KR101982001B1 - Method for selecting of anti-pd-1 or anti-pd-l1 antibodies using alloimmunity or xenoimmunity - Google Patents

Abstract

The present invention relates to an anti-PD-1 or anti-PD-L1 antibody screening method using allogeneic or heterologous rejection. Antibodies can be selected at low cost by an antibody screening method according to the present invention, which is a model that simulates PD-1 and PD-L1 interactions between T cells and cancer cells.

Description

METHOD FOR SELECTING ANTI-PD-1 OR ANTI-PD-LI ANTIBODIES USING ALLOIMMUNITY OR XENOIMMUNITY BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-

The present invention relates to an anti-PD-1 or anti-PD-L1 antibody screening method using allogeneic or heterologous rejection.

T-cell depletion is a condition of T-cell dysfunction that occurs in many chronic infections and cancers. This is defined by poor T-cell functional function, sustained expression and functional function of inhibitory receptors or memory T-cells and other transcriptional conditions. Depletion interferes with infection and optimal regulation of tumors.

The PD-1 pathway is an important immunosuppressive modulator of T-cell depletion. Blocking of this pathway can induce T-cell activation, expansion and enhanced functional function. Thus, PD-1 negatively regulates T cell responses. PD-1 was identified as a marker of T cells depleted in chronic infection, and blocking PD-1: PD-1L interaction partially restored T cell function.

Both TIM-3 and PD-1 can function as negative regulators of T cell responses and the combined targeting of the TIM-3 and PD-1 pathways regulate tumor growth rather than targeting either pathway alone . In addition, anti-PD-1 antibodies have been disclosed in the past, and pharmaceutical compositions comprising such antibodies, and methods of using such antibodies to restore T-cell function in T-cells, Have been used to treat infectious diseases (Korean Patent Application No. 2017-7014305). However, there are many difficulties in selecting excellent antibodies for such treatment.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide an anti-PD-1 or anti-PD-L1 antibody screening method using homologous or heterologous rejection, The purpose.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to " one embodiment " or " embodiment " means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment " or " an embodiment " in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, the term " homologous or heterologous rejection reaction " refers to a phenomenon in which, when an external object other than the human body enters the human body, the immune system that identifies and removes or attacks the object Immune rejection. There are two types of rejection: homologous rejections, such as those that occur between humans and humans, and intergeneric rejections, such as those that occur between humans and other animals. In particular, acute rejection is a reaction caused by T-cells (T-cells) among components of the immune system. Among the foreign substances, T cells recognize the antigen provided by the antigen presenting cell, and function as a cell mediated immunity to the foreign substance. The main histocompatibility complex (MHC) is important for the immune response by T cells. The main histocompatibility complex is a protein on the surface of the cell. different. Therefore, if the main organizational complex is the same as the one possessed by the main organizational complex, it is judged to be an external substance if it is not judged to be an external substance. In other words, the main histocompatibility complex uses the difference of the immune system as the criterion that distinguishes it from others.

In the present specification, "mixed lymphocyte reaction (MLR)" (MLR), when mixed with PBMC at 1: 1 ratio, causes homologous immune response and increases T cell division. MLR has been developed to pre-screen for allogeneic reactions in organ transplants, but it has been used as a way to create a situation that gives strong TCR stimulation in immunology experiments. In this MLR process, the expression of PD-1 in normal T cells was observed to increase. On the other hand, PD-L1 is expressed in various antigen presenting cells (APC) including monocytes of "stimulating cells". At this time, in the MLR reaction, the cells for stimulation are intended to give only irritation without disruption, so that radiation is irradiated to prevent cell division. Immediately after mixing stimulating cells with "reactive cells", even if the cells receive radiation, the cells do not die immediately, but the cells start to die from 3 to 4 days after the reaction, and the number decreases. The dragon cell dies and only the "reaction cell" remains. That is, the "stimulating cells" and the "reaction cells" cause a heterologous immune response, and the "stimulating cells" have a function of inducing PD-1 by stimulating TCR after a certain period of time by radiation, &Quot; Reactive cell " is activated by TCR by " stimulating cell " to express PD-1, and eventually PD-1 is expressed.

PD-1 should be processed in the state that PD-1 and PD-L1 are all expressing. However, even if PD-1 is added at the timing when the TCR signal is increased (see section 1 of FIG. 1 (A) PD-1 is inserted immediately after the maximal expression of PD-1 (see section 2 of FIG. 1 (A)) since the expression of PD-1 in T cells is low and no use is made Even if APC cells die, they do not exist and there is no PD-L1 expression. In order to solve the above problem, the irritating cells previously administered on the sixth day were re-administered to give additional stimulation. Thus, the environment for re-stimulation of TCR is established and PD-1 of T cells is already up. Newly stimulated cells express PD-L1 due to a heterologous immune response. In addition, the anti-PD-1 antibody may be treated at the time of administration of the cell for stimulation, and the performance of the antibody can be evaluated by measuring the cleavage of the T cell from this time.

In the present specification, T cells recognize an antigen whose major histocompatibility complex (MHC) appears on the cancer cell surface through a T cell receptor (TCR). This first signal is not sufficient to trigger a T cell response and requires a second signal delivered by the B7 co-stimulatory molecule B7-1 (or CD80) and B7-2 (or CD86). Programmed cell death 1 (PD-1) inhibitory receptors are expressed by T cells during long-term antigen exposure and lead to negative regulation of T cells when coupled with PD-L1 and PD-L2, which are predominantly expressed in inflammatory tissues and tumors do. The PD-1 interaction occurs in the effector phase of T cell response in peripheral tissues. When the signal regulated by PD-1 and PD-L1 is blocked, T cells are specifically activated for cancer-specific. In addition to specific antigen recognition via TCR, T cell activation is regulated through a balance of positive and negative signals provided by the co-stimulatory receptors. These surface proteins are generally members of the TNF receptor or B7 superfamily. Antibody blocking of functional and stimulant molecules against activated co-stimulatory molecules can enhance T-cell stimulation and promote tumor destruction. The anti-PD-1 antibody blocks the binding of PD-1 and PD-L1, thereby blocking the inhibitory signal of PD-1, thereby restoring the function of the T cell and inducing an increase in cleavage. Therefore, in order to confirm the performance of anti-PD-1 antibody, PD-1 and antigen-presenting cell (APC) PD-L1 must sufficiently express and bind to T cells. Expression of PD-1 and expression of PD-L1 are all required to evaluate the action of anti-PD-1.

As used herein, the term " biological sample " refers to all samples containing genomic DNA of the individual to be analyzed, preferably blood, plasma, serum, bone marrow, The sample can be a cell, a saliva, a sputum, a hair, a urine and the like, more preferably a sample which can be a body fluid, hair root, blood, plasma, serum and the like but can identify a gene locus.

As used herein, the term " cancer " refers to any undesired cell proliferation (or any disease that exhibits unwanted cell proliferation in itself), a neoplasm or a tumor, or a risk of undesired cell proliferation, neoplasia, Or increase the postmark. Cancer can be benign or malignant, and can be primary or secondary (metastatic). The neoplasm or tumor may be any abnormal growth or proliferation of the cells and may be located in any tissue. Examples of tissues include adrenals, adrenal medulla, anal, cecum, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (with or without brain) cerebellum, cervix, colon, duodenum, endometrium, (Eg kidney epithelium), gallbladder, esophagus, neuroglia, heart, ileum, plant, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphatic arch, maxilla, mediastinum, mesentery, endometrium, nasopharyngeal , Pancreas, parotid, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicular, thymus, thyroid, tongue, tonsil, organ, uterus, vulva, Includes white blood cells. The tumor to be treated may be a neural or non-neoplastic tumor. Neurotic tumors may originate from the central or peripheral nervous system and may be, for example, glioma, hematoblastoma, meningioma, neurofibroma, superficial myoma, schwannoma, nerve fibrosarcoma, astrocytoma and rare prominent glia species. Non-neuronal cancers / tumors may originate from any other non-neuronal tissue, and examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, NHL, Hodgkin lymphoma, chronic myelogenous leukemia (CML) (AML), myelodysplastic syndrome (MDS), skin T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermal carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymus Carcinoma, NSCLC, blood cancer and sarcoma.

As used herein, " chemotherapy " refers to the treatment of cancer by drugs or ionizing radiation (e.g., radiation therapy using X-rays or gamma-rays). In a preferred embodiment, chemotherapy refers to treatment with a drug. The drug may be a chemical, such as a small molecule drug, an antibiotic, a DNA intercalating agent, a protein inhibitor (such as a kinase inhibitor) or a biological agent such as an antibody, an antibody fragment, a nucleic acid or a peptide, , RNA), peptides, polypeptides or proteins. The drug may be formulated as a pharmaceutical composition or medicament. Formulations may include one or more drugs, such as one or more active agents, with one or more pharmaceutically acceptable diluents, excipients or carriers. Treatment may include administration of one or more drugs. The drug may be administered concurrently or sequentially, alone or in combination with other treatments, depending on the condition being treated. For example, chemotherapy may be a co-therapy comprising the administration of two drugs, one or more of which may be intended to treat cancer. The chemotherapy may be administered in one or more routes of administration, for example, parenterally, intravenously, orally, subcutaneously, intradermally or intratumorally. Chemotherapy may be administered depending on the treatment regimen. The treatment regimen may be a scheduled timetable, schedule, plan, or schedule of chemotherapy administration that may be manufactured by a physician or a health care practitioner, and may be tailored to suit the patient in need of treatment.

As used herein, a "subject" may be any animal or human as the subject to be treated. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient. A subject may be diagnosed with a disease or condition requiring treatment, or suspect that it has such a condition or condition.

Stimulating a T cell for a reaction having a second MHC class with a first stimulating cell having a first MHC class to thereby elevate PD-1 expression of the T cell for the reaction; Adding a second stimulating cell having a first or third MHC class to the T cell for response in which PD-1 expression is elevated to produce a mixed reaction product; Adding an antibody of PD-1 antibody or PD-L1 to be tested to the mixed reaction product; Quantitatively detecting the reactivity of the PD-1 and PD-1 antibodies or the PD-L1 and PD-L1 antibodies; And comparing the relative effect from the detection step to select a PD-1 antibody or a PD-L1 antibody, wherein the first, second, and third classes induce an immunological rejection reaction with each other in different classes, ≪ / RTI >

In one embodiment of the present invention, the first stimulating cell is a cell capable of promoting the expression of PD-1. In another embodiment of the present invention, the cells for the first and second stimulation cells express PD-L1. In another embodiment of the present invention, elevation of PD-I expression occurs due to TCR activation. In another embodiment of the present invention, the cell expressing PD-L1 is APC. In another embodiment of the present invention, the first stimulus cell having the first MHC class, the reaction T cell having the second MHC class, and the second stimulation cell are derived from a biological sample. In another embodiment of the present invention, the cell capable of promoting PD-1 expression is PBMC. In another embodiment of the present invention, the cell for the first or second stimulation cell is inhibited by physiotherapy or chemotherapy.

The interaction of PD-1 and PD-L1 between actual cancer cells and T cells can be simulated by the anti-PD-1 or anti-PD-L1 antibody screening method using allogeneic or heterologous rejection according to the present invention. The antibody can be selected at low cost by the method of selecting an antibody according to the present invention.

FIG. 1 is a schematic diagram illustrating the use of an anti-PD-1 antibody in a lymphocyte according to an embodiment of the present invention.
FIG. 2 is a schematic diagram illustrating a mixed lymphocyte reaction for immuno checkpoint blocker verification according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the reactivity of mixed lymphocytes in proportion to changes in antibody concentration according to an embodiment of the present invention. FIG.
FIG. 4 is a view showing an example of antibody screening by showing that there is a difference in the response of mixed lymphocytes using different anti-PD-1 antibodies according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

As described above, the present invention was carried out by applying a mixed lymphocyte reaction (MLR). FIG. 2 shows this, and FIG. 2 is a schematic diagram showing a mixed lymphocyte reaction for the immuno check-point blocker verification according to one embodiment of the present invention by applying MLR.

Example

Example  1: induction phase

Mononuclear cells (PBMC) were separated from the peripheral blood of a healthy subject by phacoem concentration gradient separation and stored in liquid nitrogen in a frozen medium to prepare a first PBMC. In the same manner, mononuclear cells were separated from a second subject or a second group of subjects different from the first subject and stored in liquid nitrogen in a freezing medium to prepare a second PBMC. However, in order to reduce the variation among individuals, several PBMCs were mixed at the same rate, and at least 5 persons were required.

The second PBMCs were gamma irradiated at 3000 rad to prevent splitting. The first PBMCs were stained with CellTrace red dye, and the second PBMCs were stained with CFSE. More specifically, each of the first and second PBMCs was suspended in CFSE buffer (PBS + 5% FBS) (1-2 × 10 ⁶ cells / mL) and CellTrace Red dye and CFSE reagent were added to each. At this time, the concentrations were adjusted to 1 μM and 5 μM, respectively, incubated at 37 ° C. for 20 minutes, and then diluted by adding CFSE buffer of 5 times the volume of the buffer to be dyed. And further incubated at room temperature for 5 minutes in a diluted state. Thereafter, the pellet was removed by centrifugation, and the buffer solution was discarded.

The stained PBMCs were resuspended in RPMI 1640, 10% FBS, Penicillin / streptomycin medium. The first and second PBMCs were put into each well of a 24-well plate at a rate of ³ × 10 ^6, and mixed well. Then, the cells were stored in a 37 ° C CO ₂ incubator, and after 3-4 days, the wells were replaced with fresh medium by about half. After 6 days, cells of the plate were collected.

Example 2  : Restoration phase

PBMC collected in Example 1 were stained with CellTrace Violet to obtain a third PBMC. The detailed procedure except for the type and concentration of the reagents was the same as in Example 1, and the concentration of the reagent was set to be 5 μM. CFSE staining of second gamma-irradiated PBMC of Example 1 was performed to obtain fourth PBMC.

A 96 well U bottom plate was prepared and treated with PD-1 antibody to verify the effect. At this time, the third and fourth PBMCs were resuspended in RPMI1640, 10% FBS, and Penicillin / streptomycin medium. And then to two types each 1X10 ⁵ cells into each well, kept at 37 ℃ CO ₂ incubator, and analyzes the division by flow cytometry 5 days after the instrument were selected antibodies.

In the examples of the present invention, CellTRACE Violet, CellTrace Red, CFSE are used as the staining reagent for PBMC, but the present invention is not limited thereto. The reagents used in the present invention can be all reagents capable of observing cell division such as CFSE, and different colors may be used to distinguish between different groups. In addition, in Example 2, it is most important to confirm the difference in cleavage after antibody treatment. Therefore, it is essential to use two different dyeing reagents, but in Embodiment 1, the CellTrace red staining step may be omitted as needed. The CFSE used in Examples 1 and 2 has a larger significance as a fluorescent marker for distinguishing the first and second objects than for the purpose of confirming cleavage. In addition, here, cell division does not occur due to irradiation. Therefore, in addition to the type of CFSE, any fluorescence reagent capable of tracking cells in a flow cytometer can be used as a substitute. When radiation is applied to prevent cell division such as the second PBMC or the fourth PBMC, a radiation irradiation apparatus such as Cesium or X-rays is required, but the present invention is not limited thereto. In the mixed lymphocyte reaction (MLR), anticancer agents such as mitomycin C can be used in addition to the above-mentioned methods. However, care should be taken in using these anticancer drugs as they may affect the assay later if they are not thoroughly cleaned.

Example  3: Intensity change of mixed lymphocyte reaction according to antibody concentration change

In carrying out the procedure according to Example 2, the anti-PD-1 antibody was administered to a 96 well U bottom plate, wherein the difference in the activity of the antibody was found to be proportional to the intensity of the mixed lymphocyte. As the concentration of the anti-PD-1 antibody administered as shown in Fig. 3 was increased (Fig. 3 (A)), the proportion of disrupted CD8 ⁺ T cells increased proportionally (Fig. 3 (B)). In the antibody screening process, we confirmed that this assay can quantitatively compare antibody titers.

Example  4: Mixed lymphocyte reaction for antibody screening Example

Example 1 and Example 2 were carried out, and an antibody screening process was practically carried out through a mixed lymphocyte reaction. To this end, the anti-PD-1 antibodies of different clones were administered to the 96 well U bottom plate in the procedure of Example 2 to confirm the intensity of the mixed lymphocytes (Fig. 4 (A)). As shown in the graph shown in FIG. 4 (B), it was confirmed that the ratio of CD8 ⁺ T cells that divide between the first PD-1 antibody and the second PD-1 antibody was different.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

Stimulating a T cell for a reaction having a second MHC class with a first stimulating cell having a first MHC class to raise the expression of PD-1 in the T cell for the reaction;
Adding a second stimulating cell having a first or third MHC class to the T cell for response in which PD-1 expression is elevated to produce a mixed reaction product;
Adding an antibody of PD-1 antibody or PD-L1 to be tested to the mixed reaction product;
Quantitatively detecting the reactivity of the PD-1 and PD-1 antibodies or the PD-L1 and PD-L1 antibodies; And
Comparing the relative effect from the detection step to select PD-1 antibody or PD-L1 antibody,
Wherein the first, second, and third classes induce immunodeficiency reactions with each other in different classes.
The method according to claim 1,
Wherein the first stimulating cell is a cell capable of promoting the expression of PD-1.
The method according to claim 1,
Wherein the cells for the first and second stimulation cells express PD-L1.
The method according to claim 1,
Wherein elevation of PD-1 expression occurs due to TCR activation.
The method of claim 3,
Wherein the cell expressing PD-L1 is APC.
The method according to claim 1,
Wherein the first stimulation cell having the first MHC class, the T cell for the reaction having the second MHC class, and the second stimulation cell are derived from a biological sample.
3. The method of claim 2,
Wherein the cell capable of promoting PD-1 expression is PBMC.
The method according to claim 1,
Wherein the cell for the first or second stimulation is inhibited by physiotherapy or chemotherapy.

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