阅读说明：本技术 用于治疗癌症的多克隆抗体和抗pd1或抗pdl1抗体的联合 (Combination of polyclonal antibodies and anti-PD 1 or anti-PDL 1 antibodies for the treatment of cancer ) 是由 O·迪沃 B·瓦恩霍维 于 2020-04-28 设计创作，主要内容包括：本发明涉及针对癌细胞的非人哺乳动物多克隆抗体；与至少一种选自下组的单克隆抗体的联合：抗PD1和抗PDL1单克隆抗体,其用于预防和/或治疗哺乳动物患者的癌症。也预期这种联合。(The present invention relates to non-human mammalian polyclonal antibodies directed against cancer cells; in combination with at least one monoclonal antibody selected from the group consisting of: anti-PD 1 and anti-PDL 1 monoclonal antibodies for use in the prevention and/or treatment of cancer in a mammalian patient. Such combinations are also contemplated.)

1. -a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies,

for use in the prevention and/or treatment of cancer in a mammalian patient.

2. The combination for its use according to claim 1, wherein the polyclonal antibody does not contain:

-a first antigenic determinant selected from the group consisting of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose; and

-a second antigenic determinant, which is different from the first antigenic determinant and is selected from the group of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

3. The combination for its use according to claim 1 or 2, wherein said polyclonal antibody does not contain two antigenic determinants (i) N-ethanediol neuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

4. The combination for its use according to any one of the preceding claims, wherein the polyclonal antibody is immunoglobulin G (IgG).

5. The combination for its use according to any one of the preceding claims, wherein the monoclonal antibody is an anti-PDL 1 antibody.

6. The combination for its use according to any one of the preceding claims, wherein the non-human mammal is selected from the group consisting of: rodents such as mice, rats, guinea pigs, and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; a goat; sheep; bovines, such as cows; pigs, such as piglets and castrated pigs; a camelid; horses and primates.

7. The combination for its use according to any one of the preceding claims, wherein the non-human mammal is selected from the group consisting of: rodents, such as mice, rats, guinea pigs and hamsters, and lagomorphs, such as rabbits.

8. The combination for its use according to any one of the preceding claims, wherein the mammalian patient is selected from the group consisting of: rodents such as mice, rats, guinea pigs, and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; a goat; sheep; bovines, such as cows; pigs, such as piglets and castrated pigs; a camelid; a horse; primates and humans.

9. The combination for its use according to any one of claims 1-8, wherein the mammalian patient is a human.

10. The combination for its use according to any one of the preceding claims, wherein the cancer cells are selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, leukemia, myeloma, melanoma, oral or oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer, adenoid cystic cancer, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, bile duct cancer, bone cancer, brain cancer, central nervous system tumor, cervical cancer, esophageal cancer, eye cancer, eyelid cancer, gastrointestinal cancer, HIV/AIDS-related cancer, tear adenocarcinoma, laryngeal cancer or hypopharyngeal cancer, leukemia, liver cancer, meningioma, nasopharyngeal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, parathyroid cancer, penile cancer, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic tumor and thymus cancer, vaginal cancer, and vulval cancer.

11. The combination for its use according to any one of the preceding claims, wherein the cancer cells are from liver cancer.

12. The combination for its use according to any one of the preceding claims, wherein the cancer cells are from a cold tumor.

13. The combination for its use according to any one of the preceding claims, wherein the non-human mammalian polyclonal antibody and the at least one monoclonal anti-PD 1 or anti-PDL 1 antibody are administered to a mammalian patient in the same composition or in separate compositions, preferably in separate compositions.

14. The combination for its use according to any one of the preceding claims, wherein the combination further comprises at least one additional anti-cancer drug different from the non-human mammalian polyclonal antibody and monoclonal anti-PD 1 or anti-PDL 1 antibody.

15. A combination for its use according to claim 14, wherein said at least one additional anticancer drug is selected from monoclonal antibodies, in particular from the group consisting of: anti-CD 137, anti-CTLA 4, anti-TIM-3, anti-B7-H3, anti-CD 134, anti-CD 154, anti-LAG-3, anti-CD 227, anti-BTNA 3, anti-CD 39, anti-CD 73, anti-CD 115, anti-sirpa, anti-SIRP γ, anti-CD 28, anti-NCR, anti-NKp 46, anti-NKp 30, anti-NKp 44, anti-NKG 2D, and anti-DNAM-1 monoclonal antibodies.

16. -a non-human mammalian polyclonal antibody directed against cancer cells; -in combination with at least one monoclonal antibody selected from the group consisting of: anti-PD 1 and anti-PDL 1 monoclonal antibodies, said combination being particularly as defined in any one of claims 2-7, 14 and 15.

Technical Field

The present invention relates to the field of immunology and more particularly to the combination of polyclonal antibodies with monoclonal anti-PD 1 and/or anti-PDL 1 antibodies, and their use in human medicine.

In particular, the invention relates to the use of a polyclonal non-human mammalian antibody in combination with monoclonal anti-PD 1 and/or anti-PDL 1 antibodies in the treatment of cancer.

Background

Cancer is the second leading cause of death in the world, and 960 ten thousand people are estimated to die of cancer in 2018. Around one sixth of all deaths worldwide are caused by cancer. In this field, it is common that no or insufficient active treatment is available. Accordingly, there is a recognized need to develop new and improved therapies for treating cancer.

Immunotherapy represents a real hope for treating a large number of cancers, particularly cancers that are currently not effectively cured by conventional therapies. This treatment consists mainly in the administration of monoclonal antibodies directed against tumor cells or against activators or inhibitors of checkpoints of the anti-cancer immune response.

Antibodies directed against tumors (passive immunotherapy) can act in two complementary ways:

-complement dependent by cytotoxicity (CDC) or killer cell dependent (ADCC) or phagocyte dependent (ADCP).

By inducing an adaptive immune response. Indeed, it has been demonstrated that opsonin action of the target and local production of complement molecules (C3a, C5a) (see Strainic et al, Immunity,2008 Mar; 28(3):425-35) activate T lymphocyte costimulation and increase survival. Thus, passive administration of an anti-tumor antibody can release factors from complement as a first step and, in a second step, alleviate the T cell response.

However, 40% to 85% of patients are resistant to treatment based on the use of monoclonal antibodies. Escape mechanisms include immunoselection mechanisms, i.e., the ability of tumor cells to lose antigen recognition by the immune system and immune subversion mechanisms (induction of specific drug resistance). The appearance of less immunogenic tumor variants may be particularly detrimental in treatments based on the use of monoclonal antibodies, which are specific for unique epitopes.

Thus, treatments based on the use of polyclonal antibodies targeting different epitopes on tumor cells may allow to minimize escape mechanisms.

However, despite potential efficacy, polyclonal antibodies are rarely used to treat tumors, particularly due to the high toxicity risk of the patient. This toxicity is mainly based on the following facts: many cell surface antigens are expressed on many different cell types in an organism, which can lead to major cross-reactivity with non-cancerous cells. Toxicity is also associated with expression of Neu5GC and alpha-1, 3-galactose carbohydrates on animal immunoglobulins, which elicit potent anti-Neu 5GC and anti-alpha-1, 3-galactose immune responses in humans associated with allergy, seropathy, and immune complex formation. Furthermore, these cross-reactivities are also associated with another problem (i.e., it "dilutes" the effective antibodies in the organism) that the large administered polyclonal antibody population binds in a different site than the actual cancer target. This loss of effective antibody in the targeted tumor requires the administration of larger amounts of polyclonal antibody in the patient, which of course leads to an aggravation of the associated toxic effects.

Thus, there remains a need in the art to provide effective and improved therapies for treating cancer with reduced or no side effects.

Resistance of many tumors to treatment based on the use of monoclonal antibodies may also result from the presence of "cold tumors" (Bonaventura et al, 2019; front. immune.; 10; 168), i.e. tumors that for various reasons contain less infiltrating immune cells, in particular infiltrating T cells, and therefore do not elicit a strong response of the immune system, making them difficult to treat with immunotherapy. In fact, in such tumors, the response rate to therapeutic monoclonal antibodies targeting immune checkpoints commonly used in therapeutic antibodies (e.g., treating tumors) is still relatively low and thus ineffective.

Thus, there remains a need in the art to provide effective treatments that allow for the treatment of cold tumors.

There is also a need in the art to provide means to convert cold tumours into "hot tumours", i.e. tumours comprising sufficient levels or even high levels of infiltrating T cells as explained above.

There is a particular need in the art to provide means to reduce the resistance of "cold" tumours to monoclonal antibodies targeting immune checkpoints by improving (i.e. increasing the ability of these tumours to be infiltrated by immune cells, and in particular T cells and bone marrow cells, more in particular T cells).

Summary of The Invention

According to a first aspect thereof, the invention relates to:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies for use in the prevention and/or treatment of cancer in a mammalian patient.

The non-human mammalian polyclonal antibodies in combination according to the present invention are directed against cancer cells corresponding to or even belonging to the cancer to be prevented or treated in a mammalian patient.

The inventors show for the first time that the combined in vivo administration of a polyclonal antibody against cancer, in particular against liver cancer, and a monoclonal anti-PDL 1 antibody provides an unexpectedly high efficiency against targeted tumors. In particular, the inventors observed a synergistic effect of these active substances on the treatment of tumors.

In addition, after several weeks of treatment, the surviving animals were evaluated histopathologically or for toxicity with no evidence of visible disease (no autoimmunity detected, i.e. no pain, no weight loss, no animal discoloration, no alopecia or diarrhea).

Accordingly, the inventors sought to identify a new, effective, improved and non-toxic therapy for the treatment of cancer.

In a specific embodiment, the polyclonal antibody in combination according to the invention does not contain:

-a first antigenic determinant selected from the group consisting of (i) N-ethanediol neuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose; and

-a second antigenic determinant, which is different from the first antigenic determinant and is selected from the group of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

More particularly, the polyclonal antibody may be free of two antigenic determinants (i) N-ethanediol neuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

In a specific embodiment, the polyclonal antibody is immunoglobulin g (igg).

In a further embodiment, the monoclonal antibody combined according to the invention is an anti-PDL 1 antibody.

In another embodiment, the non-human mammal is selected from the group consisting of: rodents such as mice, rats, guinea pigs, and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; a goat; sheep; bovines, such as cows; pigs (swine), such as piglets (pig) and castrated pigs (hog); a camelid; horses and primates.

In particular, the non-human mammal may be selected from the group consisting of: rodents, such as mice, rats, guinea pigs and hamsters, and lagomorphs, such as rabbits.

More particularly, the non-human mammal may be selected from the group consisting of: lagomorphs, goats, sheep, piglets, horses and bovines.

In particular embodiments, the mammalian patient is selected from the group consisting of: rodents such as mice, rats, guinea pigs, and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; a goat; sheep; bovines, such as cows; pigs, such as piglets and castrated pigs; a camelid; a horse; primates and humans.

In particular, the mammalian patient may be selected from the group consisting of: rodents, such as mice, rats, guinea pigs, and hamsters.

In particular, the mammalian patient may be a human.

In particular embodiments, the mammalian patient may be selected from the group consisting of: rodents such as mice, rats, guinea pigs, and hamsters; and a human.

In another embodiment, the cancer cell is selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, leukemia, myeloma, melanoma, oral or oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer, adenoid cystic cancer, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, bile duct cancer, bone cancer, brain cancer, central nervous system tumor, cervical cancer, esophageal cancer, eye cancer, eyelid cancer, gastrointestinal cancer, HIV/AIDS-related cancer, tear adenocarcinoma, laryngeal cancer or hypopharyngeal cancer, leukemia, liver cancer, meningioma, nasopharyngeal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, parathyroid cancer, penile cancer, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic tumor and thymus cancer, vaginal cancer, and vulval cancer.

In particular, the cancer cells are from liver cancer.

In particular embodiments, the non-human mammalian polyclonal antibody and the at least one monoclonal anti-PD 1 or anti-PDL 1 antibody are administered to the mammalian patient in the same composition or in separate compositions, preferably in separate compositions.

In another embodiment, the combination according to the invention further comprises at least one additional anti-cancer drug different from:

-said non-human mammalian polyclonal antibody; and

monoclonal anti-PD 1 or anti-PDL 1 antibody.

In particular, the at least one additional anti-cancer drug may be a monoclonal antibody other than monoclonal anti-PD 1 or anti-PDL 1 antibody.

In particular, the at least one additional anti-cancer drug may be a monoclonal antibody selected from the group consisting of: anti-CD 137, anti-CTLA 4, anti-TIM-3, anti-B7-H3, anti-CD 134, anti-CD 154, anti-LAG-3, anti-CD 227, anti-BTNA 3, anti-CD 39, anti-CD 73, anti-CD 115, anti-sirpa, anti-SIRP γ, anti-CD 28, anti-NCR, anti-NKp 46, anti-NKp 30, anti-NKp 44, anti-NKG 2D, and anti-DNAM-1 monoclonal antibodies.

Another object of the invention relates to the combination of:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-at least one monoclonal antibody selected from the group consisting of: anti-PD 1 and anti-PDL 1 monoclonal antibodies.

Drawings

FIG. 1 shows a schematic view of a: a graph illustrating the antisera titer determined by ELISA is shown. Serum was derived from Hepa1.6 cell line (liver thin)Cell carcinoma) in mice, and was serially diluted and reacted with purified Hepa1.6 cells.

Sera were collected from preimmunized rabbits (Rabbit-J0) 7 days after the first subcutaneous injection (Rabbit-I1 +7) and 7 days after the second subcutaneous injection (Rabbit-I2 + 7).

Ordinate of the curve: optical Density (DO 450nm)

Abscissa of the circle: serial dilutions of the collected sera. From left to right: 1: 20; 1: 60; 1: 180; 1: 540; 1: 1620; 1: 4860; 1: 14580; 1:43740.

FIG. 2: shows injection of 2.5.10 from the portal artery in 8-week-old C57/BL6J immunocompetent mice⁶Clinical scores measured during the first to the following 30 days of each Hepa1.6 cell. Different treatments were applied 4 days after the injection of Hepa1.6 cells (D4).

Clinical score 1 corresponds to animals developing setae and slightly swollen abdomen. Clinical score 2 corresponds to the animals developing abdominal swelling and facial edema. Clinical score 3 corresponds to the animals developing abdominal swelling and herniated eyes. A clinical score of 4 corresponds to animals with high abdominal swelling (> 9 cm diameter), collapse, hump, solitary (resulting in individual sacrifice).

Four different treatment groups were formed.

The first group (group 1; n-7; control) received: control isotype (3G 8: irrelevant monoclonal antibody (mAb) -isotype IgG1) (8mg/kg, 2 times weekly, 28 days) + rabbit pre-immune polyclonal antibodies (20mg/kg, 2 times weekly, 28 days).

The second group (group 2; n-8; comparative), received: control isotype (3G 8: irrelevant monoclonal antibody (mAb) -isotype IgG 1; 8mg/kg, 2 times weekly, 28 days) + polyclonal anti-Hepa 1.6 cell antibody isolated from immune rabbit serum (20mg/kg, 2 times weekly, 28 days).

Third group (group 3; n ═ 8), received: monoclonal anti-PDL-1 antibody (8mg/kg, 2 times per week, 28 days) + polyclonal anti-Hepa 1.6 cell antibody isolated from immune rabbit serum (20mg/kg, 2 times per week, 28 days).

The fourth group (group 4; n-7; comparative) received: monoclonal anti-PDL-1 antibody (8mg/kg 2 times weekly, 28 days) + rabbit pre-immune polyclonal antibody (20mg/kg 2 times weekly, 28 days).

The mean clinical values obtained are shown in the figure.

Ordinate: clinical scoring

The abscissa: and (5) day.

FIG. 3: represents the percentage of survival of 8 week old C57/BL6J immunocompetent mice from the day one of their treatment, which was injected in the portal artery with 2.5.10⁶This occurred 4 days after each Hepa1.6 cell and during the following 50 days.

The indicated groups 1, 2, 3 and 4 are the same as the groups defined in fig. 2.

Ordinate: percentage of survival

The abscissa: and (5) day.

FIG. 4: represents the immunohistological results obtained by displaying CD3+ T cells (first line) or LY6G + bone marrow cells (second line) into a tumor biopsy obtained by subcutaneous implantation of B16F10 melanoma cells into Balb/C mice. Two groups are involved:

group 1 (control pAb; n ═ 7), receiving: pre-immune polyclonal antibodies in rabbits (20mg/kg, 2 times weekly, 28 days).

Group 2 (anti-B16 pAb; n ═ 8), receiving: the IgG fraction of the anti-tumor polyclonal antibody obtained from rabbits immunized with mouse B16F10 melanoma cells was infused biweekly (12.5mg/kg, twice weekly) from day 4 (D4) to day 30 (D30).

Detailed Description

1. Definition of

For a more complete understanding of the present invention, the following definitions are set forth. This definition is intended to cover grammatical equivalents.

The term "antibody" is used herein in its broadest sense. "antibody" refers to any polypeptide comprising at least: (i) an Fc region and (ii) a binding polypeptide domain derived from an immunoglobulin variable region. Thus, antibodies include, but are not limited to: full-length immunoglobulins, antibodies, antibody conjugates and fragments of each. The terms "antibody" and "immunoglobulin" are used interchangeably herein.

The term "antibody" encompasses the above polypeptides, which may or may not comprise an antigenic determinant selected from the group consisting of: (i) n-ethanediol neuraminic acid (Neu5Gc) and/or (ii) alpha-1, 3-galactose, in particular may or may not comprise two antigenic determinants (i) N-ethanediol neuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

As used herein, "polyclonal antibody" refers to a mixture of antibodies that recognize different epitopes of a given antigen and/or cell. Polyclonal antibodies encompass those comprised in or derived from a bodily fluid, especially serum or plasma, of a mammalian organism, particularly a non-human mammalian organism.

In the case of human immunoglobulins, light chains are divided into kappa and lambda light chains. The reassociation is divided into μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, respectively.

As used herein, "IgG" refers to a polypeptide belonging to the class of antibodies substantially encoded by a recognized immunoglobulin gamma gene. In humans, IgG comprises subclasses or isotypes IgG1, IgG2, IgG3, and IgG 4. In mice, IgG comprises IgG1, IgG2a, IgG2b, IgG 3. Full-length IgG consists of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, C γ 1 (also known as CH1), C γ 2 (also known as CH2), and C γ 3 (also known as CH 3).

By "physiologically acceptable medium" according to the invention is meant a carrier or excipient which is physiologically acceptable to the mammalian patient to be treated, while retaining the therapeutic properties of the compound with which it is administered. An exemplary pharmaceutically acceptable medium is physiological saline. Other physiologically acceptable media are known to those skilled in the art in the context of the present invention.

As used herein, "antibody-dependent cell-mediated cytotoxicity" (or ADCC) refers to a cell-mediated immune mechanism in which effector cells of the immune system actively lyse target cells that have been bound by specific antibodies. ADCC is mostly mediated by NK cells, but also by other immune cells (e.g. neutrophils and eosinophils). Typically, ADCC results from activation of NK cells. Activation of NK cells involves the binding of its Fc receptor to the Fc region of IgG that binds to antigens present on the surface of target cells. This interaction induces the release of cytokines and cytotoxic granules by NK cells. To assess the ability of an antibody to induce ADCC, procedures such as de Romeuf et al Br J haematol.2008mar; 140(6) 635-43.

As used herein, an "antigenic determinant" (or epitope) as applied to a polyclonal non-human mammal antibody refers to the structural components of the antigenic molecule, which includes antigenic proteins and antigenic carbohydrates, responsible for its specific interaction with the antibody molecule elicited by the same or related antigens. Broadly, an "antigenic determinant" as applied herein to a polyclonal non-human mammal antibody is also used to collectively refer to an antigenic molecule comprising a plurality of epitopes that are readily recognized by the antibody molecule as being raised by the same or related antigens. Illustratively, the antigenic molecule N-ethanediol neuraminic acid (Neu5Gc) may be referred to herein as an "antigenic determinant", although the antigenic molecule may comprise more than one epitope recognized by an antibody raised by Neu5Gc or a molecule containing Neu5 Gc.

In blood, "serum" is a component derived from plasma, from which cells (white blood cells and red blood cells) and coagulation factors are removed. Serum includes all proteins and all electrolytes, antibodies, antigens, hormones and any substances (e.g. drugs and microorganisms) that are not used for blood coagulation (coagulation), and which are ultimately also exogenous.

The terms "malignant cell," "cancer cell," and "tumor cell" are used interchangeably herein. As used herein, "tumor cell" refers to a cell that is spontaneously hyperproliferative in vivo. Examples of tumor cells include (1) sarcomas such as osteosarcoma and soft tissue sarcoma, (2) carcinomas such as breast cancer, lung cancer, bladder cancer, thyroid cancer, prostate cancer, colon cancer, colorectal cancer, pancreatic cancer, stomach cancer, liver cancer, uterine cancer, cervical cancer and ovarian cancer, (3) lymphomas such as hodgkin's lymphoma and non-hodgkin's lymphoma, (4) neuroblastoma, (5) melanoma, (6) myeloma, (7) wilms tumor, (8) leukemias such as Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL), (9) glioma and (10) retinoblastoma.

As used herein, the term "cancer" refers to uncontrolled, abnormal growth of cells, and includes within its scope all well-known diseases caused by uncontrolled and abnormal growth of cells. Non-limiting examples of common cancers include bladder cancer, breast cancer, ovarian and gastric cancers, cervical cancer, colon cancer, endometrial cancer, head and neck cancer, lung cancer, melanoma, multiple myeloma, leukemia (e.g., myeloid, lymphocytic, myelocytic, and lymphocytic leukemias), non-hodgkin's lymphoma, prostate cancer, rectal cancer, malignant melanoma, and in particular pancreatic cancer.

In particular, the cancer cell of the present invention may be a cell included in a cancer selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral or oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer, adenoid cystic cancer, adrenal tumors, amyloidosis, anal cancer, appendiceal cancer, bile duct cancer, bone cancer, brain cancer, central nervous system tumors, cervical cancer, esophageal cancer, eye cancer, eyelid cancer, gastrointestinal cancer, HIV/AIDS-related cancers, tear adenocarcinoma, laryngeal cancer or hypopharyngeal cancer, leukemia, liver cancer, meningioma, nasopharyngeal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, parathyroid cancer, penile cancer, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma and thymus cancer, vaginal cancer, and vulval cancer.

In a specific embodiment, the cancer or tumor of the invention is a cold tumor. "Cold tumors" are terms commonly used to designate "non-inflammatory" tumors, i.e., tumors characterized by a lack of T cell infiltration. They are well known challenging tumours because adaptive immune responses are not established or maintained. They are in contrast to "hot tumors" or "inflammatory tumors" which are tumors in which the number of infiltrating T cells is sufficient to trigger an adaptive response to them.

In particular, cold tumors are selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral or oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer, adenoid cystic cancer, adrenal tumors, amyloidosis, anal cancer, appendiceal cancer, bile duct cancer, bone cancer, brain cancer, central nervous system tumors, cervical cancer, esophageal cancer, eye cancer, eyelid cancer, gastrointestinal cancer, HIV/AIDS-related cancers, tear adenocarcinoma, laryngeal cancer or hypopharyngeal cancer, leukemia, liver cancer, meningioma, nasopharyngeal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, parathyroid cancer, penile cancer, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma and thymus cancer, vaginal cancer, and vulval cancer.

2. Association according to the invention

In view of the above-mentioned objects, and in particular in order to provide a new anticancer treatment, the improved efficiency of which is independent of any observable toxicity in the treated organism, the inventors conceived the following combination:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies.

The anti-PD 1 monoclonal antibody according to the invention may for example be selected from the group consisting of: nivolumab, pembrolizumab, and cimiraprizumab.

The anti-PDL 1 monoclonal antibody according to the invention may, for example, be selected from the group consisting of: alemtuzumab, avimtuzumab and dulacizumab.

In a preferred embodiment, the monoclonal antibody combined according to the invention is an anti-PDL 1 antibody.

The combination according to the invention further comprises a non-human mammalian polyclonal antibody directed against the cancer cells.

Polyclonal antibodies are directed against the same cancer cell, i.e., they are all directed against different epitopes of different or the same antigen present on the surface of the same cancer cell.

Polyclonal antibodies according to the invention can be obtained using a method comprising the following steps:

a) immunizing a non-human mammal with a cancer cell of interest; and

b) collecting the antibodies contained in the body fluid of said non-human mammal of step a).

The step of immunizing a non-human mammal against the target cancer cells consists in injecting said cells into the non-human mammal.

In EP 0335804 in particular a protocol is described for obtaining a good level of immunity of non-human transgenic mammals to cells.

Such a regimen may consist in particular in the immunization of an animal, such as a rabbit, horse or piglet, preferably a piglet, with repeated administration of cancer cells according to known methods.

For example, several administrations, 4.10 each, are given intravenously or subcutaneously with or without adjuvant⁴-10⁹Cells, administered at least one week apart. Approximately one week after the last immunization, serum was collected from the immunized animals and isolated according to known methods.

The step of collecting the antibody may for example consist in removing a part of the blood of the immunized non-human mammal from which the antibody of interest is collected.

According to a particular embodiment, the body fluid may be selected from the group consisting of: plasma and serum.

The protocol for obtaining blood, more particularly plasma or serum, is within the general knowledge of the person skilled in the art.

According to a preferred embodiment, and as described above, the method for obtaining a polyclonal antibody of the present invention may further comprise the step of purifying the polyclonal antibody from said body fluid.

The purification step is advantageous because it allows in particular to overcome possible undesired side effects associated with the presence of various cellular contaminants in the body fluid, which contaminants may be involved in the formation of corresponding contaminating antibodies by the immunized non-human mammal.

The purification step is also advantageous in that it allows to obtain polyclonal antibodies with the desired purity.

The purification steps fall within the common general knowledge of a person skilled in the art. All possible modifications of any conventional purification scheme also fall within the common general knowledge of the skilled person.

As suitable methods for purifying these polyclonal antibodies of interest, those for purifying antibodies with an affinity support for protein G or protein a to which an antigen is coupled, such as those sold by the companies ProteoGenix, Cell Biolabs, inc. or CliniSciences or disclosed in EP 1601697, JP 7155194 or US 6,870,034, may be exemplified.

Mention may also be made of an affinity support for selectively immobilizing a polyclonal antibody of interest from the bloodstream, comprising a solid support having immobilized aptamers that specifically bind said antibody of interest from the bloodstream. This process is disclosed in particular in WO 2010/094901.

According to a particular embodiment, the polyclonal antibody according to the invention does not contain a first antigenic determinant selected from the group consisting of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

According to a specific embodiment, the polyclonal antibody further does not contain a second antigenic determinant different from said first antigenic determinant, and wherein said second antigenic determinant is selected from the group consisting of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

Thus, a polyclonal antibody may not contain the two antigenic determinants (i) N-ethanediol neuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

Polyclonal antibodies that do not contain these antigenic determinants are believed to have reduced immunogenic properties in humans compared to polyclonal antibodies that contain these antigenic determinants.

Neu5Gc is known in the art to be immunogenic in humans (Noguchi A. et al, J. biochem. Tokyo (1995),117(1): 59-62;). Furthermore, it is known that patients develop severe Immune Complexes (IC) following infusion of most animal immunoglobulin vector antibodies developed against the Neu5Gc epitope (Merrick JM et al, int. allergy appl. Immunol.,1978, Vol.57: 477-480; Aggarwal S. et al, Nat Biotechnol.2008; 26: 1227-1233; Arnold JN et al, Annu Rev Immunol.2007; 25: 21-50; Durocher Y et al, Curr Opin Biotechnol.2009; 20: 700-707; Higgins E et al, Glycoconj. 2009).

It is also known in the art that the enzyme α -1, 3-galactosyltransferase (α 1,3GT or GGTA1) synthesizes α -1, 3-galactose (α 1,3Gal) epitopes (Gal α 1,3Gal β 1, 4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig to human xenotransplantation.

According to these embodiments, the polyclonal antibody according to the invention can be obtained using a method comprising the following steps:

a) providing a non-human mammal lacking a genetic alteration of a first gene selected from the group consisting of: (i) a gene encoding a functional cytidine-5' -monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional alpha-1, 3-galactosyltransferase;

b) immunizing the genetically altered non-human mammal against human cells; and

c) collecting the antibodies contained in the genetically altered non-human mammal's body fluid of step b).

Preferably, the genetically altered non-human mammal is a CMAH and/or GGTA1 knockdown non-human transgenic mammal (or a CMAH and/or GGTA1 KO non-human mammal) that includes a CMAH and GGTA1 double knockdown non-human transgenic mammal.

As used herein, a "knockout non-human transgenic mammal" refers to a non-human transgenic mammal in which the alleles of one or more target genes have been altered, e.g., by homologous recombination or other insertion or deletion.

In some embodiments, the gene is disrupted. By "disrupting a gene" is meant that a portion of the gene encoding is altered so as to affect the transcription and/or translation encoded by that segment of the gene, for example by knockout techniques or to render that segment unreadable by insertion of additional genes via desired proteins or by insertion of regulatory sequences that regulate the transcription of existing sequences.

In some embodiments, all cells of the non-human transgenic mammal include a disrupted gene.

In certain embodiments, the knockout non-human transgenic mammal is a non-human transgenic mammal in which alleles of one or more target genes have been disabled.

In some embodiments, both alleles of the target gene are non-functional. Such embodiments include what are commonly referred to as "gene knockouts," "gene knockins," and any other modification of one or more native alleles of a native target gene that renders the gene non-functional. Such a non-human transgenic mammal may be used as a source for the production of polyclonal antibodies according to the invention.

Methods for obtaining a non-human mammal lacking genetic alterations of genes selected from the group consisting of: (i) a gene encoding a functional cytidine-5' -monophosphate N-acetylneuraminic acid hydrolase; and/or (ii) a gene encoding a functional alpha- (1,3) -galactosyltransferase.

A method for obtaining a CMAH knock-out non-human transgenic mammal is described in particular in WO2006/133356, which more particularly discloses the production of an animal product free of 5-ethanediol neuraminic acid (Neu5Gc) for human use, comprising the steps of: preparing a genetically altered non-human mammal lacking a functional cytidine-5' -monophosphate N-acetylneuraminic acid hydrolase (CMAH) gene; and extracting at least one animal product from the genetically altered non-human mammal.

Methods for obtaining GAL knockout non-human transgenic mammals are well known to those skilled in the art (Cooper DK et al, genetic engineered seeds, Lancet 1993,342: 682; Lai L et al, Science 2002,295: 1089; Sachs DH et al, Current Opinion in organic Transplantation,2009,14: 148-.

Methods for obtaining GAL knockout non-human transgenic mammals are described in particular in US7,547,816.

Antibodies to mammalian cells, particularly cancer mammalian cells, can be readily obtained by immunizing a non-human mammal by administering an immunogenic composition comprising cancer cells of interest, as is known in the art.

The methods for identifying or characterizing these particular forms of polyclonal antibodies according to the invention, which do not contain a first antigenic determinant selected from the group consisting of: (i) n-glycolneuraminic acid (Neu5Gc) and (ii) alpha-1, 3-galactose.

Methods that can be used by those skilled in the art to identify or characterize polyclonal antibodies according to the present invention include enzyme-linked immunosorbent assays (ELISAs) in which anti-Neu 5Gc antibody and/or anti-Gal antibody are used as detection molecules.

As an anti-Neu 5Gc antibody for evaluating the deletion of Neu5Gc epitope, mention may be made of the Gc-Free Basic Kit sold by the company sialx, Inc.

As anti-Gal antibodies for the purpose of indicating deletion of the alpha-1, 3-galactose epitope, the experimental protocol disclosed in Jianq-Qiang Wang et al (J.am.chem.Soc.,1999,121:8181) or those sold by the company Sigma-Aldrich under the name WH0051083M1Sigma can be considered.

In particular, the combination according to the invention may further comprise at least one additional anticancer drug different from:

-said non-human mammalian polyclonal antibody; and

monoclonal anti-PD 1 or anti-PDL 1 antibody.

In particular, the at least one additional anti-cancer drug may be a monoclonal antibody other than monoclonal anti-PD 1 or anti-PDL 1 antibody.

In particular, the at least one additional anti-cancer drug may be a monoclonal antibody selected from the group consisting of: anti-CD 137, anti-CTLA 4, anti-TIM-3, anti-B7-H3, anti-CD 134, anti-CD 154, anti-LAG-3, anti-CD 227, anti-BTNA 3, anti-CD 39, anti-CD 73, anti-CD 115, anti-sirpa, anti-SIRP γ, anti-CD 28, anti-NCR, anti-NKp 46, anti-NKp 30, anti-NKp 44, anti-NKG 2D, and anti-DNAM-1.

3. Medical use according to the invention

As described above, according to a first aspect thereof, the present invention relates to:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies for use in the prevention and/or treatment of cancer in a mammalian patient.

Both these polyclonal and monoclonal antibodies are described in the present specification.

As mentioned before, the non-human mammalian polyclonal antibodies in combination according to the present invention are directed against cancer cells corresponding to or even belonging to the cancer to be prevented or treated in a mammalian patient.

For the treatment of a patient in need thereof, e.g. as mentioned above, a therapeutically effective dose of a combination according to the invention may be administered.

As used herein, a "therapeutically effective dose" refers to a dose that produces an administration effect. The exact dosage will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques.

The dosage range may be 0.001-100mg polyclonal antibody according to the invention per kg body weight (mg/kg) or more, e.g. 0.1, 1.0, 10 or 50mg/kg body weight. In particular, the dosage range of the polyclonal antibody according to the invention may be 0.1-100mg/kg, or more particularly 1-10 mg/kg.

The dose may also be 0.001-100mg of anti-PD 1 and/or anti-PDL 1 monoclonal antibody according to the invention per kg body weight (mg/kg) or greater, for example 0.1, 1.0, 10 or 50mg/kg body weight. In particular, the dose range of the anti-PD 1 and/or anti-PDL 1 monoclonal antibodies according to the invention may be 0.1-100mg/kg, or more particularly 1-10 mg/kg.

The dosage and frequency of administration can be adjusted according to the host response and the frequency of injection due to better drug resistance.

For illustrative purposes only, the frequency of co-administration according to the invention may be daily administration for 5-15 consecutive days.

As is known in the art, adjustments for protein degradation, systemic delivery versus local delivery, as well as age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be necessary and can be readily determined by routine experimentation by one skilled in the art.

The combined administration of the invention can be carried out in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, parenterally, intranasally, intraperitoneally, intraocularly, rectally, vaginally, transdermally, topically (e.g., as a gel), intraperitoneally, intramuscularly, intrapulmonary, or intrathecally.

In a particular embodiment, the combination according to the invention is in a form suitable for administration by intravenous or parenteral route.

As described above, the present invention relates to:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies for use in the prevention and/or treatment of cancer in a mammalian patient.

The non-human mammalian polyclonal antibody directed against cancer cells and the at least one monoclonal antibody selected from the group consisting of anti-PD 1 and anti-PDL 1 monoclonal antibodies may be combined in one and the same composition, or may be used in the form of separate compositions, which may be administered simultaneously or sequentially.

In a specific embodiment, the non-human mammalian polyclonal antibody directed against the cancer cells and the at least one monoclonal antibody selected from the group consisting of: anti-PD 1 and anti-PDL 1 monoclonal antibodies.

When a non-human mammalian polyclonal antibody directed against cancer cells and at least one monoclonal antibody selected from the group consisting of: anti-PD 1 and anti-PDL 1 monoclonal antibodies, which compositions may be administered to a mammalian patient simultaneously or separately by the same route or by different routes.

By simultaneous, it is understood that the compositions may be applied at the same time or at most on the same day or days.

By separate, it is understood that the compositions may be administered at least several days apart, e.g., at least two days apart.

In some embodiments, the composition of polyclonal antibodies according to the invention and the composition of anti-PD 1 and/or anti-PDL 1 monoclonal antibodies are in liquid form.

In some embodiments, the composition of polyclonal antibodies according to the invention and the composition of anti-PD 1 and/or anti-PDL 1 monoclonal antibodies are in solid form, including lyophilized form.

These compositions can be formulated according to standard methods, such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; 20 th edition, 2005).

The above compositions comprise physiologically/pharmaceutically acceptable media/excipients. The terms "physiologically acceptable" and "pharmaceutically acceptable" are used interchangeably herein.

Pharmaceutically acceptable Excipients or media which can be used are described in particular in the Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; 6 th revised edition, 2009).

The combination of the invention may be administered with other therapies or therapeutic agents, including, for example, small molecules, other biologies, radiation therapy, surgery, etc., which differ from the additional anti-cancer drugs described above and also differ from:

-said non-human mammalian polyclonal antibody; and

monoclonal anti-PD 1 or anti-PDL 1 antibody.

According to a particular embodiment, the combination according to the invention may further comprise, as further therapeutic agent, at least one immunosuppressive drug, such as a glucocorticoid, a cytostatic agent (azathioprine, methotrexate), an antibody or a drug acting on immunophilin (cyclosporine, tacrolimus, rapamycin).

These additional therapeutic agents may be administered to the mammalian patient in the same composition as the non-human mammalian polyclonal antibody against cancer cells or at least one monoclonal antibody selected from the group consisting of anti-PDl and anti-PDLl monoclonal antibodies, or in a separate composition.

In particular, these other therapeutic agents are administered in a separate composition from the non-human mammalian polyclonal antibody or at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies.

In particular embodiments, the present invention relates to:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: use of an anti-PDl and anti-PDLl monoclonal antibody in the manufacture of a medicament for the prevention and/or treatment of cancer in a mammalian patient.

In another embodiment, the present invention relates to a method of preventing and/or treating cancer in a mammalian patient comprising administering to said mammalian patient:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies.

The invention also relates to:

-a non-human mammalian polyclonal antibody directed against cancer cells; and

-a combination of at least one monoclonal antibody selected from the group consisting of: anti-PDl and anti-PDLl monoclonal antibodies for use as a medicament.

The invention is further illustrated by the following examples.

Detailed Description

Example 1: ELISA control of polyclonal antibodies obtained by immunizing rabbits with cancer cell lines

Rabbits were immunized with cells from the murine tumor cell line, Hepa1.6 (hepatocellular carcinoma) to obtain the corresponding anti-tumor polyclonal antibodies.

The tumor cells were injected subcutaneously twice, two weeks apart.

Sera were actually collected in preimmunized rabbits (Rabbit-J0) 7 days after the first subcutaneous injection (Rabbit-I1 +7) and 7 days after the second subcutaneous injection (Rabbit-I2 +7) to test the ability of the antibodies to recognize tumor cells for the immunized rabbits.

More specifically, based on the Hepa1.6 cells used to immunize rabbits, the antibody response of the immunized rabbits was measured using a cell ELISA test. Collected serum was purified at 1: 20; 1: 60; 1: 180; 1: 540; 1: 1620; 1: 4860; 1: 14580; serial dilutions of 1:43740 were made into 0.05% PBS-bovine serum albumin and incubated for 2 hours at room temperature. After centrifugation and washing of the ELISA plate (taking care not to aspirate the cells), peroxidase-labeled goat anti-rabbit antibody (dilution 1/1000) was added, and incubated for 1 hour and washed again. Then, 50. mu.l of TMB was added and incubated for 15 minutes in the dark. After addition of 10. mu.l of HCL, the optical density was read at 450 nM.

The results are shown in FIG. 1.

Note that:

it can be seen that the polyclonal antibodies obtained in the collected sera effectively recognized the targeted tumor cells of interest after the second injection.

Example 2: in vivo use of pooled polyclonal antibodies in syngeneic and orthotopic mouse models

Mouse models of hepa1.6 hepatocellular carcinoma have been described in Gauttier et al, Int J cancer.2014dec 15; 135(12) 2857-67.

This model was homologous and in situ in immunocompetent C57/BL6J mice.

2.5.10 resuspended in PBS⁶One hepa1.6 cell was injected into the portal vein of an 8-week-old male mouse, which, if left untreated, resulted in hepatic hypertension and death of the animal within 12-15 days.

After 4 days (D4), different treatments were administered:

a first group (group 1; n ═ 7; control) receiving: control isotype (3G 8: irrelevant monoclonal antibody (mAb) -isotype IgG1) (8mg/kg, 2 times weekly, 28 days) + rabbit pre-immune polyclonal antibodies (20mg/kg, 2 times weekly, 28 days).

A second group (group 2; n ═ 8; comparative), receiving: control isotype (3G 8: irrelevant monoclonal antibody (mAb) -isotype IgG 1; 8mg/kg, 2 times weekly, 28 days) + polyclonal anti-Hepa 1.6 cell antibody isolated from immune rabbit serum (20mg/kg, 2 times weekly, 28 days).

-a third group (group 3; n ═ 8) accepting: monoclonal anti-PDL-1 antibody (8mg/kg, 2 times per week, 28 days) + polyclonal anti-Hepa 1.6 cell antibody isolated from immune rabbit serum (20mg/kg, 2 times per week, 28 days).

A fourth group (group 4; n ═ 7; comparative) which received: monoclonal anti-PDL-1 antibody (8mg/kg 2 times weekly, 28 days) + rabbit pre-immune polyclonal antibody (20mg/kg 2 times weekly, 28 days).

The polyclonal antibody used corresponds to the IgG antibody obtained according to example 1, which was purified from rabbit serum using methods well known to those skilled in the art, including affinity columns on protein a.

After these treatments, different groups of clinical scores were observed within 30 days after tumor cell injection.

The results obtained are shown in FIG. 2.

Clinical scores from 0-4 are as follows:

clinical score 1 corresponds to animals developing setae and slightly swollen abdomen;

clinical score 2 corresponds to the animals developing abdominal swelling and facial edema;

clinical score 3 corresponds to the animals developing abdominal swelling and herniated eyes;

a clinical score of 4 corresponds to the animals developing abdominal height swelling (> 9 cm diameter), collapse, humpback, solitary (leading to individual sacrifice).

Note that:

as expected, in untreated control group 1, all mice died at day 15 after cell injection (as shown in fig. 3).

The results observed were similar for groups 2 and 4, corresponding to the administration of purified polyclonal or monoclonal anti-PDL 1 antibody obtained as described above. These two separate treatments resulted in a reduction in clinical score to about 3 after 25 days.

In contrast, a synergistic effect was observed in the group 3 animals treated with the combined polyclonal antibody of the invention and anti-PDL 1 monoclonal antibody, since they maintained a clinical score of 0-1 until day 30 (end of experiment).

Surprisingly, careful observation of group 3 mice within a few days after this experiment did not observe any toxicity, particularly any signs of autoimmunity (i.e. no pain, no weight loss, no animal discoloration, no depilation or diarrhoea).

Example 3: doxok of the inventionThe cloned antibody allows the transformation of cold tumors into hot tumors

Rabbits were immunized with mouse B16F10 melanoma cells to obtain the corresponding anti-tumor polyclonal antibody.

It is well known that the B16F10 melanoma cell line is a good example of a "cold" tumor because of, for example, resistance to anti-PDL 1 antibodies, where the effect of such antibodies on tumor regression is very limited (Chen et al, Cancer Immunol res.2014; 3(2): 149-60; Ueha et al, 2015; 3(6): 631-40).

These IgG fractions from rabbits raised against polyclonal antibodies produced by B16F10 cells were infused into Balb/C mice subcutaneously implanted with B16F10 melanoma cells (D0) every two weeks (12.5mg/kg twice a week) from day 4 (D4) to day 30 (D30) (group 2-n ═ 8).

A control group of animals (group 1-n ═ 7) also implanted subcutaneously with B16F10 melanoma cells received only pre-immune polyclonal antibodies from rabbits (20mg/kg, 2 times per week, 28 days).

At 30 days after injection of the cells (for group 2) or 18 days after said injection for group 1 (since all individuals from this group have died), tumor biopsies are taken and analyzed by immunohistology.

CD3+ T cells and LY6G + bone marrow cells are shown as shown in figure 4.

Significantly higher T cell and myeloid cell infiltration was observed in tumors of animals receiving polyclonal antibodies of the invention against said tumors.

Thus, it can be seen that the polyclonal antibodies of the present invention induce infiltration of CD3+ T cells and bone marrow immune cells into the tumor bed.

Thus, it converts the known "cold" tumor to a "hot" tumor, thereby allowing anti-PD 1/anti-PDL 1 to effectively exert their effect in preventing tumor cells from inactivating T cells that can now enter the tumor, thereby enabling the immune system to be fully directed against the tumor.