WO2020171171A1 - Anti-hla-dr antibody, and use thereof for cancer therapy - Google Patents

Abstract

[Problem] The present invention addresses the problem of providing a substance that binds to HLA-DR antigen and can exert a cytotoxic activity on cancer cells expressing the HLA-DR antigen, and also providing a therapeutic agent for tumors that contains the substance binding to HLA-DR antigen.　[Solution] The present inventors found that the aforesaid problem can be solved by providing a substance binding to HLA-DR antigen, said substance having heavy chain complementarity-determining regions (CDRs) 1 to 3 and light chain CDRs 1 to 3 with specific amino acid sequences, in particular, an antibody or a humanized antibody derivative against HLA-DR antigen.　Selected drawing: none

Description

Anti-HLA-DR antibody and its use for cancer treatment

The present invention relates to an antibody having a cytotoxic activity specifically against a cancer expressing HLA-DR, and a composition and a method for cancer treatment and cancer test containing the antibody.

Cancer is the leading cause of death in the Japanese people, with one in two suffering from cancer once in their lives and one in three dying due to cancer (2013 Ministry of Health, Labor and Welfare data. ). Therefore, although new cancer therapeutic agents are being developed both in Japan and overseas, satisfactory therapeutic results have not yet been obtained.

The HLA-DR antigen has been reported to be highly expressed in many cancers (leukemia, malignant lymphoma, glioblastoma, melanoma, breast cancer, colon cancer, lung cancer, etc.) and is considered to be a target for cancer treatment. Therefore, preparation of an antibody against this antigen has been attempted, a plurality of antibodies have been commercially available, and preparation of a new antibody has also been attempted (Patent Documents 1 and 2).

So far, although drugs that target HLA-DR, such as IMMU-114 (humanized L243 antibody) and Hu1D10, have been developed (Patent Document 1), their efficacy is part of Phase 1 trials. Only partial response was observed in the cases, and no results that meet the needs of the medical field have been obtained (Non-Patent Document 1).

In addition, deaths due to undeniable adverse events such as acute renal failure, gastrointestinal bleeding, septic shock, aseptic meningitis, and atypical hemolytic uremic syndrome were also reported in these studies. Active development has not been continued. Furthermore, these developed products have not been confirmed to be effective for solid cancer (Non-Patent Document 2).

Furthermore, we have produced antibodies that bind to multiple HLA molecules (HLA-DP, HLA-DQ, HLA-DR) and are examining the cytotoxicity of these antibodies in 3 types of Hodgkin lymphoma cells. This antibody has a characteristic effect of making a huge hole in a cancer cell in a very short time, and has a damaging activity independent of effector cells or complement (Patent Document 2). However, no information is known as to whether this research group is developing with this antibody.

Patent 5214252 Patent 5884139

The present invention provides a binding substance for an HLA-DR antigen capable of exerting cytotoxicity against a cancer cell expressing the HLA-DR antigen, and a binding substance for such an HLA-DR antigen. It is an object to provide a therapeutic drug for a tumor containing

The present inventors have developed a binding substance for HLA-DR antigens, which has heavy chain complementarity determining regions (CDRs) 1 to 3 and light chain CDRs 1 to 3 of a specific amino acid sequence, particularly an antibody or human form against HLA-DR antigens. It has been clarified that the above problems can be solved by providing an antibody derivative, and the present invention has been completed. The present inventors have also revealed that cancer can be treated by utilizing such a binding substance for the HLA-DR antigen.

More specifically, the present application provides the following aspects in order to solve the aforementioned problems:
[1]: Complementarity determining region of heavy chain, CDR1 (SEQ ID No.: 1), CDR2 (SEQ ID No.: 2), CDR3 (SEQ ID No.: 3),
Light chain complementarity determining regions, CDR1 (SEQ ID No.: 4), CDR2 (SEQ ID No.: 5), CDR3 (SEQ ID No.: 6),
An antibody or a humanized antibody derivative having a binding property to an HLA-DR antigen and inducing toxicity to a cancer cell, comprising:
[2]: The human antibody derivative is selected from humanized antibody variants selected from humanized antibodies, chimeric antibodies, multivalent antibodies, and multispecific antibodies or functional fragments thereof, [1] Antibody or humanized antibody derivative;
[3]: The antibody or human antibody derivative according to [1] or [2], wherein the functional fragment is F(ab′)2;
[4]: The amino acid sequence of the heavy chain variable region VH domain of the antibody or human antibody derivative is SEQ ID No.: 8, SEQ ID No.: 12, SEQ ID No.: 16, SEQ ID No.: 20, Any of [1] to [3] selected from SEQ ID No.: 24, SEQ ID No.: 28, SEQ ID No.: 32, SEQ ID No.: 36, and SEQ ID No.: 40 The antibody or humanized antibody derivative described in 1 above;
[5]: The amino acid sequence of the light chain variable region VL domain of the antibody or human antibody derivative is SEQ ID No.: 10, SEQ ID No.: 14, SEQ ID No.: 18, SEQ ID No.: 22, Any one of [1] to [4] selected from SEQ ID No.: 26, SEQ ID No.: 30, SEQ ID No.: 34, SEQ ID No.: 38, and SEQ ID No.: 42 The antibody or humanized antibody derivative described in 1 above;
[6]: The antibody or humanized antibody derivative according to any one of [1] to [5], which induces cytotoxicity to cancer cells but not to normal cells;
[7]: The antibody or humanized antibody derivative according to any one of [1] to [6], wherein the cancer cell is selected from the group consisting of Hodgkin lymphoma, lung cancer, and melanoma;
[8]: The antibody or humanized antibody derivative according to any one of [1] to [7], which is bound to a drug to form an antibody drug complex (ADC).
[9]: A pharmaceutical composition for treating cancer, comprising the antibody or humanized antibody derivative according to any one of [1] to [8];
[10]: The pharmaceutical composition according to [9], wherein the cancer is selected from the group consisting of Hodgkin lymphoma, lung cancer, and melanoma;
[11]: a step of contacting the cancer cells collected from the subject with the antibody or humanized antibody derivative according to any one of [1] to [8] in vitro,
Under culture conditions, a step of measuring whether the cell viability of cancer cells is reduced, or a step of measuring whether the secretion of immunostimulatory substances is enhanced,
A method of measuring cytotoxicity against cancer cells, comprising:
[12]: In the presence of peripheral blood lymphocytes of the same subject, it is measured whether the cell viability of cancer cells is reduced or whether immune cells derived from peripheral blood lymphocytes are activated, [ [11] The method for measuring cytotoxicity against cancer cells according to [11];
[13]: The antibody or humanized antibody derivative according to any one of [1] to [8] is administered to the subject based on the cytotoxicity of cancer cells collected from the subject in vitro. The method for measuring cytotoxicity against cancer cells according to [11] or [12], which comprises measuring the increase in cytotoxicity against cancer cells in the case of
[14]: Cancer when the antibody or humanized antibody derivative according to any one of [1] to [8] is administered to the subject due to enhanced secretion of immunostimulatory substances in vitro A method for measuring cytotoxicity against cancer cells according to [11] or [12], which comprises measuring an increase in cytotoxicity against cells.
[15]: Cytotoxicity of the antibody or human antibody derivative according to any one of [1] to [8] to cancer cells collected from a subject, secretion of an immunostimulator, or immunity A measurement kit containing the above-mentioned antibody for measuring cell activation in vitro.

The binding substance for the HLA-DR antigen of the present invention, particularly an antibody or a humanized antibody derivative, reduces or eliminates a tumor for the treatment of a cancer expressing the HLA-DR antigen and for inhibiting the growth of cancer cells. Can be used for In addition, since this binding substance, particularly an antibody or a human antibody derivative can bind to the HLA-DR antigen, immunization of the HLA-DR antigen in vivo as a test application of cancer cells expressing the HLA-DR antigen. It can be used for biological detection (ELISA, Western blotting, flow cytometry, etc.).

In addition, whether or not the cell viability of the cancer cells is decreased by bringing the cancer cells collected from the subject into contact with the binding substance of the present invention, particularly an antibody or a humanized antibody derivative under culture conditions, or immunity By measuring whether or not the secretion of the activator is enhanced, when the binding substance of the present invention, particularly the antibody or the humanized antibody derivative is administered to a subject having cancer cells, the cancer cells are treated in the subject. It can be examined whether it has cytotoxicity against. When the binding substance of the present invention, particularly an antibody or a humanized antibody derivative, is administered to a subject having cancer cells, it is necessary to determine whether or not the subject has cytotoxicity to the cancer cells under culture conditions. Under the presence of peripheral blood lymphocytes of the same subject, by measuring whether the cell viability of cancer cells is reduced or whether immune cells derived from peripheral blood lymphocytes are activated, You can look it up.

FIG. 1 is a diagram showing the cell survival rate after treating two Hodgkin lymphoma cell lines (L428 cells and KM-H2 cells) with antibody LN-3. FIG. 2 is a diagram showing cell death induction (relative value based on dead cell staining) after treating a lung cancer cell line (Calu-1) with antibody LN-3. Figure 3 shows the cell survival rate of HLA-DR expressing cells ((A) L428 cells, (B) KM-H2 cells) and non-expressing cells ((C) Jurkat cells) treated with antibody LN-3. FIG. FIG. 4 shows the amino acid sequences ((A) heavy chain, (B) light chain) of the variable region of antibody LN-3. FIG. 5-1 is a diagram showing (A) antibody binding strength to each peptide by epitope mapping using a peptide array designed based on the human HLA-DRβ1 derived sequence. FIG. 5-2 is a diagram showing a region in the HLA-DRβ1 three-dimensional structure of the (B) antibody-binding peptide by epitope mapping using a peptide array designed based on the human HLA-DRβ1-derived sequence. FIG. 6 is a diagram showing that the BP1206 chimeric antibody has higher cytotoxicity than the antibody LN-3. FIG. 7-1 is a diagram showing the effect of BP1206 chimeric antibody on tumor growth in a L428 cell subcutaneous transplant model ((A) tumor volume, (B) body weight). FIG. 7-2 is a diagram showing the effect of BP1206 chimeric antibody on tumor growth in a L428 cell subcutaneous transplant model ((C) tumor weight at autopsy after 42 days). FIG. 8-1 is a graph showing the dose response of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((A) tumor volume, (B) body weight). FIG. 8-2 is a graph showing the dose responsiveness of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((C) tumor weight at necropsy after 42 days). FIG. 9 is a diagram showing the effect of a surrogate antibody (anti-mouse MHC class II antibody) against the BP1206 chimeric antibody on tumor growth in a mouse T cell lymphoma cell line E.G7 cell subcutaneous transplant model ((A) tumor Volume, (B) body weight). FIG. 10-1 is a diagram showing that immunity induction to the model antigen (Ova) occurs in the mouse individual body of the anti-mouse MHC class II antibody administration group. FIG. 10-2 is a diagram showing that administration of anti-mouse MHC class II antibody did not alter the composition of dendritic cells (DC) in the spleen of mouse individuals. FIG. 11 is a diagram showing a cell presence ratio when PBMCs of healthy subjects were treated with the BP1206 chimeric antibody, as compared with those before treatment. FIG. 12 is a diagram showing that the antibody of the present invention has cytotoxic activity against two types of human melanoma-derived cell lines and two types of human lung cancer-derived cell lines. FIG. 13 is a diagram showing that the antibody of the present invention has cytotoxic activity against three types of human melanoma-derived cell lines, and the combined effect of the antibody of the present invention and vemurafenib. Figure 14 is a diagram showing cell death induction after treatment of a melanoma cell line with a BP1206 chimeric antibody (relative value based on viable cell mass measurement) ((A) HT144 cells, BP1206 chimeric antibody in combination with cisplatin, ( (B) HT144 cells, BP1206 antibody in combination with Vemurafenib, (C) A375 cells, BP1206 antibody in combination with Vemurafenib). FIG. 15 is a diagram showing changes in cell shape when a Hodgkin lymphoma cell line (L428 cell) was treated with a BP1206 chimeric antibody. FIG. 16 shows the effect of BP1206 chimeric antibody on HMGB1 secretion in the L428 cell subcutaneous transplant model (left) and infiltration of the antibody into tumor tissue (right). FIG. 17-1 is a diagram showing a variable region amino acid sequence ((A) heavy chain) of a humanized antibody. FIG. 17-2 is a diagram showing a variable region amino acid sequence ((B) light chain) of a humanized antibody. FIG. 18 is a diagram showing cell death induction (relative value based on dead cell staining) when a Hodgkin lymphoma cell line (L428 cell) was treated with a BP1206 chimeric antibody or a BP1206 humanized antibody. FIG. 19 is a diagram showing that expression of the HLA-DR antigen was observed in nearly half of the tumor tissues examined, although there were variations in the amount of expression and the proportion of expressing cells. .. FIG. 20 shows that in various cancer types, a correlation was observed between the expression level of the target antigen of the antibody of the present invention in cell lines and the induction of cell death by the addition of the antibody of the present invention. FIG. 6 shows that the higher the target antigen expression level in E. coli, the higher the cell death inducing activity.

The terms used in the present invention are defined as follows:
(A) HLA-DR: one of the major molecules that make up HLA (human leukocyte antigen) Class II, which is an antigen-presenting molecule;
(B) Human antibody derivative: a human antibody variant selected from a humanized antibody or a chimeric antibody, or a functional fragment thereof;
(C) humanized antibody: an antibody produced in a non-human animal body by recombination of a portion other than the heavy chain and light chain complementarity determining regions (CDR) into a human antibody gene;
(D) chimeric antibody: an antibody produced recombinantly so that the variable region of the antibody produced in the non-human animal body is bound to the constant region of the human antibody gene;
(E) Functional fragment of human antibody variant: means a part (partial fragment) of a human antibody variant (humanized antibody or chimeric antibody), which retains the action of the antibody on the antigen ( For example, specifically includes F(ab')2, Fab', Fab, single chain Fv (scFv) and the like).

The present invention provides, in one aspect, a binding substance for an HLA-DR antigen, particularly an antibody or a humanized antibody derivative, which has a binding property for an HLA-DR antigen and induces toxicity to cancer cells. You can The binding substance of the present invention, particularly an antibody or a humanized antibody derivative, is used for treatment of cancer cells expressing HLA-DR antigen, for inhibiting the growth of cancer cells, and for reducing or eliminating tumors. be able to.

Antibodies and Human Antibody Derivatives In one aspect, the present invention provides antibodies or human antibody derivatives having binding properties to HLA-DR antigens. The antibody or human-type antibody derivative of the present invention comprises a total of 6 positions of complementarity determining regions (CDRs) 1 to 3 of heavy chain and CDRs 1 to 3 of light chain which are the same as those of the antibody having the binding property to HLA-DR antigen. It is characterized by having the amino acid sequence of. Examples of such amino acid sequences of CDRs at 6 positions of an antibody having binding properties to HLA-DR antigens include CDR1 (SEQ ID No.: 1) and CDR2 (SEQ ID No.: 2) of heavy chain. , CDR3 (SEQ ID No.: 3), and light chain CDR1 (SEQ ID No.: 4), CDR2 (SEQ ID No.: 5), CDR3 (SEQ ID No.: 6) Include an antibody or human-type antibody derivative specified by a combination of CDRs other than the above-mentioned combination of CDRs, as long as it has the characteristic of binding to the HLA-DR antigen.

The antibody or humanized antibody derivative of the present invention is also characterized in that it has the ability to induce toxicity to cancer cells expressing the HLA-DR antigen. The toxicity to the cancer cells expressing the HLA-DR antigen may occur in vitro or in vivo. Whether or not it has the ability to induce cytotoxicity to cancer cells depends on the ability to actually induce cytotoxicity to cancer cells from among antibodies having binding properties to HLA-DR antigen. It can be obtained by screening.

The screening of the ability of the antibody or human antibody derivative to induce cytotoxicity to cancer cells can be performed in vivo or in vitro. In-vivo screening is performed by transplanting target cancer cells into animals such as immunodeficient mice such as nude mice and SCID mice, and tumors in the body when the antibody or humanized antibody derivative of the present invention is administered. This can be done by measuring the change in mass size. Screening performed in vitro can be performed by contacting a target cancer cell with the antibody or human antibody derivative of the present invention under culture conditions and examining whether the cancer cell causes cell death.

The cytotoxicity of the antibody of the present invention or the human antibody derivative against cancer cells is that the antibody or the human antibody derivative of the present invention bound to the cancer cell is a natural killer cell (NK cell), macrophage, neutrophil, Antibody-dependent cellular cytotoxicity induced by stimulating effector cells selected from the group consisting of eosinophils, complement-dependent cytotoxicity induced by stimulating the complement system, or these external factors It may be due to any aspect of physical destruction of cells due to independent antibody binding.

When simply referred to as “antibody” in the present invention, the antibody may be derived from any animal species of mammals, and the species from which the antibody is derived are not limited to humans, and include mouse, rat, guinea pig, hamster, It may be a rabbit or the like.

In the present invention, it is also a human antibody derivative of the above-mentioned antibody, as long as it has a binding property to HLA-DR antigen and has a functional characteristic of inducing toxicity to cancer cells. Good. When referred to as a human antibody derivative in the present invention, in one aspect, it has the amino acid sequence of the CDR at 6 positions of the above antibody, and the amino acid sequence of the constant region derived from a human antibody, and the other amino acid sequences are derived from the original antibody. It is possible to provide a derivative of an antibody, which is characterized by being a combination of the amino acid sequence of and the amino acid sequence of human antibody. Examples of such antibody derivatives include humanized antibodies in which amino acid sequences derived from human antibodies have been substituted for the regions other than the complementarity determining region (CDR) of the above-mentioned "antibody", or the variable regions of the above antibodies are the constant regions of human antibodies. Chimeric antibodies such as those linked to regions, multivalent antibodies in which one type of antibody has multiple antigen binding sites, and multispecific antibodies in which one type of antibody has multiple specificities (bispecific antibodies) are included However, other than these are also included.

In one aspect, examples of the amino acid sequence of the heavy chain variable region VH domain of the antibody or humanized antibody derivative of the present invention include: SEQ ID No.:8, SEQ ID No.:12, SEQ ID No.:16, SEQ Amino acid sequence described in either ID No.: 20, 20, SEQ ID No.: 24, SEQ ID No.: 28, SEQ ID No.: 32, SEQ ID No.: 36, or SEQ ID No.: 40 Can be raised.

In one aspect, examples of the amino acid sequence of the light chain variable region VL domain of the antibody or humanized antibody derivative of the present invention include SEQ ID No.: 10, SEQ ID No.: 14, SEQ ID No.: 18, SEQ Amino acid sequence described in any of ID No.:22, SEQ ID No.:26, SEQ ID No.:30, SEQ ID No.:34, SEQ ID No.:38, or SEQ ID No.:42. Can be raised.

In the present invention, the human antibody derivative of the present invention also includes the functional fragment of the above-mentioned antibody or human antibody derivative. Functional fragments of the antibody or human antibody derivative of the present invention include F(ab')2, Fab', Fab, single-chain Fv (scFv) and the like. The functional fragment of the present invention may be any of these as long as it is characterized by being capable of inducing toxicity to cancer cells, and for example, F(ab')2 fragment or the like can be used. , Can be used as such a functional fragment.

Obtaining Antibody or Human Antibody Derivative The antibody or human antibody derivative of the present invention can be obtained by culturing antibody-producing cells collected from the animal body of the above-mentioned species of origin, but the antibody or human antibody derivative can also be obtained. It is also possible to design a vector for protein expression containing a DNA sequence capable of defining the amino acid sequence of, to introduce the vector into cells for protein production, and obtain it recombinantly.

The DNA sequence capable of defining the amino acid sequence of the antibody or human antibody derivative of the present invention is a method for obtaining it from cells producing the desired antibody or human antibody derivative, and the animal species used in the expression system based on the amino acid sequence. It can be prepared by a method of designing based on the optimized codon or a method of using these methods in combination.

Obtained by incorporating the prepared DNA sequence into an expression vector suitable for the cell type for protein expression (such as CHO cells) for which the antibody or human antibody derivative is to be expressed, and introducing it into the cell type for protein expression Can be obtained using a method well known to those skilled in the art.

The DNA sequences defining the amino acid sequences of the heavy chain variable region VH domain of the antibody or human antibody derivative of the present invention exemplified above are, for example, as follows:

The DNA sequence defining the amino acid sequence of the light chain variable region VL domain of the antibody or human antibody derivative of the present invention exemplified above is, for example, as follows:

Use of Antibody or Human Antibody Derivative The antibody or human antibody derivative of the present invention is characterized in that it has the ability to induce cytotoxicity against cancer cells described above, and in one embodiment, for treating or preventing cancer. A pharmaceutical composition comprising the antibody or humanized antibody derivative of the present invention, which induces toxicity to cancer cells in a subject in need thereof, can be provided.

Cancer cells that can be targeted in the present invention include, for example, leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Burkitt lymphoma, malignant lymphoma, diffuse lymphoma, follicular lymphoma), myeloma (including multiple myeloma), melanoma, lung cancer, breast cancer, colon cancer, kidney cancer, gastric cancer, ovarian cancer, pancreas Cancer, cervical cancer, endometrial cancer, endometrial cancer, esophageal cancer, liver cancer, head and neck cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate Cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, capsular tumor, male embryoma, endometrial hyperplasia, endometriosis, germinoma, fibrosarcoma, Kaposi's sarcoma, hemangiomas, cavernous hemangiomas, Hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, blastoma, osteogenic sarcoma, smooth muscle Included are cells from a cancer selected from the group consisting of tumors, thyroid sarcomas, and Wilms tumors. In the embodiment of the present invention, the cancer cell is preferably a cancer cell that expresses an HLA-DR antigen, for example Hodgkin lymphoma, lung cancer, melanoma, cervical cancer, endometrial cancer, ovarian cancer, head. Cells derived from cervical cancer are preferred.

The antibody or humanized antibody derivative of the present invention is characterized in that it induces cytotoxicity to target cancer cells as described above, but does not induce cytotoxicity to normal cells. That is, when administered to a living body, cytotoxicity is induced only in cancer cells that express the target HLA-DR antigen, and in the case of normal cells that express the HLA-DR antigen, clinically, It is required not to induce potentially problematic cytotoxicity.

In the present invention, the antibody or humanized antibody derivative of the present invention can be provided as a composition in combination with another antibody or another drug such as an anticancer agent. In one embodiment, the antibody or humanized antibody derivative of the present invention can be bound to a drug to form an antibody drug complex (ADC).

In the present invention, a formulation containing the antibody or humanized antibody derivative of the present invention together with a physiologically acceptable diluent or carrier can also be provided. Suitable carriers include buffers (phosphate buffer, citrate buffer, acetate buffer, etc.), salts (sodium chloride, etc.), sugars (glucose, trehalose, mannitol, sorbitol, etc.), additives (arginine, etc.). Amino acids, surfactants such as polysorbates, etc.), but are not limited to these. Alternatively, the antibody or humanized antibody derivative of the present invention can be reconstituted and used by freeze-drying (freeze-drying) and adding a buffer aqueous solution as described above when necessary. The preparation containing the antibody or human antibody derivative of the present invention can be administered in various dosage forms, for example, a parenteral preparation such as an injection or a drip.

The dose of the antibody or humanized antibody derivative of the present invention varies depending on the symptoms, age, body weight, etc., but usually for parenteral administration, 0.01 mg to 1000 mg per dose, preferably 1 mg to 10 mg per body weight per day. Depending on the type of cancer, it can be administered via an appropriate administration route such as intraperitoneal injection, subcutaneous injection, intramuscular injection, intratumoral injection, or intravenous injection.

The present invention is based on the above-mentioned characteristic of having an ability to induce cytotoxicity to cancer cells, and in another aspect, a subject in need of treatment or prevention of cancer is treated with the antibody or human of the present invention. Methods of treating or preventing cancer in a subject can also be provided that include administering an effective amount of a type antibody derivative. Treatment or prevention of cancer with the antibody or human antibody derivative of the present invention occurs when the antibody or human antibody derivative causes cytotoxicity to cancer cells in the body.

When the antibody or humanized antibody derivative of the present invention is administered to a living body for treating or preventing cancer, an adjuvant (for example, Clin. Microbiol) is used so that cell-mediated immunity or humoral immunity is effectively established in vivo. Rev., 7:277-289, 1994, etc.), or liposomal preparations, particulate preparations bound to beads with a diameter of several μm, preparations bound with lipids, etc. It can also be administered in the form of dosage forms.

In Vitro Use of Antibodies and Human-Type Antibody Derivatives The antibody or human-type antibody derivative of the present invention is for detecting the HLA-DR antigen in a sample based on the characteristic that it has a binding property to the HLA-DR antigen. Can be used for Specifically, the antibody or humanized antibody derivative of the present invention is a purification method using an antibody such as an immunoprecipitation method, an agglutination reaction, or a magnetic bead method for a sample containing cancer cells collected from a subject. It can be used in carrying out various detection methods that can be performed using an antibody, such as an ELISA method, a Western blot, an immunoassay such as immunohistochemistry, and immunocytochemistry such as flow cytometry. In each case, the antibody or humanized antibody derivative of the present invention can be detected using a detection label generally known to those skilled in the art (eg, fluorescence, DAB, enzyme, etc.).

The antibody or human antibody derivative of the present invention also has a binding property to the HLA-DR antigen and induces toxicity to cancer cells, based on the characteristics of the antibody or human antibody derivative of the present invention. , Can be used to measure cytotoxicity against cancer cells in a subject. The method for measuring the cytotoxicity against cancer cells in such a subject includes the following steps:
A step of contacting a cancer cell collected from a subject with an antibody of the present invention or a humanized antibody derivative under culture conditions (that is, in vitro),
Under culture conditions, a step of measuring whether the cell viability of cancer cells is reduced, or a step of measuring whether the secretion of immunostimulatory substances is enhanced,
Examples of the method for measuring cytotoxicity against cancer cells using the antibody or humanized antibody derivative of the present invention containing

In this method, whether or not the cell viability of cancer cells is reduced by contact with the antibody of the present invention or a humanized antibody derivative under culture conditions is determined in the presence of peripheral blood lymphocytes of the same subject. It can be carried out by measuring whether or not the cell viability of cancer cells is reduced by contact with the antibody of the invention or the humanized antibody derivative. By this method, by measuring the cytotoxicity of the cancer cells collected from the subject under culture conditions, when the antibody or humanized antibody derivative of the present invention is administered to the subject, ( in vivo) cytotoxicity can be measured.

In addition, whether or not the secretion of the immunostimulatory substance is enhanced as a result of activation of peripheral blood lymphocyte-derived immune cells by the antibody of the present invention or the humanized antibody derivative under culture conditions is determined by the same test. It can be carried out by measuring whether or not immune cells derived from peripheral blood lymphocytes contacted with the antibody or humanized antibody derivative of the present invention are activated in the presence of peripheral blood lymphocytes in the body. By this method, by measuring the increase in the secretion of the immunostimulatory substance, when the antibody or humanized antibody derivative of the present invention is administered to the subject, cytotoxicity in the subject (in vivo) It is possible to measure activation of immune cells such as lymphocytes possessed and the resulting increase in cytotoxicity to cancer cells.

The present invention also comprises in vitro measurement of cytotoxicity against cancer cells collected from a subject, secretion of an immunostimulatory substance, or activation of immune cells, including the antibody or humanized antibody derivative of the present invention. A measurement kit for doing so can also be provided.

The present invention will be specifically described below with reference to examples. The examples given below do not limit the invention in any way.

Example 1: Identification of antibody clone having cytotoxicity In this example, an antibody having cytotoxicity against human cancer cell-derived cells was identified from among existing anti-HLA-DR antibodies.

Specifically, we evaluated the cytotoxicity of the commercially available mouse-derived anti-HLA-DR antibody LN-3 (MAB13305, Ab Ab) to human Hodgkin lymphoma cell lines.

As human Hodgkin lymphoma cell lines, L428 cells (DSMZ, ACC197) and KM-H2 cells (DSMZ, ACC8) that express HLA-DR were used. 2x10e6 cells/ml of cells in RPMI1640 medium (Wako Pure Chemical 189-02025) containing 10% FBS (Equitech-bio.Inc SFBM30-0500) and 1% penicilin-streptmycin (Nacalai Tesque 09367-34). Was prepared into 100 μl each in a Fischer tube (Thermo Fisher, 04-978-145), and antibody LN-3 was added to a final concentration of 1 μg/ml. After incubating in a 37°C water bath for 2 hours, cells were collected by centrifugation, and cells that had undergone apoptosis (dead cells) were stained with Annexin VPC (Biolegend 640941) and 7-AAD (Biolegend 420404), It was subjected to flow cytometric analysis. Cells in which neither Annexin V APC nor 7-AAD were stained were judged as live cells, and the survival rate was calculated.

Antibody LN-3 showed the action of reducing the survival rate of L428 cells to 11.3% and the survival rate of KM-H2 cells to 23.2% (Fig. 1). Here, the survival rate was calculated with the survival rate of each cell treated with the isotype control antibody as 100%. L428 cells and KM-H2 cells, as a result of examination by flow cytometry, since they show the same level of HLA-DR expression, the results of this Example, due to the difference in affinity between the antibody and cells, It was expected that the cytotoxic activity of the antibodies would be different.

Example 2: Evaluation of cytotoxicity against solid cancer cells In this example, the cytotoxicity of existing anti-HLA-DR antibodies against solid cancer cells was examined.

As the solid cancer cells to be evaluated, the lung cancer cell line Calu-1 (ATCC HTB-46), in which high expression of HLA-DR was confirmed in the presence of IFNγ, was adopted.

Calu-1 cells were prepared to 1×10 ⁵ cells/ml using MyCoy's 5a Medium (SH30200.01, Hyclone) containing 10% FBS, and IFN-γ was added to a final concentration of 2 ng/ml. Then, 100 μl of each was seeded on a 96-well plate. IncuCyte (registered trademark) Annexin V Red Reagent (Essen Bio, 4641) and antibody LN-3 (final concentration 1 μg/ml) were added and subjected to analysis by an Incucyte real-time analyzer (Essen Bio). Since apoptotic cells were stained with Annexin V Red Reagent, cell death induction was evaluated based on the change in fluorescence intensity after 15 hours.

In antibody LN-3, cell death induction was observed in many cells including cancer cells, which was higher than that of cycloheximide, which has a cell growth inhibitory effect, and the cytotoxicity of the antibody to Calu-1 was confirmed. Further, the cytotoxicity was enhanced by antibody cross-linking by the addition of anti-mouse IgG antibody, suggesting that the mechanism of action of the antibody may depend on the polymerization of the target molecule on the cell membrane (Fig. 2). ).

Example 3: Evaluation of target specificity In this example, the antibody LN-3, which was found to be cytotoxic in Example 2, was evaluated for target specificity of cytotoxicity.

A cytotoxicity test was performed on L428 cells, KM-H2 cells, which are human Hodgkin lymphoma cell lines that express HLA-DR, and Jurkat cells (ATCC TIB-152), which are HLA-DR non-expressing cells. Cytotoxicity was measured by the same method as in Example 1 except for the antibody concentration.

The antibody LN-3 shows dose-dependent cytotoxicity against HLA-DR expressing cells (Fig. 3(A) and (B) for L428 cells and KM-H2 cells, respectively) and to Jurkat cells. On the other hand, it showed no cytotoxicity (FIG. 3(C)), indicating that the induced cytotoxicity was HLA-DR specific (FIG. 3).

Example 4: Determination of antibody amino acid sequence In this example, the amino acid sequence of antibody LN-3 was analyzed by mass spectrometry. Samples for mass spectrometric analysis were prepared by two methods, in gel digestion and in solution digestion.

In in-gel digestion, after performing SDS-PAGE on a sample of antibody LN-3 under reducing conditions, the band corresponding to the antibody component was excised, deglycosylated and reductively alkylated, and treated with 6 proteases (Pepsin , Trypsin, AspN, Chymotrypsin, LysC, Elastase) were used to fragment the antibody protein into peptides.

In in-solution digestion, a sample of antibody LN-3 was reductively alkylated in solution, and then the protein was fragmented into peptides with 6 proteases (Pepsin, Trypsin, AspN, Chymotrypsin, LysC, and Elastase).

MS/MS analysis was performed on the fragmented peptide using Thermo Fisher Q-Exactive, and the sequence was determined by de novo. The leucine and the isoleucine having the same mass were distinguished from each other by using w-ion having different masses generated by HCD cleavage of a peptide fragment having the amino acid at the N-terminus.

The determined amino acid sequence is shown in Fig. 4. In this figure, FIG. 4(A) shows the heavy chain variable region amino acid sequence of antibody LN-3, and FIG. 4(B) shows the light chain variable region amino acid sequence of antibody LN-3.

Example 5: Determination of epitope In this example, the epitope in the amino acid sequence of HLA-DR to which antibody LN-3 binds was analyzed by peptide array.

Since the antibody LN-3 targets the HLA-DRβ1 antigen, the reported amino acid sequence of HLA-DRβ1 (sequence ID: P04229 HLA class II, histocompatibility antigen, extracellular domain of DRB1-1 beta chain (amino acids 30- 227)), a total of 62 peptides with a 15-residue length from the N-terminus of the protein were provided with a 3-residue offset (12-residue overlap) and printed on a glass slide to form a peptide array. It was made. The glass slide was divided into 20 sections in advance, and all the peptides (62) were spotted in each section at N=3. After adding antibody clones to each section of the peptide array and incubating, a secondary antibody (anti-mouse antibody, Antimouse IgG (H+L), Thermo_84545) labeled with fluorescence was reacted and detected with a fluorescence scanner.

From the fluorescence intensity obtained from each peptide spot, the antibody binding strength of antibody LN-3 to each peptide fragment was quantified (Fig. 5(A)). As a result, the binding of the antibody LN-3 to the peptides at four positions ((1) to (4) in FIG. 5(A)) was confirmed, and the sequence was identified as a sequence recognizable by the antibody clone. The HLA-DRβ1 portions of (1) to (4) are shown on the three-dimensional structure of HLA-DRβ1 (FIG. 5(B)).

Example 6: Production of chimeric antibody In this example, a human Fc chimeric recombinant antibody (hereinafter referred to as BP1206 chimeric antibody) designed based on the antibody LN-3 was expressed and purified.

As a vector for expression, pCI-neo has a light chain expression element (EF-1α promoter, secretion signal, light chain variable region, light chain constant region linked in tandem), heavy chain expression element (EF-1α promoter, secretion) A plasmid was constructed by ligating both the signal, the heavy chain variable region, and the heavy chain constant region in tandem).

The DNA sequences of the heavy chain variable region and the light chain variable region are based on the amino acid sequences of the antibody variable regions (Fig. 4, SEQ ID No.: 8 for heavy chains, SEQ ID No.: 10 for light chains). In addition, it was designed based on the codon optimized for the hamster expression system (SEQ ID No.:7 and SEQ ID No.:9). As the amino acid sequences of the heavy chain constant region portion and the light chain constant region portion, the known amino acid sequences of the heavy chain constant region portion and light chain constant region portion of a human antibody, or a DNA sequence defining a variant thereof can be used. In this example, a variant (IgG1-lala) having the L4A and L5A substitutions in the CH2 region of human IgG1 as the amino acid sequences of the heavy chain constant region portion and the light chain constant region portion (SEQ1 ID No.: 44, respectively) ), and the sequence of human IgG kappa (SEQ ID No.: 46), and the DNA sequences encoding the respective amino acid sequences were used (SEQ ID No.: 43 and SEQ ID No.: 45, respectively).

 

ExpiCHO cells (invitrogen A2910002) were transfected with the plasmid using the Gibco ^™ ExpiCHO ^™ Expression System (GIbco A29129), cultured for 14 days, and the culture supernatant was collected. The antibody was obtained by purifying the culture supernatant using a protein A column (MonoSpin ProA, GL Science 7510-11314).

Using the same method as in Example 1, it was examined whether the obtained chimeric recombinant antibody BP1206 chimeric antibody had cytotoxicity in vitro. The antibody LN-3 was used as a positive control, and the isotype control antibody was used as a negative control. The results are shown in Figure 6. As also shown by this result, it was shown that the BP1206 chimeric antibody had higher cytotoxicity than the antibody LN-3.

Example 7: Drug effect on cancer-bearing mice (1)
In this example, the antitumor effect of the BP1206 chimeric antibody obtained in Example 6 on tumor in vivo was examined.

Hodgkin lymphoma cell line L428 cells (5×10e6 cells) were transplanted subcutaneously into the flank of a mouse NOD/Shi-scid, IL-2RγKOJic (generic name: NOG mouse, in vivo science). When the tumor mass reached 100 mm3, it was divided into groups based on the tumor diameter, and the BP1206 chimeric antibody or isotype control antibody was made into a solution at 10 mg/kg, and a total of 4 times at a frequency of once every 4 days. It was intraperitoneally administered (n=10). Body weight and tumor diameter were measured twice a week for 42 days from the first administration day of the BP1206 chimeric antibody. Further, after the observation period of 42 days was over, the tumor was excised and its weight was measured.

When the mean tumor volume calculated from the tumor diameter was evaluated, the isotype control antibody group continued to increase until Day 42, while the BP1206 chimeric antibody group continued to decrease, and compared to the isotype control antibody group after Day 5. It showed a statistically significant low value (Fig. 7(A)). Further, in one example of the BP1206 chimeric antibody group, tumor cells in the body disappeared.

When the average body weight value was evaluated, both the isotype control antibody group and the BP1206 chimera antibody group increased, and there was no statistically significant difference between the two groups (Fig. 7(B)). However, when the average tumor weight after the observation period of 42 days was evaluated, the BP1206 chimeric antibody group showed a statistically significant low value as compared with the isotype control antibody group (FIG. 7(C)). ).

Example 8: Drug effect on cancer-bearing mice (2) (dose-response test)
In this example, the dose-responsiveness of the anti-tumor effect of the BP1206 chimeric antibody obtained in Example 6 on tumor in vivo was examined.

Hodgkin lymphoma cell line L428 cells (5x10e6 cells) were transplanted subcutaneously into the flank of mouse NOD/Shi-scid, IL-2RγKOJic. When the tumor mass reaches 100 mm3, divide into groups based on the tumor diameter, and use BP1206 chimeric antibody at 10 mg/kg, 5 mg/kg, 1 mg/kg and isotype control antibody at 10 mg/kg. Then, the rats were intraperitoneally administered once every 4 days for a total of 4 times (n=10). Body weight and tumor diameter were measured twice a week for 42 days from the first administration day of the BP1206 chimeric antibody. Further, after the observation period of 42 days was over, the tumor was excised and its weight was measured.

When the average tumor volume calculated from the tumor diameter was evaluated, the isotype control antibody group continued to increase until Day 42, while it decreased in the BP1206 chimeric antibody 10 mg/kg administration group and 5 mg/kg administration group, and the isotype It showed a statistically significant lower value than the control antibody group. On the other hand, the BP1206 chimeric antibody 1 mg/kg administration group showed a statistically significant low value as compared with the isotype control antibody group on the 35th day during the observation period (FIG. 8(A)).

When the average weight value was evaluated, it increased in all groups, and no statistically significant difference was observed among the 4 groups (Fig. 8(B)). On the other hand, the mean tumor weight after the 42-day observation period was statistically significantly lower in the BP1206 chimeric antibody 10 mg/kg and 5 mg/kg groups than in the isotype control antibody group. As shown in FIG. 8(C), the tumor disappeared from the body in 3 of the 10 mg/ml BP1206 chimeric antibody administration groups.

Example 9: Drug effect on cancer-bearing mice (3) (confirmation using surrogate antibody)
In this example, using a surrogate antibody against the BP1206 chimeric antibody obtained in Example 6 (antibody that crosses animal antigen, anti-mouse MHC class II antibody), the effect on cancer cells of the BP1206 chimeric antibody , But was affected by species differences between the host and the transplanted cancer cells.

Mouse C57BL/6 (CLEA Japan, 7 weeks old, female) was subcutaneously implanted with the mouse T cell lymphoma cell line E.G7 cell mutant (5×10e6 cells) in the flank. The cells used in this example are mutants prepared by forcibly expressing CIITA, which is a transcription factor that induces the expression of MHC Class II, in EG7 cells (ATCC (registered trademark) CRL-2113 ^™ ).

Three days after transplantation of E.G7 cells, one group will receive anti-mouse MHC class II antibody (M5/114 clone, Bioxcell) that is a surrogate antibody, and another group will receive 10 mg/kg of isotype control antibody. The mice were intraperitoneally administered once every 4 days for a total of 4 times (n=6). Phosphate buffer solution (PBS) was administered to the remaining group as a negative control on the same schedule. Body weight and tumor diameter were measured twice a week for 21 days from the first administration of anti-mouse MHC class II antibody.

When the average tumor volume calculated from the tumor diameter was evaluated, the isotype control antibody-administered group and the PBS-administered group continued to increase significantly until Day 21, while the anti-mouse MHC class II antibody-administered group showed an increase in volume. It was relatively suppressed, and showed a statistically significant low value at Day 10 as compared with the isotype control antibody administration group and the PBS administration group (FIG. 9(A)). In addition, tumor cells in the body disappeared in one of the anti-mouse MHC class II antibody-administered groups.

When we evaluated the mean body weight, it increased slightly in each of the anti-mouse MHC class II antibody administration group, the isotype control antibody administration group, and the PBS administration group, and there was no statistically significant difference between the three groups. (Fig. 9(B)).

Next, we examined the immunity induction against tumor cells in the individual mice of the anti-mouse MHC class II antibody-administered group. After collecting the mouse spleen and disrupting the tissue using GentleMacs (Milteny), APC-CMV-tetramer (MBL, TS-0020-2C), BV421-anti-CD3 antibody (Biolegend, 300434) and FITC-anti-CD8 The cells were stained with an antibody (Biolegend, 300906) and subjected to FACS analysis. Peptide-specific T cell induction was evaluated based on the proportion of tetramer positive cells in CD3 and CD8 positive cells.

As a result, immunity induction against the model antigen (Ova) (Fig. 10(A)) was confirmed in the individual mice of the anti-mouse MHC class II antibody-administered group. Although this immunity induction did not show statistical significance compared with the isotype control antibody-administered group, the highest immunity induction was confirmed in individuals with complete tumor disappearance, suggesting a correlation between tumor suppression and immunity induction. It was From these results, the validity as a target of MHC Class II was suggested.

Since HLA-DR is expressed in dendritic cells (DC) in addition to tumor cells, the effect of the antibody of the present invention on dendritic cells was evaluated. DC can be classified into cDC1 and cDC2, and it is known that cDC1 particularly contributes to the process of recognizing a foreign antigen and activating TIL. In this study, the effect of antibody administration on DC in spleen was determined based on the ratio of DC to total cells in the spleen, the ratio of cDC1 cells in DC, and the ratio of cDC2 cells in mice treated with antibody. Was evaluated. Spleens collected from mice were disrupted with GentleMACS (Milteny) and fluorescently labeled antibodies against DC markers MHC Class II and CD11a, cDC1 marker XCR1 and cDC2 marker CD172a (PE-Cy7 labeled anti-MHC Class II (respectively). Biolegend, 107630) FITC-labeled anti-CD11a (biolegend, 117306), APC-labeled anti-XCR (biolegend, 148206), PE-labeled anti-CD172a (biolegend, 144011) were used for FACS analysis. -The DC ratio was calculated based on the expression of DR and CD11a-positive cells, and the ratio of cDC1 and cDC2 in DC was calculated based on the expression ratio of XCR1 and CD172a. The percentages of cDC1 cells and cDC2 cells were 2.17 ± 0.1% and 2.0 ± 0.1%, 70.64 ± 2.6% and 73.4 ± 1.3%, 11.04 ± in the isotype control antibody-administered group and the surrogate antibody-administered group, respectively. It was 1.3% and 11.4±1.3%.

As a result, in the anti-mouse MHC class II antibody-administered group, the ratio of dendritic cells (DC) to the total cells in the mouse spleen and the ratio of cDC1 cells in DC were compared with the isotype control antibody-administered group. , And the proportion of cDC2 cells were not significantly different (FIG. 10(B)). Therefore, it was revealed that the type of antibody to be administered had no effect on DC in the spleen and no on-target toxicity was observed.

Example 10: Effect on normal cells In this example, the effect of the BP1206 chimeric antibody on normal cells expressing HLA-DR was evaluated.

B cells included in peripheral blood mononuclear cells (PBMC) are normal cells that express HLA-DR, and are considered to be cells that can be affected clinically when anti-HLA-DR antibody is administered. From this, the effect on the same cells was evaluated. As a comparative control, the effect on T cells that did not express HLA-DR was also analyzed.

Normal human human-derived peripheral blood mononuclear cells (PBMC; Precision Bioservices) were prepared at 10e8 cells/ml, dispensed 100 μl (10e7 cells each in cell number) into a Fischer tube, and BP1206 chimeric antibody at a final concentration of 1 μg/ml. It was added so as to become ml. After incubating for 2 hours in a 37°C water bath, the cells are transferred to a 96 well U bottom plate and collected, and then FITC-CD19 (Biolegend 302205), BV421-CD3 (biolegend 300434), Annexin V APC and 7-AAD Stained. Viable cells were detected by flow cytometric analysis based on Annexin VAPC and 7-AAD staining (staining of apoptotic cells).

For each of B cells (CD19 positive cells) and T cells (CD3 positive cells) in living cells, the ratio to the number of cells when the isotype control antibody was added was calculated (Fig. 11). The BP1206 chimeric antibody reduced the abundance of B cells (CD19 positive cells) by about 60%, while not affecting the abundance of T cells (CD3 positive cells).

In addition, it has been reported that the marketed antibody rituximab (a monoclonal antibody consisting of anti-human CD20 human/mouse chimeric antibody) reduces the abundance ratio of B cells by 90% (Blood. 2010 Jun 24; 115(25 ): 5180-5190), suggesting that the B cell suppression of the BP1206 chimeric antibody was less than that of rituximab.

Example 11: Evaluation of cytotoxicity against solid cancer cells (1)
In this example, the cytotoxic effect of the BP1206 chimeric antibody on solid cancer cells of various origins was examined.

As solid cancer cells, in this example, two human melanoma-derived cell lines (HT144 (ATCC, HTB-63) and A375 (ATCC, CRL-1619)) and two human lung cancer-derived cell lines (COR- L105 (ECACC, 92031918) and LU65 (JCRB, 0079)) were used to assess the cytotoxic effect of the BP1206 chimeric antibody on these cells using a three-dimensional culture method suitable for culturing these cells. ..

As medium, for each cell:
・For culture of HT144 cells, MyCoy's 5A medium (GE, SH30200.01)
・For the culture of A375 cells, Dulbecco's Modified Eagle's Medium (DMEM) (Wako, containing 2 mM GlutaMAX (Thermo, 35050061), 1 mM Sodium Pyruvate (Thermo, 11360070), 1500 mg/L Sodium bicarvonate (Thermo, 25080094) (Wako, 045-30285)
・ RPMI1640 (Wako, 189-02025) for culturing COR-L105
・For culture of LU65, RPMI1640 (Wako, 189-02025)
Was used with 10% FBS (gibco, 10270-106) added.

Colony culture was performed using a 24-well plate. Add 0.3 mL of 0.5% agar-containing medium to the well to form a Bed layer, and add 0.3 mL of 0.3% agar-containing medium containing cells and test substance on it, and incubate at 37°C in 5% CO ₂ incubator for 5 days. I went.

The ratio of viable cells was measured using MTT reagent (Cell Proliferation Kit I, Sigma, 11465007001) or Calcein-AM (Dojindo, C396). In the MTT measurement, 10 μl of MTT reagent (Cell Proliferation Kit I, Sigma, 11465007001) was added to 100 μl of cell culture supernatant, and the mixture was incubated at 37° C. in a 5% CO ₂ incubator for 4 hours. After adding 100 μl of the solubilizing solution and allowing it to stand still at 37° C. in a 5% CO ₂ incubator overnight, the absorption at 550 nm was measured by a microplate reader to calculate the viable cell amount. In Calcein-AM measurement, Calcein-AM solution was added to the culture supernatant after the reaction so that the final concentration was 2 μM and suspended, and the fluorescence intensity was measured by transferring to a 96-well black plate (ex /em =490/ 520).

Figure 12 shows the results when the colony culture method was adopted. In both of the two types of human melanoma-derived cells (HT144 and A375) and the two types of human lung cancer-derived cells (COR-L105 and LU65) examined in this Example, the survival rate of tumor cells was obtained by adding the BP1206 chimeric antibody. It has become clear that Among these cells, especially for human melanoma-derived cells, HT144 cells, a remarkable effect of reducing the ratio of viable cells to about 40% was shown.

Example 12: Evaluation of cytotoxicity against solid cancer cells (2)
In this example, the cytotoxic effect of BP1206 chimeric antibody on solid cancer cells of various origins and the combined effect with anticancer agents were examined.

As solid cancer cells, in this example, three human melanoma-derived cell lines ((Hs852.T (ATCC CRL-7585), C32 (ATCC CRL-1585) and SK-MEL-24 (ATCC HTB-71) )) was used to evaluate the cytotoxic effect of the BP1206 chimeric antibody on these cells using a flat culture or a suspension system.

As medium, for each cell:
・For culture of Hs 852.T cells, L-Glutamine-containing Dulbecco's Modified Eagle's Medium (DMEM) and High Glucose (Wako, 044-29765)
・For C32 cell culture, Eagle's Minimum Essential Medium (EMEM) (Wako, 055-08975)
・For SK-MEL-24 culture, Eagle's Minimum Essential Medium (EMEM) (Wako, 055-08975)
Was used with 10% FBS (gibco, 10270-106) added.

In flat culture, seed each cell on a 96-well plate (C32 cells: 5 × 10 ³ cells/well, Hs 852.T cells: 7.7 × 10 ³ cells/well) and add each test drug there After culturing for one day, MTT test and LDH test were performed using the culture supernatant.
In the suspension system, remove the adherent cells from the culture flask and add 1 × 10 ⁶ cells/mL suspension to 0.1 mL of the test drug and PI (final concentration 2 μM), and use a thermal cycler at 37°C. And reacted for 2 hours. Calcein-AM solution (final concentration 2 μM) was added to the reaction solution to suspend it, and the suspension was transferred to a 96-well black plate to measure the fluorescence intensity.

For C32 cells, we investigated the combined effect of Vemurafenib (SIGMA, S1267), which is used as a standard therapeutic drug for melanoma. In the case of flat culture, Vemurafenib 1000 nM was added 24 hours after the start of cell culture. In the case of the suspension system, Vemurafenib 1000 nM was added at the same timing as the addition of the BP1206 chimeric antibody.

The ratio of viable cells was determined using MTT reagent (Cell Proliferation Kit, I, Sigma, 11465007001) or Calcein-AM (Dojindo, C396), and dead cell number was PI (Propidium Iodide, Dojindo, P378) or LDH (Dojindo, CK17) was used for each measurement. Measurement of the percentage of viable cells using MTT reagent or Calcein-AM was performed as described in Example 11. The number of dead cells was measured using PI by adding PI to the reaction solution to 1.5 μM in advance, and after the reaction was completed, the culture supernatant was transferred to a 96 well black plate and the fluorescence intensity was measured ( ex /em =535/617).

The LDH test is a test method that quantitatively evaluates cell death based on the activity of LDH (lactate dehydrogenase) that leaks out of cells due to cell membrane damage accompanying cell death. Specifically, lactic acid which is a substrate of LDH, NAD+ which is a cofactor, and WST-8 tetrazolium salt which is a chromogenic substrate are added to the culture supernatant, and WST-8 is produced by NADH produced in the catalytic process of the enzyme. The amount of dead cells was indirectly quantified from the absorbance (450 nm) after coloring using the principle that tetrazolium salt was reduced and colored.

The results are shown in Figure 13. In all of the three human melanoma-derived cell lines (Hs852.T cells, C32 cells and SK-MEL-24 cells) examined in this Example, addition of the BP1206 chimeric antibody reduced the survival rate of tumor cells. It became clear that they are doing. Furthermore, among these cells, we confirmed the combined effect of BP1206 chimeric antibody and vemurafenib on C32 cells, but under these conditions, the proportion of viable cells was up to 39% in flat culture, and suspension culture In the case of, it showed a remarkable effect of reducing it to 59%.

Example 13: Evaluation of dose response of cytotoxicity to solid cancer cells (three-dimensional culture method)
In this example, the dose response of the cytotoxic effect of BP1206 chimeric antibody on solid cancer cells was examined.

Using two types of melanoma cell lines (HT144 (ATCC, HTB-63) and A375 (ATCC, CRL-1619)) as solid cancer cells, the cytotoxic effect of the BP1206 chimeric antibody on these cells was further investigated. Evaluation was performed using a three-dimensional culture method, which is a condition close to the environment.

As culture medium, MyCoy's 5A medium (GE Healthcare, SH30200.01) containing 0.5% Agar (Difco, 526-00054) and 10% FBS (gibco, 10270-106) was used to culture HT144 cells. DMEM (Wako Pure Chemical Industries, 044-29765) containing 0.5% Agar and 10% FBS was used for culturing. First, the above medium was dispensed on a 24-well plate at 0.3 ml/well, cooled at 4° C. and solidified to form a bed layer. After returning to room temperature, cells and BP1206 chimeric antibody were prepared at 6×10 ⁴ cells/ml using the same medium as the bed layer, and added at 0.3 ml/well on the bed layer. When examining the combined effect of cisplatin or vemurafenib, the specified dose is cisplatin (SIGMA, PHR1624, 2 μM) or vemurafenib (SIGMA, S1267, HT144 cells 300 nM, A375 cells 600 nM) together with the BP1206 chimeric antibody. The mixture was mixed in the same manner, and similarly added at 0.3 ml/well on the bed layer. After cooling at 4°C to solidify, the temperature was returned to room temperature, and the cells were cultured at 37°C in a 5% CO ₂ incubator for 5 days.

10 μl of MTT reagent (Cell Proliferation Kit I, Sigma, 11465007001) was added to 100 μl of the medium, and the mixture was incubated at 37° C. in a 5% CO ₂ incubator for 4 hours. After adding 100 μl of the solubilizing solution and allowing it to stand still at 37° C. in a 5% CO ₂ incubator overnight, the absorption at 550 nm was measured by a microplate reader to calculate the viable cell amount.

Figure 14 shows the results of cell death induction (relative value based on viable cell mass measurement) after treating cells with the BP1206 chimeric antibody. In this Figure 14, (A) HT144 cells, BP1206 chimeric antibody and cisplatin combination, (B) HT144 cells, BP1206 chimeric antibody and Vemurafenib combination, (C) A375 cells, BP1206 chimeric antibody and Vemurafenib combination, The cell death rate induced by is shown. The BP1206 chimeric antibody showed a dose-dependent survival inhibitory effect on both cell lines (Fig. 14). The survival inhibitory effect was enhanced more than additively by using cisplatin or vemurafenib in combination with the BP1206 chimeric antibody.

Example 14: Observation of cell morphological change associated with cell death induction In this example, when the BP1206 chimeric antibody induces cell death of cancer cells, cell morphological changes caused by cancer cells were shown. ..

L428 cells were suspended in the medium and seeded in a PCR tube (Nippon Genetics, FG-008FC) at a concentration of 50,000 cells/50 μl, and then the BP1206 chimeric antibody solution (50 μl) was added to a final concentration of 10 μg/mL. And incubated at 37°C for 1 hour. The cells were collected by centrifugation, suspended in a 1% glutaraldehyde solution (Nacalai, 1700392) and fixed overnight, and then the cell shape was analyzed with a tabletop electron microscope Miniscope TM3030Plu (Hitachi) (Fig. 15).

Aggregation of cells was observed by addition of the BP1206 chimeric antibody, and cell death was induced thereafter, suggesting that polymerization may be a trigger for cell death induction.

Example 15: Immunological cell death (Immunogenic cell death) induction evaluation In this example, when the BP1206 chimeric antibody was administered to tumor-bearing mice, immunological cell death in tumor (Immunogenic cell death, hereinafter also referred to as ICD) Based on the secretion of the marker (described), the ICD-inducing ability of the antibody was evaluated.

Mouse CB17.Cg-PrkdcscidLystbg-J/CrlCrlj (generic name: SCID-Beige) was subcutaneously transplanted with Hodgkin lymphoma cell line L428 cells (5×10e6 cells) into the flank, and the tumor mass exceeded 100 mm ³ . BP1206 chimeric antibody (10 mg/kg), isotype control antibody (10 mg/kg) or PBS was administered once to an individual (administration route: intraperitoneal, dosage form: solution). The number of experimental cases was 3 for the BP1206 chimeric antibody administration group and 3 for the isotype control antibody administration group, and 1 for the PBS administration group.

Tumors were excised 4 days after the last administration of the antibody, immersed in 4% paraformaldehyde for 48 hours for fixation, and then subjected to immunostaining using anti-HMGB1 antibody and anti-human IgG antibody. HMGB1 is a protein that originally exists in the nucleus, but is known to be released into the cytoplasm with cell death, and since it serves as an ICD marker, it can be labeled with an anti-HMGB1 antibody to produce a BP1206 chimeric antibody. The effect on HMGB1 secretion can be investigated. In addition, immunostaining using an anti-human IgG antibody was performed for the purpose of confirming whether the BP1206 chimeric antibody actually infiltrated into the tissue in the animal body.

Dyeing was performed according to the following procedure. After preparing thin slices, submerge in BOND Dewax Solution (Reica, AR9222) and 100% ethanol to deparaffinize them, wash with 0.01M PBS, and use BOND Epitope Retrieval Solution1 (Reica, AR9961). Then, the antigen activation treatment was performed at 98°C for 30 minutes. After washing with 0.01M PBS, react with anti-HMGB1 antibody (GeneTex, GTX628834, 750-fold diluted) or rabbit anti-human IgG (H+L) antibody (Bethyl, A80-118A, 10,000-fold diluted) at room temperature for 30 minutes I went.

After washing with 0.01M PBS, it was reacted with Novolink Polymer (Reica, DS9800) for 8 minutes at room temperature. After washing with 0.01M PBS, it was reacted with Peroxidase Block (RE7101-CE, Reica) for 5 minutes at room temperature. After washing with 0.01M PBS, it was reacted with Mixed DAB Refine (Reica, DS9800) at room temperature for 10 minutes. After washing with 0.01M PBS, reaction with hematoxin for 10 minutes at room temperature, washing with deionized water, dehydration treatment with 100% ethanol and xylene, and microscopic observation.

The results are shown in Figure 16. 16(A) and (B) are BP1206 chimeric antibody-administered mice, (C) and (D) are isotype control antibody-administered mice, and (E) and (F) are PBS-administered mice, respectively. (C) and (E) are whole tumor views, and (B), (D) and (F) are enlarged views of strongly stained sites. In each figure, the effect of BP1206 chimeric antibody on HMGB1 secretion in the L428 cell subcutaneous transplant model (left) and tumor infiltration (right) are shown.

From these results, in the histological images (A) and (B) (enlarged view) of the BP1206 chimera antibody-administered mouse, HMGB1 secretion was observed in the tumor lysis (necrotic focus) region caused by the BP1206 chimera antibody administration. It was confirmed that administration of the chimeric antibody induced ICD and released HMGB1 from the cell nucleus. From this, it was shown that the BP1206 chimeric antibody actually infiltrated into the tissue in the animal body. On the other hand, in the histological images (C) and (D) (enlarged view) of the isotype control antibody-administered mouse and the histological images (E) and (F) (enlarged view) of the PBS-administered mouse, the release of HMGB1 from the cell nucleus was observed. It did not occur and it was confirmed that tumor lysis (necrosis) did not occur.

Example 16: Humanization of antibody In this example, a humanized recombinant antibody (hereinafter referred to as BP1206 humanized antibody) was designed based on the structure of the BP1206 chimeric antibody prepared in Example 6, and its expression/purification I went.

Humanization of the antibody was designed using Tabhu (http://circe.med.uniroma1.it/tabhu/), which is public software. Using this software, sequences having characteristics similar to those of the BP1206 chimeric antibody in terms of sequence and three-dimensional structure were extracted from the database, and 5 types of sequences (3 types of human antibody: AF107234, AF107237, 3AB363085, 2 types of humanized antibody: 3L7F) Antibodies, 3EOA antibodies) were picked up as candidate sequences, and the sequences other than the CDR regions of these antibodies were graphed with the sequences of the CDR regions of the BP1206 chimeric antibody (F0 series of each sequence).

Next, we performed backmutation of some of the amino acid residues of the 5 antibody sequences from 3 human antibodies to the mouse sequence (F1 series). The target amino acid residues for backmutation were selected with an upper limit of 10 amino acid residues for each sequence, based on the large structural difference between humanized antibody and mouse antibody (higher TubHu score). The details followed the method described in the non-patent document (Bioinformatics. 2015 Feb 1; 31(3):434-5). Through this work, a total of eight BP1206 humanized antibody sequences were produced (Fig. 17).

Example 17: Functional evaluation of humanized antibody In this Example, the cytotoxic activity of the eight types of BP1206 humanized antibodies prepared in Example 13 against cancer cells was clarified.

The BP1206 humanized antibody prepared in Example 13 was evaluated for in vitro cytotoxicity against Hodgkin lymphoma cell line L428 cells. L428 cells were prepared in RPMI medium containing 10% FBS at 1×10 ⁶ cells/ml, and Propidium iodide (PI; DOJINDO, FE159) was added to a final concentration of 2 μM, then 50,000 cells/50 μl The cells were seeded in a PCR tube (Fast gene, FG-1700) at the concentration of. Eight types of BP1206 humanized antibody solutions (50 μl) obtained in Example 13 were added to each tube (final antibody concentration 10 μg/mL) and incubated at 37° C. for 1 hour. After suspending the cell solution by pipetting, the fluorescence intensity of PI was measured with a microplate reader to measure the dead cell ratio (ex/em=535 nm/617 nm). Eight types of BP1206 humanized antibodies were shown to retain 30-70% of the cell death-inducing activity of the BP1206 chimeric antibody (FIG. 18).

Example 18: Search for indications for solid cancer In this example, indications for solid cancer of the BP1206 humanized antibody obtained in the present invention were searched.

Cervical cancer (n=102), endometrial cancer (n=102), head and neck cancer (n=102), ovarian cancer (n=102), lung cancer (n=102) , Melanoma (n=48), the expression of HLA-DR antigen in each patient-derived tumor tissue was examined from the viewpoint of HLA-DR antigen expression level and HLA-DR expressing cell ratio. It examined using the antibody.

The above-mentioned sample was spotted on a slide glass and carried out using a tumor tissue microarray (manufactured by Pantomics). After activating the antigens on the microarray by the pressure cooker method, they are sequentially reacted with the LN3 antibody, peroxidase-labeled polymer, and chromogenic substrate (diaminobenzidine (DAB)), and the expression of HLA-DR antigen is evaluated based on the color development of DAB. did.

The results are shown in Figure 19. It was revealed that expression of the HLA-DR antigen was observed in nearly half of the cells, although there were variations in the expression amount and the proportion of expressing cells in all cancer types (Fig. 19).

Example 19: Correlation between target expression and cytotoxicity In this example, various cancer cell lines are used to express target HLA-DR antigen and cytotoxicity that can be induced against the cells. The correlation between the two was investigated.

16 cell lines:
・Kasumi-1: Derived from human acute myelogenous leukemia (AML) (ATCC CRL-2724);
・OCI-LY-19 cells: derived from human diffuse large B-cell lymphoma (DLBCL) (DSMZ, ACC 528);
・Nalm-6 cells: derived from human acute lymphocytic leukemia (ALL) (DSMZ, ACC 128);
・KMS-26 cells: derived from human multiple myeloma (MM) (JCRB1187);
・KG-1 cells: derived from human acute myeloblastic leukemia (AML) (ATCC CCL-246);
-Rmos cells: derived from human Burkitt lymphoma (BL) (DSMZ, ACC-603);
・KMS-11 cells: derived from human multiple myeloma (MM) (JCRB1179);
・JJN-3 cells: derived from human multiple myeloma (MM) (DSMZ, ACC 541);
・Mino cells: derived from human mantle cell lymphoma (MCL) (ATCC CRL-3000);
・NU-DHL-1 cells: derived from human diffuse large B-cell lymphoma (DLBCL) (DSMZ, ACC 583);
・MUTZ-5 cells: Human acute lymphocytic leukemia (ALL) origin (DSMZ, ACC 490);
・Daudi cells: derived from human Burkitt lymphoma (BL) (ATCC CRL-213);
・GRANTA-519 cells: derived from human diffuse large B-cell lymphoma (DLBCL) (DSMZ, ACC 342);
-L428 cells: derived from human Hodgkin lymphoma (HL) (DSMZ, ACC 197);
-Pfeiffer cells: derived from human diffuse large B-cell lymphoma (DLBCL) (ATCC CRL-2632);
・Raji cells: derived from human Burkitt lymphoma (BL) (JCRB9012);
Was used to examine the expression level of HLA-DR antigen in each cell and the cell death-inducing activity when the BP1206 humanized antibody was added.

Each cell was cultured by the method specified by the supplier. The cells were suspended in PBS, 5×10 ⁵ cells were collected in a PCR tube, BP1206 antibody was added to a final concentration of 10 μg/ml, and the mixture was incubated at 37° C. for 2 hours in a thermal cycler. The cells were washed with PBS, stained with 7-AAD (Biolegend, 400625), and subjected to FACS analysis. The ratio of 7-AAD unstained cells was calculated as the ratio of viable cells. The results are shown as a bar graph in FIG.

On the other hand, the target HLA-DR antigen expression level of the cell line was analyzed by FACS (BD Verse) after labeling each cell with a labeled anti-human HLA-DR antibody (Biolegend, 307615). The expression level of the HLA-DR antigen is shown by dots in FIG.

As a result, a correlation was observed between the target expression level in each cancer cell line and the induction of cell death by the addition of the BP1206 humanized antibody, and the higher the target HLA-DR antigen expression level in the cell line, the higher the expression level. It was also found that the cell death-inducing activity is also high.

The HLA-DR antigen-binding substance of the present invention, particularly an antibody or a humanized antibody derivative, is used to treat HLA-DR antigen-expressing cancer cells, to inhibit the growth of cancer cells, and thus to reduce or eliminate tumors. Can be made. In addition, since this binding substance, particularly an antibody or a human antibody derivative can bind to the HLA-DR antigen, immunization of the HLA-DR antigen in vivo as a test application of cancer cells expressing the HLA-DR antigen. It can be used for biological detection (ELISA, Western blotting, flow cytometry, etc.).

In addition, whether or not the cell viability of the cancer cells is decreased by bringing the cancer cells collected from the subject into contact with the binding substance of the present invention, particularly an antibody or a humanized antibody derivative under culture conditions, or immunity By measuring whether or not the secretion of the activator is enhanced, when the binding substance of the present invention, particularly the antibody or the humanized antibody derivative is administered to a subject having cancer cells, the cancer cells are treated in the subject. It is possible to investigate the degree of cytotoxicity against the.

Claims (15)

1. Heavy chain complementarity determining regions, CDR1 (SEQ ID No.: 1), CDR2 (SEQ ID No.: 2), CDR3 (SEQ ID No.: 3),
   Light chain complementarity determining regions, CDR1 (SEQ ID No.: 4), CDR2 (SEQ ID No.: 5), CDR3 (SEQ ID No.: 6),
   An antibody or a humanized antibody derivative having a binding property to an HLA-DR antigen and inducing toxicity to a cancer cell, comprising:
2. The antibody or human according to claim 1, wherein the humanized antibody derivative is selected from a humanized antibody variant selected from a humanized antibody, a chimeric antibody, a multivalent antibody, and a multispecific antibody, or a functional fragment thereof. Type antibody derivative.
3. The antibody or human antibody derivative according to claim 1 or 2, wherein the functional fragment is F(ab')2.
4. The amino acid sequence of the heavy chain variable region VH domain of the antibody or human antibody derivative is SEQ ID No.:8, SEQ ID No.:12, SEQ ID No.:16, SEQ ID No.:20, SEQ ID No. :, 24, SEQ ID No.: 28, SEQ ID No.: 32, SEQ ID No.: 36, and SEQ ID No. 40, the antibody according to any one of claims 1 to 3. Or a human antibody derivative.
5. The amino acid sequence of the light chain variable region VL domain of the antibody or humanized antibody derivative is SEQ ID No.: 10, SEQ ID No.: 14, SEQ ID No.: 18, SEQ ID No.: 22, SEQ ID No. :, 26, SEQ ID No.: 30, 30, SEQ ID No.: 34, SEQ ID No.: 38, and SEQ ID No.: 42, The antibody according to any one of claims 1 to 4. Or a human antibody derivative.
6. The antibody or humanized antibody derivative according to any one of claims 1 to 5, which induces cytotoxicity against cancer cells but not normal cells.
7. The antibody or humanized antibody derivative according to any one of claims 1 to 6, wherein the cancer cell is selected from the group consisting of Hodgkin lymphoma, lung cancer, and melanoma.
8. The antibody or humanized antibody derivative according to any one of claims 1 to 7, which is bound to a drug to form an antibody drug complex (ADC).
9. A pharmaceutical composition for treating cancer, which comprises the antibody or the human antibody derivative according to any one of claims 1 to 8.
10. 10. The pharmaceutical composition according to claim 9, wherein the cancer is selected from the group consisting of Hodgkin lymphoma, lung cancer and melanoma.
11. A step of contacting a cancer cell collected from a subject with an antibody or a humanized antibody derivative according to any one of claims 1 to 8 in vitro,
    Under culture conditions, a step of measuring whether the cell viability of cancer cells is reduced, or a step of measuring whether the secretion of immunostimulatory substances is enhanced,
    A method for measuring cytotoxicity against cancer cells, which comprises:
12. In the presence of peripheral blood lymphocytes of the same subject, whether the cell viability of cancer cells is reduced or whether immune cells derived from peripheral blood lymphocytes are activated are measured, The method according to claim 11. For measuring cytotoxicity against cancer cells.
13. From the in vitro cytotoxicity of cancer cells collected from a subject, cancer cells when the antibody or humanized antibody derivative according to any one of claims 1 to 8 is administered to the subject. 13. The method for measuring cytotoxicity against cancer cells according to claim 11 or 12, which comprises measuring the increase in cytotoxicity against cancer cells.
14. Due to the enhanced secretion of the immunostimulatory substance in vitro, the cytotoxicity against cancer cells when the antibody or humanized antibody derivative according to any one of claims 1 to 8 is administered to the subject The method for measuring cytotoxicity against cancer cells according to claim 11 or 12, wherein the enhancement is measured.
15. The cytotoxicity of the antibody or humanized antibody derivative according to any one of claims 1 to 8 against cancer cells collected from a subject, the secretion of an immunostimulator, or the activation of immune cells, A measurement kit containing the above antibody for in vitro measurement.
    
    

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