# US 2019/0170752 A1 · Method for predicting efficacy of immune checkpoint inhibitors in cancer patients

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- Number: US 2019/0170752 A1
- Document ID: US20190170752A1
- Status: Published application
- Kind: A1
- Application number: 16/323,410
- Filed: 2017-07-31
- Published: 2019-06-06
- Earliest claimed: 2016-08-08
- Assignees: BeiGene, Ltd.
- Applicants: BeiGene, Ltd.
- Inventors: Lusong Luo; Chengliang Dai; Zhijian Sun
- Web page: https://patentgrep.com/patent/US20190170752A1

## CPC

- G01N 33/575: for cancer
- A61P 35/00: Antineoplastic agents
- C07K 16/2818: {against CD28 or CD152}
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- G01N 2500/10: involving cells
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Disclosed is a method for predicting clinical response of a cancer patient to treatment with an immune checkpoint inhibitor, or a combination of two or more thereof, or with a combination of an immune checkpoint inhibitor and other antitumor drugs, by introducing bispecific T cell engager (BiTE) into the platform of lymphocytes containing T cells in vitro, which are cultured on feeder cells or in Matrigel®.

## Claims

### Claim 1

An ex vivo or in vitro method for predicting clinical response of cancer cells treated by an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, comprising
1) Contacting a co-culture system comprising the cancer cells and lymphocytes containing T cells with an bispecific T-cell engage antibody in the presence or absence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs; and
2) Determining whether the release of IFN-γ due to the activation of T cells by the bispecific T-cell engage antibody is increased in the presence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs as compared to the one absence thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cancer cells are primary tumor cells from a cancer patient, or cancer cells of a cancer cell line.

### Claim 3 (depends on 1)

The method of claim 1, wherein the co-culture system is obtained by co-culturing the cancer cells with lymphocytes containing T cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the increased release of IFN-γ is determined by measuring human IFN-γ from supernatant in the absence or presence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, to produce a cIFN-γ amount and a tIFN-γ amount, respectively, wherein a tIFN-γ amount higher than a cIFN-γ amount indicates that the patient is more likely to respond to the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 5 (depends on 4)

The method of claim 4 wherein the increased release of IFN-γ is determined by the value of the tIFN-γ/cIFN-γ ratio, i.e., by dividing the tIFN-γ amount by the cIFN-γ amount, wherein a tIFN-γ/cIFN-γ ratio number higher than 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 or more indicates that the patient is more likely to respond to the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 6 (depends on 1)

The method of claim 1, for predicting clinical response of a cancer patient to treatment by an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, comprising:
(a) co-culturing primary tumor cells from a cancer patient, or cancer cells of a cancer cell line, preferably one corresponding to the cancer in the patient, with lymphocytes containing T cells, to obtain a co-culture system;
(b) adding a bispecific T-cell engage into the co-culture system of (a), and dividing into control groups and treatment groups;
(c) adding an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs into the treatment groups of (b); and
(d) measuring human IFN-γ from supernatant of the control groups and the treatment groups, to produce a cIFN-γ amount and a tIFN-γ amount, respectively, wherein a tIFN-γ amount higher than a cIFN-γ amount indicates that the patient is more likely to respond to the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 7

A method for screening a cancer patient who may benefit from treatment by an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, comprising:
1) Contacting a co-culture system comprising primary tumor cells from the cancer patient and lymphocytes containing T cells with an bispecific T-cell engage antibody in the presence or absence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs; and
2) Determining whether the release of IFN-γ due to the activation of T cells by the bispecific T-cell engage antibody is increased in the presence of the i immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs as compared to the one absence thereof.

### Claim 8 (depends on 7)

The method of claim 7, comprising:
(a) co-culturing primary tumor cells from a cancer patient with lymphocytes containing T cells, to obtain a co-culture system;
(b) adding a bispecific T-cell engage into the co-culture system of (a), and dividing into control groups and treatment groups;
(c) adding an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs into the treatment groups of (b); and
(d) measuring human IFN-γ from supernatant of the control groups and the treatment groups, to produce a cIFN-γ amount and a tIFN-γ amount, respectively, wherein a tIFN-γ amount higher than a cIFN-γ amount indicates that the patient is more likely to benefit from the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 9 (depends on 8)

The method of claim 8, wherein a tIFN-γ/cIFN-γ ratio is determined by dividing the tIFN-γ amount by the cIFN-γ amount, wherein a tIFN-γ/cIFN-γ ratio number higher than 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 or more indicates that the patient is more likely to benefit from the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 10 (depends on 7, 8, 9)

A method for treating cancer by an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs in a patient, wherein said patient has been screened by the method according to any one of claims 7-9 as a patient, who may benefit from treatment by said immune checkpoint inhibitor or said combination of two or more thereof or said combination of an immune checkpoint inhibitor and other antitumor drugs.

### Claim 11

A method for screening an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, effective in the treatment of a cancer of interest, comprising:
1) Contacting a co-culture system comprising primary tumor cells from the cancer patient and lymphocytes containing T cells with an bispecific T-cell engage antibody in the presence or absence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs; and
2) Determining whether the release of IFN-γ due to the activation of T cells by the bispecific T-cell engage antibody is increased in the presence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs as compared to the one absence thereof.

### Claim 12 (depends on 11)

The method of claim 11, comprising:
(a) co-culturing cancer cells, preferably cancer cells of a cancer cell line corresponding to the cancer of interest, or primary cancer cells from one or more cancer patients, with lymphocytes containing T cells, to obtain a co-culture system;
(b) adding a bispecific T-cell engage into the co-culture system of (a), and dividing into control groups and treatment groups;
(c) adding an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs into the treatment groups of (b); and
(d) measuring human IFN-γ from supernatant of the control groups and the treatment groups, to produce a cIFN-γ amount and a tIFN-γ amount, respectively, wherein a tIFN-γ amount higher than a cIFN-γ amount indicates that the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs is effective in the treatment of the cancer of interest.

### Claim 13 (depends on 12)

The method of claim 12, wherein a tIFN-γ/cIFN-γ ratio is determined by dividing the tIFN-γ amount by the cIFN-γ amount, wherein a tIFN-γ/cIFN-γ ratio number higher than 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 or more indicates that the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs is effective in the treatment of the cancer of interest.

### Claim 14

A bispecific T-cell engage antibody for use in a method for predicting clinical response of a cancer patient to treatment by an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs, wherein
1) the bispecific T-cell engage antibody is contacted with a co-culture system comprising primary tumor cells from the cancer patient and lymphocytes containing T cells in the presence or absence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs; and
2) the release of IFN-γ due to the activation of T cells by the bispecific T-cell engage antibody is measured to determine whether the release of IFN-γ is increased in the presence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs as compared to the one absence thereof.

### Claim 15

A bispecific T-cell engage antibody for use in a method for screening an immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs effective in the treatment of a cancer of interest, wherein
1) the bispecific T-cell engage antibody is contacted with a co-culture system comprising primary tumor cells from the cancer patient and lymphocytes containing T cells in the presence or absence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs; and
2) the release of IFN-γ due to the activation of T cells by the bispecific T-cell engage antibody is measured to determine whether the release of IFN-γ is increased in the presence of the immune checkpoint inhibitor or a combination of two or more thereof or a combination of an immune checkpoint inhibitor and other antitumor drugs as compared to the one absence thereof.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the bispecific T-cell engage is a bispecific single chain antibody construct containing at least two domains, wherein one of the domains specifically binds to antigens selected from human CD3, CD4, or CD8 antigen, and the second domain specifically binds to human tumor antigens selected from EpCAM antigen, EGFR antigen, CEA antigen, Her2 antigen, CD19 or CD20.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the bispecific T-cell engage is an anti-EpCAM×CD3 bispecific T-cell engage, e.g., comprising an amino sequence of SEQ NO 1.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the cancer is a solid tumor or a liquid tumor.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the cancer or the solid tumor is selected from gastric cancer, colon cancer, colorectal cancer, breast cancer, ovary cancer or a combination thereof.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the immune checkpoint inhibitor is an antibody or a fragment antigen binding thereof, or a chemical molecule drug.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the immune checkpoint inhibitor is a chemical molecule drug, which inhibits one or more checkpoint proteins selected from human PD-1, TIM-3, A2AR, B7-H3(CD276), B7-H4(VTCN1), BTLA, CTLA-4, IDO, KIR, LAG3, or VISTA.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the immune checkpoint inhibitor is an antibody or a fragment antigen binding thereof, which specifically binds to one or more checkpoint proteins selected from human PD-1, TIM-3, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, CTLA-4, IDO, LAG3, or VISTA.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the immune checkpoint inhibitor is an antibody or a fragment antigen binding thereof, which specifically binds to PD-1.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, Mab-1 comprising an amino sequence of SEQ NO 2.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the other anti-tumor drug may have various modes of actions selected from immune therapies, targeted therapies, chemotherapies, vaccines, radiotherapies or combination thereof.

### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the other anti-tumor drug is selected from a chemical molecule drug, a proteasome inhibitor, a histone deacetylase inhibitor, a glucocorticoid, a steroid, a targeted therapy agent or combination thereof.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the other anti-tumor drug is a B-raf inhibitor.

### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the other anti-tumor drug is 5-(((1R,1aS,6bR)-1-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1a,6b-dihydro-1H-cyclopropa[b]benzofuran-5-yl)oxy)-3,4-dihydro-1,8-naphthyridin-2(1H)-one or a pharmaceutically acceptable salt thereof.

### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the other anti-tumor drug is 5-(((1R,1aS,6bR)-1-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1a,6b-dihydro-1H-cyclopropa[b]benzofuran-5-yl)oxy)-3,4-dihydro-1,8-naphthyridin-2(1H)-one Sesqui-Maleate.

### Claim 30 (depends on 1, 2)

The method of any one of claim 1 or 2, wherein the primary tumor cells are derived from a tumor biopsy, or a tumor tissue sample of surgery.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the primary tumor cells are cultured on feeder cells, or in Matrigel®.

### Claim 32 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the primary tumor cells were re-suspended with a medium comprising a substance listed as follows, before culturing,
1) F12/DMEM 1:1 medium 500 mL;
2) Fetal Bovine Serum 50 mL;
3) Insulin-Transferrin-Selenium (ITS-G) 0.5×;
4) Penicillin-Streptomycin 1×;
5) HEPES 1×;
6) Glutamine 2 mM;
7) MEM Non-Essential Amino Acids Solution 1×; and
8) Sodium pyruvate 1×.

### Claim 33 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the lymphocytes containing T cells are derived from peripheral blood.

### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the lymphocytes containing T cells are derived from allogeneic PBMCs, or autologous PBMCs.

### Claim 35 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 13 or 14, wherein the lymphocytes containing T cells are derived from tissue selected from allogeneic TILs, autologous TILs, allogeneic lymphocytes from lymph node, autologous lymphocytes, ascites from lymph node, allogeneic spleen, autologous spleen, allogeneic ascites, or autologous ascites.

### Claim 36 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the lymphocytes containing T cells can be replaced by purified CD3+ cells or CD4+ cells.

### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the PBMCs can be replaced by purified CD3+ cells or CD4+ cells.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 5, wherein the PBMCs are pre-activated by an antibody specifically binding to human CD3, CD4, or CD8 antigen.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the PBMCs are pre-activated by an anti-CD3 antibody.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the control groups are groups further adding an isotype control antibody.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 13 or 14, wherein the control groups are groups further adding an isotype control IgG.

### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the tIFN-γ/cIFN-γ ratio is determined by simultaneously measuring tIFN-γ amount and cIFN-γ amount with flow cytometry, ELISPOT or ELISA.

### Claim 43 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-13 and the bispecific T-cell engage antibody of claim 14 or 15, wherein the tIFN-γ/cIFN-γ ratio is determined by simultaneously measuring tIFN-γ amount and cIFN-γ amount with ELISA.