatment, the apoptosis signal increases; in the case of co-treatment with anti-CD 300c monoclonal antibodies and immunotherapy such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies and anti-CD 47 antibodies, the levels of cleaved caspase9 and p21 are increased and the level of cyclin D1 is decreased. From these results, it was identified that co-treatment with an anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-CD 47 antibody can induce apoptosis of cancer cells better than the case of treatment with an anti-CD 300c monoclonal antibody alone.

Experimental example 8.2 identification of growth inhibitory Effect of cancer cell lines

To identify the inhibition of cancer cell growth caused by co-administration of anti-CD 300c monoclonal antibody CL7 and cancer immunotherapy, a comparison of cancer cell growth inhibition was performed using a549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). Specifically, cells were grown at 2X 10 in the absence of Fetal Bovine Serum (FBS) ⁴ Cells/well (A549) or 3X 10 ⁴ Cells/well (MDA-MB-231) were dispensed into 96-well plates and the cells were plated at 6X 10 under 0.1% fetal bovine serum ³ Cells/well (A549) or 1X 10 ⁴ Cells/well (MDA-MB-231) were dispensed into 96-well plates. Subsequently, 10. Mu.g/mL of anti-CD 300c monoclonal antibody was usedCells were treated individually or in combination and incubated for 5 days. For the control group, the same amount of Phosphate Buffer (PBS) was used for treatment. Then, the absorbance was measured at OD 450nm by treatment with CCK-8 (DOJINDO). The results are shown in FIG. 57 (A549) and FIG. 58 (MDA-MB-231).

As shown in fig. 57, for the a549 cell line, it was identified that a 17% higher cell growth inhibition was observed with the anti-CD 300c monoclonal antibody alone and a 34% higher cell growth inhibition was observed with the anti-CD 300c monoclonal antibody and IMFINZI together, compared to the control, in the FBS-free condition.

As shown in fig. 58, the MDA-MB-231 cell line was identified to observe 19% higher cell growth inhibition in the case of treatment with anti-CD 300c monoclonal antibody alone compared to the control under 0.1% fbs conditions; a 45% higher inhibition of cell growth was observed with co-treatment with anti-CD 300c monoclonal antibody and anti-CD 47 antibody; and a higher cell growth inhibition of 51% was observed with co-treatment with anti-CD 300c monoclonal antibody, anti-CD 47 antibody and IMFINZI. A higher inhibition of cell growth of 19% was observed with anti-CD 300c monoclonal antibody alone compared to the control under conditions of 0.1% fbs; a 22% higher inhibition of cell growth was observed with co-treatment with anti-CD 300c monoclonal antibody and anti-CD 47 antibody; and a 32% higher inhibition of cell growth was observed with co-treatment with anti-CD 300c monoclonal antibody, anti-CD 47 antibody and IMFINZI.

From these results, it was identified that the growth of cancer cells was further inhibited in the case of co-treatment with an anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-CD 47 antibody, compared to the case of treatment with an anti-CD 300c monoclonal antibody alone.

Experimental example 9 identification of (synergistic) increased anti-cancer effects in vivo by combination (colon cancer mouse model)

Experimental example 9.1 identification of in vivo cancer growth inhibition

To identify the in vivo anticancer effect of the anti-CD 300c monoclonal antibody CL7, 2×10 was injected subcutaneously ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in an SPF facility. On day 12 after implantation of the colon cancer cell line (D12), the tumor size was 50mm ³ -100 mm ³ anti-CD 300c monoclonal antibody and anti-PD-1 antibody purchased from BioXcell, alone or in combination, and an equal amount of Phosphate Buffered Saline (PBS) was administered as a control group. A schematic of the experimental procedure is shown in FIG. 59, specifically, mice were injected with various antibodies (CL 7:10mg/kg; and anti-PD-1 antibody: 10 mg/kg) by intraperitoneal injection, twice weekly for two weeks (4 times in D12, D15, D19 and D22). Tumor volumes were measured for 25 days. The results are shown in FIG. 60.

As can be seen from fig. 60, it was identified that although cancer growth was inhibited even in the experimental group to which the anti-CD 300c monoclonal antibody was administered alone compared to the control group, cancer growth was inhibited more effectively in the case of co-treatment with the anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody compared to the treatment with the anti-CD 300c monoclonal antibody alone.

Experimental example 9.2 identification of an increase in tumor infiltrating lymphocytes in an in vivo tumor microenvironment

To identify the effect of anti-CD 300c monoclonal antibodies on Tumor Infiltrating Lymphocytes (TILs) in Tumor Microenvironment (TME), mice were euthanized on day 25 of the experiment performed in the same manner as experimental example 9.1, injected intravascularly with 1% Paraformaldehyde (PFA), and then perfused to obtain cancer tissue. The obtained cancer tissues were fixed with 1% pfa and sequentially dehydrated with 10%, 20% and 30% sucrose solutions. Dehydrated cancer tissue was frozen in OCT compound (optimal cutting temperature compound) and then cut into 50 μm thickness using a cryomicrotome. The tissue was incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was again filtered through a nylon mesh to make them single cells. To suppress non-specific responses in single cell suspensions, single cell suspensions were blocked with CD16/32 antibody (Invitrogen) for 1 hour, cell viability was checked, and staining of cd8+ T cells and cd31+ tumor vascular cells (which are tumor infiltrating lymphocyte markers) was performed.

Results identified an increase in cd8+ T cells in the experimental group co-administered with anti-CD 300c monoclonal antibody and anti-PD-1, anti-PD-L1, anti-CTLA-4 or anti-CD 47 antibodies compared to the experimental group administered with anti-CD 300c monoclonal antibody alone. From these results, it was found that in the case of co-treatment with an anti-CD 300c monoclonal antibody and an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody or an anti-CD 47 antibody, the number of tumor-infiltrating lymphocytes was increased compared with the case of treatment with an anti-CD 300c monoclonal antibody alone, thereby exerting an anticancer effect.

Experimental example 9.3 identification of the in vivo role of increasing M1 macrophages

To identify whether the anti-CD 300c monoclonal antibody increased M1 macrophages in the cancer tissue of the mouse model, cancer tissue sections prepared in the same manner as experimental example 9.2 were stained with antibodies against the M1 macrophage marker iNOS and the M2 macrophage marker CD206, and examined by FACS.

As a result, as shown in fig. 61, it was identified that M1 macrophages were partially increased in the experimental group treated with the anti-PD-1 antibody, whereas M1 macrophages were significantly increased in the experimental group treated with the anti-CD 300c monoclonal antibody, but almost no M2 macrophages were observed, as compared to the control group. In addition, a further increase in M1 macrophages in the experimental group co-administered with anti-CD 300c monoclonal antibody and anti-PD-1 antibody was also identified. From these results, it was identified that the differentiation into M1 macrophages was effectively promoted in the case of co-treatment with an anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody or an anti-CD 47 antibody, compared to the case of treatment with an anti-CD 300c monoclonal antibody alone.

Experimental example 9.4 identification of immune-boosting effects in CD8+ T cells

To identify whether anti-CD 300c monoclonal antibody CL7 promotes cd8+ T cell immunity in a mouse tumor model, mice were euthanized, intravascular injected with 1% Paraformaldehyde (PFA), and then perfused to obtain cancer tissue on day 25 of the experiment performed in the same manner as experimental example 9.1. The obtained cancer tissues were fixed with 1% pfa and sequentially dehydrated with 10%, 20% and 30% sucrose solutions. Dehydrated cancer tissue was frozen in OCT compound (optimal cutting temperature compound) and then cut into 50 μm thickness using a cryomicrotome. Then, cd8+ and iNOS were stained.

As shown in fig. 62, it was identified that cd8+ T cells were partially increased in the experimental group treated with the anti-PD-1 antibody, and cd8+ T cells were significantly increased in the experimental group treated with the anti-CD 300c monoclonal antibody, compared to the control group. In addition, cd8+ T cells were further increased in the experimental group where the anti-CD 300c monoclonal antibody and the anti-PD-1 antibody were co-administered, as compared to the group where the anti-PD-1 antibody was administered alone. Based on these results, it was found that the anti-CD 300c monoclonal antibody increased the number of cd8+ T cells more effectively when used in combination with conventional cancer immunotherapy.

Experimental example 9.5 identification of an increasing Effect of immune cell Activity in vivo

To identify whether co-administration of an anti-CD 300c monoclonal antibody and immunotherapy increased the activity of immune cells, spleens were obtained in the same manner as experimental example 9.1 from mice co-administered with an anti-CD 300c monoclonal antibody and an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-KIR antibody, an anti-LAG 3 antibody, an anti-CD 137 antibody, an anti-OX 40 antibody, an anti-CD 276 antibody, an anti-CD 27 antibody, an anti-GITR antibody, an anti-TIM 3 antibody, an anti-41 BB antibody, an anti-CD 226 antibody, an anti-CD 40 antibody, an anti-CD 70 antibody, an anti-ICOS antibody, an anti-CD 40L antibody, an anti-BTLA antibody, an anti-TCR antibody, an anti-TIGIT antibody, or an anti-CD 47 antibody. The spleens obtained were FACS stained with various markers capable of detecting T cell activity and NKT cell activity and examined by MFI as described in experimental example 9.2.

The results identified that T cell activation markers Gzma, icos, CD and Ifng increased, while NKT cell activation markers CD11, CD38 and CXCR6 increased significantly with co-administration of anti-CD 300c monoclonal antibody and immunotherapy as described above. From these results, T cells and NKT cells were identified to be further activated in the case of co-treatment with anti-CD 300c antibody and immunotherapy, compared to the case of treatment with anti-CD 300c antibody alone.

Experimental example 9.6 identification of in vivo Treg cell inhibition

To identify a change in Treg cell pattern that elicited an immunosuppressive response in the case of co-administration of an anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-KIR antibody, an anti-LAG 3 antibody, an anti-CD 137 antibody, an anti-OX 40 antibody, an anti-CD 276 antibody, an anti-CD 27 antibody, an anti-GITR antibody, an anti-TIM 3 antibody, an anti-41 BB antibody, an anti-CD 226 antibody, an anti-CD 40 antibody, an anti-CD 70 antibody, an anti-ICOS antibody, an anti-CD 40L antibody, an anti-BTLA antibody, an anti-TCR antibody, an anti-TIGIT antibody, or an anti-CD 47 antibody, T cells were extracted from spleen tissue prepared in the same manner as experimental example 9.2, and FACS stained to examine the number of FOXP3 expressing Treg cells in cd3+ T cells.

Based on the proportion of Treg cells in cd3+ cells, it was identified that a significant reduction in the proportion of Treg cells was observed with co-administration of anti-CD 300c monoclonal antibody and immunotherapy, as compared to the case of each immunotherapy alone, wherein a significant reduction in the proportion of Treg cells indicates induction of activation of T cells that attack cancer cells.

Experimental example 10 identification of (synergistic) increased anti-cancer effects in vivo by combination (melanoma mouse model)

Experimental example 10.1 identification of in vivo cancer growth inhibition

To identify whether the anti-CD 300c monoclonal antibody CL7 was effective in cancers other than the CT26 colorectal cancer mouse model, additional experiments were performed with the melanoma mouse model. Will be 7X 10 ⁵ Is subcutaneously injected into 8-week-old male C57BL/6 mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in an SPF facility. Tumors were given on day 8 after melanoma cell line transplantation50mm in size ³ -100 mm ³ 25mg/kg CL7, 10mg/kg alpha-PD-1 and 4mg/kg alpha-CTLA 4 are intraperitoneally injected into the mice of (E). Fig. 63 shows a schematic diagram of an experimental method, more specifically, mice were injected with each of an anti-CD 300c monoclonal antibody, an anti-PD-1 antibody, an anti-CD 300c monoclonal antibody+an anti-PD-1 antibody (Combo) and an anti-CD 300c monoclonal antibody+an anti-PD-1 antibody+an anti-CTLA-4 antibody (triplex), twice a week for 2 weeks (total 4 times), and with Phosphate Buffered Saline (PBS) in the same amount as the anti-CD 300c monoclonal antibody, as a control group. The cancer size was measured for 20 days.

As a result, as shown in fig. 64, it was identified that cancer growth was inhibited even when the anti-CD 300c monoclonal antibody was administered alone, but in the group where the anti-CD 300c monoclonal antibody and the anti-PD-1 antibody + anti-CTLA-4 antibody were co-administered, cancer growth was more effectively inhibited.

Experimental example 10.2 identification of cytotoxic T cell increase

To identify the in vivo effect of the anti-CD 300c monoclonal antibody CL7 in combination with the anti-PD-1 antibody and/or the anti-CTLA-4 antibody on cd8+ T cells in the B16F10 melanoma model, a mouse tumor model was prepared as in experimental example 10.1 and each test substance was injected at the same concentration.

Tumor tissue was collected from 6 mice per group. The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution (from Invitrogen) to check cell viability, and with cd8+ antibody and cd4+ antibody, then the data were read with CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in fig. 65, it was identified that the number of cd8+ T cells was increased in the CT26 cancer model (experimental example 6.2) even in the B16F10 melanoma model after co-administration of the anti-CD 300c monoclonal antibody and the immunotherapy (group D and group T). Herein, group D represents a combination of CL7 and an anti-PD-1 antibody, and group T represents a combination of CL7, an anti-PD-1 antibody, and an anti-CTLA-4 antibody.

Experimental example 10.3 identification of an increase in cytotoxic T cells relative to regulatory T cells

To identify the in vivo effect of the anti-CD 300c monoclonal antibody CL7 in combination with the anti-PD-1 antibody and/or the anti-CTLA-4 antibody on regulatory T cells in the B16F10 melanoma model, a mouse tumor model was prepared as in example 10.1, and each test substance was injected at the same concentration. The experimental groups were as follows: (i) a group to which CL7 is administered, (ii) a group to which an anti-PD-1 antibody is administered, (iii) a group to which CL7 and an anti-PD-1 antibody are administered (group D), and (iv) a group to which CL7, an anti-PD-1 antibody, and an anti-CTLA 4 antibody are administered (group T).

Tumor tissue was collected from 6 mice per group. The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 antibody (from Invitrogen) and the cells were stained with staining solution to check cell viability, and with antibodies against Treg marker proteins CD25 and Foxp3, cd3+ antibodies and cd8+ antibodies. The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in fig. 66, it was identified that the number of cd8+ T cells was increased relative to regulatory T cells in the CT26 cancer model (experimental example 6.5) after co-administration of the anti-CD 300c monoclonal antibody and immunotherapy (group D and group T), even in the B16F10 melanoma model. Herein, group D represents a combination of CL7 and an anti-PD-1 antibody, and group T represents a combination of CL7, an anti-PD-1 antibody, and an anti-CTLA-4 antibody.

Experimental example 10.4 identification of increased tumor-associated macrophages (TAMs)

To identify the in vivo effects of the anti-CD 300c monoclonal antibody CL7 in combination with anti-PD-1 antibody and/or anti-CTLA-4 antibody on macrophages in a B16F10 melanoma model, a mouse tumor model was prepared as in experimental example 10.1, and each test substance was injected at the same concentration.

Tumor tissue was collected from 6 mice per group. The collected tumor tissue was incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37℃for 1 hour. Then, the resultant was filtered through a 70 μm cell filter, the erythrocytes were subjected to lysis, and then the resultant was filtered again through a nylon mesh. Subsequently, the single cell suspension was blocked with CD16/32 (Invitrogen) antibody, and the cells were stained with a staining solution to examine cell viability, and with antibodies against F4/80 and iNOS. The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in fig. 67, it was identified that the expression level of tumor-associated M1 type macrophages was increased even in the B16F10 melanoma model (group D and group T) after co-administration of the anti-CD 300c monoclonal antibody and immunotherapy as in the CT26 cancer model (experimental example 6.1). Herein, group D represents a combination of CL7 and an anti-PD-1 antibody, and group T represents a combination of CL7, an anti-PD-1 antibody, and an anti-CTLA-4 antibody.

Taken together, the results shown in fig. 65, 66 and 67 have the following meanings. From the perspective of anti-CD 300c monoclonal antibody CL7 to treat cancer in a B16F10 melanoma mouse model with the same mechanisms as in a CT26 colorectal cancer mouse model (fig. 40, 41 and 42), it is predicted that co-administration of CL7 and immunotherapy may play the same role in various cancers.

Experimental example 11 identification of colorectal cancer mouse model to achieve complete remission by Co-administration

To identify in vivo anticancer effects caused by co-administration of anti-CD 300c monoclonal antibody CL7 and anti-PD-1 and/or anti-CTLA-4 antibodies, 2X 10 was injected subcutaneously ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models. Animal feeding and experimentation were performed in an SPF facility. On day 11 after implantation of the colon cancer cell line (D11), the tumor size was 50mm ³ -100 mm ³ anti-CD 300c monoclonal antibodies, anti-PD-1 antibodies, and anti-CTLA-4 antibodies, each purchased from BioXcell, were administered alone or in combination, and Phosphate Buffered Saline (PBS) in the same amount as CL7 was injected as a control group.Specifically, at D11, D14 and D18, the mice were injected with each antibody (CL 7:25mg/kg; anti-PD-1 antibody: 10mg/kg; anti-CTLA-4 antibody: 4 mg/kg) alone or in combination by intraperitoneal injection, and tumor volumes were measured.

As a result, as shown in fig. 68a, it was identified that although cancer growth was inhibited even in the experimental group to which the anti-CD 300c monoclonal antibody was administered alone compared to the control group, cancer growth was further inhibited in the case of co-treatment with the anti-CD 300c monoclonal antibody and an immunotherapy such as an anti-PD-1 antibody and an anti-CTLA-4 antibody compared to the case of treatment with the anti-CD 300c monoclonal antibody alone. In particular, triple co-administration of cl7+αpd-1+αctla-4 reduced tumor size by 90%.

Furthermore, as shown in fig. 68b, 50% Complete Remission (CR) was achieved in the case of co-administration (anti-CD 300c monoclonal antibody and anti-PD-1 antibody) and 70% Complete Remission (CR) was achieved in the case of triple co-administration (anti-CD 300c monoclonal antibody + anti-PD-1 antibody + anti-CTLA-4 antibody), indicating that co-administration produced excellent anticancer effect.

Experimental example 12 identification of improved Long-term survival due to Co-administration

The mice tested in experimental example 13 were examined for long-term survival. As a result, as shown in fig. 69, it was found that the long-term survival rate was also improved when the immunotherapy such as the anti-CD 300c monoclonal antibody, the anti-PD-1 antibody, and the anti-CTLA-4 antibody was used in combination, as compared with the treatment using the anti-CD 300c monoclonal antibody alone.

Experimental example 13 identification of cancer recurrence prevention effect caused by Co-administration

To identify the in vivo cancer recurrence prevention effect caused by co-administration of anti-CD 300c monoclonal antibody CL7 and immunotherapy, 2X 10 was injected subcutaneously ⁵ Colorectal cancer cell line of cells (CT 26) was transplanted into 8-week-old BALB/c mice to prepare syngeneic mouse tumor models, and then experiments were performed as described in experimental example 11 to obtain fully remitted mice. The mice thus obtained were transplanted again with 2X 10 ⁵ Colorectal cancer cell line of cells (CT 26) and observed for 30 days.

As a result, as shown in fig. 70, it was identified that no cancer recurrence or metastasis occurred in the group achieving complete remission by co-administration of the anti-CD 300c monoclonal antibody and immunotherapy. From these results, it can be predicted that individuals who completely alleviate cancer by co-administering an anti-CD 300c monoclonal antibody and an anti-PD-1 antibody (cl7+αpd-1; combination) or co-administering an anti-CD 300c monoclonal antibody, an anti-PD-1 antibody and an anti-CTLA-4 antibody (cl7+αpd-1+αctla-4; triplet) have a systemic protective immune response through continued immune memory, which can inhibit recurrence or metastasis of cancer.

Experimental example 14 identification of the immune memory Effect caused by Co-administration

To analyze effector memory T cells in mice that have been completely relieved in experimental example 13, the mice were sacrificed and spleens were collected therefrom. Spleen cells were then obtained therefrom and stained with antibodies (from Invitrogen) against CD44 and CD62L, which are markers associated with T cell activity. The data were then read with a CytoFLEX flow cytometer and analyzed with FlowJo software.

The results are shown in fig. 71, identifying significant increases in effector memory T cells with co-treatment with anti-CD 300c monoclonal antibodies and immunotherapy. This shows that mice that have been completely relieved have immune memory by increasing effector memory T cells after co-administration (combination) of CL7 and anti-PD-1 antibody or triple co-administration (triple) of CL7, anti-PD-1 antibody and anti-CTLA-4 antibody as predicted in experimental example 13, and thus can inhibit the growth of other cancer cells even if these cells are present.

EXAMPLE 4 Co-administration of anti-CD 300c monoclonal antibodies (CL 10, SL 18) and immunotherapy

Each of the anti-CD 300c monoclonal antibodies (CL 10, SL 18) prepared in example 1 was used in combination with other immunotherapies, e.g., anti-PD-L1 antibodies And anti-PD-1 antibody->The results were observed.

The manufacturer of each immunotherapy was as follows:(AstraZeneca); and->(Merck Sharp&Dohme)。

Experimental example 15 identification of increased ability to differentiate into M1 macrophages

To identify whether the anti-CD 300c monoclonal antibody CL10 or SL18 promotes macrophage differentiation into M1 macrophages, THP-1 cells were cultured at 1X 10 ⁴ Cells/well were dispensed onto 96-well plates and then treated with 10 μg/mL CL10 or SL 18. In addition, to identify effects caused by co-treatment of CL10 or SL18 and immunotherapy, anti-CD 300c monoclonal antibodies were used with 10. Mu.g/mLAnd/or +.>THP-1 cells were treated in combination. Subsequently, in CO ₂ Incubate for 48 hours in incubator, then use ELISA Kit (Human TNF-. Alpha.Quantikine Kit, R&D Systems) detects the level of TNF- α, a differentiation marker for M1 macrophages. The results are shown in fig. 72a (CL 10 combination) and fig. 72b (SL 18 combination).

As shown in FIGS. 72a and 72b, it was identified that the use of CL10 or SL18 andor->In the case of co-treatment of THP-1 cells, the expression level of TNF-alpha was increased. In particular, it was identified that in co-administration of CL10 or SL18 and two antibodies +.>And->The highest levels of TNF-alpha expression were observed.

Experimental example 16: identification of cancer cell proliferation inhibition

To identify the inhibition of cancer cell growth caused by co-administration of anti-CD 300c monoclonal antibody CL10 or SL18 and immunotherapy, a549 (human lung cancer cell line) cells were used to compare cell growth inhibition. Specifically, cells were treated with 2X 10 under 0% FBS ⁴ Cells/well were distributed into 96-well plates and the cells were plated at 6X 10 under 0.1% FBS ³ Cells/well were distributed in 96-well plates. Cells are then treated with CL10 or SL18 alone, or with CL10 or SL18 andand/orCells were co-processed and each was used at a concentration of 10. Mu.g/mL. Incubation was performed for 5 days. Then, treatment was performed with 30. Mu.L/well of CCK-8 (DOJINDO). In CO ₂ Incubate for 4 hours in incubator, and measure absorbance at OD450nm per hour. The results are shown in fig. 73a (CL 10 combination) and fig. 73b (SL 18 combination).

As shown in FIGS. 73a and 73b, it was identified that under 0.1% FBS conditions, the A549 cells were treated with CL10 or SL18 and compared to the cases where the A549 cells were treated with CL10 or SL18 aloneOr->Further increased inhibition of cancer cell proliferation was observed with co-treatment of a549 cells, and the highest cancer cell proliferation was observed with co-treatment of a549 cells with CL10 or SL18 and two antibodies (IMFINZI and keyruda) Inhibition.

From experimental example 15 and this experimental example, it can be seen that the remaining anti-CD 300c monoclonal antibodies produced in example 1, including CL10 and SL18, also exert efficacy through the same mechanism of action as CL7, and have increased efficacy through combination with immunotherapy.

Statistical processing

For the results obtained by the experiments, one-way ANOVA followed by Bonferroni post hoc test (Bonferroni's post-hoc) was used for comparative analysis between experimental groups. When the p value is 0.05 or less, the difference between groups is significant.

<110> Centrics Bio Inc

<120> anti-CD 300C monoclonal antibody and biomarker thereof for preventing or treating cancer

<130> OPA23246

<150> KR 10-2021-0062311

<151> 2021-05-13

<150> KR 10-2021-0062312

<151> 2021-05-13

<150> KR 10-2021-0062313

<151> 2021-05-13

<150> KR 10-2021-0114297

<151> 2021-08-27

<150> KR 10-2022-0042680

<151> 2022-04-06

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Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe Thr Phe

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Phe Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg

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Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg

1 5 10

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<220>

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Ser Ile Ser Gly Gly Gly Tyr Gly Thr Tyr Tyr Ala Asp

1 5 10

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Tyr Cys Ala Arg Ser Thr Val Trp Ala Phe Asp Ile Trp

1 5 10

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Cys Ser Gly Asn Asn Ile Gly Ser Lys Ser Val His Trp

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Asp Val Ser Lys Arg Pro Ser Glu Arg Pro Asp

1 5 10

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Tyr Cys Gln Ser Phe Asp Ser Ser Gly Thr Trp Ile Phe

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<220>

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<212> DNA

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<220>

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tgcagcagct acactagcag cagcactgtg atc 33

<210> 67

<211> 12

<212> PRT

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<220>

<223> CL6 heavy chain CDR1

<400> 67

Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg

1 5 10

<210> 68

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<220>

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<400> 68

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 69

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<400> 69

Tyr Cys Ala Val Ser Gly Ala Gly Arg Gly Phe Phe Asp Tyr Trp

1 5 10 15

<210> 70

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<220>

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Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp

1 5 10 15

<210> 71

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<220>

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Glu Asp Asn Lys Arg Pro Ser Gly Val Pro

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Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Val Ile Phe

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<210> 73

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<220>

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<210> 76

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<400> 76

tgcagtggta acaatatcgg tactagacgc gtg 33

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<210> 79

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Phe Thr Phe Ser Arg Tyr Ala Met Ser Trp Val Arg

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Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

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Tyr Cys Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile Trp

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Cys Ser Gly Asn Asn Ile Gly Thr Arg Arg Val His Trp

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Ser Lys Asn Asn Arg Pro Ser Gly Val Pro

1 5 10

<210> 84

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> CL7 light chain CDR3

<400> 84

Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val Phe

1 5 10

<210> 85

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR1

<400> 85

agcagctacg caatgagctg ggtcaga 27

<210> 86

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR2

<400> 86

gcaattagcg gtagcggtgg tagcacttac tacgcagac 39

<210> 87

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR3

<400> 87

tgcgcacgta gcggtcgtta cgcagacttg acatctgggg ga 42

<210> 88

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> CL8 light chain CDR1

<400> 88

tgcagcggta gcaacagcaa catcggtaac aactacgtg 39

<210> 89

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> CL8 light chain CDR2

<400> 89

aacaacaagc gtcctagtgg tgtg 24

<210> 90

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> CL8 light chain CDR3

<400> 90

tgcagcagct acactagcag cagcactgtg atgttc 36

<210> 91

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR1

<400> 91

Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 92

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR2

<400> 92

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 93

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> CL8 heavy chain CDR3

<400> 93

Tyr Cys Ala Arg Ser Gly Arg Tyr Ala Asp Leu Thr Ser Gly

1 5 10

<210> 94

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CL8 light chain CDR1

<400> 94

Cys Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp

1 5 10 15

<210> 95

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CL8 light chain CDR2

<400> 95

Asp Asn Asn Lys Arg Pro Ser Gly Val Pro

1 5 10

<210> 96

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL8 light chain CDR3

<400> 96

Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Val Met Phe

1 5 10

<210> 97

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR1

<400> 97

agcagctact actggagctg ggtcaga 27

<210> 98

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR2

<400> 98

gcaattagcg gtagcggtgg tagcacttac tacgcagac 39

<210> 99

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR3

<400> 99

tgcgcacgta tcgacgtgta cggtttcgac atctgggga 39

<210> 100

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> CL9 light chain CDR1

<400> 100

tgcagcggta gcactagcaa catcggtact aactacgtg 39

<210> 101

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> CL9 light chain CDR2

<400> 101

aacaacaacc gtcctagtgg tgtg 24

<210> 102

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> CL9 light chain CDR3

<400> 102

tgccagactt gggacagcag cactgacgta gtg 33

<210> 103

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR1

<400> 103

Phe Thr Phe Ser Ser Tyr Tyr Trp Ser Trp Val Arg

1 5 10

<210> 104

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR2

<400> 104

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 105

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL9 heavy chain CDR3

<400> 105

Tyr Cys Ala Arg Ile Asp Val Tyr Gly Phe Asp Ile Trp

1 5 10

<210> 106

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CL9 light chain CDR1

<400> 106

Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Asn Tyr Val Tyr Trp

1 5 10 15

<210> 107

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CL9 light chain CDR2

<400> 107

Asp Asn Asn Asn Arg Pro Ser Gly Val Pro

1 5 10

<210> 108

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL9 light chain CDR3

<400> 108

Tyr Cys Gln Thr Trp Asp Ser Ser Thr Asp Val Val Phe

1 5 10

<210> 109

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR1

<400> 109

tcagcagcta cggtatgcat tgggtcagac a 31

<210> 110

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR2

<400> 110

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 111

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR3

<400> 111

actgcgcaag cggttacggt ctgatggacg tgtggggaca 40

<210> 112

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> CL10 light chain CDR1

<400> 112

gctgcactcg tagcagcggt atcatcgcaa gcaactacgt gca 43

<210> 113

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> CL10 light chain CDR2

<400> 113

cgcaacaacc agcgccctag tggtgtg 27

<210> 114

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> CL10 light chain CDR3

<400> 114

actgcagcag ctacgcaggt aacaacaacc tggtgttcgg 40

<210> 115

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR1

<400> 115

Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg

1 5 10

<210> 116

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR2

<400> 116

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 117

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> CL10 heavy chain CDR3

<400> 117

Tyr Cys Ala Ser Gly Tyr Gly Leu Met Asp Val Trp

1 5 10

<210> 118

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CL10 light chain CDR1

<400> 118

Cys Thr Arg Ser Ser Gly Ile Ile Ala Ser Asn Tyr Val Gln Trp

1 5 10 15

<210> 119

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CL10 light chain CDR2

<400> 119

Arg Asn Asn Gln Arg Pro Ser Gly Val Pro

1 5 10

<210> 120

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CL10 light chain CDR3

<400> 120

Tyr Cys Ser Ser Tyr Ala Gly Asn Asn Asn Leu Val Phe

1 5 10

<210> 121

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR1

<400> 121

ttcagcacct atggcatgca ttgggttcgc 30

<210> 122

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR2

<400> 122

agcgccatca gcggcagcgg cggcagcacc tattatgccg at 42

<210> 123

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR3

<400> 123

tactgtgccc gcggcctgag cggccttgat tattgg 36

<210> 124

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> SK11 light chain CDR1

<400> 124

tgccgctcca gccagggcat caccaactat ctggcctgg 39

<210> 125

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> SK11 light chain CDR2

<400> 125

ctgatctatg atgccagcaa ccgcgccacc ggcatc 36

<210> 126

<211> 45

<212> DNA

<213> artificial sequence

<220>

<223> SK11 light chain CDR3

<400> 126

tattattgtc agcagagcta tagcacccct ctgaccttcg gtcag 45

<210> 127

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR1

<400> 127

Phe Thr Phe Ser Thr Tyr Gly Met His Trp Val Arg

1 5 10

<210> 128

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR2

<400> 128

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 129

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK11 heavy chain CDR3

<400> 129

Tyr Cys Ala Arg Gly Leu Ser Gly Leu Asp Tyr Trp

1 5 10

<210> 130

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK11 light chain CDR1

<400> 130

Cys Arg Ser Ser Gln Gly Ile Thr Asn Tyr Leu Ala Trp

1 5 10

<210> 131

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK11 light chain CDR2

<400> 131

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

1 5 10

<210> 132

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK11 light chain CDR3

<400> 132

Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe

1 5 10

<210> 133

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR1

<400> 133

ccttcagcag ctatgccatg cattgggttc gcca 34

<210> 134

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR2

<400> 134

tgagcgccat cagcggcagc ggcggcgata cctatcatgc cgatagcgt 49

<210> 135

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR3

<400> 135

actactgtac ccgcggcctg agcggctttg attattgggg 40

<210> 136

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK12 light chain CDR1

<400> 136

catgccgcgc cagccagagc atcagcagct atctgaactg gta 43

<210> 137

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK12 light chain CDR2

<400> 137

tgctgatcta tgatgccagc aaccgcgccc ctggcatccc 40

<210> 138

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK12 light chain CDR3

<400> 138

tgtattattg tcagcagagc tatagcatcc ctatcacctt cggtcaggg 49

<210> 139

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR1

<400> 139

Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg

1 5 10

<210> 140

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR2

<400> 140

Ala Ile Ser Gly Ser Gly Gly Asp Thr Tyr His Ala Asp

1 5 10

<210> 141

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK12 heavy chain CDR3

<400> 141

Tyr Cys Thr Arg Gly Leu Ser Gly Phe Asp Tyr Trp

1 5 10

<210> 142

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK12 light chain CDR1

<400> 142

Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

1 5 10

<210> 143

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK12 light chain CDR2

<400> 143

Asp Ala Ser Asn Arg Ala Pro Gly Ile Pro

1 5 10

<210> 144

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK12 light chain CDR3

<400> 144

Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Ile Thr Phe

1 5 10

<210> 145

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR1

<400> 145

ttcagcgatt atgccatgag ctgggttcgc 30

<210> 146

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR2

<400> 146

agcatcagca gcagcagcag ctatatctac tataccgata gc 42

<210> 147

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR3

<400> 147

tactgtgccc gcggcggcta tggctttgat tattgg 36

<210> 148

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> SK13 light chain CDR1

<400> 148

tgccgcgcca gccagagcat cagcagctat ctgaactgg 39

<210> 149

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> SK13 light chain CDR2

<400> 149

ctgatctata gcgccagcag ccgcccacag ggcatc 36

<210> 150

<211> 45

<212> DNA

<213> artificial sequence

<220>

<223> SK13 light chain CDR3

<400> 150

tattattgtc agcagtatga tgatctgcct tttaccttcg gtcag 45

<210> 151

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR1

<400> 151

Phe Thr Phe Ser Asp Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 152

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR2

<400> 152

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Thr Asp

1 5 10

<210> 153

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK13 heavy chain CDR3

<400> 153

Tyr Cys Ala Arg Gly Gly Tyr Gly Phe Asp Tyr Trp

1 5 10

<210> 154

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK13 light chain CDR1

<400> 154

Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

1 5 10

<210> 155

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK13 light chain CDR2

<400> 155

Ser Ala Ser Ser Arg Pro Gln Gly Ile Pro

1 5 10

<210> 156

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK13 light chain CDR3

<400> 156

Tyr Cys Gln Gln Tyr Asp Asp Leu Pro Phe Thr Phe

1 5 10

<210> 157

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR1

<400> 157

ccttcagcaa ctttgcgatc gcctgggttc gcca 34

<210> 158

<211> 46

<212> DNA

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR2

<400> 158

gcgccatcag cggccgcggc accagcacct attatgccga tagcgt 46

<210> 159

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR3

<400> 159

actactgtgc ccgcggcgtg agcggctttg atagctgggg 40

<210> 160

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK14 light chain CDR1

<400> 160

catgccgcgc cagccagagc atcagcagcc atctggcctg gta 43

<210> 161

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK14 light chain CDR2

<400> 161

tgctgatcta tgataccagc aaccgcgcca ccggcatccc 40

<210> 162

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK14 light chain CDR3

<400> 162

tgtactattg tcagcagagc tatagcaccc cttttacctt cggtcaggg 49

<210> 163

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR1

<400> 163

Phe Thr Phe Ser Asn Phe Ala Ile Ala Trp Val Arg

1 5 10

<210> 164

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR2

<400> 164

Ala Ile Ser Gly Arg Gly Thr Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 165

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK14 heavy chain CDR3

<400> 165

Tyr Cys Ala Arg Gly Val Ser Gly Phe Asp Ser Trp

1 5 10

<210> 166

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK14 light chain CDR1

<400> 166

Cys Arg Ala Ser Gln Ser Ile Ser Ser His Leu Ala Trp

1 5 10

<210> 167

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK14 light chain CDR2

<400> 167

Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro

1 5 10

<210> 168

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK14 light chain CDR3

<400> 168

Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe Thr Phe

1 5 10

<210> 169

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR1

<400> 169

ccttcagcag ctatgccatg cattgggttc gcca 34

<210> 170

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR2

<400> 170

tgagcgccat caacggcagc ggcggcagca cctattatgc cgatagcgt 49

<210> 171

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR3

<400> 171

actactgtgc ccgcggcctg cagggctttg attattgggg aca 43

<210> 172

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK15 light chain CDR1

<400> 172

catgccaggc cagccaggat atcaccaact atctgaactg gta 43

<210> 173

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK15 light chain CDR2

<400> 173

tgctgatcta tgatgccagc agcctggaaa ccggcatccc 40

<210> 174

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK15 light chain CDR3

<400> 174

tgtattattg tcagcagagc tatagcaccc ctatcacctt cggtcaggg 49

<210> 175

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR1

<400> 175

Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg

1 5 10

<210> 176

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR2

<400> 176

Ala Ile Asn Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 177

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK15 heavy chain CDR3

<400> 177

Tyr Cys Ala Arg Gly Leu Gln Gly Phe Asp Tyr Trp

1 5 10

<210> 178

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK15 light chain CDR1

<400> 178

Cys Gln Ala Ser Gln Asp Ile Thr Asn Tyr Leu Asn Trp

1 5 10

<210> 179

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK15 light chain CDR2

<400> 179

Asp Ala Ser Ser Leu Glu Thr Gly Ile Pro

1 5 10

<210> 180

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK15 light chain CDR3

<400> 180

Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile Thr Phe

1 5 10

<210> 181

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR1

<400> 181

ccttcagcag ctatgccatg agctgggttc gcca 34

<210> 182

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR2

<400> 182

tgagcgccat caacggcagc ggcggcagca ccctgtatgc cgatagcgt 49

<210> 183

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR3

<400> 183

actactgtgc ccgcggcgtg agcggctttg atagctgggg 40

<210> 184

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK16 light chain CDR1

<400> 184

catgccgcat cagccagagc atcagcagct atctgaactg gta 43

<210> 185

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK16 light chain CDR2

<400> 185

tgctgatcta tgatgccagc ctgcgcgcca ccggcatccc 40

<210> 186

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK16 light chain CDR3

<400> 186

tgtattattg tcagcagagc tataaaaccc ctatcacctt cggtcaggg 49

<210> 187

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR1

<400> 187

Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 188

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR2

<400> 188

Ala Ile Asn Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp

1 5 10

<210> 189

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK16 heavy chain CDR3

<400> 189

Tyr Cys Ala Arg Gly Val Ser Gly Phe Asp Ser Trp

1 5 10

<210> 190

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK16 light chain CDR1

<400> 190

Cys Arg Ile Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

1 5 10

<210> 191

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK16 light chain CDR2

<400> 191

Asp Ala Ser Leu Arg Ala Thr Gly Ile Pro

1 5 10

<210> 192

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK16 light chain CDR3

<400> 192

Tyr Tyr Cys Gln Gln Ser Tyr Lys Thr Pro Ile Thr Phe

1 5 10

<210> 193

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR1

<400> 193

ccttcagcag ctattattgg agctgggttc gcca 34

<210> 194

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR2

<400> 194

tgagcaccat caccggcagc ggcggcagca ccgattatgc caacagcgt 49

<210> 195

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR3

<400> 195

actactgtgc caccggcggc ggcatctttg actattgggg 40

<210> 196

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> SK17 light chain CDR1

<400> 196

catgccaggc cagccagacc atcagcaact atctgaactg gta 43

<210> 197

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> SK17 light chain CDR2

<400> 197

tgctgatcta tgatgccagc aaccgcgcca ccggcatccc 40

<210> 198

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> SK17 light chain CDR3

<400> 198

tgtattattg tcagcagtac aacagctatc ctcctagctt cggtcaggg 49

<210> 199

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR1

<400> 199

Phe Thr Phe Ser Ser Tyr Tyr Trp Ser Trp Val Arg

1 5 10

<210> 200

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR2

<400> 200

Thr Ile Thr Gly Ser Gly Gly Ser Thr Asp Tyr Ala Asn

1 5 10

<210> 201

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK17 heavy chain CDR3

<400> 201

Tyr Cys Ala Thr Gly Gly Gly Ile Phe Asp Tyr Trp

1 5 10

<210> 202

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SK17 light chain CDR1

<400> 202

Cys Gln Ala Ser Gln Thr Ile Ser Asn Tyr Leu Asn Trp

1 5 10

<210> 203

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SK17 light chain CDR2

<400> 203

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

1 5 10

<210> 204

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SK17 light chain CDR3

<400> 204

Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro Ser Phe

1 5 10

<210> 205

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR1

<400> 205

tcaccttcag cgattatcat atgcattggg ttcg 34

<210> 206

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR2

<400> 206

tcagcagcag cggcggctat acctattatg ccga 34

<210> 207

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR3

<400> 207

cccgatcgat acgcctgcct ctggattatt gggg 34

<210> 208

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> SL18 light chain CDR1

<400> 208

gcggcaacaa catcggcagc aaaggcgtgc attggta 37

<210> 209

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SL18 light chain CDR2

<400> 209

atgaagatag caaacgccct agcggcgtgc gtga 34

<210> 210

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> SL18 light chain CDR3

<400> 210

gctatgatag caccaaaggc gtggtgtttg gtgg 34

<210> 211

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR1

<400> 211

Phe Thr Phe Ser Asp Tyr His Met His Trp Val Arg

1 5 10

<210> 212

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR2

<400> 212

Thr Ile Ser Ser Ser Gly Gly Tyr Thr Tyr Tyr Ala Glu

1 5 10

<210> 213

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SL18 heavy chain CDR3

<400> 213

Tyr Cys Ala Arg Ser Ile Arg Leu Pro Leu Asp Tyr Trp

1 5 10

<210> 214

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SL18 light chain CDR1

<400> 214

Cys Ser Gly Asn Asn Ile Gly Ser Lys Gly Val His Trp

1 5 10

<210> 215

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> SL18 light chain CDR2

<400> 215

Glu Asp Ser Lys Arg Pro Ser Gly Val Arg

1 5 10

<210> 216

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> SL18 light chain CDR3

<400> 216

Tyr Cys Gln Ser Tyr Asp Ser Thr Lys Gly Val Val Phe

1 5 10

<210> 217

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR1

<400> 217

tcagcagcta cggtatgcat tgggtcagac aggc 34

<210> 218

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR2

<400> 218

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 219

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR3

<400> 219

actgcgtgcg tggttacggt gcaatggacg tgtggggaca 40

<210> 220

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR1

<400> 220

gctgcactcg tagcagcggt agcatcgcaa gcaactacgt gca 43

<210> 221

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR2

<400> 221

accgcaacaa ccagcgccct agtggtgtgc c 31

<210> 222

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR3

<400> 222

actgcagcag ctacactact agcagcactc tggtgttcgg 40

<210> 223

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR1

<400> 223

Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg

1 5 10

<210> 224

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR2

<400> 224

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 225

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain CDR3

<400> 225

Tyr Cys Val Arg Gly Tyr Gly Ala Met Asp Val Trp

1 5 10

<210> 226

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR1

<400> 226

Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln Trp

1 5 10 15

<210> 227

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR2

<400> 227

Arg Asn Asn Gln Arg Pro Ser Gly Val Pro

1 5 10

<210> 228

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain CDR3

<400> 228

Tyr Cys Ser Ser Tyr Thr Thr Ser Ser Thr Leu Val Phe

1 5 10

<210> 229

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR1

<400> 229

tcagcagcta cgcaatgcat tgggtcagac aggc 34

<210> 230

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR2

<400> 230

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 231

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR3

<400> 231

actgcgcaag cggctacggt ctgatggacg tatggggaca 40

<210> 232

<211> 46

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR1

<400> 232

gctgcactgg tactagcagc gacgtgggta actacaacct ggtgag 46

<210> 233

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR2

<400> 233

acagcaacaa ccagcgccct agtggtgtgc c 31

<210> 234

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR3

<400> 234

actgcagcag ctacactggt agcaacgctc tgttgttcgg 40

<210> 235

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR1

<400> 235

Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg

1 5 10

<210> 236

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR2

<400> 236

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 237

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain CDR3

<400> 237

Tyr Cys Ala Ser Gly Tyr Gly Leu Met Asp Val Trp

1 5 10

<210> 238

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR1

<400> 238

Cys Thr Gly Thr Ser Ser Asp Val Gly Asn Tyr Asn Leu Val Ser Trp

1 5 10 15

<210> 239

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR2

<400> 239

Ser Asn Asn Gln Arg Pro Ser Gly Val Pro

1 5 10

<210> 240

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain CDR3

<400> 240

Tyr Cys Ser Ser Tyr Thr Gly Ser Asn Ala Leu Leu Phe

1 5 10

<210> 241

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR1

<400> 241

tcagcagcta cgcaatgagc tgggtcagac a 31

<210> 242

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR2

<400> 242

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 243

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR3

<400> 243

actgcgcacg ctggcattac agcttcgact actggggaca 40

<210> 244

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR1

<400> 244

gctgccgtgg taacaacatc ggtagcaagc gtgtgca 37

<210> 245

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR2

<400> 245

acagctacaa ccaccgtcct agcggtgtgc c 31

<210> 246

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR3

<400> 246

actgcaacac ttgggacgac agcctggagg gtcctgtgtt cgg 43

<210> 247

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR1

<400> 247

Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 248

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR2

<400> 248

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 249

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain CDR3

<400> 249

Tyr Cys Ala Arg Trp His Tyr Ser Phe Asp Tyr Trp

1 5 10

<210> 250

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR1

<400> 250

Cys Arg Gly Asn Asn Ile Gly Ser Lys Arg Val His Trp

1 5 10

<210> 251

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR2

<400> 251

Ser Tyr Asn His Arg Pro Ser Gly Val Pro

1 5 10

<210> 252

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain CDR3

<400> 252

Tyr Cys Asn Thr Trp Asp Asp Ser Leu Glu Gly Pro Val Phe

1 5 10

<210> 253

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR1

<400> 253

tcagcggcta cgcaatgagc tgggtcagac a 31

<210> 254

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR2

<400> 254

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 255

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR3

<400> 255

actgcgcacg tagtcctagc ggtctgttcg actactgggg aca 43

<210> 256

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR1

<400> 256

gctgcggtgg taacaacatc ggtagcaagc gtgtgca 37

<210> 257

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR2

<400> 257

acaacactag caacaagcat agcggtgtgc c 31

<210> 258

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR3

<400> 258

actgcagcag ctacctacag cagcactctc tgttcgg 37

<210> 259

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR1

<400> 259

Phe Thr Phe Ser Gly Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 260

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR2

<400> 260

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 261

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain CDR3

<400> 261

Tyr Cys Ala Arg Ser Pro Ser Gly Leu Phe Asp Tyr Trp

1 5 10

<210> 262

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR1

<400> 262

Cys Gly Gly Asn Asn Ile Gly Ser Lys Arg Val His Trp

1 5 10

<210> 263

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR2

<400> 263

Asn Thr Ser Asn Lys His Ser Gly Val Pro

1 5 10

<210> 264

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain CDR3

<400> 264

Tyr Cys Ser Ser Tyr Leu Gln Gln His Ser Leu Phe

1 5 10

<210> 265

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR1

<400> 265

agcagctacg caatgagctg ggtcagaca 29

<210> 266

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR2

<400> 266

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 267

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR3

<400> 267

actgcacacg tttcgtgggt gcaatcggtg cattcgacta ctggggaca 49

<210> 268

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR1

<400> 268

gctgcagtgg taacaacatc ggtagccgta gcgtgca 37

<210> 269

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR2

<400> 269

accgcaacaa ccagcgccct agtggtgtgc c 31

<210> 270

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR3

<400> 270

actgcgcagc atgggacgac agcctgagcg gtcctgtgtt cgg 43

<210> 271

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR1

<400> 271

Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 272

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR2

<400> 272

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 273

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain CDR3

<400> 273

Tyr Cys Thr Arg Phe Val Gly Ala Ile Gly Ala Phe Asp Tyr Trp

1 5 10 15

<210> 274

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR1

<400> 274

Cys Ser Gly Asn Asn Ile Gly Ser Arg Ser Val His Trp

1 5 10

<210> 275

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR2

<400> 275

Arg Asn Asn Gln Arg Pro Ser Gly Val Pro

1 5 10

<210> 276

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain CDR3

<400> 276

Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val Phe

1 5 10

<210> 277

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR1

<400> 277

tcagccatta cgcaatgagc tgggtcagac a 31

<210> 278

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR2

<400> 278

ctgcaattag cggtagcggt ggtagcactt actacgcaga cag 43

<210> 279

<211> 55

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR3

<400> 279

actgcgcacg tggttgggac agccctactc tgacatactt cgacagctgg ggaca 55

<210> 280

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR1

<400> 280

gctgcagcgg tactagcagc aacatcggta acaacgacgt gag 43

<210> 281

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR2

<400> 281

accaggacac taagcgtcct agcggtgtgc c 31

<210> 282

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR3

<400> 282

actgcgcagc atgggacgac agcctgagcg gtcctgtgtt cgg 43

<210> 283

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR1

<400> 283

Phe Thr Phe Ser His Tyr Ala Met Ser Trp Val Arg

1 5 10

<210> 284

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR2

<400> 284

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

1 5 10

<210> 285

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain CDR3

<400> 285

Tyr Cys Ala Arg Gly Trp Asp Ser Pro Thr Leu Thr Tyr Phe Asp Ser

1 5 10 15

Trp

<210> 286

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR1

<400> 286

Cys Ser Gly Thr Ser Ser Asn Ile Gly Asn Asn Asp Val Ser Trp

1 5 10 15

<210> 287

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR2

<400> 287

Gln Asp Thr Lys Arg Pro Ser Gly Val Pro

1 5 10

<210> 288

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain CDR3

<400> 288

Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val Phe

1 5 10

<210> 289

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR1

<400> 289

agcagctacg gtatgcattg ggtcaga 27

<210> 290

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR2

<400> 290

gcaatcagcg gtagcggtgg ttacacttac tacgcagac 39

<210> 291

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR3

<400> 291

tgcgcacgct ggcattacag cttcgactac tgggga 36

<210> 292

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR1

<400> 292

tgcagcggta gcagcagcaa catcggtaac aactacgtg 39

<210> 293

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR2

<400> 293

cgcaacaacc agcgccctag tggtgtg 27

<210> 294

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR3

<400> 294

tgccagagct acgacaacag caacgtgctg ttc 33

<210> 295

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR1

<400> 295

Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg

1 5 10

<210> 296

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR2

<400> 296

Ala Ile Ser Gly Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp

1 5 10

<210> 297

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain CDR3

<400> 297

Tyr Cys Ala Arg Trp His Tyr Ser Phe Asp Tyr Trp

1 5 10

<210> 298

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR1

<400> 298

Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp

1 5 10 15

<210> 299

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR2

<400> 299

Arg Asn Asn Gln Arg Pro Ser Gly Val Pro

1 5 10

<210> 300

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain CDR3

<400> 300

Tyr Cys Gln Ser Tyr Asp Asn Ser Asn Val Leu Phe

1 5 10

<210> 301

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> CK1 heavy chain variable region

<400> 301

gaagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc cgctatgcca tgacctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcagc atgagcggca ccggcggcac cacctattat 180

gccgatagcg tgaaaggtcg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcgcc 300

tatggctttg atcattgggg acaaggtact ctggtgaccg tgagcagc 348

<210> 302

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> CK1 light chain variable region

<400> 302

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcgccagcca gagcatcggc aactatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcaacc tggaaaccgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agtagcgcca tcccttatac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 303

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CK1 heavy chain variable region

<400> 303

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Met Ser Gly Thr Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Tyr Gly Phe Asp His Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 304

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> CK1 light chain variable region

<400> 304

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Ala Ile Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 305

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> CK2 heavy chain variable region

<400> 305

gaagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatggca tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcagcggca gcggcaccag catctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcggc 300

accgcctttg attattgggg acaaggtact ctggtgaccg tgagcagc 348

<210> 306

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> CK2 light chain variable region

<400> 306

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcgccagcca gagatcagac aactatctgg cctggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcaacc gcgccaccgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agctatagca ccccttttac cttcggtcag 300

ggcactaaag tggaaaccaa a 321

<210> 307

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CK2 heavy chain variable region

<400> 307

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Thr Ser Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 308

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> CK2 light chain variable region

<400> 308

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Ser Asp Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Thr Lys

100 105

<210> 309

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> CK3 heavy chain variable region

<400> 309

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatgcca tcagctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc accagcggca gcggccgcgc cacctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc gcgcgatacc 300

tggtgggaag gctattttga tctgtgggga caaggtactc tggtgaccgt gagcagc 357

<210> 310

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> CK3 light chain variable region

<400> 310

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc aggccagcca tatcagcacc catctgaact ggtatcagca gaaaccaggt 120

caggctccac gtctgctgat ctatggcgcc agcagccgcg ccaccggcat ccctgatcgc 180

ttctcaggat ctggaagcgg taccgatttt accctgacca tcagccgcct ggaacctgag 240

gactttgccg tgtattattg tcagcagtat aacacctatc ctcctacctt cggtcagggc 300

actaaagtgg aaatcaaa 318

<210> 311

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> CK3 heavy chain variable region

<400> 311

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Thr Ser Gly Ser Gly Arg Ala Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Trp Trp Glu Gly Tyr Phe Asp Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 312

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> CK3 light chain variable region

<400> 312

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser His Ile Ser Thr His Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 313

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> CL4 heavy chain variable region

<400> 313

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcggc agcaactata tgagctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcacc atcagcggca gcggcaccag cacctattat 180

gccgatagct tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcatg 300

tggggcatgg atgtgtgggg acaaggtact ctggtgaccg tgagcagc 348

<210> 314

<211> 312

<212> DNA

<213> artificial sequence

<220>

<223> CL4 light chain variable region

<400> 314

cagagcgtgc tgacccagcc tcctagcgcc tccggtacac caggacagcg cgtgactatt 60

agctgtaccg gcaaacatcg gcacaccgtg aactggtacc agctactgcc tggaactgca 120

cctaagctgc tgatctatct ggatagcgaa cgccctagcg gcgtacctga tcgctttagc 180

ggtagcaaat caggcaccag cgccagcctg gccatcagcg gccttcgctc cgaagatgaa 240

gccgattatt attgtcagag ctatgatagc agcagcgtgg tgtttggtgg cggtaccaag 300

ctgaccgtgc tg 312

<210> 315

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CL4 heavy chain variable region

<400> 315

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Gly Ser Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Met Trp Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 316

<211> 104

<212> PRT

<213> artificial sequence

<220>

<223> CL4 light chain variable region

<400> 316

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Lys His Arg His Thr Val Asn Trp

20 25 30

Tyr Gln Leu Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Leu Asp

35 40 45

Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser

50 55 60

Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Ser Val Val Phe Gly

85 90 95

Gly Gly Thr Lys Leu Thr Val Leu

100

<210> 317

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> CL5 heavy chain variable region

<400> 317

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatgcca tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcagc atcagcggcg gcggctatgg cacctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcagcacc 300

gtgtgggcct ttgatatctg gggacaaggt actctggtga ccgtgagcag c 351

<210> 318

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> CL5 light chain variable region

<400> 318

cagagcgtgc tgacccagcc tcctagcgcc tccggtacac caggacagcg cgtgactatt 60

agctgtagcg gcaacaacat cggcagcaaa agcgtgcatt ggtaccagca actgcctgga 120

actgcaccta agctgctgat ctatgatgtg agcaaacgcc ctagcgagcg tcctgatcgc 180

tttagcggta gcaaatcagg caccagcgcc agtctggcca tcagcgacct tcgctccgaa 240

gatgaagccg attattattg tcagagcttt gatagcagcg gcacctggat ctttggtggc 300

ggtaccaagc tgaccgtgct g 321

<210> 319

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> CL5 heavy chain variable region

<400> 319

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Gly Gly Tyr Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Val Trp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 320

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> CL5 light chain variable region

<400> 320

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Val Ser Lys Arg Pro Ser Glu Arg Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Asp Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Gly Thr Trp

85 90 95

Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 321

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> CL6 heavy chain variable region

<400> 321

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc agtcagtggt 300

gcaggtcgtg gtttcttcga ctactgggga caaggtactc tggtcactgt ctcctca 357

<210> 322

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> CL6 light chain variable region

<400> 322

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagcg gtagcagcag caacattggt agcaactacg tgtactggta tcagcaactc 120

ccaggcaccg ctcctaagct cctgatttac gaggacaaca agcgtcctag tggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgcagc agctacacta gcagcagcac tgtgatcttc 300

ggcggtggga ccaaactgac cgtccta 327

<210> 323

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> CL6 heavy chain variable region

<400> 323

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Ser Gly Ala Gly Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 324

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> CL6 light chain variable region

<400> 324

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

85 90 95

Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 325

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> CL7 heavy chain variable region

<400> 325

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc cgctacgcaa tgagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtagcagc 300

cagggtatct tcgacatctg gggacaaggt actctggtca ctgtctcctc a 351

<210> 326

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> CL7 light chain variable region

<400> 326

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagtg gtaacaatat cggtactaga cgcgtgcatt ggtatcagca actcccagac 120

accgctccta agctcctgat ttacagtaag aacaaccgtc ctagtggtgt gcctgatcgc 180

ttttctgggt ccaagtctgg cacctcagcc tctctggcta tcagtggact tcgctccgag 240

gacgaggctg actattactg cgcagcatgg gacgacagcc tgagcggtcc tgtgttcggc 300

ggtgggacca aactgaccgt ccta 324

<210> 327

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> CL7 heavy chain variable region

<400> 327

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 328

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> CL7 light chain variable region

<400> 328

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly Thr Arg Arg Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Asp Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Lys Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly

85 90 95

Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 329

<211> 354

<212> DNA

<213> artificial sequence

<220>

<223> CL8 heavy chain variable region

<400> 329

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacgcaa tgagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcaaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtagcggt 300

cgttacgcag acttgacatc tgggggacaa ggtactctgg tcactgtctc ctca 354

<210> 330

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> CL8 light chain variable region

<400> 330

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagcg gtagcaacag caacatcggt aacaactacg tgagctggta tcagcaactc 120

ccagacaccc ctcctaagct cctgatttac gacaacaaca agcgtcctag tggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgcagc agctacacta gcagcagcac tgtgatgttc 300

ggcggtggga ccaaactgac cgtccta 327

<210> 331

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> CL8 heavy chain variable region

<400> 331

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Arg Tyr Ala Asp Leu Thr Ser Gly Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 332

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> CL8 light chain variable region

<400> 332

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Asp Thr Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

85 90 95

Thr Val Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 333

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> CL9 heavy chain variable region

<400> 333

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctactact ggagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtatcgac 300

gtgtacggtt tcgacatctg gggacaaggt actctggtca ctgtctcctc a 351

<210> 334

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> CL9 light chain variable region

<400> 334

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagcg gtagcactag caacatcggt actaactacg tgtactggta tcagcaactc 120

ccaggcaccg ctcctaagct cctgatttac gacaacaaca accgtcctag tggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgccag acttgggaca gcagcactga cgtagtgttc 300

ggcggtggga ccaaactgac cgtccta 327

<210> 335

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> CL9 heavy chain variable region

<400> 335

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Asp Val Tyr Gly Phe Asp Ile Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 336

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> CL9 light chain variable region

<400> 336

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Ser Ser Thr

85 90 95

Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 337

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> CL10 heavy chain variable region

<400> 337

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc aagcggttac 300

ggtctgatgg acgtgtgggg acaaggtact ctggtcactg tctcctca 348

<210> 338

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> CL10 light chain variable region

<400> 338

tctgtgctga ctcagccacc ttcagcatct ggtactccag gtcagcgcgt caccatcagc 60

tgcactcgta gcagcggtat catcgcaagc aactacgtgc agtggtatca gcaactccca 120

ggcaccgctc ctaagctcct gatttaccgc aacaaccagc gccctagtgg tgtgcctgat 180

cgcttttctg ggtccaagtc tggcacctca gcctctctgg ctatcagtgg acttcgctcc 240

gaggacgagg ctgactatta ctgcagcagc tacgcaggta acaacaacct ggtgttcggc 300

ggtgggacca aactgaccgt ccta 324

<210> 339

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CL10 heavy chain variable region

<400> 339

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Tyr Gly Leu Met Asp Val Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 340

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> CL10 light chain variable region

<400> 340

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ile Ile Ala Ser Asn

20 25 30

Tyr Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Asn Asn

85 90 95

Asn Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 341

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> SK11 heavy chain variable region

<400> 341

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc acctatggca tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcagcggca gcggcggcag cacctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcctg 300

agcggccttg attattgggg acaaggtact ctggtgaccg tgagcagc 348

<210> 342

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK11 light chain variable region

<400> 342

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gctccagcca gggcatcacc aactatctgg cctggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcaacc gcgccaccgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agctatagca cccctctgac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 343

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK11 heavy chain variable region

<400> 343

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Ser Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 344

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK11 light chain variable region

<400> 344

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Gly Ile Thr Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 345

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> SK12 heavy chain variable region

<400> 345

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatgcca tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcagcggca gcggcggcga tacctatcat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtac ccgcggcctg 300

agcggctttg attattgggg acaaggtact ctggtgaccg tgagcagc 348

<210> 346

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK12 light chain variable region

<400> 346

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcgccagcca gagcatcagc agctatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcaacc gcgcccctgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agctatagca tccctatcac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 347

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK12 heavy chain variable region

<400> 347

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Asp Thr Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Leu Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 348

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> SK12 light chain variable region

<400> 348

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Phe Ser Asp

100 105 110

<210> 349

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> SK13 heavy chain variable region

<400> 349

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc gattatgcca tgagctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcagc atcagcagca gcagcagcta tatctactat 180

accgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcggc 300

tatggctttg attattgggg acaaggtacc ctggtgaccg tgagcagc 348

<210> 350

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK13 light chain variable region

<400> 350

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcgccagcca gagcatcagc agctatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatagc gccagcagcc gcccacaggg catccccgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag tatgatgatc tgccttttac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 351

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK13 heavy chain variable region

<400> 351

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 352

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK13 light chain variable region

<400> 352

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Arg Pro Gln Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asp Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 353

<211> 346

<212> DNA

<213> artificial sequence

<220>

<223> SK14 heavy chain variable region

<400> 353

gaagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc aactttgcga tcgcctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcagcggcc gcggcaccag cacctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcgtg 300

agcggctttg atagctgggg acaaggtact ctggtgaccg tgagca 346

<210> 354

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK14 light chain variable region

<400> 354

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcgccagcca gagcatcagc agccatctgg cctggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat accagcaacc gcgccaccgg catccctgat 180

cgcttctcag gatctgggag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtacta ttgtcagcag agctatagca ccccttttac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 355

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK14 heavy chain variable region

<400> 355

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Ala Ile Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Arg Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Ser Gly Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 356

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK14 light chain variable region

<400> 356

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser His

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 357

<211> 349

<212> DNA

<213> artificial sequence

<220>

<223> SK15 heavy chain variable region

<400> 357

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatgcca tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcaacggca gcggcggcag cacctattat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagacga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcctg 300

cagggctttg attattgggg acaaggtact ctggtgaccg tgagcagca 349

<210> 358

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK15 light chain variable region

<400> 358

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc aggccagcca ggatatcacc aactatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcagcc tggaaaccgg catccctgat 180

cgtttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agctatagca cccctatcac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 359

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK15 heavy chain variable region

<400> 359

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asn Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Thr Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Gln Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 360

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK15 light chain variable region

<400> 360

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 361

<211> 349

<212> DNA

<213> artificial sequence

<220>

<223> SK16 heavy chain variable region

<400> 361

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctatgcca tgagctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcgcc atcaacggca gcggcggcag caccctgtat 180

gccgatagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgcggcgtg 300

agcggctttg atagctgggg acaaggtact ctggtgaccg tgagcagcg 349

<210> 362

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK16 light chain variable region

<400> 362

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc gcatcagcca gagcatcagc agctatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcctgc gcgccaccgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag agctataaaa cccctatcac cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 363

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK16 heavy chain variable region

<400> 363

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asn Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Ser Gly Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 364

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK16 light chain variable region

<400> 364

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ile Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Leu Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Lys Thr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 365

<211> 349

<212> DNA

<213> artificial sequence

<220>

<223> SK17 heavy chain variable region

<400> 365

gaagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc agctattatt ggagctgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcacc atcaccggca gcggcggcag caccgattat 180

gccaacagcg tgaaaggccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc caccggcggc 300

ggcatctttg actattgggg acaaggtact ctggtgaccg tgagcagcg 349

<210> 366

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SK17 light chain variable region

<400> 366

gaaatcgtgc tgacccagag ccctggcacc ctgagcctga gccctggcga acgcgcaaca 60

ctgtcatgcc aggccagcca gaccatcagc aactatctga actggtatca gcagaaacca 120

ggtcaggctc cacgtctgct gatctatgat gccagcaacc gcgccaccgg catccctgat 180

cgcttctcag gatctggaag cggtaccgat tttaccctga ccatcagccg cctggaacct 240

gaggactttg ccgtgtatta ttgtcagcag tacaacagct atcctcctag cttcggtcag 300

ggcactaaag tggaaatcaa a 321

<210> 367

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> SK17 heavy chain variable region

<400> 367

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Thr Gly Ser Gly Gly Ser Thr Asp Tyr Ala Asn Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gly Gly Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 368

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SK17 light chain variable region

<400> 368

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Thr Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 369

<211> 352

<212> DNA

<213> artificial sequence

<220>

<223> SL18 heavy chain variable region

<400> 369

cgagtgcagc tgctggaaag tggaggtgga ctggtgcagc ctggcggcag cctgcgcctg 60

agctgtgccg ccagcggatt caccttcagc gattatcata tgcattgggt tcgccaagca 120

cctggcaaag gcctggaatg ggtgagcacc atcagcagca gcggcggcta tacctattat 180

gccgaaagcg tgaaaagccg ctttaccatc agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgccgaggac accgcagtct actactgtgc ccgatcgata 300

cgcctgcctc tggattattg gggacaaggt actctggtga ccgtgagcag ca 352

<210> 370

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> SL18 light chain variable region

<400> 370

cagagcgtgc tgacccagcc tcctagcgcc tccggtacac caggacagcg cgtgactatt 60

agctgtagcg gcaacaacat cggcagcaaa ggcgtgcatt ggtatcagca actgcctgga 120

actgcaccta agctgctgat ctatgaagat agcaaacgcc ctagcggcgt gcgtgatcgc 180

tttagcggta gcaaatcagg caccagcgcc agcctggcca tcagcggcct tcgctccgaa 240

gatgaagccg attattattg tcagagctat gatagcacca aaggcgtggt gtttggtggc 300

ggtaccaagc tgaccgtgct g 321

<210> 371

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> SL18 heavy chain variable region

<400> 371

Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

His Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Ser Ser Gly Gly Tyr Thr Tyr Tyr Ala Glu Ser Val

50 55 60

Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ile Arg Leu Pro Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 372

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> SL18 light chain variable region

<400> 372

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly Ser Lys Gly Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Val Arg Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Thr Lys Gly Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 373

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain variable region

<400> 373

gaggtgcagc tgttggagtc tggtggaggc ttggtacagt ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgt gcgtggttac 300

ggtgcaatgg acgtgtgggg acaaggtact ctggtcactg tctcctca 348

<210> 374

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain variable region

<400> 374

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcactc gtagcagcgg tagcatcgca agcaactacg tgcagtggta tcagcaactc 120

ccaggcaccg ctcctaagct cctgatttac cgcaacaacc agcgccctag tggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgcagc agctacacta ctagcagcac tctggtgttc 300

ggcggtggga ccaaactgac cgtccta 327

<210> 375

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 heavy chain variable region

<400> 375

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Tyr Gly Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 376

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A10 light chain variable region

<400> 376

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn

20 25 30

Tyr Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Ser Ser

85 90 95

Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 377

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain variable region

<400> 377

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacgcaa tgcattgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcaaaggac actgccgtgt attactgcgc aagcggctac 300

ggtctgatgg acgtatgggg acaaggtact ctggtcactg tctcctca 348

<210> 378

<211> 330

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain variable region

<400> 378

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcactg gtactagcag cgacgtgggt aactacaacc tggtgagctg gtatcagcaa 120

ctcccaggca ccgctcctaa gctcctgatt tacagcaaca accagcgccc tagtggtgtg 180

cctgatcgct tttctgggtc caagtctggc acctcagcct ctctggctat cagtggactt 240

cgctccgagg acgaggctga ctattactgc agcagctaca ctggtagcaa cgctctgttg 300

ttcggcggtg ggaccaaact gaccgtccta 330

<210> 379

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 heavy chain variable region

<400> 379

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Tyr Gly Leu Met Asp Val Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 380

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_A12 light chain variable region

<400> 380

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Asn Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Gly Ser

85 90 95

Asn Ala Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 381

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain variable region

<400> 381

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacgcaa tgagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgctggcat 300

tacagcttcg actactgggg acaaggtact ctggtcactg tctcctca 348

<210> 382

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain variable region

<400> 382

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgccgtg gtaacaacat cggtagcaag cgtgtgcatt ggtatcagca actcccaggc 120

accgctccta agctcctgat ttacagctac aaccaccgtc ctagcggtgt gcctgatcgc 180

ttttctgggt ccaagtctgg cacctcagcc tctctggcta tcactggact tcgctccgag 240

gacgaagctg actattactg caacacttgg gacgacagcc tggagggtcc tgtgttcggc 300

ggtgggacca aactgaccgt ccta 324

<210> 383

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 heavy chain variable region

<400> 383

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp His Tyr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 384

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_E6 light chain variable region

<400> 384

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Gly Asn Asn Ile Gly Ser Lys Arg Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Tyr Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Thr Trp Asp Asp Ser Leu Glu Gly

85 90 95

Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 385

<211> 352

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain variable region

<400> 385

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc ggctacgcaa tgagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtagtcct 300

agcggtctgt tcgactactg gggacaaggt actctggtca ctgtctcctc ag 352

<210> 386

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain variable region

<400> 386

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcggtg gtaacaacat cggtagcaag cgtgtgcatt ggtatcagca actcccaggc 120

accgctccta agctcctgat ttacaacact agcaacaagc atagcggtgt gcctgatcgc 180

ttttctgggt ccaagtctgg cacctcagcc tctctggcta tcagtggact tcgctccgag 240

gacgaggctg actattactg cagcagctac ctacagcagc actctctgtt cggcggtggg 300

accaaactaa ccgtccta 318

<210> 387

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 heavy chain variable region

<400> 387

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Ser Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 388

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_F4 light chain variable region

<400> 388

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Gly Gly Asn Asn Ile Gly Ser Lys Arg Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asn Thr Ser Asn Lys His Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Leu Gln Gln His Ser Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 389

<211> 360

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain variable region

<400> 389

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc ctcttccgcc 60

tcctcctgtg cagcctccgg attcactttc agcagctacg caatgagctg ggtcagacag 120

gcaccaggta agggactgga gtgggtctct gcaattagcg gtagcggtgg tagcacttac 180

tacgcagaca gcgtgaaggg tcgcttcacc atctcacgcg acaactccaa gaacaccctg 240

tacctgcaga tgaacagcct tcgcgcagag gacactgccg tgtattactg cacacgtttc 300

gtgggtgcaa tcggtgcatt cgactactgg ggacaaggta ctctggtcac tgtctcctca 360

360

<210> 390

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain variable region

<400> 390

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagtg gtaacaacat cggtagccgt agcgtgcatt ggtatcagca actcccaggc 120

accgctccta agctcctgat ttaccgcaac aaccagcgcc ctagtggtgt gcctgatcgc 180

ttttctgggt ccaagtctgg cacctcagcc tctctggcta tcagtggact tcgctccgag 240

gacgaggctg actattactg cgcagcatgg gacgacagcc tgagcggtcc tgtgttcggc 300

ggtgggacca aactgaccgt ccta 324

<210> 391

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 heavy chain variable region

<400> 391

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Ser Ser Ala Ser Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

20 25 30

Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Thr Arg Phe Val Gly Ala Ile Gly Ala Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 392

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Cb301_H2L1_G11 light chain variable region

<400> 392

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Asn Ile Gly Ser Arg Ser Val

20 25 30

His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly

85 90 95

Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 393

<211> 363

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain variable region

<400> 393

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc cattacgcaa tgagctgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca attagcggta gcggtggtag cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgtggttgg 300

gacagcccta ctctgacata cttcgacagc tggggacaag gtactctggt cactgtctcc 360

tca 363

<210> 394

<211> 330

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain variable region

<400> 394

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagcg gtactagcag caacatcggt aacaacgacg tgagctggta tcagcaactc 120

ccaggcaccg ctcctaagct cctgatttac caggacacta agcgtcctag cggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgcgca gcatgggacg acagcctgag cggtcctgtg 300

ttcggcggtg ggaccaaact gaccgtccta 330

<210> 395

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 heavy chain variable region

<400> 395

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp Asp Ser Pro Thr Leu Thr Tyr Phe Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 396

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_B5 light chain variable region

<400> 396

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly Asn Asn

20 25 30

Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Asp Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 397

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain variable region

<400> 397

gaggtgcagc tgttggagtc tggtggaggc ttggtacagc ctggaggttc tcttcgcctc 60

tcctgtgcag cctccggatt cactttcagc agctacggta tgcattgggt cagacaggca 120

ccaggtaagg gactggagtg ggtctctgca atcagcggta gcggtggtta cacttactac 180

gcagacagcg tgaagggtcg cttcaccatc tcacgcgaca actccaagaa caccctgtac 240

ctgcagatga acagccttcg cgcagaggac actgccgtgt attactgcgc acgctggcat 300

tacagcttcg actactgggg acaaggtact ctggtcactg tctcctca 348

<210> 398

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain variable region

<400> 398

cagtctgtgc tgactcagcc accttcagca tctggtactc caggtcagcg cgtcaccatc 60

agctgcagcg gtagcagcag caacatcggt aacaactacg tgagctggta tcagcaactc 120

ccaggcaccg ctcctaagct cctgatttac cgcaacaacc agcgccctag tggtgtgcct 180

gatcgctttt ctgggtccaa gtctggcacc tcagcctctc tggctatcag tggacttcgc 240

tccgaggacg aggctgacta ttactgccag agctacgaca acagcaacgt gctgttcggc 300

ggtgggacca aactgaccgt ccta 324

<210> 399

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 heavy chain variable region

<400> 399

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp His Tyr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 400

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> C301_OPALTL_E6 light chain variable region

<400> 400

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser Asn

85 90 95

Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 401

<211> 489

<212> DNA

<213> artificial sequence

<220>

<223> CD300c ECD

<400> 401

ggctattttc ctctgagcca ccccatgacc gtggcgggcc ccgtgggggg atccctgagt 60

gtgcagtgtc gctatgagaa ggaacacagg accctcaaca aattctggtg cagaccacca 120

cagattctcc gatgtgacaa gattgtggag accaaagggt cagcagggaa aaggaatggc 180

cgagtgtcca tcagggacag tcctgcaaac ctcagcttca cagtgaccct ggagaatctc 240

acagaggagg acgcaggcac ctactggtgt ggggtggata caccgtggct ccgagacttt 300

catgatccca ttgtcgaggt tgaggtgtcc gtgttcccgg ccgggacgac cacagcctcc 360

agcccccaga gctccatggg cacctcaggt cctcccacga agctgcccgt gcacacctgg 420

cccagcgtga ccagaaagga cagccccgaa cccagcccac accctggctc cctgttcagc 480

aatgtccgc 489

<210> 402

<211> 163

<212> PRT

<213> artificial sequence

<220>

<223> CD300c ECD

<400> 402

Gly Tyr Phe Pro Leu Ser His Pro Met Thr Val Ala Gly Pro Val Gly

1 5 10 15

Gly Ser Leu Ser Val Gln Cys Arg Tyr Glu Lys Glu His Arg Thr Leu

20 25 30

Asn Lys Phe Trp Cys Arg Pro Pro Gln Ile Leu Arg Cys Asp Lys Ile

35 40 45

Val Glu Thr Lys Gly Ser Ala Gly Lys Arg Asn Gly Arg Val Ser Ile

50 55 60

Arg Asp Ser Pro Ala Asn Leu Ser Phe Thr Val Thr Leu Glu Asn Leu

65 70 75 80

Thr Glu Glu Asp Ala Gly Thr Tyr Trp Cys Gly Val Asp Thr Pro Trp

85 90 95

Leu Arg Asp Phe His Asp Pro Ile Val Glu Val Glu Val Ser Val Phe

100 105 110

Pro Ala Gly Thr Thr Thr Ala Ser Ser Pro Gln Ser Ser Met Gly Thr

115 120 125

Ser Gly Pro Pro Thr Lys Leu Pro Val His Thr Trp Pro Ser Val Thr

130 135 140

Arg Lys Asp Ser Pro Glu Pro Ser Pro His Pro Gly Ser Leu Phe Ser

145 150 155 160

Asn Val Arg

<210> 403

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Heavy chain CDR1

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=G or S

<220>

<221> MISC_FEATURE

<222> (5)

<223> Xaa=S, R, or D

<220>

<221> MISC_FEATURE

<222> (6)

<223> Xaa=N or Y

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa=Y, A, G, or H

<220>

<221> MISC_FEATURE

<222> (9)

<223> Xaa=S, or H

<400> 403

Phe Thr Phe Xaa Xaa Xaa Xaa Met Xaa Trp Val Arg

1 5 10

<210> 404

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Heavy chain CDR2

<220>

<221> MISC_FEATURE

<222> (1)

<223> xaa=t or a

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=G or S

<220>

<221> MISC_FEATURE

<222> (7)

<223> xaa=t or G

<220>

<221> MISC_FEATURE

<222> (8)

<223> Xaa=S or Y

<220>

<221> MISC_FEATURE

<222> (13)

<223> xaa=d or E

<400> 404

Xaa Ile Ser Xaa Ser Gly Xaa Xaa Thr Tyr Tyr Ala Xaa

1 5 10

<210> 405

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Heavy chain CDR3

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=R or S

<220>

<221> MISC_FEATURE

<222> (5)

<223> Xaa=G or S

<220>

<221> MISC_FEATURE

<222> (6)

<223> Xaa=M, S, Y, or I

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa=W, Q, G, or R

<220>

<221> MISC_FEATURE

<222> (8)

<223> xaa=g or L

<220>

<221> MISC_FEATURE

<222> (9)

<223> Xaa=M, I, or P

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa=D, F, or L

<220>

<221> MISC_FEATURE

<222> (11)

<223> xaa=v or D

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa=I, Y, or is absent

<400> 405

Tyr Cys Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp

1 5 10

<210> 406

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Light chain CDR1

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa=T or S

<220>

<221> MISC_FEATURE

<222> (3)

<223> xaa=g or R

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=K, N, or S

<220>

<221> MISC_FEATURE

<222> (5)

<223> Xaa=H, N, or S

<220>

<221> MISC_FEATURE

<222> (6)

<223> Xaa=R, I, or G

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa=H, G, or I

<220>

<221> MISC_FEATURE

<222> (8)

<223> Xaa=T, I, or S

<220>

<221> MISC_FEATURE

<222> (9)

<223> Xaa=R, A, K, or absence of

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa=R, S, G, or is absent

<220>

<221> MISC_FEATURE

<222> (11)

<223> Xaa=N or absence of

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa=Y or absence of

<220>

<221> MISC_FEATURE

<222> (14)

<223> Xaa=N, H, or Q

<400> 406

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Trp

1 5 10 15

<210> 407

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Light chain CDR2

<220>

<221> MISC_FEATURE

<222> (1)

<223> Xaa=L, S, R, or E

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa=D, K, or N

<220>

<221> MISC_FEATURE

<222> (3)

<223> Xaa=S or N

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=E, N, Q, or K

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa=P or R

<400> 407

Xaa Xaa Xaa Xaa Arg Pro Ser Gly Val Xaa

1 5 10

<210> 408

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> anti-CD 300c monoclonal antibody or antigen-binding fragment thereof

Light chain CDR3

<220>

<221> MISC_FEATURE

<222> (3)

<223> Xaa=Q, A, or S

<220>

<221> MISC_FEATURE

<222> (4)

<223> Xaa=S or A

<220>

<221> MISC_FEATURE

<222> (5)

<223> xaa=y or W

<220>

<221> MISC_FEATURE

<222> (6)

<223> xaa=d or a

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa=S, D, or G

<220>

<221> MISC_FEATURE

<222> (8)

<223> Xaa=S, N, or T

<220>

<221> MISC_FEATURE

<222> (9)

<223> Xaa=S, L, N, or K

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa=V, S, N, or G

<220>

<221> MISC_FEATURE

<222> (11)

<223> Xaa=G, L, V, or is absent

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa=P or absence of

<400> 408

Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Phe

1 5 10

Claims

1. An anti-CD 300c monoclonal antibody, or antigen-binding fragment thereof, comprising:

(i) A heavy chain variable region comprising: CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 7. SEQ ID NO: 19. SEQ ID NO: 31. SEQ ID NO: 43. SEQ ID NO: 55. SEQ ID NO: 67. SEQ ID NO: 79. SEQ ID NO: 91. SEQ ID NO: 103. SEQ ID NO: 115. SEQ ID NO: 127. SEQ ID NO: 139. SEQ ID NO: 151. SEQ ID NO: 163. SEQ ID NO: 175. SEQ ID NO: 187. SEQ ID NO: 199. SEQ ID NO: 211. SEQ ID NO: 223. SEQ ID NO: 235. SEQ ID NO: 247. SEQ ID NO: 259. SEQ ID NO: 271. SEQ ID NO:283 and SEQ ID NO:295;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 32. SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO: 92. SEQ ID NO: 104. SEQ ID NO: 116. SEQ ID NO: 128. SEQ ID NO: 140. SEQ ID NO: 152. SEQ ID NO: 164. SEQ ID NO: 176. SEQ ID NO: 188. SEQ ID NO: 200. SEQ ID NO: 212. SEQ ID NO: 224. SEQ ID NO: 236. SEQ ID NO: 248. SEQ ID NO: 260. SEQ ID NO: 272. SEQ ID NO:284 and SEQ ID NO:296; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 9. SEQ ID NO: 21. SEQ ID NO: 33. SEQ ID NO: 45. SEQ ID NO: 57. SEQ ID NO: 69. SEQ ID NO: 81. SEQ ID NO: 93. SEQ ID NO: 105. SEQ ID NO: 117. SEQ ID NO: 129. SEQ ID NO: 141. SEQ ID NO: 153. SEQ ID NO: 165. SEQ ID NO: 177. SEQ ID NO: 189. SEQ ID NO: 201. SEQ ID NO: 213. SEQ ID NO: 225. SEQ ID NO: 237. SEQ ID NO: 249. SEQ ID NO: 261. SEQ ID NO: 273. SEQ ID NO:285 and SEQ ID NO:297; and

(ii) A light chain variable region comprising: CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. SEQ ID NO: 22. SEQ ID NO: 34. SEQ ID NO: 46. SEQ ID NO: 58. SEQ ID NO: 70. SEQ ID NO: 82. SEQ ID NO: 94. SEQ ID NO: 106. SEQ ID NO: 118. SEQ ID NO: 130. SEQ ID NO: 142. SEQ ID NO: 154. SEQ ID NO: 166. SEQ ID NO: 178. SEQ ID NO: 190. SEQ ID NO: 202. SEQ ID NO: 214. SEQ ID NO: 226. SEQ ID NO: 238. SEQ ID NO: 250. SEQ ID NO: 262. SEQ ID NO: 274. SEQ ID NO:286 and SEQ ID NO:298;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 11. SEQ ID NO: 23. SEQ ID NO: 35. SEQ ID NO: 47. SEQ ID NO: 59. SEQ ID NO: 71. SEQ ID NO: 83. SEQ ID NO: 95. SEQ ID NO: 107. SEQ ID NO: 119. SEQ ID NO: 131. SEQ ID NO: 143. SEQ ID NO: 155. SEQ ID NO: 167. SEQ ID NO: 179. SEQ ID NO: 191. SEQ ID NO: 203. SEQ ID NO: 215. SEQ ID NO: 227. SEQ ID NO: 239. SEQ ID NO: 251. SEQ ID NO: 263. SEQ ID NO: 275. SEQ ID NO:287 and SEQ ID NO:299; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 12. SEQ ID NO: 24. SEQ ID NO: 36. SEQ ID NO: 48. SEQ ID NO: 60. SEQ ID NO: 72. SEQ ID NO: 84. SEQ ID NO: 96. SEQ ID NO: 108. SEQ ID NO: 120. SEQ ID NO: 132. SEQ ID NO: 144. SEQ ID NO: 156. SEQ ID NO: 168. SEQ ID NO: 180. SEQ ID NO: 192. SEQ ID NO: 204. SEQ ID NO: 216. SEQ ID NO: 228. SEQ ID NO: 240. SEQ ID NO: 252. SEQ ID NO: 264. SEQ ID NO: 276. SEQ ID NO:288 and SEQ ID NO:300.

2. the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein (i) the heavy chain variable region comprises:

CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 7. SEQ ID NO: 19. SEQ ID NO: 43. SEQ ID NO: 55. SEQ ID NO: 67. SEQ ID NO: 79. SEQ ID NO: 103. SEQ ID NO: 115. SEQ ID NO: 127. SEQ ID NO: 139. SEQ ID NO: 151. SEQ ID NO: 163. SEQ ID NO:199 and SEQ ID NO:211;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO: 104. SEQ ID NO: 116. SEQ ID NO: 128. SEQ ID NO: 140. SEQ ID NO: 152. SEQ ID NO: 164. SEQ ID NO:200 and SEQ ID NO:212; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 9. SEQ ID NO: 21. SEQ ID NO: 45. SEQ ID NO: 57. SEQ ID NO: 69. SEQ ID NO: 81. SEQ ID NO: 105. SEQ ID NO: 117. SEQ ID NO: 129. SEQ ID NO: 141. SEQ ID NO: 153. SEQ ID NO: 165. SEQ ID NO:201 and SEQ ID NO:213; and

(ii) The light chain variable region comprises:

CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. SEQ ID NO: 22. SEQ ID NO: 46. SEQ ID NO: 58. SEQ ID NO: 70. SEQ ID NO: 82. SEQ ID NO: 106. SEQ ID NO: 118. SEQ ID NO: 130. SEQ ID NO: 142. SEQ ID NO: 154. SEQ ID NO: 166. SEQ ID NO:202 and SEQ ID NO:214;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 11. SEQ ID NO: 23. SEQ ID NO: 47. SEQ ID NO: 59. SEQ ID NO: 71. SEQ ID NO: 83. SEQ ID NO: 107. SEQ ID NO: 119. SEQ ID NO: 131. SEQ ID NO: 143. SEQ ID NO: 155. SEQ ID NO: 167. SEQ ID NO:203 and SEQ ID NO:215, respectively; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 12. SEQ ID NO: 24. SEQ ID NO: 48. SEQ ID NO: 60. SEQ ID NO: 72. SEQ ID NO: 84. SEQ ID NO: 108. SEQ ID NO: 120. SEQ ID NO: 132. SEQ ID NO: 144. SEQ ID NO: 156. SEQ ID NO: 168. SEQ ID NO:204 and SEQ ID NO:216.

3. the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein (i) the heavy chain variable region comprises:

CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 43. SEQ ID NO: 79. SEQ ID NO:115 and SEQ ID NO:211;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 44. SEQ ID NO: 80. SEQ ID NO:116 and SEQ ID NO:212; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 45. SEQ ID NO: 81. SEQ ID NO:117 and SEQ ID NO:213; and

(ii) The light chain variable region comprises:

CDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 46. SEQ ID NO: 82. SEQ ID NO:118 and SEQ ID NO:214;

CDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 47. SEQ ID NO: 83. SEQ ID NO:119 and SEQ ID NO:215, respectively; and

CDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 48. SEQ ID NO: 84. SEQ ID NO:120 and SEQ ID NO:216.

4. the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:43, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:44, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 45; and

the light chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:46, and said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:47, and said CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 48.

5. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:79, and said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:80, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:81, and

The light chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:82, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:83, and said CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 84.

6. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:115, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:116, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:117, and

the light chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:118, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:119, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:120, and a sequence of amino acids shown in seq id no.

7. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:211, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:212, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:213, and

the light chain variable region comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence of SEQ ID NO:214, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO:215, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:216, and a sequence of amino acids shown in seq id no.

8. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID No: 303. 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395 and 399; and

the light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID No: 304. 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, and 400.

9. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

the heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID No: 315. 327, 339 and 371; and

the light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID No:316 328, 340 and 372.

10. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof has cross-species reactivity.

11. The anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof is cross-reactive with human and mouse CD300c antigens.

12. An anti-CD 300c monoclonal antibody, or antigen-binding fragment thereof, comprising:

a heavy chain variable region comprising CDR1 to CDR3, said CDR1 to CDR3 comprising amino acid sequences represented by formulae (1) to (3), respectively, and

a light chain variable region comprising CDR1 to CDR3, said CDR1 to CDR3 comprising amino acid sequences represented by formulae (4) to (6), respectively,

FTFX1X2X3X4MX5WVR(1)

in the above-mentioned formula (i), the water,

x1=g or S

X2= S, R or D

X3=n or Y

X4= Y, A, G or H

X5=s or H

X1ISX2SGX3X4TYYAX5(2)

In the above-mentioned formula (i), the water,

x1=t or a

X2=g or S

X3=t or G

X4=s or Y

X5=d or E

YCAX1X2X3X4X5X6X7X8X9W(3)

In the above-mentioned formula (i), the water,

x1=r or S

X2=g or S

X3= M, S, Y or I

X4= W, Q, G or R

X5=g or L

X6= M, I or P

X7= D, F or L

X8=v or D

X9= I, Y or absence of

CX1X2X3X4X5X6X7X8X9X10X11VX12W(4)

In the above-mentioned formula (i), the water,

x1=t or S

X2=g or R

X3= K, N or S

X4= H, N or S

X5= R, I or G

X6= H, G or I

X7= T, I or S

X8= R, A, K or absence of

X9= R, S, G or absence of

X10=n or absence of

X11=y or absence of

X12= N, H or Q

X1X2X3X4RPSGVX5(5)

In the above-mentioned formula (i), the water,

x1= L, S, R or E

X2= D, K or N

X3=s or N

X4= E, N, Q or K

X5 = P or R

YCX1X2X3X4X5X6X7X8X9X10VF(6)

In the above-mentioned formula (i), the water,

x1= Q, A or S

X2=s or a

X3=y or W

X4=d or a

X5= S, D or G

X6= S, N or T

X7= S, L, N or K

X8= V, S, N or G

X9= G, L, V or absence of

X10=p or absent.

13. A pharmaceutical composition for preventing or treating cancer, comprising the anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 as an active ingredient.

14. The pharmaceutical composition of claim 13, wherein the cancer comprises any one or more selected from the group consisting of: colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

15. The pharmaceutical composition of claim 13, wherein the cancer is a solid cancer.

16. The pharmaceutical composition of claim 13, wherein the cancer comprises any one or more selected from the group consisting of: colorectal cancer, lung cancer, melanoma, and breast cancer.

17. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition inhibits proliferation, survival, metastasis, recurrence or therapy resistance of cancer.

18. The pharmaceutical composition of claim 13, further comprising one or more cancer immunotherapies.

19. The pharmaceutical composition of claim 18, wherein the cancer immunotherapy comprises any one or more selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG 3, anti-CD 137, anti-OX 40, anti-CD 276, anti-CD 27, anti-GITR, anti-TIM 3, anti-41 BB, anti-CD 226, anti-CD 40, anti-CD 70, anti-ICOS, anti-CD 40L, anti-BTLA, anti-TCR, and anti-TIGIT.

20. The pharmaceutical composition of claim 18, wherein the cancer immunotherapy comprises any one or more selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4 and anti-CD 47 antibodies.

21. The pharmaceutical composition of claim 18, wherein the cancer immunotherapy comprises any one or more selected from the group consisting of: divaruzumab (durvalumab), pamglizumab (pembrolizumab), nivolumab (nivolumab), αcd47, and ipilimumab (ipilimumab).

22. The pharmaceutical composition of claim 18, wherein the anti-CD 300c antibody or antigen-binding fragment thereof and the cancer immunotherapy are prepared separately and administered simultaneously or separately in a sequential manner.

23. A method for preventing or treating cancer comprising administering an anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12 to an individual in need of prevention or treatment of cancer.

24. The method of claim 23, further comprising administering one or more cancer immunotherapies.

25. The method of claim 23, further comprising determining the expression level of CD300c protein based on a biological sample or data of the individual prior to administering the anti-CD 300c antibody or antigen-binding fragment thereof.

26. The method of claim 25, further comprising determining the therapeutic responsiveness of the anti-CD 300c antibody or antigen-binding fragment thereof based on the determined marker expression level.

27. The method of claim 23, further comprising determining the expression level of one or more markers selected from the group consisting of:

bst2, cd40, cd70, cd86, ccl8, xcl, ccr7, cd80, cd206, msr1, arg1, vegfa, pdgfrb, col a1, hif1a, vcam1, icam1, gzma, gzmb, icos, cd69, ifng, tnf, cd d1, cd1d2, cd38, cxcr6, xcr1, tbx21, stat1, stat4, cxcr3, IL-12b, IL-4, IL-6, IL-13, PD-1, PD-L1, CTLA-4, lag3, tim3, icos, ox40, gitr, hvem, CD, CD28, cma1, timd4, bcl6, cxcl5 and Ccl21a.

28. The method of claim 27, further comprising selecting one or more other cancer immunotherapies for use in combination with an anti-CD 300c antibody or antigen-binding fragment thereof based on the determined expression level of the marker.

29. The method of claim 28, wherein the marker comprises one or more selected from the group consisting of: PD-1, PD-L1, CTLA-4, lag3, tim3, icos, ox40, gitr, hvem, CD27 and CD28.

30. The method of claim 27, wherein the marker comprises one or more selected from the group consisting of: vegfa, pdgfrb, col4a1, hif1a, bst2, CCL8, xcl1, CCR7, CD80, tbx21, stat1, stat4, ifng, cxcr3, IL-6, gzma, icos, cd69, cd1d1, cd38, cxcr6, ox40, gitr, CD27 and CD28.

31. The method of claim 30, further comprising determining that the therapeutic responsiveness of the anti-CD 300c antibody or antigen-binding fragment thereof is good or excellent if the expression level of one or more markers selected from vegfa, pdgfrb, col a1, hif1a and IL-6 is statistically significantly reduced compared to an individual not receiving the anti-CD 300c antibody or antigen-binding fragment thereof.

32. The method of claim 30, further comprising determining that the therapeutic responsiveness of the anti-CD 300-300c monoclonal antibody or antigen-binding fragment thereof is good or excellent if the expression level of one or more markers selected from Bst2, CCL8, xcl, CCR7, CD80, tbx21, stat1, stat4, ifng, cxcr3, gzma, icos, cd69, CD1d1, CD38, cxcr6, ox40, gitr, CD27, and CD28 is statistically significantly increased compared to an individual not receiving the anti-CD 300-300c antibody or antigen-binding fragment thereof.

33. A kit for preventing or treating cancer, comprising:

a composition comprising an anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12; and

instructions for using the antibody or antigen binding fragment thereof.

34. The kit of claim 33, wherein the instructions comprise instructions for use of the antibody or antigen binding fragment thereof in combination with one or more additional cancer therapies.

35. The kit of claim 33, wherein the instructions comprise instructions for measuring the expression level of CD300c protein using a biological sample or data obtained from an individual prior to administration of the antibody or antigen binding fragment thereof.

36. A method of providing information for preventing or treating cancer comprising determining the expression level of CD300c protein based on biological samples or data obtained from an individual in need of prevention or treatment of cancer.

37. The method of claim 36, wherein the information for preventing or treating cancer comprises information about any one or more of: therapeutic responsiveness of a therapeutic agent associated with a CD300c protein (e.g., an anti-CD 300c antibody or antigen binding fragment thereof), selection of a therapeutic agent, selection of an individual to be treated, prognosis of the individual, and survival of the individual.

38. A kit for providing information for preventing or treating cancer, comprising a material for measuring the expression level of CD300c protein using a biological sample or data obtained from an individual in need of prevention or treatment of cancer.

39. An isolated nucleic acid molecule encoding an anti-CD 300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12.

40. An expression vector comprising a nucleic acid molecule according to claim 39.

41. A host cell comprising a nucleic acid molecule according to claim 39.

42. A method for producing an anti-CD 300c monoclonal antibody, or antigen-binding fragment thereof, comprising culturing the host cell of claim 41.