WO2023143590A1 - Tumor combination therapy using anti-il-11 antibody - Google Patents

Abstract

The present invention provides a drug combination for treating tumors. The drug combination comprises: an anti-IL-11 antibody or a fragment thereof; and an anti-tumor drug, wherein the anti-tumor drug can cause local IL-11 expression up-regulation and/or signaling pathway activation in a tumor microenvironment by killing tumor cells. The present invention also provides a synergistic drug administration method for the anti-IL-11 antibody or the fragment thereof and the anti-tumor drug.

Description

Tumor Combination Therapy Using Anti-IL-11 Antibody

Cross References to Related Applications

This patent application claims the priority right of the Chinese invention patent application with application number CN202210112529.7 filed on January 29, 2022, the entire content of which is hereby incorporated by reference.

technical field

The invention relates to the field of biopharmaceuticals, in particular, the invention relates to a tumor combination therapy using an anti-interleukin-11 (Interleukin-11, IL-11) antibody.

Background technique

According to the "World Cancer Report 2020" released by the World Health Organization's International Agency for Research on Cancer (IARC), there will be 19.29 million new cancer cases worldwide in 2020, of which about 10.07 million new cancer cases will be diagnosed in men, and the most common type is lung cancer. , prostate cancer, colorectal cancer, stomach cancer and liver cancer; about 9.23 million new cancers were diagnosed among women, the most common being breast cancer, colorectal cancer, lung cancer, cervical cancer and thyroid cancer. Cancer has become a major public health burden and economic problem worldwide.

At present, immunotherapy has achieved excellent therapeutic effects in various tumors or cancers such as melanoma and non-small cell lung cancer, especially for programmed death-1 (PD-1), programmed death ligand 1 (PD-L1 ) antibodies are considered the latest breakthrough in cancer immunotherapy. Early preclinical evidence showed that the activation of PD-1 and PD-L1 inhibited the activation and proliferation of tumor antigen-specific T cells, promoted tumorigenesis, and negatively regulated T cell immune function; blocking this interaction Activates the immune system to fight cancer.

To date, various pharmaceutical companies have conducted about 500 clinical studies on different types of antibodies, involving as many as 20 solid and hematological malignancies. Taking antibody drugs targeting PD-1 as an example, the FDA has approved a variety of antibodies for blocking PD-1 signaling to treat various types of cancer; in addition, there are other antibodies for blocking PD-1 signaling that are being clinically In test. However, monotherapy with such immuno-oncology (IO) antibody drugs is not always satisfactory, for example, not all patients respond to PD-1/PD-L1 inhibitor therapy, and some patients Drug resistance emerged after treatment.

In view of the clinical limitations of monotherapy with IO drugs, more and more combination therapies have emerged. The guiding principle for most combination therapies is to improve tumor antigen presentation or rescue dysfunctional Immune effector cells to enhance the efficacy of target blockade. The combined use of different cancer therapies can improve response rates.

Contents of the invention

The inventors of the present invention found that anti-interleukin-11 (IL-11) antibodies can effectively enhance the efficacy of anti-tumor drugs, such as immune checkpoint agonists or inhibitors.

Therefore, the object of the present invention is to provide a combination therapy of an antibody targeting IL-11 or a fragment thereof and other antitumor drugs for treating tumors.

The term "treating" or "treating a tumor" as used in the present invention refers to one or more of the following: delaying or inhibiting tumor growth, reducing tumor cell load or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and/or Or disappear, prevent tumor recurrence, prolong the survival duration of the individual, etc.

The term "antibody" used in the present invention covers any known antibody form capable of specifically binding or targeting an antigen or target, including naturally occurring, artificially obtained or artificially constructed functional antibody proteins.

The term "antibody fragment" as used in the present invention encompasses various functional or active fragments of antibodies, such as antigen-binding fragments thereof.

The technical scheme of the present invention is as follows.

In one aspect, the present invention provides a pharmaceutical combination comprising:

(1) Anti-IL-11 antibodies or fragments thereof; and

(2) Antineoplastic drugs.

Without being bound by any theory, the anti-tumor drugs can cause local upregulation of IL-11 expression and/or activation of signaling pathways by killing tumor cells in the tumor microenvironment.

In the drug combination provided by the present invention, the anti-IL-11 antibody or its fragment can block or inhibit the activation of IL-11 downstream signaling pathway by binding to IL-11. In particular, the anti-IL-11 antibody or fragment thereof is capable of binding the antigen IL-11, especially human IL-11, with high affinity. According to a specific embodiment of the present invention, the present invention provides the following antibodies: a murine antibody obtained by using human IL-11 or murine IL-11 as an immunogen, and a human-derived antibody obtained by humanizing the murine antibody Humanized antibodies, and antibodies obtained by optimizing the sequence of the humanized antibodies using yeast display technology. The anti-IL-11 antibody or its fragment can block or inhibit the activation of IL-11 downstream signaling pathway by binding to IL-11.

Specifically, the anti-IL-11 antibodies or fragments thereof provided by the present invention include:

Heavy chain variable region (VH), which includes complementarity determining regions (CDRs) 1 (H-CDR1), 2 (H-CDR2) and 3 (H-CDR3); and, the light chain variable region (VL), which includes CDRs 1 (L-CDR1), 2 (L -CDR2) and 3 (L-CDR3).

In some specific embodiments, in the anti-IL-11 antibody or fragment thereof:

(1) The heavy chain variable region comprises heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2) and heavy chain CDR3 (H-CDR3) derived from the heavy chain variable region shown in the following amino acid sequence ):

the amino acid sequence shown in SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15 or SEQ ID NO.16; and

The light chain variable region comprises light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2) and light chain CDR3 (L-CDR3) derived from the light chain variable region shown in the following amino acid sequence:

The amino acid sequence shown in SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.17 or SEQ ID NO.18;

or,

(2) The heavy chain variable region comprises heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2) and heavy chain CDR3 (H-CDR3) derived from the heavy chain variable region shown in the following amino acid sequence ):

the amino acid sequence shown in SEQ ID NO.5, SEQ ID NO.9 or SEQ ID NO.19; and

The light chain variable region comprises light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2) and light chain CDR3 (L-CDR3) derived from the light chain variable region shown in the following amino acid sequence:

The amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.10 or SEQ ID NO.20.

Each amino acid sequence in the above group (1) and group (2) is the amino acid sequence of the heavy chain variable region or the light chain variable region of the anti-IL-11 antibody provided in the present invention, respectively. Using the definition tools (such as Chothia, Kabat, IMGT, Contact, etc.) of the complementarity determining regions in antibody heavy chain or light chain variable regions known in the art, those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained in each. Chain CDRs. According to a specific embodiment of the present invention, Kabat tool can be used to divide CDRs in the variable region sequence.

Preferably, in the anti-IL-11 antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region respectively comprise the heavy chain variable region and the light chain shown in the following amino acid sequence pairing Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 and light chain CDR1, light chain CDR2 and light chain CDR3 of the variable region:

(1)

(1-1) SEQ ID NO.7+SEQ ID NO.8;

(1-2) SEQ ID NO.11+SEQ ID NO.12;

(1-3) SEQ ID NO.15+SEQ ID NO.17; or

(1-4) SEQ ID NO.16+SEQ ID NO.18;

or,

(2)

(2-1) SEQ ID NO.5+SEQ ID NO.6;

(2-2) SEQ ID NO.9+SEQ ID NO.10; or

(2-3) SEQ ID NO.19+SEQ ID NO.20.

As mentioned above, the Kabat tool can be used to divide the CDRs in each variable region sequence in the above amino acid sequence pairing; in the case of the above pairing, the combination of CDRs of the heavy chain variable region and the light chain variable region is contained in In the anti-IL-11 antibody or fragment thereof provided by the present invention.

Correspondingly, in the anti-IL-11 antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region comprise a combination of heavy chain CDRs and light chain CDRs selected from the following (H-CDR1 , H-CDR2, H-CDR3; and, L-CDR1, L-CDR2, L-CDR3):

(1)

(1-1) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31 in sequence; and, comprising sequentially shown in SEQ ID L-CDR1, L-CDR2, L-CDR3 of the amino acid sequences of NO.32, SEQ ID NO.33, and SEQ ID NO.34;

(1-2) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.29, SEQ ID NO.35, and SEQ ID NO.31 in turn; and, comprising sequentially shown in SEQ ID L-CDR1, L-CDR2, L-CDR3 of the amino acid sequences of NO.36, SEQ ID NO.33, and SEQ ID NO.34;

or,

(2)

(2-1) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25 in sequence; and, comprising sequences shown in SEQ ID L-CDR1, L-CDR2, L-CDR3 of the amino acid sequences of NO.26, SEQ ID NO.27, SEQ ID NO.28; or

(2-2) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.25 in sequence; and, comprising sequences shown in SEQ ID L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequences of NO.39, SEQ ID NO.27, and SEQ ID NO.28.

Preferably, the antibody or fragment thereof provided by the present invention is an anti-human interleukin-11 (hIL-11) antibody or fragment thereof. The amino acid sequence of human IL-11 is exemplarily provided in GenBank: AAH12506.1.

Preferably, the anti-IL-11 antibody or fragment thereof provided by the present invention at least comprises a heavy chain variable region and The light chain variable regions both include the above CDRs and the framework region (framework region, FR) therebetween, and the arrangement of each region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Further preferably, in the anti-IL-11 antibody or fragment thereof provided by the present invention:

(1)

The heavy chain variable region comprises an amino acid sequence selected from:

The amino acid sequence shown in SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15 or SEQ ID NO.16 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or

The light chain variable region comprises an amino acid sequence selected from:

The amino acid sequence shown in SEQ ID NO.8, SEQ ID NO.12 or SEQ ID NO.17 or SEQ ID NO.18 or an amino acid sequence having at least 75% identity to said amino acid sequence;

or,

(2)

The heavy chain variable region comprises an amino acid sequence selected from:

The amino acid sequence shown in SEQ ID NO.5, SEQ ID NO.9 or SEQ ID NO.19 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or

The light chain variable region comprises an amino acid sequence selected from:

The amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 10 or SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to said amino acid sequence.

At most 25% difference in amino acid sequence resulting from said "at least 75% identity" may be present in any framework region in the heavy chain variable region or light chain variable region, or in the antibodies or fragments thereof of the present invention In any domain or sequence other than the heavy chain variable region and the light chain variable region. The differences may result from amino acid deletions, additions or substitutions at any position, where the substitutions may be conservative or non-conservative. Said "at least 75% identity" encompasses any percentage identity between at least 75% identity and 100% identity, such as 75%, 80%, 85%, 90%, even 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

According to a specific embodiment of the present invention, in the anti-IL-11 antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region comprise a combination of amino acid sequences selected from the following:

(1)

(1-1) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence; and the amino acid sequence shown in SEQ ID NO.8, or the amino acid sequence with the amino acid sequence Amino acid sequences with at least 75% identity;

(1-2) The amino acid sequence shown in SEQ ID NO.11, or having at least an amino acid sequence of 75% identity; and, the amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 75% identity to said amino acid sequence;

(1-3) the amino acid sequence shown in SEQ ID NO.15, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.17, or an amino acid sequence with said amino acid sequence Amino acid sequences having at least 75% identity to the sequence; and

(1-3) the amino acid sequence shown in SEQ ID NO.16, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.18, or an amino acid sequence with said amino acid sequence Amino acid sequences with at least 75% identity to the sequence;

or,

(2)

(2-1) The amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.6, or an amino acid sequence with said amino acid sequence Amino acid sequences with at least 75% identity to the sequence;

(2-2) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.10, or an amino acid sequence with said amino acid sequence Amino acid sequences having at least 75% identity to the sequence; and

(2-3) The amino acid sequence shown in SEQ ID NO.19, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.20, or an amino acid sequence with said amino acid sequence Amino acid sequences having at least 75% identity to the sequence.

In terms of antigen, the anti-IL-11 antibody or fragment thereof of the present invention is an anti-IL-11 antibody or an antigen-binding fragment thereof. Preferably, the antibody is a mouse antibody, a rabbit antibody, a human antibody, a chimeric antibody or a fully or partially humanized antibody. The antibody can also be a derivatized antibody, for example, an antibody obtained by CDRs transplantation, affinity maturation, point mutation transformation, chemical modification, etc. on the basis of the original murine monoclonal antibody, wherein the chemical modification includes glycosylation, Acetylation, PEGylation, phosphorylation, amidation, protease cleavage, linking with cellular ligands or effector molecules, protection and/or blocking of active reactive groups, etc. Preferably, the antigen-binding fragment of the anti-IL-11 antibody can be scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv fragment of any form of the antibody.

In addition to the variable region, the anti-IL-11 antibody or fragment thereof provided by the present invention also includes a heavy chain constant region (CH) and/or a light chain constant region (CL), preferably a human or mouse heavy chain region. chain constant region and/or light chain constant region. Preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.

According to a specific embodiment of the present invention, the anti-IL-11 antibody is a monoclonal antibody, preferably are murine, chimeric or humanized monoclonal antibodies. According to a specific embodiment of the present invention, the monoclonal antibody comprises the heavy chain constant region sequence of mouse IgG1, such as shown in SEQ ID NO.3; and/or comprises the murine light chain constant region, such as shown in SEQ ID NO. 4. According to a specific embodiment of the present invention, the monoclonal antibody comprises a human heavy chain constant region and a light chain constant region, for example shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

According to a specific embodiment of the present invention, the anti-IL-11 antibody of the present invention is a monoclonal antibody. Preferably, the anti-IL-11 antibody provided by the present invention is immunoglobulin, for example, the type of said immunoglobulin is human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is of human IgG1 or IgG4 subtype.

In the drug combination provided by the present invention, another component is the antitumor drug.

In the context of the present invention, "anti-tumor" drugs proposed by the present invention refer to drugs for the treatment of tumors, preferably cancers. Preferably, the antitumor drug is a drug capable of treating the following tumors or cancers: lung cancer (such as non-small cell lung cancer), breast cancer, classical Hodkin's lymphoma, gastric cancer, liver cancer (such as primary liver cancer), melanoma, d - MMR-mutated or MSI-H malignancies, cervical cancer, head and neck neoplasms (e.g., head and neck squamous cell carcinoma), urethral bladder cancer, primary mediastinal B-cell lymphoma, renal cell carcinoma, colorectal cancer, and urothelium One or more cancers; more preferably, the cancer is selected from the group consisting of colorectal cancer, liver cancer, lung cancer, breast cancer, urothelial cancer, melanoma or head and neck tumors.

In particular, the anti-tumor drugs proposed in the present invention are immuno-oncology (IO) antibody drugs, preferably immune checkpoint inhibitors and/or agonists. According to a specific embodiment of the present invention, the anti-tumor drug is an inhibitor and/or agonist against PD-1 or PD-L1. Further, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof (such as an antigen-binding fragment) or an antibody-drug conjugate targeting PD-1 or PD-L1. Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8. According to a specific embodiment of the present invention, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).

According to a specific embodiment of the present invention, the anti-tumor drug is an anti-PD1 antibody or an anti-PD-L1 antibody. The anti-PD1 antibody such as Pabolizumab (Pabolizumab), nivolumab (nivolumab), lambrolizumab, camrelizumab (Camrelizumab), tislelizumab (Tislelizumab), sintilimumab Anti-(Sintilimab), Toripalimab (Toripalimab), Serpulimab (Serpulimab), Pucotenlimab (Pucotenlimab), Penpulimab (Penpulimab), etc. " part of the WBP336C provided. The anti-PD-L1 antibody, such as Durvalumab, Atezolizumab (Atezolizumab), Envafolimab, Sugemalimab, etc. Alternatively, the anti-tumor drug is an antibody against chemokine receptor CCR8, such as TPP15285 provided in the "Specific Embodiments" section of this application.

Regarding the use of the two components included in the pharmaceutical combination provided by the present invention, the anti-IL-11 antibody or its fragment and the anti-tumor drug can be administered simultaneously or sequentially. Alternatively, the pharmaceutical combination is in the form of a pharmaceutical composition (that is, the two components are in the same system), and the anti-IL-11 antibody or its fragment is administered simultaneously with the anti-tumor drug. According to the specific embodiment of the present invention, the drug combination provided by the present invention can be used to treat tumors. The tumor may be cancer, such as liver cancer or colorectal cancer.

In another aspect, the present invention provides the use of the above-mentioned drug combination comprising an anti-IL-11 antibody or a fragment thereof and an anti-tumor drug in the preparation of a drug for treating tumors.

In the use of this aspect provided by the present invention, the tumor is cancer. Preferably, the cancer is liver cancer or colorectal cancer.

In another aspect, the present invention provides the use of the above-mentioned anti-IL-11 antibody or fragment thereof in the preparation of a drug for enhancing the efficacy of an anti-tumor drug.

In the application of this aspect provided by the present invention, the anti-IL-11 antibody or its fragment can block or inhibit the activation of IL-11 downstream signal transduction pathway by binding to IL-11. Specifically, anti-IL-11 antibodies or fragments thereof are as described above.

In the application of this aspect provided by the present invention, the efficacy of anti-tumor drugs refers to one or more of the following conditions achieved when treating tumor-bearing subjects: delaying or inhibiting tumor growth, reducing tumor cell load or tumor burden, promote tumor regression, cause tumor shrinkage, necrosis and/or disappearance, prevent tumor recurrence, prolong the survival duration of an individual, and the like.

Without being bound by any theory, the anti-tumor drugs can cause local upregulation of IL-11 expression and/or activation of signaling pathways by killing tumor cells in the tumor microenvironment.

Preferably, the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist. According to a specific embodiment of the present invention, the anti-tumor drug is an inhibitor and/or agonist against PD-1 or PD-L1. Further, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof (such as an antigen-binding fragment) or an antibody-drug conjugate targeting PD-1 or PD-L1. Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8. According to a specific embodiment of the present invention, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).

According to a specific embodiment of the present invention, the anti-tumor drug is anti-PD1 antibody or anti-PD-L1 Antibody. The anti-PD1 antibody such as Pabolizumab (Pabolizumab), nivolumab (nivolumab), lambrolizumab, camrelizumab (Camrelizumab), tislelizumab (Tislelizumab), sintilimumab Anti-(Sintilimab), Toripalimab (Toripalimab), Serpulimab (Serpulimab), Pucotenlimab (Pucotenlimab), Penpulimab (Penpulimab), etc. " part of the WBP336C provided. The anti-PD-L1 antibodies are, for example, Durvalumab, Atezolizumab, Envafolimab, Sugemalimab and the like. Alternatively, the anti-tumor drug is an antibody against chemokine receptor CCR8, such as TPP15285 provided in the "Specific Embodiments" section of this application.

In yet another aspect, the present invention provides the use of the anti-IL-11 antibody or fragment thereof as described above in the preparation of a medicament for use in combination with an anti-tumor drug.

In the application of this aspect provided by the present invention, the anti-IL-11 antibody or its fragment can block or inhibit the activation of IL-11 downstream signal transduction pathway by binding to IL-11. Specifically, anti-IL-11 antibodies or fragments thereof are as described above.

Without being bound by any theory, the anti-tumor drugs can cause local upregulation of IL-11 expression and/or activation of signaling pathways by killing tumor cells in the tumor microenvironment.

Preferably, the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist. According to a specific embodiment of the present invention, the anti-tumor drug is an inhibitor and/or agonist against PD-1 or PD-L1. Further, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof (such as an antigen-binding fragment) or an antibody-drug conjugate targeting PD-1 or PD-L1. Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8. According to a specific embodiment of the present invention, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).

According to a specific embodiment of the present invention, the anti-tumor drug is an anti-PD1 antibody or an anti-PD-L1 antibody. The anti-PD1 antibody such as Pabolizumab (Pabolizumab), nivolumab (nivolumab), lambrolizumab, camrelizumab (Camrelizumab), tislelizumab (Tislelizumab), sintilimumab Anti-(Sintilimab), Toripalimab (Toripalimab), Serpulimab (Serpulimab), Pucotenlimab (Pucotenlimab), Penpulimab (Penpulimab), etc. " part of the WBP336C provided. The anti-PD-L1 antibodies are, for example, Durvalumab, Atezolizumab, Envafolimab, Sugemalimab and the like. Alternatively, the anti-tumor drug is an antibody against chemokine receptor CCR8, for example, the application "with TPP15285 provided in the Body Embodiments section.

In yet another aspect, the present invention provides a method for treating tumors, said method comprising administering to a subject in need thereof:

(1) Anti-IL-11 antibodies or fragments thereof; and

(2) Antineoplastic drugs.

In the method of this aspect provided by the invention, the method is used to treat a tumor, preferably a cancer. Preferably, the cancer is liver cancer or colorectal cancer.

Without being bound by any theory, the anti-tumor drugs can cause local upregulation of IL-11 expression and/or activation of signaling pathways by killing tumor cells in the tumor microenvironment.

In the method of this aspect provided by the present invention, the anti-IL-11 antibody or fragment thereof can block or inhibit the activation of IL-11 downstream signal transduction pathway by binding to IL-11. Specifically, anti-IL-11 antibodies or fragments thereof are as described above.

In the method of this aspect provided by the present invention, the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist. According to a specific embodiment of the present invention, the anti-tumor drug is an inhibitor and/or agonist against PD-1 or PD-L1. Further, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof (such as an antigen-binding fragment) or an antibody-drug conjugate targeting PD-1 or PD-L1. Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8. According to a specific embodiment of the present invention, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment) or an antibody-drug conjugate targeting CCR8.

According to a specific embodiment of the present invention, the anti-tumor drug is an anti-PD1 antibody or an anti-PD-L1 antibody. The anti-PD1 antibody such as Pabolizumab (Pabolizumab), nivolumab (nivolumab), lambrolizumab, camrelizumab (Camrelizumab), tislelizumab (Tislelizumab), sintilimumab Anti-(Sintilimab), Toripalimab (Toripalimab), Serpulimab (Serpulimab), Pucotenlimab (Pucotenlimab), Penpulimab (Penpulimab), etc. " part of the WBP336C provided. The anti-PD-L1 antibodies are, for example, Durvalumab, Atezolizumab, Envafolimab, Sugemalimab and the like. Alternatively, the anti-tumor drug is an antibody against chemokine receptor CCR8, such as TPP15285 provided in the "Specific Embodiments" section of this application.

In the method of this aspect provided by the present invention, the subject is a mammal, preferably a primate or a rodent, more preferably a human. According to a specific embodiment of the present invention, the subject Patients with liver cancer or colorectal cancer.

As for the use of the two components in the method provided by the present invention in the subject, the anti-IL-11 antibody or fragment thereof and the anti-tumor drug can be administered to the subject simultaneously or sequentially. Alternatively, the anti-IL-11 antibody or fragment thereof and the anti-tumor drug are administered simultaneously, preferably in a drug delivery system such as a pharmaceutical composition. Administration can be by parenteral administration (e.g., subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, or intrasynovial injection or infusion) ; kidney dialysis infusion; local perfusion) the anti-IL-11 antibody or its fragment and the anti-tumor drug.

Compared with the prior art, the present invention is based on the significant synergistic effect of anti-IL-11 antibodies and other anti-tumor drugs, especially antibodies against immune checkpoint PD-1/PD-L1, and proposes two drugs targeting Combination therapy for related diseases. Without being bound by any theory, it is speculated that antitumor drugs can cause upregulation of IL-11 expression or pathway activation in the tumor microenvironment by killing tumor cells, thereby reducing the level of IL-11 in the tumor microenvironment through IL-11 antibodies or Inhibiting its signaling pathway has the effect of enhancing the efficacy of anti-tumor drugs and further synergizing anti-tumor effects.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 shows the analysis results of IL-11 stimulation of reporter gene expression in STAT3/IL-11RA/GP130-HEK293 cells.

Figure 2 shows the tumor growth of subcutaneous transplanted tumors in C57BL/6 mice bearing MC38 tumor cells, wherein 2A: tumor volume; 2B: tumor weight.

Figure 3 shows the tumor growth of subcutaneous transplanted tumors in C57BL/6 mice bearing Hepa 1-6 tumor cells, wherein 3A: tumor volume; 3B: tumor weight.

Figure 4 shows the tumor growth of subcutaneous transplanted tumors in C57BL/6 mice bearing Hepa 1-6 tumor cells, wherein 4A: tumor volume; 4B: tumor weight.

Figure 5 shows the tumor growth of subcutaneous xenografts in C57BL/6 mice bearing MC38-hPD-L1 tumor cells.

Figure 6 shows the tumor growth of subcutaneous transplanted tumors in C57BL/6 mice bearing Hepa 1-6 tumor cells, wherein 6A: tumor volume; 6B: tumor weight.

Figure 7 shows the tumor growth of subcutaneous xenograft tumors in Balb/C mice bearing CT26 tumor cells, where 7A: tumor volume; 7B: tumor weight.

Figure 8 shows the expression of B-hPD-L1 plus/hCD47 MC38 tumor cells B-Tumor growth of subcutaneous transplanted tumors in hPD-L1/hCD47/hSIRPα humanized mice, where 8A: tumor volume; 8B: tumor weight.

Figure 9 shows the tumor growth of subcutaneous transplanted tumors in C57BL/6 mice bearing Hepa 1-6 tumor cells, wherein 9A: tumor volume; 9B: tumor weight.

Figure 10 shows the tumor growth of subcutaneous xenograft tumors in C57BL/6 mice bearing MC38 tumor cells, where 10A: tumor volume; 10B: tumor weight.

Figure 11 shows the tumor growth of subcutaneous xenograft tumors in C57BL/6 mice bearing MC38 tumor cells, wherein 11A: tumor volume; 11B: tumor weight.

Detailed ways

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only for illustrating the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

In the following examples, different anti-IL-11 antibodies were used in combination with anti-PD-(L)1 antibodies to observe the anti-tumor efficacy in tumor models of different origins.

(1) Antibody

In addition to the anti-IL-11 antibodies provided in the Examples, the present invention employs the following antibodies.

1. Anti-IL-11 antibody 3C6-mFc: For the sequence, please refer to the patent publication WO2019/238882 A1;

Heavy chain variable region sequence (3C6-VH; SEQ ID NO.1)

Light chain variable region sequence (3C6-VL: SEQ ID NO.2)

2. Anti-mouse PD-1 antibody WBP336C (muWBP336c): For the sequence, please refer to the patent publication WO2019/062755A1;

Heavy chain variable region (WBP336C-VH: SEQ ID NO.21)

Light chain variable region (WBP336C-VL: SEQ ID NO.22)

3. Anti-PD-L1 mAb: Atezolizumab.

4. Anti-CCR8 antibody TPP15285: see the patent publication WO2021152186A1 for the sequence;

In WO2021152186A1, the heavy chain variable region of TPP15285 is shown in SEQ ID NO.229, and the light chain variable region is shown in SEQ ID NO.233.

5. For the anti-CD47/PD-L1 antibody 2MW1531, see the patent publication CN114478770A for the sequence:

In CN114478770A, the anti-PD-L1 light and heavy chain variable region of 2MW1531 is shown in SEQ ID NO.1/SEQ ID NO.3; the anti-CD47 light and heavy chain variable region is shown in SEQ ID NO.5/SEQ ID NO.8; The chain constant region is shown in SEQ ID NO.7/SEQ ID NO.10.

(2) The constant region sequence of the antibody

The antibodies of the present invention are constructed using the constant regions of murine antibodies and human antibodies, respectively.

Mouse antibody heavy chain constant region (SEQ ID NO.3)

Mouse antibody light chain constant region (SEQ ID NO.4)

Human antibody heavy chain constant region (SEQ ID NO.13)

Human antibody light chain constant region (SEQ ID NO.14)

(3) Judgment of synergy

In the embodiments of the present invention, the following King's formula is used to calculate the q value to judge whether the combined drug has a synergistic effect:

q=E(A+B)/(EA+EB－EA×EB),

Among them, E(A+B) is the tumor inhibition rate (TGI%) of the combination of the two drugs, EA and EB are the tumor inhibition rates of each drug alone, q<1 indicates that the combination of the two drugs has an antagonistic effect, and q>=1 indicates that the two drugs have an antagonistic effect. Drug combination has synergistic effect.

Embodiment 1 Obtaining of anti-IL-11 antibody

Mice were immunized with IL-11 (Juheli, recombinant human interleukin-11 for injection, Qilu Pharmaceutical Factory, S20053046) as an immunogen. The serum titer of immunized mice was detected by ELISA method, and mice with high titer were selected for shock immunization, and then spleen cells were separated and fused with myeloma cells, and cultured as single cells. Take the culture supernatant for ELISA detection, analyze the binding of the supernatant to human IL-11 (AAH12506.1, Pro22-Leu199), mouse IL-11 (NP_032376.1, Pro22-Leu199), and select hybridomas with good binding cell.

After the hybridoma cells are cultured to a certain number, the cells are harvested, RNA is extracted and reverse transcription PCR is performed to obtain the light and heavy chain variable region genes of the mouse antibody and perform sequence determination.

According to the amino acid sequence of the obtained murine antibody, the coding gene is optimized and synthesized, and the enzyme cleavage sites are designed at both ends of the coding gene; then the gene encoding the variable region of the antibody and the constant region of the heavy and light chain of the murine antibody are encoded by enzyme digestion and connection The gene fragment (amino acid sequence is SEQ ID NO.3, SEQ ID NO.4) is fused and cloned into a eukaryotic transient expression vector to obtain a recombinant murine antibody expression vector. The recombinant expression vector was transfected into HEK293 cells for recombinant expression, and the expression supernatant was purified using a Protein G affinity chromatography column to obtain anti-IL-11 murine antibodies mu18A10m and mu8C8m. The sequences are as follows (the CDR regions are divided according to the Kabat rules, such as underlined; same below).

Mouse anti-mu18A10m:

mu18A10m-VH (SEQ ID NO.5; CDRs: SEQ ID NO.23/24/25)

mu18A10m-VL (SEQ ID NO.6; CDRs: SEQ ID NO.26/27/28)

Mouse anti-mu8C8m:

mu8C8m-VH (SEQ ID NO.7; CDRs: SEQ ID NO.29/30/31)

mu8C8m-VL (SEQ ID NO.8; CDRs: SEQ ID NO.32/33/34)

The above-mentioned murine antibodies were humanized, including transplanting the light and heavy chain CDR regions to human antibody templates with high homology, and performing necessary back mutations to obtain humanized antibody hz18A10 (heavy chain can be Variable region hz18A10-VH, shown in SEQ ID NO.9; light chain variable region hz18A10-VL, shown in SEQ ID NO.10) and hz8C8 (heavy chain variable region hz8C8-VH, shown in SEQ ID NO.11 ; light chain variable region hz8C8-VL, shown in SEQ ID NO.12). Further, using the humanized antibodies hz18A10 and hz8C8 as parent antibodies, the single-chain antibody (scFv) mutation library displayed by yeast was constructed, and mutant sequences with similar or improved affinity to the parent antibodies were obtained through sorting.

According to the above, it is fused with the gene fragment encoding the heavy and light chain constant region of the mouse antibody (SEQ ID NO.3, SEQ ID NO.4), or fused with the gene fragment encoding the heavy and light chain constant region of the human antibody (SEQ ID NO. ID NO.13, SEQ ID NO.14) were fused and expressed to obtain a modified antibody against IL-11.

The variable region sequences of the obtained anti-IL-11 modified antibodies are shown in Table 1 and Table 2, respectively.

Table 1. Heavy and light chain variable region sequences of modified antibodies

Table 2. Heavy and light chain variable region sequences of engineered antibodies

Example 2 Characterization of IL-11 Antibody

Anti-IL-11 antibody 3C6-hFc was used as a positive control (the heavy chain and light chain variable region sequences of 3C6-hFc are shown in SEQ ID NO.1 and SEQ ID NO.2 above). The biological activity of the engineered antibody was characterized as follows. The constant regions of the control antibody and the engineered antibody were human antibody heavy chain constant region SEQ ID NO.13 and light chain constant region SEQ ID NO.14.

(1) Binding affinity analysis of IL-11 antibody to IL-11

The Octet QKe system instrument of Fortebio Company was used to measure the affinity of the modified antibody by using the AHC bioprobe that captures the human Fc segment to capture the Fc segment of the antibody, and the recombinantly expressed anti-IL-11 antibody 3C6-hFc at the same concentration was used as a positive control.

Specifically, the antibody was diluted with HBS-EP+(GE) buffer, flowed through the surface of AMC probe (Cat: 18-5088, PALL), and the antibody was captured on the surface of the probe; then the human IL diluted with HBS-EP+ buffer -11 (300nM) was used as the mobile phase to react with the antibody captured on the surface of the probe. The binding time was 300s and the dissociation time was 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding model was fitted by software to calculate the kinetic constant of antigen-antibody binding.

(2) Activity analysis of IL-11 antibody blocking formation of IL-11/IL-11Rα/GP130 tribody complex

The blocking activity of the engineered antibody on the formation of IL-11/IL-11Rα/GP130 tribody complex was analyzed by competition ELISA, and the same concentration of recombinantly expressed anti-IL-11 antibody 3C6-hFc was used as a positive control.

First, IL-11Rα-His (NP_004503.1, Met 1-Val 363, C-terminus-6×His Tag) was added to the enzyme-linked plate and coated overnight; the next day to prepare samples, firstly, a fixed concentration of IL-11 (Cat: 12225-HNCE, Sino Biological) (0.4 μg/ml) and serially diluted engineered antibody (working concentration 75, 25, 8.33, 2.78, 0.926, 0.309, 0.103 μg/ml) were mixed and co-incubated, and finally gp130-hFc (NP_034690.3, Met1-Glu617, C-terminus-mFc) at a concentration of 1 μg/ml was mixed and added to the enzyme-linked plate. After co-incubation, wash and add HRP-labeled anti-human Fc secondary antibody for color development and detection.

The results are shown in Table 3 and Table 4.

Table 3. Determination results of modified antibodies

Table 4. Determination results of modified antibodies

(3) Reporter gene system analysis of the effect of modified antibody on STAT3 signaling pathway

(1) Construction of IL-11Rα/GP130/STAT3-luc HEK293 reporter gene system

In order to analyze the cytological activity of the antibody, an IL-11Rα/STAT3-luc HEK293 reporter gene system was constructed. In HEK293 cells, the vector containing the STAT3-Luciference reporter gene system (Nanjing Kebai) was transfected, and then the stable cell line STAT3-Luc/HEK293 integrated with the STAT3-Luciference gene was obtained through pressurized screening; On the basis of strains, transfected into a vector containing the full-length human IL-11Rα gene (Yiqiao Shenzhou), and then through pressurized screening, a cell bank that can stably express human IL-11Rα (IL-11Rα/STAT3-luc HEK293Pool ); Finally, monoclonal isolation and screening were carried out on the cell bank to obtain IL-11Rα/STAT3-luc HEK293 monoclonal, and the monoclonal that could be stably passaged was selected and preserved as a cell line. HEK293 cells constitutively express GP130, together with stably transfected IL-11Rα/STAT3-luc constitute the IL-11RA/GP130/STAT3-luc HEK293 reporter gene system.

When measuring the activity, the cells were seeded in a 96-well plate, and IL-11 was diluted to an appropriate concentration and serially diluted. At the same time, a blank well was set, added to the cells, incubated for 24 hours, and then the expression of the reporter gene was detected. When processing data, the color development value of all wells is subtracted from the color development value of the blank well, and the obtained data is used as reagent. volume-effect curve. The results showed (FIG. 1) that IL-11 could stimulate the transcriptional expression of the reporter gene with a clear dose effect. The EC50 of IL-11 stimulating the transcriptional expression of STAT3 reporter gene was 0.11ng/ml.

(2) Reporter gene system to determine the effect of modified antibody on STAT3 signaling pathway

Using the IL-11RA/GP130/STAT3-luc HEK293 reporter gene system, the activity of the antibody to inhibit the transcription and expression of the IL-11-stimulated cell reporter gene was detected. Seed the cells in a 96-well plate, dilute IL-11 to 0.3ng/ml, and incubate with serially diluted antibody (10μg/ml, 3-fold serial dilution of 10 gradients) for 30 minutes, then add to the cells and incubate for 6 hours , and then add the detection reagent in the Luciferase assay kit (Novizan, D1201-02bio-lite) and read the signal value (RLUsample).

In the experiment, 3C6-hFc was used as a positive control antibody, irrelevant human IgG (NC-hIgG) was used as a negative control antibody, PBS with IL-11 added was used as a positive blank control (RLUhigh), and PBS was used as a negative blank control (RLUlow) . Calculate the inhibition rate of each well according to the reading value corresponding to each gradient concentration well, and the inhibition rate calculation formula is as follows:

Inhibition %=(RLUhigh-RLUsample)/(RLUhigh-RLUlow)×100%

Finally, with the antibody concentration as the abscissa and the average value of the percentage inhibition rate as the ordinate, the dose-effect curve was drawn using the GraphPad Prism 9 four-parameter formula to obtain the IC50 value of each antibody.

Referring to the above method, the activity of the modified antibodies to inhibit IL-11-stimulated cell reporter gene transcription and expression was analyzed. The results showed (Table 5) that most of the modified antibodies maintained the same blocking activity as the parental antibodies.

Table 5. The results of the blocking activity of the engineered antibody against the reporter gene system

Example 3 Therapeutic effect of anti-IL-11 antibody combined with anti-PD-1 antibody in colorectal cancer

6-week-old female C57BL/6 mice (Speyford (Beijing) Biotechnology Co., Ltd.) were subcutaneously inoculated with 3×10 ⁶ MC38 mouse colon cancer cells, and were randomly divided into groups when the tumor grew to about 70 mm ³ . Only/group, grouping and administration dosage, frequency are as table 6, every week intraperitoneal administration twice (when adopting two kinds of reagents, be administration at the same time, hereinafter the same), administration 3 times altogether, administration is measured simultaneously Tumor volume and mouse weight, when the weight of the mice decreased by more than 15%, or when the tumor volume of a single animal exceeded ^3000mm3 or the average tumor volume of a group of animals exceeded ^2000mm3 , the experiment on the relevant mice was stopped, and the mice were euthanized. The results are shown in Figure 2.

Table 6. C57BL/6 mice bearing MC38 tumor grouping and administration dose and frequency

Among them, the anti-IL-11 antibodies mu8C8 and WBP336C are constructed using mouse heavy chain and light chain constant regions (SEQ ID NO.3 and SEQ ID NO.4), wherein the heavy chain and light chain variable region sequences of mu8C8 are shown in SEQ ID Shown in NO.15 and SEQ ID NO.17; Same below.

The tumor inhibition rate of each group is shown in Figure 2A, and the q value was calculated according to King's formula>1.

As shown in group experiments in C57BL/6 mice subcutaneously implanted with MC38 tumors, the combination of anti-IL-11 antibody and anti-PD1 antibody more clearly inhibited Tumor growth, and has statistical significance (*P<0.05, **P<0.01, ***P<0.001); and the two have obvious synergistic effect.

Example 4 The therapeutic effect of anti-IL-11 antibody combined with anti-PD-1 antibody in liver cancer

6-8 week old female C57BL/6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5×106 Hepa ^1-6 mouse liver cancer tumor cells (ATCC, CRL1930) until the tumor grew to 40-60mm ³ were randomly divided into 6 rats/group, the grouping, dosage and frequency of administration were shown in Table 7, each group was administered intraperitoneally twice a week, a total of 3 administrations, and the tumor volume and size were measured at the same time. Mouse body weight, when the mouse body weight drops by more than 15%, or the tumor volume of a single animal exceeds 3000mm ³ or a When the average tumor volume of animals in the same group exceeds 2000 mm ³ , the experiment on the relevant mice is stopped, and the mice are euthanized. The results are shown in Figure 3.

Table 7. Grouping of C57BL/6 mice bearing Hepa1-6 tumors and administration dose and frequency

Among them, the anti-IL-11 antibody mu18A10 was constructed using mouse heavy chain and light chain constant regions (SEQ ID NO.3 and SEQ ID NO.4), and its heavy chain and light chain variable region sequences were as shown in SEQ ID NO.19 and Shown in SEQ ID NO.20; Same below.

The tumor inhibition rate of each group is shown in Figure 3A, and the q value was calculated according to King's formula>1.

As shown in the grouping experiment results in C57BL/6 mice subcutaneously transplanted with liver cancer cell Hepa 1-6 tumor model, anti-PD-1 antibody showed dose-dependent anti-tumor efficacy, when anti-PD-1 antibody was combined with 20 mg/kg The combination of anti-IL-11 antibodies showed a synergistic anti-tumor effect, and this synergistic anti-tumor effect showed a synergistic effect when different doses of anti-PD-1 antibodies were used in combination.

Example 5 Dose- effect relationship of anti-IL-11 antibody combined with anti-PD-1 antibody in the treatment of liver cancer

6-8 week old female C57BL/6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5×106 Hepa ^1-6 mouse liver cancer tumor cells (ATCC, CRL1930) until the tumor grew to 50-60mm ³ were randomly divided into 6 rats/group. The grouping, dosage and frequency of administration were shown in Table 8. Each group was administered intraperitoneally twice a week for a total of 3 times. Tumor volume and size were measured at the same time. Mouse body weight. When the body weight of the mice dropped by more than 15%, or when the tumor volume of a single animal exceeded ^3000mm3 or the average tumor volume of a group of animals exceeded ^2000mm3 , the experiment on the relevant mice was stopped, and the mice were euthanized. The results are shown in Figure 4.

Table 8. Grouping of C57BL/6 mice bearing Hepa1-6 tumors, dosage and frequency of administration

The tumor inhibition rate of each group is shown in Figure 4A, and the q value was calculated according to King's formula>1.

As shown in the grouping experiment results in C57BL/6 mice subcutaneously implanted with Hepa 1-6 tumor model, different doses of anti-IL-11 antibody combined with 2 mg/kg anti-PD-1 antibody showed a dose-dependent synergistic anti-PD-1 antibody. Tumor effect, and the high-dose combination group was significantly better than anti-PD-1 antibody and anti-IL-11 antibody alone (*P<0.05, **P<0.01, ***P<0.001).

Example 6 Therapeutic effect of anti-IL-11 antibody combined with anti-PD-L1 antibody in colorectal cancer

6-8 week old female C57BL/6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5× ¹⁰⁵ mouse colorectal cancer MC38-hPD-L1 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Cell Resource Center), when the tumor grows to ^about 50-80mm3, it will be randomly divided into 6 rats/group. , measure the tumor volume and mouse body weight at the same time, when the mouse body weight drops by more than 15%, or when the tumor volume of a single animal exceeds ^3000mm3 or the average tumor volume of a group of animals exceeds ^2000mm3, the experiment on the relevant mice is stopped, Euthanize the mouse. The results are shown in Figure 5.

Table 9. Grouping of C57BL/6 mice bearing MC38 subcutaneously transplanted tumors, dosage and frequency of administration

The tumor inhibition rate of each group is shown in Figure 5, and the q value was calculated according to King's formula>1.

As shown in the results of group experiments in C57BL/6 mice subcutaneously implanted with MC38-hPD-L1 tumor model, the combination of anti-PD-L1 antibody and anti-IL-11 antibody showed a dose-dependent synergistic anti-tumor trend.

Example 7 Therapeutic effect of anti-IL-11 antibody combined with anti-PD-L1 antibody in liver cancer

6-week-old male C57BL/6 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 1×106 Hepa ^1-6 mouse liver cancer cells, and were randomly divided into groups when the tumors grew to ^about 49mm3. 6 mice/group, the grouping, dosage and frequency of administration are shown in Table 10, and each group was administered intraperitoneally twice a week, for a total of 4 administrations. The tumor volume and the weight of the mice were measured at the same time as the administration. When the weight of the mice dropped by more than 15 %, or when the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals exceeds 2000mm3, the experiment on the relevant mice is stopped, and the mice are euthanized. The results are shown in Figure 6.

Table 10. Grouping of C57BL/6 mice bearing Hepa 1-6 tumors and administration dose and frequency

Among them, the anti-IL-11 antibody mu8C8F was constructed using mouse heavy chain and light chain constant regions (SEQ ID NO.3 and SEQ ID NO.4), wherein the heavy chain and light chain variable region sequences of mu8C8F are shown in SEQ ID NO. Shown in 16 and SEQ ID NO.18; Same below.

The tumor inhibition rate of each group is shown in Fig. 6A, and the q value was calculated according to King's formula > 1.

The results showed that the combination of mu8C8F and Atezolizumab antibody clearly inhibited the tumor growth compared with the control group mice using the isotype antibody (*P<0.05,**P<0.01,*** P<0.001).

Example 8 Therapeutic effect of anti-IL-11 antibody combined with anti-PD-1 antibody in colorectal cancer

6-week-old male Balb/C mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5×10 ⁵ CT26 mouse colon cancer cells. When the tumors grew to about 72 mm ³ , they were randomly divided into 6 groups. / group, grouping and administration dosage, frequency are shown in Table 11, and each group is intraperitoneally administered twice a week, and is administered 4 times in total, and the tumor volume and mouse body weight are measured at the same time as the administration, when the mouse body weight drops more than 15%. , or when the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals exceeds 2000mm3, stop the experiment on the relevant mice, and give the mice euthanasia. The results are shown in Figure 7.

Table 11.Balb/C mice bearing CT26 tumor grouping and administration dose, frequency

The tumor inhibition rate of each group is shown in Fig. 7A, and the q value was calculated according to King's formula > 1.

The results showed that the combination of mu8C8F and anti-PD-1 antibody clearly inhibited the tumor growth compared with the control group mice using the isotype antibody, and it was statistically significant (*P<0.05, **P<0.01 ,***P<0.001).

Example 9 Therapeutic effect of anti-IL-11 antibody combined with anti-CD47/PD-L1 antibody in colon cancer

Take B-hPD-L1/hCD47/hSIRPα humanized mice aged 5-8 weeks (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), and subcutaneously inoculate ⁵ ×105 B-hPD-L1 plus/hCD47 MC38 mice Colon cancer cells (mouse colon cancer MC38 cells were purchased from Sunran Shanghai Biotechnology Co., Ltd. Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. genetically modified MC38, knocked out mouse Pdl1 and Cd47 genes, and expressed human PD-L1 and CD47 protein, and named as B-hPD-L1 plus/hCD47MC38), when the tumor grows to about ^89mm3 , it will be randomly divided into 6 groups, and the grouping, dosage and frequency of administration are shown in Table 12. Each group Administer intraperitoneally twice a week, 4 times in total, measure the tumor volume and mouse body weight at the same time, when the mouse body weight drops by more than 15%, or the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals When the size exceeds 2000 mm ³ , the experiment on the relevant mice is stopped, and the mice are euthanized. The results are shown in Figure 8.

Table 12. B-hPD-L1/hCD47/hSIRPα Humanized Mice Bearing B-hPD-L1 plus/hCD47 MC38 Tumor Grouping, Dosage and Frequency

The tumor inhibition rate of each group is shown in Figure 8A, and the q value was calculated according to King's formula>1.

The results showed that the combination of mu8C8F and 2MW1531 antibody clearly inhibited tumor growth compared with the control group mice with isotype antibody (*P<0.05,**P<0.01,*** P<0.001).

Example 10 Therapeutic effect of anti-IL-11 antibody combined with anti-PD-1 antibody in liver cancer

6-week-old male C57BL/6 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5×10 ⁶ Hepa 1-6 mouse liver cancer cells, and were randomly divided into groups when the tumors grew to about 50 mm ³ . 6 mice/group, the grouping, dosage and frequency of administration are shown in Table 13, and each group was intraperitoneally administered twice a week for a total of 3 times, and the tumor volume and mouse weight were measured at the same time. %, or when the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals exceeds 2000mm3, the experiment on the relevant mice is stopped, and the mice are euthanized. The results are shown in Figure 9.

Table 13. Grouping of C57BL/6 mice bearing Hepa1-6 tumors and administration dose and frequency

Among them, the anti-IL-11 antibody hz8C8F is constructed using human heavy chain and light chain constant regions (SEQ ID NO.13 and SEQ ID NO.14), wherein the sequences of the heavy chain and light chain variable regions of hz8C8F are shown in SEQ ID NO. Shown in 15 and SEQ ID NO.17; Same below.

The tumor inhibition rate of each group is shown in Fig. 9A, and the value of q>1 was calculated according to King's formula.

The results showed that the combination of hz8C8F and muWBP336C antibody clearly inhibited the tumor growth compared with the control mice with isotype antibody (*P<0.05,**P<0.01,*** P<0.001).

Example 11 The therapeutic effect of anti-IL-11 antibody combined with anti-CCR8 antibody in colon cancer

6-week-old female C57BL/6 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with 3×10 ⁶ MC38 mouse colon cancer cells. When the tumor grew to about 70 mm ³ , they were randomly divided into groups. Only/group, grouping, dosage and frequency of administration are shown in Table 14. Each group is administered intraperitoneally twice a week, 4 times in total, and the tumor volume and mouse weight are measured at the same time. When the mouse body weight drops by more than 15% or the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals exceeds ^2000mm3 . The experiments on the relevant mice were terminated and the mice were euthanized. The results are shown in Figure 10.

Table 14. C57BL/6 mice bearing MC38 tumor grouping and administration dose and frequency

Among them, the anti-CCR8 antibody TPP15285 was constructed using human heavy chain and light chain constant regions (SEQ ID NO.13 and SEQ ID NO.14).

The tumor inhibition rate of each group is shown in Fig. 10A, and the q value was calculated according to King's formula > 1.

The results showed that the combination of mu8C8F and anti-CCR8 antibody TPP15285 clearly inhibited tumor growth in a dose-dependent manner compared to control mice treated with isotype antibodies.

Example 12 The therapeutic effect of anti-IL-11 antibody combined with anti-PD1 antibody in colon cancer

6-week-old female C57BL/6 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with 3×10 ⁶ MC38 mouse colon cancer cells. When the tumor grew to about 150mm3, they were randomly divided into groups, 6 mice / group, grouping and administration dosage, frequency are shown in Table 15, and each group is intraperitoneally administered twice a week, and is administered 3 times in total, and the tumor volume and mouse body weight are measured at the same time as the administration, when the mouse body weight drops more than 15%. , or when the tumor volume of a single animal exceeds 3000mm3 or the average tumor volume of a group of animals exceeds ^2000mm3 , the experiment on the relevant mice is stopped, and the mice are euthanized. The results are shown in Figure 11.

Table 15. Grouping of C57BL/6 mice bearing MC38 tumors and administration dose and frequency

Among them, the anti-IL-11 antibody hz18A10F is constructed using human heavy chain and light chain constant regions (SEQ ID NO.13 and SEQ ID NO.14), wherein the sequences of the heavy chain and light chain variable regions of hz18A10F are shown in SEQ ID NO. 19 and shown in SEQ ID NO.20.

The tumor inhibition rate of each group is shown in Fig. 11A. The q value of the combination of hz8C8F and muWBP336c calculated according to the King's formula was >1, while the q value of the combination of hz18A10F and muWBP336c was <1.

The results showed that the combination of hz8C8F and hz18A10F and anti-PD1 antibody muWBP336c could better inhibit the growth of MC38 tumors compared with the control mice using isotype antibodies, and the combination of mu8C8F and muWBP336C was more effective than that of muWBP336C alone. There were significant differences compared with drugs (*P<0.05, **P<0.01, ***P<0.001).

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (21)

1. A drug combination comprising:

   (1) Anti-interleukin-11 (IL-11) antibodies or fragments thereof; and

   (2) Antineoplastic drugs.
2. The drug combination according to claim 1, wherein the anti-tumor drug can cause local IL-11 expression up-regulation and/or signal transduction pathway activation by killing tumor cells in the tumor microenvironment; and/or

   The anti-IL-11 antibody or its fragment can block or inhibit the activation of IL-11 downstream signal transduction pathway by binding to IL-11;

   Preferably, the anti-IL-11 antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region and the light chain variable region, the heavy chain variable region The chain variable region and the light chain variable region comprise a combination of heavy and light chain CDRs selected from the group consisting of (H-CDR1, H-CDR2, H-CDR3; and, L-CDR1, L-CDR2, L-CDR3 ):

   (1) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31 in turn; and, comprising sequentially shown in SEQ ID NO. 32. L-CDR1, L-CDR2, L-CDR3 of the amino acid sequences of SEQ ID NO.33 and SEQ ID NO.34;

   (2) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.29, SEQ ID NO.35, and SEQ ID NO.31 in turn; and, comprising sequentially shown in SEQ ID NO. 36. L-CDR1, L-CDR2, L-CDR3 of the amino acid sequences of SEQ ID NO.33 and SEQ ID NO.34;

   (3) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25 in turn; and, comprising sequentially shown in SEQ ID NO. 26. L-CDR1, L-CDR2, L-CDR3 of the amino acid sequence of SEQ ID NO.27, SEQ ID NO.28; or

   (4) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.25 in turn; and, comprising sequentially shown in SEQ ID NO. 39. L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequences of SEQ ID NO.27 and SEQ ID NO.28.
3. The pharmaceutical combination according to claim 1 or 2, wherein the anti-IL-11 antibody or fragment thereof is an anti-human interleukin-11 (hIL-11) antibody or fragment thereof;

   Preferably, the anti-IL-11 antibody or fragment thereof comprises at least the heavy chain variable region and the light chain variable region, each of which comprises the combination of the CDRs of claim 3 and the framework between the CDRs Region (framework region, FR), the arrangement of each region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
4. The pharmaceutical combination according to any one of claims 1 to 3, wherein the heavy chain variable region and the light chain variable region of the anti-IL-11 antibody or fragment thereof comprise amino acid sequences selected from combination:

   (1) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with said amino acid sequence; and the amino acid sequence shown in SEQ ID NO.8, or having at least 75% identity with said amino acid sequence Amino acid sequences with 75% identity;

   (2) the amino acid sequence shown in SEQ ID NO.11, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.12, or an amino acid sequence with said amino acid sequence Amino acid sequences of at least 75% identity; and

   (3) the amino acid sequence shown in SEQ ID NO.15, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.17, or an amino acid sequence with said amino acid sequence Amino acid sequences with at least 75% identity;

   (4) the amino acid sequence shown in SEQ ID NO.16, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.18, or an amino acid sequence with said amino acid sequence Amino acid sequences with at least 75% identity;

   (5) the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.6, or an amino acid sequence with said amino acid sequence Amino acid sequences with at least 75% identity;

   (6) the amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.10, or an amino acid sequence with said amino acid sequence Amino acid sequences of at least 75% identity; and

   (7) the amino acid sequence shown in SEQ ID NO.19, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO.20, or an amino acid sequence with said amino acid sequence Amino acid sequences of at least 75% identity.
5. The pharmaceutical combination according to any one of claims 1 to 4, wherein the anti-IL-11 antibody or fragment thereof is an anti-IL-11 antibody or an antigen-binding fragment thereof;

   The antibody is a mouse antibody, a rabbit antibody, a human antibody, a chimeric antibody or a fully or partially humanized antibody; or a derivatized antibody;

   The antigen-binding fragment is any fragment of an antibody such as scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv;

   Preferably, the anti-IL-11 antibody is a monoclonal antibody, preferably comprising mouse or human heavy chain constant region and light chain constant region.
6. The drug combination according to any one of claims 1 to 5, wherein the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist.
7. The drug combination according to any one of claims 1 to 6, wherein the anti-tumor drug is an inhibitor and/or agonist against PD-1 or PD-L1;

   Preferably, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof or an antibody-drug conjugate targeting PD-1 or PD-L1;

   Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8; preferably, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).
8. The drug combination according to any one of claims 1 to 7, wherein the anti-IL-11 antibody or fragment thereof is used simultaneously or sequentially with the anti-tumor drug;

   Alternatively, the pharmaceutical combination is in the form of a pharmaceutical composition.
9. Use of the pharmaceutical combination according to any one of claims 1 to 8 in the preparation of a medicament for treating tumors.
10. Use of the anti-interleukin-11 (IL-11) antibody or fragment thereof as defined in any one of claims 1 to 8 in the preparation of a medicament for enhancing the efficacy of an antitumor drug.
11. The use according to claim 10, characterized in that the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist.
12. The use according to claim 10 or 11, characterized in that the antitumor drug is an inhibitor and/or agonist against PD-1 or PD-L1;

    Preferably, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof or an antibody-drug conjugate targeting PD-1 or PD-L1;

    Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8; preferably, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).
13. The use according to any one of claims 10 to 12, characterized in that the tumor is liver cancer or colorectal cancer.
14. Use of the anti-IL-11 antibody or fragment thereof as defined in any one of claims 1 to 8 in the preparation of a medicament for use in combination with an antitumor drug.
15. The use according to claim 14, characterized in that the anti-tumor drug is a drug that can cause local upregulation of IL-11 expression and/or activation of signal transduction pathways by killing tumor cells in the tumor microenvironment;

    Preferably, the anti-tumor drug is an immuno-oncology (IO) antibody drug, preferably an immune checkpoint inhibitor and/or agonist.
16. The use according to claim 14 or 15, characterized in that the antitumor drug is an inhibitor and/or agonist against PD-1 or PD-L1;

    Preferably, the anti-tumor drug is an anti-PD-1 or PD-L1 antibody or a fragment thereof or an antibody-drug conjugate targeting PD-1 or PD-L1;

    Alternatively, the anti-tumor drug is an inhibitor of chemokine receptor CCR8; preferably, the anti-tumor drug is an anti-CCR8 antibody or a fragment thereof (such as an antigen-binding fragment).
17. A method for treating tumors, the method comprising administering the drug combination according to any one of claims 1 to 8 to a subject in need thereof.
18. The method according to claim 17, wherein the method is used to treat tumors, preferably cancers;

    Preferably, the cancer is liver cancer or colorectal cancer.
19. The method according to claim 17 or 18, wherein the subject is a mammal, preferably a primate or a rodent, more preferably a human;

    Preferably, the subject is a liver cancer patient or a colorectal cancer patient.
20. The method according to any one of claims 17 to 19, wherein the anti-IL-11 antibody or fragment thereof and the anti-tumor drug are administered to the subject simultaneously or sequentially;

    Alternatively, the anti-IL-11 antibody or fragment thereof and the anti-tumor drug are administered simultaneously, preferably in a drug delivery system such as a pharmaceutical composition.
21. The method according to any one of claims 17 to 20, which comprises parenteral administration (e.g. subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular , intraurethral, intracranial, intratumoral or intrasynovial injection or infusion; renal dialysis infusion; local perfusion) the anti-IL-11 antibody or its fragment and the anti-tumor drug.