CN111876381A

CN111876381A - T cell model and application thereof in-vitro human PD-1 antibody efficacy evaluation

Info

Publication number: CN111876381A
Application number: CN202010711055.9A
Authority: CN
Inventors: 张红娟; 黄宇君; 王晶晶; 欧阳雪松; 李其翔
Original assignee: Crown Bioscience Inc Taicang
Current assignee: Crown Bioscience Inc Taicang
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-11-03

Abstract

The invention relates to the field of biological medicines, in particular to a T cell model and application thereof in-vitro evaluation of drug effects of humanized PD-1 antibodies; the invention promotes the in vivo MC-38 tumor epitope specific immune response and the generation of memory T cells by inoculating the MC-38 tumor to the hugEMPD-1 mouse, and establishes the MC-38 epitope specific T cell bank by amplifying the MC-38 tumor epitope specific T cells in vitro. And then, a good experiment platform is provided for evaluating the drug effect of the human-derived hPD-1/PD-L1 antibody by co-culturing an MC-38 cell (MC-38-hPD-L1) carrying a tumor specific antigen and simultaneously over-expressing a human PD-L1 molecule and an epitope specific T cell, and simulating the activation of the tumor antigen-TCR specific T cell in a tumor microenvironment and an inhibition experiment based on a T cell surface PD-1 molecule in the tumor microenvironment in vitro.

Description

T cell model and application thereof in-vitro human PD-1 antibody efficacy evaluation

Technical Field

The invention relates to the field of biological medicines, in particular to a T cell model and application thereof in-vitro evaluation of drug effects of humanized PD-1 antibodies.

Technical Field

With the intensive research on tumor immunity and the study on the interaction mechanism between tumor cells and Immune cells in the tumor microenvironment in the last decade, more and more basic researches and clinical trials show that the clinical effect of an Immune Checkpoint Inhibitor (ICI) in antitumor therapy is significant. The most effective immune checkpoint targets reported to date are PD-1(Programmed Death-1) and CTLA-4(cytotoxic T lymphocytoantigen 4), and antibodies against PD-1 have been approved for first-line treatment of various cancers such as melanoma, lung, kidney, head and neck 1.

PD-1 is a type of transmembrane glycoprotein with the molecular weight of 50-55kD, belongs to a CD28 superfamily, is an important immunosuppressive molecule expressed on the surface of a T cell, participates in negative feedback regulation of T cell activation and promotes T cell exhaustion (T cell X haustion), and in a mouse strain susceptible to autoimmune diseases, such as MRL mice, PD-1 knockout can induce autoimmune diseases 2, such as fatal myocarditis, systemic lupus erythematosus and the like. The ligands for PD-1 include PD-L1(CD274, B7-H1) and PD-L2(CD273, B7-DC), expressed in a broad spectrum on a variety of antigen presenting cells, such as dendritic cells and macrophages; tumor cells also highly express PD-L1 and are regulated by various cytokines such as IFN-r. After the PD-L1/PD-L2 ligand binds to PD-1 receptor on the surface of T cells, tyrosine phosphorylation of PD-1 intracellular domain is promoted, tyrosine phosphatase SHP2 is recruited, thereby inhibiting phosphorylation and signaling at the downstream of TCR, and negatively regulating the activation of T cells and cytokine production 3. Research on blocking antibodies aiming at PD-1, PD-L1 and PD-L2 shows that the function of killer CD8+ T Cells (CTLs) infiltrated in a tumor microenvironment can be effectively improved by inhibiting a PD-1/PD-L1 channel, the secretion of inflammatory cytokines such as IFN-r, TNF-a and IL-2 is promoted, the re-entry of exhausted T cells into a cell cycle is further promoted, and the tumor killing effect is enhanced.

The in vitro efficacy test currently used for preclinical evaluation of PD-1/PD-L1 blocking antibodies mainly includes Mixed Lymphocyte Reaction (MLR), SEB (staphylococcus enterotoxin B) superantigen-induced T cell activation, cytomegalovirus activation test (cytomegalovirus stimulation assays)2 and the like. Among them, the most widely used is mixed lymphocyte reaction, which is T cell activation mediated by allogeneic immune rejection, and due to the diversity of MHC alloantigens (alloantigen) and the difference in the ability and activation strength of different donor T cells to recognize MHC alloantigens, the mixed lymphocyte reaction experiments of different batches have great difference (variation). Secondly, after the mixed lymphocyte reaction is mainly carried out by detecting cytokine secreted by T cells or the amplification of the T cells after the donor DC cells and the tested T cells are co-cultured for 3-5 days, the tumor killing capacity of the T cells and the blocking effect of PD-1/PD-L1 inhibitory antibodies can not be directly reflected. In addition, T cell activation in the tumor microenvironment is triggered by the binding of tumor antigen presented specifically by tumor cell MHC molecules and TCR capable of recognizing the epitope, and is different from the TCR-alloantigen affinity (affinity and avidity) of T cell activation mediated by allogeneic antigen, so that the expression intensity of PD-1 is different, and the mixed lymphocyte reaction cannot well simulate the interaction of PD-1/PD-L1 and the inhibition capability of antibody in the tumor microenvironment.

Disclosure of Invention

The HuGEMM (human specific target protein gene knock-in mouse) model is a mouse protein in which a human specific target protein is substituted, so that the curative effect of inhibitor therapy aiming at a human body, such as human PD-1, PD-L1, human CTLA-4 antibody and the like, and related pathway proteins can be directly evaluated. HuGEMM PD-1 mice not only play an important role in vivo efficacy evaluation, but also play an important role in vitro efficacy evaluation of PD-1/PD-L1 human antibodies. The invention promotes the in vivo MC-38 tumor epitope specific immune response and the formation of memory T cells by inoculating the MC-38 tumor to the hugEMPD-1 mouse, and establishes the MC-38 epitope specific T cell bank by amplifying the MC-38 tumor epitope specific T cells in vitro. And then, a good experiment platform is provided for evaluating the drug effect of the human-derived hPD-1/PD-L1 antibody by co-culturing an MC-38 cell (MC-38-hPD-L1) carrying a tumor specific antigen and simultaneously over-expressing a human PD-L1 molecule and an epitope specific T cell, and simulating the activation of the tumor antigen-TCR specific T cell in a tumor microenvironment and an inhibition experiment based on a T cell surface PD-1 molecule in the tumor microenvironment in vitro.

In order to achieve the purpose, the invention provides the following technical scheme:

a T cell model prepared by the steps of:

(1) inoculating MC-38 tumor cells to huGEMM PD-1 mice to induce the generation of MC-38 antigen specific effector T cells and memory T cells;

(2) synthesizing MC-38 antigen-specific polypeptide;

(3) and (3) stimulating the MC-38 antigen specific effector T cells and memory T cells obtained in the step (1) by the MC-38 antigen specific polypeptide synthesized in the step (2), enriching epitope specific T cells, amplifying in vitro, establishing a cell bank, and establishing the T cell model.

Further, in the T cell model, the specific step of step (1) is:

MC-38 colon cancer tumor cells in logarithmic growth phase were inoculated into huGEMMPD-1 mice with a C57/BL6 background at an inoculum size of 1X10⁶Cell/cell; selecting tumor volume of 50-150mm after tumor inoculation for 5-30 days³The mice of (4) were subjected to aseptic isolation of spleen cells, cryopreserved in a serum cryopreservation solution containing 10% DMSO, and stored in liquid nitrogen.

Further, in a T cell model as described above, the MC-38 antigen-specific polypeptide of step (2) is selected from Adpgk and/or Rpl 18.

Further, in a T cell model as described above, the amino acid sequence of the polypeptide Adpgk is SEQ ID NO: 1; the amino acid sequence of the polypeptide Rpl18 is SEQ ID NO: 2.

further, in the T cell model, the specific step of step (3) is:

a. quickly thawing and reviving spleen cells of cryopreserved hGEMMPD-1MC-38 tumor-bearing miceAt 1x10⁶-5x10⁶Culturing at a cell density of 0.5-1.5ug/mL of Adpgk or Rpl18, 0.5-1.5ug/mL of anti-mCD28 and 1-3ng/mL of rm-IL2, 5% CO₂Culturing in 37 deg.C incubator for 3 days;

b. on the third day, cells were counted at 1-2X10⁶Resuspend the cells at/ml, replad and add fresh medium containing 1-3ng/ml rm-IL2 and 5-15ng/ml rm-IL7, and continue the culture.

c. On day six, cells were counted following the procedure of day three and counted at 1-2X10⁶Resuspending the cells at/ml, replating and adding fresh medium containing 1-3ng/ml rm-IL2 and 5-15ng/ml rm-IL7, and continuing the culture;

d. on day nine, cells were counted and resuspended in fresh medium containing 25-75ng/ml rm-IL2 at a cell concentration of 1-2X10⁶/ml。

e. On the eleventh day, cells were harvested by centrifugation, stimulated with T cell activated cocktail for 2-6h, flow tested for Live/Dead, CD8, PD-1, IFN-r, and the remaining cells were cryopreserved for use to obtain a T cell bank specific for either the Adpgk or Rpl18 epitope, the T cell model.

In another aspect of the present invention, the use of the above T cell model in the evaluation of the drug effect of the human PD-1 antibody in vitro comprises the following steps:

a) MC-38 tumor cells stimulated in vitro with 0.5-1.5ug/ml Adpgk or Rpl18 were incubated at 37 ℃ for 30 min in an incubator and unbound polypeptides were washed away with PBS;

b) labeling MC-38 tumor cells in log phase growth with 2.5uM CFSE;

c) co-culturing MC-38 tumor cells after stimulation by Adpgk or Rpl18 with T cells specific for Adpgk or Rpl18 epitope, wherein the number ratio of the T cells to the tumor cells is 1:1-5: 1; adding a PD-1 antibody drug to be evaluated into a co-culture system;

d) co-culturing for 24-48 hr, collecting cell supernatant to detect IFN-r secretion, collecting cell precipitate, flow detecting L/D, CD45 and CFSE, and detecting the ratio of dead cells in CFSE + cell to directly detect the ability of epitope specific T cell to kill tumor cell after adding PD-1 antibody medicine.

Further, in the application of the T cell model in the evaluation of the drug effect of the human PD-1 antibody in vitro, the MC-38 tumor cells are MC-38 cells which carry tumor specific antigens and simultaneously over-express human PD-L1 molecules: MC-38-hPD-L1.

Furthermore, in the application of the T cell model in the evaluation of the drug effect of the human PD-1 antibody in vitro, the PD-1 antibody drug is Keytruda analogue or an isotype control antibody IgG 4.

Furthermore, in the application of the T cell model in-vitro human PD-1 antibody drug effect evaluation, the PD-1 antibody drug is used for treating one or more of melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, urothelial cancer, gastric cancer/gastroesophageal junction adenocarcinoma, metastatic colorectal cancer, cervical cancer, hepatocellular carcinoma, renal cell carcinoma, B cell lymphoma and Merkel cell carcinoma.

The invention has the following beneficial effects:

1) the test shows that the T cells from the T cells in the tumor microenvironment are expanded in vitro, so that the characteristics of the inhibited T cells in the tumor microenvironment can be better simulated.

2) The T cell separated from the spleen of the tumor-bearing huGEMM PD-1 mouse carries the humanized PD-1 target spot, can be directly used for verifying the drug effect of the humanized PD-1 antibody drug, and avoids using a mouse substitute drug.

3) The MC-38Adpgk and Rpl18 epitope-specific T cells obtained by in vitro amplification are distinguished from normal PBMC donor-derived T cell-mediated allogeneic rejection, and are antigen-specific immune responses occurring in the tumor microenvironment.

4) The in vitro amplification of the cryopreserved MC-38Adpgk and Rpl18 epitope specific T cells can effectively control the stability between batches and avoid the difference between different batches of MCH and allogeneic rejection T cells in allogeneic rejection.

5) Based on CFSE labeling, epitope-specific T cell-mediated killing of MC-38 tumor cells is directly detected by flow, and compared with an MLR experiment for detecting killing of T cells indirectly reacted by cytokine secretion, more direct experimental evidence is provided for evaluating the capability of PD-1/PD-L1 for enhancing T cell killing.

Drawings

FIG. 1 is an overall technical flow diagram of the present invention;

FIG. 2 is an in vitro expansion of ADPGK antigen T-specific T cells;

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The technical scheme of the experiment is shown in figure 1 and mainly comprises the following 4 steps: 1hugEMM PD-1 mice were inoculated with MC-38 tumors, inducing the production of MC-38 antigen-specific effector T cells and memory T cells. 2MC-38 antigen-specific polypeptide synthesis. In vitro expansion of 3 adpck, Rpl18 epitope specific T cells and establishment of cell banks. 4 evaluation of epitope-specific T cell killing and PD-1/PD-L1 inhibitory ability based on Adpgk and Rpl 18.

Example 1

HUGEMM PD-1 mouse MC-38 tumor inoculation, induced the production of MC-38 antigen specific effector T cells and memory T cells.

The method comprises the following steps: hugEMM PD-1 mouse (B6/JGpt-Pdcd 1)^{em1Cin(hPDCD1)}/Gpt, Cat # T003095, Collection drug kang) MC-38 tumor vaccination induced the production of MC-38 antigen specific effector T cells and memory T cells. MC-38 colon cancer tumor cells in logarithmic growth phase were inoculated into huGEMMPD-1 mice with a C57/BL6 background at an inoculum size of 1X10⁶Cell/cell. Selecting the tumor volume of 100mm 10-15 days after tumor inoculation³The mice of (4) were subjected to aseptic isolation of spleen cells, cryopreserved in a serum cryopreservation solution containing 10% DMSO, and stored in liquid nitrogen.

Example 2

Synthesis of MC-38 antigen-specific polypeptide.

Studies by Mahesh Yadav and Mahesh Yadav et al found that MC-38 tumors were able to specifically present specific peptide fragments of mutations in proteins of Adpgk and Rpl18 (Adpgk: SEQ ID NO: 1 ═ ASMTNMELM, Rpl18: SEQ ID NO: 2 ═ KILTFDRL, mutated amino acids M and R bold), to the MHCI molecule, and were recognized and bound by this epitope-specific TCR, thereby promoting activation and immune response of antigen-specific killer CD8+ infiltrating into the tumor microenvironment. Therefore, we have synthesized two polypeptides as shown in Table 1 below according to literature reports.

TABLE 1 polypeptide List

Polypeptide name	Sequence (N '-C')	Length (aa)	Mass (mg)	Purity of	Decoration
						Adpgk	ASMTNMELM	9	4	>95％	Is free of
Rpl18	KILTFDRL	8	4	>95％	Is free of

Example 3

In vitro expansion of Adpgk epitope specific T cells and establishment of cell banks.

After MC-38 is inoculated into huGEMMPD-1 mice, tumor antigens, in particular the antigen epitopes (Adpgk and Rpl18) with strong immunogenicity, which are reported above, are captured by professional antigen presenting cells such as dendritic cells, are presented on MHCI molecules and migrate to lymph nodes of the mice, navT cells which are not stimulated by any antigen are activated and amplified in the lymph nodes, migrate to the tumor microenvironment through draining lymph nodes and are re-stimulated by the tumor antigens in the tumor microenvironment, are reactivated and exponentially amplified, the tumor cells are killed by the epitope specificity, most of the effector cells are subjected to apoptosis after the tumor cells are killed by the effector cells, only a few cells form memory cells and migrate to spleen. Therefore, tumors were selected at 100mm approximately 10-15 days after inoculation of our MC-38 cells³The size of the mice, which had smaller tumor volumes, indicated that there was more immune response and memory T cell formation in vivo, and the spleen was selected to expand T cells in vitro to obtain more epitope-specific T cells. Frozen spleen cells are cultured in vitro, the synthesized antigen short peptide is added, antigen presenting cells in the spleen cells such as dendritic cells, macrophages and the like are used for carrying out antigen presenting, and T cells with epitope specificity are activated in vitro. During the culture, a co-stimulatory molecule CD-28 activating Antibody (CD28 Monoclonal Antibody (37.51), functional grade, eBioscience) was added^TM16-0281-85, Thermo Fisher) helps the epitope-specific T cells to obtain a more adequate co-stimulatory signal, while recombinant mouse interleukin-2 (rm-IL-2) is added during culture to maintain the expansion provided by activated epitope-specific T cells. Six days after activation, recombinant mouse interleukin-7 (rm-IL-7) was added to the culture to continue to promote the maintenance and survival of epitope-specific T cells. Based on the above principle, the detailed steps for the in vitro expansion of Adpgk, Rpl18 epitope-specific T cells and the establishment of cell banks are as follows:

a. quickly thawing and recovering spleen cells of cryopreserved hGEMMPD-1MC-38 tumor-bearing mice at 1x10⁷Cell density culture at/mL with 1ug/mL Adpgk polypeptide, 1ug/mL anti-mCD28 and 2ng/mL rm-IL2, 5% CO₂Culturing in 37 deg.C incubator for 3 days;

b. on day three, cells were counted at 1.5X10⁶Resuspend the cells at/ml, replad and add fresh medium containing 2ng/ml rm-IL2 and 10ng/ml rm-IL7, continue culturing;

c. on day six, cells were counted following the procedure of day three and counted at 1.5X10⁶Resuspend the cells at/ml, replad and add fresh medium containing 2ng/ml rm-IL2 and 10ng/ml rm-IL7, continue culturing;

d. on day nine, cells were counted and resuspended in fresh medium containing 50ng/ml rm-IL2 at a cell concentration of 1.5X10⁶/ml。

e. On the eleventh day, cells were harvested by centrifugation and stimulated with T cell activated cocktail for 4h, flow (BDLSRFortessa)^TM) And detecting Live/Dead, CD8, PD-1 and IFN-r, and freezing the residual cells for later use to obtain a T cell bank with specific Adpgk epitope, namely the T cell model. As shown in fig. 2, a microscope picture of the in vitro expansion of the adpck antigen-specific T cells shows the T cells expanded in vitro culture on days 0, 3, 6, and 10.

The staining antibody combinations used in the above experiments are shown in table 2 below:

TABLE 2 staining antibody combinations

Example 4

Evaluation of epitope-specific T cell killing and PD-1/PD-L1 inhibitory potency based on Adpgk.

The method comprises the following steps:

a) in vitro stimulated MC-38 tumor cells with 1ug/ml Adpgk, incubated at 37 ℃ for 30 min in an incubator, and unbound polypeptides were washed off with PBS;

b) labeling MC-38 tumor cells in log phase growth with 2.5uM CFSE;

c) co-culturing MC-38 tumor cells stimulated by Adpgk and T cells specific to Adpgk epitopes, wherein the number ratio of the T cells to the tumor cells is 2.5: 1; adding 10ug/ml anti-PD-1(Keytruda analogue, Mesudanaceae) to the co-culture system;

d) co-culturing for 24-48 hr, collecting cell supernatant to detect IFN-r secretion, collecting cell precipitate, and flow detecting L/D, CD45 and CFSE to detect the dead cell ratio in CFSE + cell and to detect the capacity of epitope specific T cell to kill tumor cell after adding PD-1 antibody medicine.

The method can directly reflect the killing condition of the tumor target cells, and can collect culture supernatant to detect the secretion of cytokines, so that the method can comprehensively and accurately reflect the killing and activating capacity of T cells compared with an MLR method.

In conclusion, the T cell model provided by the invention can be used for in vitro simulation of tumor antigen-TCR specific T cell activation in a tumor microenvironment and inhibition experiments based on T cell surface PD-1 molecules in the tumor microenvironment, provides a good experimental platform for evaluating the drug effect of the humanized PD-1 antibody, provides a result as accurate as possible on the premise of saving a large amount of time and money for related drug experiments, and provides a valuable reference for in vivo experiments of subsequent drugs.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhongmei corotaceae Biotechnology (Taicang) Co., Ltd

<120> T cell model and application thereof in-vitro efficacy evaluation of humanized PD-1 antibody

<130>20200721

<160>2

<170>PatentIn version 3.3

<210>1

<211>9

<212>PRT

<213> Artificial

<220>

<223>Adpgk

<400>1

Ala Ser Met Thr Asn Met Glu Leu Met

1 5

<210>2

<211>8

<212>PRT

<213> Artificial

<220>

<223>Rpl18

<400>2

Lys Ile Leu Thr Phe Asp Arg Leu

1 5

Claims

1. A T cell model prepared by the steps of:

(2) synthesizing MC-38 antigen-specific polypeptide;

2. The T-cell model of claim 1, wherein the specific steps of step (1) are:

MC-38 colon cancer tumor cells in logarithmic growth phase were inoculated into huGEMMPD-1 mice with a C57/BL6 background at an inoculum size of 1X10⁶Cell/cell; selecting tumor volume of 50-150mm after tumor inoculation for 5-30 days³The spleen of the mouse of (1)The cells were aseptically separated, frozen in serum frozen stock containing 10% DMSO, and stored in liquid nitrogen.

3. A T-cell model according to claim 1, wherein the MC-38 antigen-specific polypeptide of step (2) is selected from Adpgk and/or Rpl 18.

4. The T-cell model of claim 3, wherein the amino acid sequence of the polypeptide Adpgk is SEQ ID NO: 1; the amino acid sequence of the polypeptide Rpl18 is SEQ ID NO: 2.

5. the T-cell model of claim 3, wherein the specific steps of step (3) are:

a. quickly thawing and recovering spleen cells of cryopreserved hGEMMPD-1MC-38 tumor-bearing mice at 1x10⁶-5 x 10⁶Culturing at a cell density of 0.5-1.5ug/mL while adding Adpgk or Rpl18, anti-mCD28 at 0.5-1.5ug/mL and rm-IL2 at 1-3ng/mL, and 5% CO₂Culturing in 37 deg.C incubator for 3 days;

b. on day three, cells were counted at 1-2X10⁶Resuspending the cells at/ml, replating and adding fresh medium containing 1-3ng/ml rm-IL2 and 5-15ng/ml rm-IL7, and continuing the culture;

d. on day nine, cells were counted and resuspended in fresh medium containing 25-75ng/ml rm-IL2 at a cell concentration of 1-2X10⁶/ml；

6. The use of a T cell model of claim 3 for the in vitro assessment of the potency of a human PD-1 antibody, comprising the steps of:

b) labeling MC-38 tumor cells in log phase growth with 2.5uM CFSE;

7. Use of a T cell model according to claim 6 for the in vitro evaluation of the pharmacological effects of human PD-1 antibodies, wherein the MC-38 tumor cells are MC-38 cells carrying a tumor-specific antigen and simultaneously overexpressing the human PD-L1 molecule: MC-38-hPD-L1.

8. The use of a T cell model according to claim 6 for the in vitro evaluation of the pharmacological effects of a human PD-1 antibody, wherein the PD-1 antibody drug is a Keytruda analog or its isotype control antibody IgG 4.

9. The use of a T-cell model according to claim 6 for the in vitro evaluation of the pharmacological effects of a human PD-1 antibody, wherein the PD-1 antibody is used for the treatment of one or more of melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, Hodgkin's lymphoma, urothelial cancer, gastric/gastroesophageal junction adenocarcinoma, metastatic colorectal cancer, cervical cancer, hepatocellular carcinoma, renal cell carcinoma, B-cell lymphoma, Merkel cell carcinoma.