WO2022228514A1 - Anti-human leukemia inhibitory factor antibody and use thereof - Google Patents

Abstract

Provided are an anti-human leukemia inhibitory factor (LIF) antibody and a pharmaceutical composition comprising the antibody. Also provided are applications of the antibody and the pharmaceutical composition in cancer treatment.

Description

Anti-human leukemia inhibitory factor antibody and use thereof technical field

The present invention relates to the fields of medicine and immunology, in particular, the present invention relates to an antibody against human leukemia inhibitory factor, a pharmaceutical composition comprising the antibody and its use in the treatment of cancer.

Background technique

Leukemia inhibitory factor (LIF) is a multifunctional cytokine that belongs to the interleukin-6 cytokine family and can be produced by various tissues. LIF is widely expressed in mammalian adults and embryos and regulates cell differentiation, proliferation and survival. In addition, it was found that the high expression level of LIF is closely related to the occurrence, development and poor prognosis of various malignant tumors. With the broad prospects of immunotherapy in the treatment of malignant tumors, LIF has also attracted increasing attention as a target for the treatment of malignant tumors.

Currently, several anti-LIF antibodies against different tumors have been developed. For example, US2018/0344759 discloses an anti-LIF antibody that can be used to treat ovarian cancer, lung cancer and colorectal cancer. CN103797031A1 discloses an anti-LIF antibody that can be used to treat glioma, leukemia, rectal cancer, bladder cancer and breast cancer. Pancreatic cancer is a gastrointestinal malignant tumor with a high degree of malignancy and is difficult to diagnose and treat. Its morbidity and mortality have increased significantly in recent years. Pancreatic cancer has a low cure rate and a 5-year survival rate of <1%, making it one of the malignant tumors with the worst prognosis.

Therefore, there is an urgent need to develop new therapeutic methods and methods that can effectively treat pancreatic cancer and improve the survival rate.

The anti-LIF antibody provided in this application meets the above requirements to a certain extent.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the present invention provides isolated murine or humanized antibodies or antigen-binding fragments thereof that specifically bind to human leukemia inhibitory factor. In addition, the present invention also provides nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof; vectors comprising the nucleic acid sequences and corresponding host cells; pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and the Use of antibodies and pharmaceutical compositions in the treatment of pancreatic cancer.

In a first aspect, the present invention provides isolated antibodies (anti-LIF antibodies) or antigen-binding fragments thereof that specifically bind to human leukemia inhibitory factor.

In a specific embodiment, the anti-LIF antibody and antigenic fragments thereof comprise one, two or three CDRs (preferably three CDRs) selected from the VH region sequences of the antibodies shown in Table II, Table IV or Table VI . In other embodiments, the anti-LIF antibodies provided by the invention comprise one, two or three CDRs (preferably three CDRs) selected from the VL region sequences of the antibodies shown in Table II, Table IV or Table VI. In some embodiments, the anti-LIF antibody provided by the present invention comprises the 6 CDR region sequences of the antibodies shown in Table II, Table IV or Table VI, that is, the antibody of the present invention comprises the sequences from SEQ ID NO.7, SEQ ID NO.11 or the 3 heavy chain variable region CDRs of SEQ ID NO.15 and the 3 light chain variable region CDRs from SEQ ID NO.8, SEQ ID NO.12 or SEQ ID NO.16. In a preferred embodiment, the CDR sequences of the anti-LIF antibodies or antigen-binding fragments thereof provided by the present invention are the CDR sequences shown in Table I.

In some embodiments, an anti-LIF antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (i) CDR1, which is represented by SEQ ID NO:1 The amino acid sequence of SEQ ID NO: 2 is composed of (ii) CDR2, which is composed of the amino acid sequence of SEQ ID NO: 2, (iii) CDR3 is composed of the amino acid sequence of SEQ ID NO: 3, and the light chain variable region includes: (i) CDR1 , which consists of the amino acid sequence of SEQ ID NO:4, (ii) CDR2, which consists of the amino acid sequence of SEQ ID NO:5, (iii) CDR3, which consists of the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, an anti-LIF antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

The heavy chain variable region:

1) comprising the 3 heavy chain variable region CDRs of the antibody shown in Table II, and having at least 75%, 76%, 77%, 78%, 79%, 80% with the amino acid sequence of SEQ ID NO: 7, 11 or 15 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, the anti-LIF antibody comprising said VH has the ability to bind LIF; or

2) comprising the 3 heavy chain variable region CDRs of the antibody shown in Table II, and having one or more substitutions (such as conservative substitutions), insertions or deletions compared with the amino acid sequence of SEQ ID NO: 7, 11 or 15 The amino acid sequence of the VH, the anti-LIF antibody comprising the VH has the ability to bind to LIF;

The light chain variable region:

1) 3 CDRs comprising the light chain variable region of the antibody shown in Table II and having at least 75%, 76%, 77%, 78%, 79%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% identical, the anti-LIF antibody comprising said VL has the ability to bind LIF; or

2) comprising the 3 CDRs of the variable region of the light chain of the antibody shown in Table II and having one or more substitutions (such as conservative substitutions), insertions or With the deleted amino acid sequence, the anti-LIF antibody comprising the VH has the ability to bind to LIF.

In a preferred embodiment, the present invention provides an anti-LIF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region VL comprises The amino acid sequence shown in SEQ ID NO: 8 or consists of it.

In preferred embodiments, the anti-LIF antibodies of the invention are humanized anti-LIF antibodies or antigen-binding fragments thereof.

In a preferred embodiment, the present invention provides a humanized anti-LIF antibody or antigen-binding fragment thereof, wherein the variable region VH of the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; the light chain can be The variable region VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 12. In another preferred embodiment, the present invention provides another humanized anti-LIF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; light The chain variable region VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, the invention also encompasses antigen-binding fragments of anti-LIF antibodies, including but not limited to Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single chain antibodies molecules (eg, scFvs); and multispecific antibodies formed from antibody fragments.

In some embodiments, the Fc region of an antibody may be modified for specific application purposes based on the state of the art.

In some embodiments, the anti-LIF antibody or antigen-binding fragment thereof is not greater than 1 x ^10-8 M, eg, 1 x ^10-8 M, 1 x ^10-9 M, 1 x ^10-10 M, 1 ×10 ^-11 M, 1 × 10 ^-12 M, 1 × 10 ^-13 M, or 1 × 10 ^-14 M or less KD binds to human leukemia inhibitory factor protein.

In a second aspect, the present invention also provides nucleic acid molecules encoding any of the above antibodies or fragments thereof. Based on the amino acid sequences of the above-mentioned antibody molecules, those skilled in the art can easily obtain the nucleic acid molecules described in the present application according to common knowledge in the art. In one embodiment, the nucleic acid molecule comprises a nucleic acid molecule obtained due to codon degeneracy.

Nucleic acid molecules encoding the antibodies can be naturally occurring nucleic acids derived from the germline or from rearrangements that occur in B cells, or can be synthetic. Synthetic nucleic acids also include nucleic acids with modified internucleoside linkages, including phosphorothioates, to increase the resistance of the nucleic acid to degradation. Nucleic acids can be genetically engineered or produced entirely synthetically by nucleotide synthesis.

In a preferred embodiment, the present invention provides a vector comprising at least one nucleic acid encoding the light chain of the monoclonal antibody of the present invention and/or at least one nucleic acid encoding the heavy chain of the monoclonal antibody of the present invention. Nucleic acids may be present in the same vector or may be present in different vectors. Preferably, the vector comprises regulatory sequences, such as promoter sequences, enhancer sequences, etc., operably linked to the nucleic acid. Preferably, the vector also contains an origin for replication and maintenance in the host cell. The vector may also comprise a nucleotide sequence encoding a signal sequence located 5&apos; to the nucleic acid encoding the light or heavy chain. The signal sequence can facilitate secretion of the encoded peptide chain into the medium.

Therefore, it should be understood that recombinant expression vectors or expression cassettes or transgenic cell lines or recombinant bacteria containing the above nucleic acid molecules also belong to the protection scope of the present invention.

In a third aspect, the present invention provides methods for producing monoclonal antibodies. In one embodiment, monoclonal antibodies are produced by culturing host cells containing the above-described expression vectors. In one embodiment, the resulting monoclonal antibody is secreted into the supernatant and can be purified by conventional chromatographic techniques.

In one embodiment, the present invention provides anti-LIF antibodies or antigen-binding fragments thereof prepared by the methods of the present invention.

In a fourth aspect, the present invention also provides a composition comprising any anti-human leukemia inhibitory factor antibody provided in the present application, preferably the composition is a pharmaceutical composition.

In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the anti-LIF antibodies and antigen-binding fragments thereof included in the composition are conjugated to a conjugation moiety. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

In one embodiment, the pharmaceutical compositions disclosed herein further comprise other active ingredients for the treatment of malignant tumors. In a specific embodiment, the other active ingredients are, for example, pyrimidine nucleoside antitumor drugs, anti-PD-1/PD-L1 antibodies, other anti-LIF antibodies, and the like. In one embodiment, the pyrimidine nucleoside antitumor drug is gemcitabine and its salts. In further specific embodiments, the gemcitabine is gemcitabine hydrochloride. In one embodiment, the anti-PD-1 antibody is an anti-PD-1 antibody known in the art. In a specific embodiment, the anti-PD-1 antibody is, for example, selected from nivolumab, pembrolizumab, Lambrolizumab, Pidilizumab and other anti-PD-1 antibodies. In one embodiment, the anti-PD-1 antibody is a commercially available antibody.

In a fifth aspect, the present invention provides a method for treating a disease or condition associated with aberrant expression of human leukemia inhibitory factor, comprising administering to a subject a therapeutically effective amount of an anti-LIF antibody of the present invention, or administering a pharmaceutical combination of the present invention thing.

In one embodiment, the disease associated with abnormal expression of human leukemia inhibitory factor is a malignant tumor that overexpresses human leukemia inhibitory factor. In another embodiment, the malignancy is, for example, pancreatic cancer.

In a sixth aspect, the present invention provides a method of detecting human leukemia inhibitory factor in a subject or sample, the method comprising: (a) contacting the subject or sample with any of the anti-LIF antibodies or fragments thereof described herein; and (b) The formation of a complex between the anti-LIF antibody or fragment thereof and LIF is detected. In a preferred embodiment, the anti-LIF antibodies and antigen-binding fragments thereof of the invention further comprise a detectable label.

In a seventh aspect, the present invention relates to the use of any anti-LIF antibody or fragment thereof described herein in the manufacture of a medicament or a kit for the treatment of a disease or condition associated with aberrant LIF expression in a subject.

In one embodiment, the present invention provides the use of a composition comprising the anti-LIF antibody in the manufacture of a medicament or a kit for treating a disease or condition associated with aberrant LIF expression in a subject.

In an eighth aspect, the present invention provides a kit comprising the antibody or composition of the present invention, such as a detection kit, a treatment kit, and the like.

Description of drawings

Figure 1 is the SDS-PAGE pattern of purified non-reducing and reducing anti-human leukemia inhibitory factor antibody.

Figure 2 shows the results of evaluating the ADA titers of murine anti-LIF antibodies and humanized anti-LIF antibodies in mice.

Figure 3 shows the results of evaluating the ADA titer of mouse anti-LIF antibody and humanized anti-LIF antibody in 21-day-old mouse serum.

Figure 4 is the results of the anti-LIF antibody of the present application and its combination with gemcitabine on the tumor inhibition of mouse pancreatic cancer, wherein, Figure 4a shows the detection results of the tumor volume of nude mice in different test groups, and Figure 4b shows the results in different test groups The detection results of the tumor growth curve of nude mice, Figure 4c shows the detection of the tumor weight results of nude mice in different test groups, Figure 4d shows the detection results of the tumor growth curves of NSG mice in different test groups, and Figure 4e shows the results of different tests The detection results of tumor volume of NSG mice in the groups, Figure 4f shows the detection results of tumor weight of NSG mice in different test groups.

Figure 5 shows the results of the anti-LIF antibody of the present application and its combination with anti-PD-1 antibody on the tumor inhibition of mouse pancreatic cancer, wherein, Figure 5a shows the volume results of mouse tumors in different test groups, and Figure 5b shows the results of small tumors in different test groups. Results of mouse tumor growth curves, Figure 5c is the results of mouse tumor weights in different test groups.

Figure 6 is the result of the applicant's anti-LIF antibody and its combination with anti-PD-1 antibody on mouse pancreatic cancer tumor inhibition, wherein, Figure 6a shows the results of mouse tumor weight in different test groups, Figure 6b The results of tumor volume in mice in different test groups are shown, and Figure 6c shows the growth curves of tumors in different test groups.

Figure 7 shows the results of humanized anti-LIF antibody-1 killing pancreatic cancer cells. Figure 7a shows the inhibitory effect of the antibody on the proliferation of PANC1 cells at the cellular level; Figure 7b (PANC1 cells) and Figure 7c (SW1990 cells) show the inhibitory effect of the antibody on the proliferation of PANC1 and SW1990 cells at the organoid level; Figure 7d shows the Inhibitory effect of antibodies on cancer cell proliferation at the level of pancreatic cancer patient-derived organoids.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods and examples described herein are illustrative only and not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from the description and drawings, and from the appended claims. Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary.

I. Definitions

For the purpose of interpreting this specification, the following definitions will be used and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

The term "and/or" should be understood to mean either or both of the alternatives.

As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps. Herein, when the term "comprising" or "comprising" is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps. For example, reference to an antibody variable region that "comprises" a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they are What is necessary is just to show the desired antigen-binding activity. An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camels may contain only heavy chains. Antibodies can be humanized or human antibodies and single domain antibodies such as VH, VHH or VL.

The terms "whole antibody", "full length antibody", "complete antibody" and "intact antibody" are used interchangeably herein to refer to a naturally occurring heavy chain (H) comprising at least two heavy chains (H) interconnected by disulfide bonds and two light chain (L) glycoproteins. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity determining regions (CDRs), with more conserved regions (framework regions (FR)) interposed therebetween. Each VH and VL consists of three CDRs and four The FRs are composed, from the amino terminus to the carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.

The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding an antigen or competing with an intact antibody (ie, with the intact antibody from which the antigen-binding fragment is derived) bind antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, diabody, single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains, eg, Fab fragments can be obtained by papain digestion of complete antibodies. In addition, digestion of complete antibodies by pepsin below the disulfide bond in the hinge region produces F(ab')2, which is a dimer of Fab', a bivalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer to a Fab' monomer. Fab' monomers are basically Fab fragments with hinge regions (for a more detailed description of other antibody fragments see: Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y. (1993)). The Fv fragment consists of the VL and VH domains of the antibody one-arm. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, using recombinant methods, they can be linked by a synthetic linker peptide that enables the production of the two domains as a single protein chain, described in The VL and VH domains of a single protein chain are paired to form a single-chain Fv. The antibody fragments can be obtained by chemical methods, recombinant DNA methods or protease digestion.

As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody derived from, for example, a single copy or clone of a eukaryotic, prokaryotic, or phage clone, ie, except for possible variants, which are usually present in small amounts The individual antibodies comprising the population are identical and/or bind the same epitope, with the exception of antibodies (eg, variant antibodies containing natural mutations or produced during the production of monoclonal antibody preparations). The modifier "monoclonal" refers to the characteristic that an antibody is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.

"Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are amino acid regions in the variable region of antibodies that are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.

In a given light chain variable region or heavy chain variable region amino acid sequence, its CDR sequence can be determined using various schemes known in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University of Bath) College London), the International ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web at imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al. , "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

For example, different defined ranges of CDR regions using Kabat and Chothia numbering.

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

A CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention). Unless otherwise stated, in the present invention, when referring to a residue position in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering system according to the Kabat ( Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The terms "Fc domain" or "Fc region" are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes; or The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of both heavy chains of IgM and IgE class antibodies. Unless otherwise stated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU index) is used for numbering.

The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (eg, B cell receptors), and B cell activation.

A "humanized antibody" is one that retains the antigen-specific reactivity of a non-human antibody (eg, a mouse monoclonal antibody), while being less immunogenic when administered to humans as a therapeutic.

"Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the equilibrium dissociation constant (K _D ). The equilibrium dissociation constant is the ratio of the dissociation rate constant to the association rate constant ( _kdis and _kon , respectively). The smaller the KD, the smaller the dissociation, and the stronger the affinity between the antibody and the antigen. Affinity can be measured by common methods known in the art, eg, KD determined in a _BIACORE instrument using surface plasmon resonance (SPR). Typically, the antibody (eg, an anti-LIF antibody of the invention) will be no higher than 1 x ^10-8 M, eg, less than about 1 x ^10-9 M, 1 x ^10-10 M, 1 x ^10-11 M, 1 x Equilibrium dissociation constant (KD) of ^10-12 M, 1 x ^10-13 M or 1 x ^10-14 M or less dissociates from antigen (eg LIF).

"Percent (%) identity" of an amino acid sequence refers to the alignment of the candidate sequence with the specific amino acid sequence shown in this specification and gaps introduced if necessary to achieve the maximum percent sequence identity, without regard to any When conservative substitutions are made as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence set forth in this specification. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, eg, at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein , 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may contain conservative modifications.

With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to the polypeptide sequence that result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are additive to and not exclusive of the polymorphic variants, interspecies homologues and alleles of the invention. The following 8 groups contain amino acids that are conservatively substituted for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N) , Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Val amino acid (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine acid (C), methionine (M) (see eg, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

"Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to carriers.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. A host cell is any type of cellular system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells and also include transgenic animals, transgenic plants, or cells within cultured plant or animal tissue.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses) that are incorporated into recombinant polynucleotides virus and adeno-associated virus).

The term "pharmaceutical composition" refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered .

The term "treatment" refers to a clinical intervention intended to alter the natural course of disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, reducing symptoms, reducing any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and relieving or improving prognosis. In some embodiments, the antibody molecules of the invention are used to delay the progression of a disease, to slow the progression of a disease, or to stop or reverse the course of a disease.

The term "anti-tumor effect" or "tumor inhibitory effect" refers to a biological effect that can be exhibited by various means including, but not limited to, for example, reduction in tumor volume, reduction in tumor weight, reduction in tumor cell number, reduction in tumor cell proliferation, or tumor cell proliferation Survival is reduced. The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.

The term "effective amount" refers to an amount or dose sufficient to obtain, or at least partially obtain, the desired effect after administration in single or multiple doses, and a "therapeutically effective amount" refers to an amount that produces the desired effect in the subject being treated, including Improvement in the subject's condition (eg, improvement in one or more symptoms) and/or delay in symptom progression, etc. A disease-prophylactically effective amount refers to an amount sufficient to prevent, prevent or delay the onset of a disease. Determining an effective amount is well within the ability of those skilled in the art, eg, a therapeutically effective amount depends on the particular disease involved; the extent or severity of the disease; the individual patient's response; the particular antibody administered; the mode of administration; Utilization characteristics; selected dosing regimen; and use of any concomitant therapy, etc. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%. The ability of the antibody molecules of the invention to inhibit measurable parameters (eg, tumor volume) can be evaluated in animal model systems predictive of efficacy in human tumors.

The term "subject" or "individual" is a primate (eg, human and non-human primates such as monkeys). In certain embodiments, the individual or subject is a human.

II. Production and Purification of Antibody Molecules of the Invention

In yet another aspect, the present invention provides a method for producing an antibody molecule of the present invention, the method comprising: culturing a host cell comprising a polypeptide chain encoding the antibody under conditions suitable for expression of the polypeptide chain; and The antibody is produced by assembling the polypeptide chain under conditions in which the polypeptide chain is assembled into the antibody molecule.

Polypeptide chains of antibody molecules of the invention can be obtained, for example, by solid state peptide synthesis (eg Merrifield solid phase synthesis) or recombinant production. For recombinant production, polynucleotides encoding any polypeptide chain and/or polypeptide chains of the antibody molecule are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.

Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

Once an expression vector comprising one or more polynucleotides of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to accomplish this, eg, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, liposome-based transfection, or other conventional techniques.

In one embodiment, host cells comprising one or more polynucleotides of the present invention are provided. In some embodiments, host cells comprising the expression vectors of the present invention are provided. Host cells suitable for replication and to support expression of the antibody molecules of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as desired, and large numbers of vector-containing cells can be grown for seeding large-scale fermenters to obtain sufficient quantities of the antibody molecules of the invention for clinical use. Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2/0, etc. For a review of suitable mammalian host cell lines for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (eds. B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.

III. PHARMACEUTICAL COMPOSITIONS, COMBINATIONS AND KITS

In one aspect, the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody molecule described herein formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion). In some embodiments, the antibody molecule of the invention is the only active ingredient in the pharmaceutical composition. In other embodiments, a pharmaceutical composition may comprise an antibody molecule described herein and more than one therapeutic agent.

In another aspect, the invention also provides pharmaceutical combinations comprising the antibody molecules described herein and more than one therapeutic agent.

The therapeutic agent suitable for use in the pharmaceutical compositions and drug combinations of the present invention may be a therapeutic agent selected from any of the following classes (i)-(iv): (i) Drugs that enhance antigen presentation (eg, tumor antigen presentation) (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects. In one embodiment, the therapeutic agent is gemcitabine. In another embodiment, the therapeutic agent is an anti-PD-1 antibody.

The compositions of the present invention may be in a variety of forms. Such forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

Identification of subjects in need of treatment is within the ability and knowledge of those skilled in the art. Those subjects in need of such treatment can be readily identified by those of clinical skill in the art using clinical tests, physical examination and medical/family history.

The effective amount of each active ingredient can be easily determined by an attending physician or a diagnosing physician who is skilled in the art mainly by considering the following factors, including but not limited to: the age and general health of the subject; the specific disease; The severity of the disease; the individual subject's response; the particular drug molecule administered; the mode of administration; the bioavailability profile of the administered formulation;

In particular, the pharmaceutical composition of the present invention or the combination of the present invention or the product of the present invention comprises about 1 to 50 mg/kg, eg, about 1 to about 30 mg/kg, about 5 to about 30 mg/kg, about 5 to about 25 mg Anti-LIF of the present application is administered at doses per kg, about 5 to about 20 mg/kg, about 10 to about 20 mg/kg, about 10 to about 15 mg/kg, about 5 to about 10 mg/kg, about 1 to about 5 mg/kg Antibody. In some embodiments, the anti-LIF antibody molecule is at about 1 mg/kg, about 3 mg/kg, or 10 mg/kg, or 15 mg/kg, about 20 mg/kg, or 25 mg/kg, about 30 mg/kg, or about 40 mg/kg Dosing.

The gemcitabine, anti-PD-1 antibody used in combination with the antibody of the invention is administered in an effective amount known or recommended in the art, eg, at about 1 to about 100 mg/kg, about 20 to about 80 mg/kg, about 30 to about 70 mg Gemcitabine is administered at doses of about 40 to about 60 mg/kg, about 1 to about 30 mg/kg, about 10 to about 20 mg/kg, about 5 to about 10 mg/kg, about 1 to about 5 mg/kg Anti-PD-1 antibodies are administered, eg, by injection (eg, subcutaneously or intravenously).

Co-administration of the present invention may administer the individual active ingredients separately, sequentially or simultaneously. If administered simultaneously, the anti-LIF antibodies of the invention and other active ingredients can be formulated, for example, in a single pharmaceutical composition. Alternatively, the various active ingredients can be formulated and administered independently.

In the context of this application, the terms "in conjunction with," "administered in combination," "administered in combination with a drug," and similar expressions are used interchangeably and are not intended to imply that a therapy or therapeutic agent must be administered and/or formulated at the same time together for delivery, although these delivery methods are within the scope of the description herein. The anti-LIF antibody can be administered concurrently with, before, or after one or more other additional therapies or therapeutic agents. Anti-LIF antibody molecules and other drugs or therapeutic regimens can be administered in any order. Generally, each agent will be administered at a dose and/or on a time schedule established for that agent. It will be further appreciated that the additional therapeutic agents used in such a combination can be administered together in a single composition or separately in different compositions. In general, additional therapeutic agents that are expected to be used in combination should be utilized at levels that do not exceed their use alone. In some embodiments, the levels used in combination will be lower than those used alone.

In one embodiment, the anti-LIF antibodies disclosed herein are administered in combination with anti-PD-1 antibodies known in the art. In some embodiments, the anti-PD-1 antibody is selected from, for example, nivolumab and other monoclonal antibodies disclosed in US 8,008,449 and WO 2006/121168; pembrolizumab, lambrolizumab disclosed in US 8,354,509 and WO 09/114335 and other anti-PD-1 antibodies; Pidilizumab and other anti-PD-1 antibodies disclosed in WO 2009/101611; anti-PD-1 antibodies disclosed in US 8,609,089, US 2010028330 and/or US 20120114649, etc.

In one embodiment, the anti-LIF antibody of the present application and the pyrimidine nucleoside anti-tumor drug or anti-PD-1/PD-L1 antibody are administered simultaneously. In another embodiment, the pyrimidine nucleoside antineoplastic drug or anti-PD-1/PD-L1 antibody is administered prior to administration of the anti-LIF antibody of the present application. In another embodiment, the pyrimidine nucleoside antineoplastic drug or anti-PD-1/PD-L1 antibody is administered after administration of the anti-LIF antibody of the present application.

The phrases "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or lyophilized forms. Sterile injectable solutions can be prepared by incorporating the active compound (ie, the antibody molecule) in the required amount in an appropriate solvent followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients. Coatings such as lecithin and the like can be used. In the case of dispersions, proper fluidity of the solution can be maintained by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody molecule of the present invention.

Kits comprising the antibody molecules described herein are also within the scope of the invention. A kit may contain one or more other elements, including, for example, instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

IV. Uses and Methods of Molecules of the Invention

In one aspect, the present invention provides in vivo, in vitro uses and methods of application of the antibody molecules of the present invention.

In some embodiments, the antibody molecule of the invention or a pharmaceutical composition comprising the antibody molecule of the invention is used as a medicament for the treatment and/or prevention of a disease in an individual or as a diagnostic tool for a disease, preferably the individual is a mammal , more preferably a human.

In some embodiments, the present invention provides methods and uses of applying the antibody molecules of the present invention to treat cancers associated with aberrant expression of LIF, which cancers may, for example, be selected from ovarian cancer, lung cancer, colorectal cancer, glioma, leukemia , rectal cancer, bladder cancer, breast cancer, pancreatic cancer. Preferably, the cancer is pancreatic cancer.

In one aspect, the present invention provides diagnostic methods for detecting the presence of relevant antigens in biological samples such as serum, blood or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions) in vitro or in vivo. The diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject under conditions that allow the interaction to occur and (ii) The formation of complexes between the antibody molecule and the sample (and optionally, a control sample) is detected. The formation of complexes indicates the presence of relevant antigens and may indicate applicability or need for treatment and/or prevention as described herein.

In some embodiments, the relevant antigen is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment following a treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (eg, RT-PCR) or in vivo imaging techniques. Typically, antibody molecules used in in vivo and in vitro detection methods are labeled, directly or indirectly, with detectable substances to facilitate detection of bound or unbound conjugates. Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (eg, nuclear magnetic resonance active) substances, and radioactive substances.

In some embodiments, the level and/or distribution of the relevant antigen is determined in vivo, eg, non-invasively (eg, detectable by detection using a suitable imaging technique (eg, positron emission tomography (PET) scan) In one embodiment, the level of the relevant antigen is determined in vivo, for example, by detecting antibody molecules of the invention that are detectably labeled with a PET reagent (eg, 18F-fluorodeoxyglucose (FDG)). and/or distribution.

In one embodiment, the present invention provides diagnostic kits comprising the antibody molecules described herein and instructions for use.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell technology within the scope of the art. biological methods.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Obtaining mouse anti-human leukemia inhibitory factor antibody

1. Expression of recombinant human leukemia inhibitory factor

The amino acid sequence of human leukemia inhibitory factor was obtained from the Protein database of NCBI, and the cDNA sequence was artificially synthesized and constructed into a PET30a expression vector according to conventional methods and transformed into Escherichia coli BL21 strain to obtain an Escherichia coli strain expressing recombinant human leukemia inhibitory factor.

Induce the expression of recombinant human leukemia inhibitory factor: inoculate the above strains into LB medium, cultivate overnight at 37°C and 200 rpm, and then inoculate in freshly prepared LB medium at a volume ratio of 1:50, continue at 37°C and 200 rpm. Shake culture. When the OD value measured at 600 nm reached 0.6, IPTG was added at a final concentration of 0.5 mM for induction, and the bacteria were harvested after shaking at 200 rpm for 20 hours at 16 °C. Add lysate (20mM Tris-HCl, 60mM NaCl, 1mM EDTA, 0.5% Triton X-100) to lysate the bacterial cells at a weight-to-volume ratio of 1:10 according to the wet weight of the bacteria, centrifuge to obtain the lysate supernatant, adjust the lysate supernatant pH to 6.0.

Purification of recombinant human leukemia inhibitory factor: the obtained lysate supernatant was sequentially carried out by anion exchange chromatography (SP sepharose fast flow column), cation exchange chromatography (Q sepharose fast flow column) and hydroxyapatite chromatography. Purification to obtain high-purity recombinant human leukemia inhibitory factor.

2. Preparation of hybridomas resistant to recombinant human leukemia inhibitory factor

80 μg of recombinant human leukemia inhibitory factor protein obtained above was mixed with an equal volume of complete Freund's adjuvant, and 8-week-old female BALB/c mice were immunized. After the first immunization, the immunization was boosted every 2 weeks for a total of 3 times. . The mouse serum was detected by ELISA method. When the titer reached ¹ :105, the mice were boosted once more, and the mice were sacrificed after 3 days, and the spleen was aseptically collected and ground on a plate to prepare a spleen single cell suspension for later use.

According to a conventional method, the obtained spleen single cell suspension was fused with mouse SP2/0 myeloma cells (purchased from ATCC) to obtain corresponding hybridoma cells. The fusions were screened for LIF-blocking positive cells by ELISA. Cells were harvested after several rounds of subcloning to obtain hybridoma cells expressing anti-LIF antibodies.

3. Sequence characteristics of anti-human leukemia inhibitory factor antibody

For the selected positive hybridomas, the total RNA was prepared, reverse transcribed into cDNA, the VH and VL genes were amplified by PCR, and the heavy chain variable region CDRs of the anti-LIF antibodies shown in Tables I-III were obtained after sequencing. Sequences and light chain variable region CDR sequences, heavy chain variable region (VH) sequences and light chain variable region (VL) sequences, heavy chain sequences and light chain sequences.

Table 1: CDR sequences of anti-LIF antibodies (based on Kabat nomenclature):

Table II: Heavy chain variable region sequences and light chain variable region sequences of anti-LIF antibodies:

Table III: Heavy and light chain sequences of anti-LIF antibodies:

Example 2. Expression and purification of murine anti-LIF antibodies

The selected positive hybridoma cells were inoculated into the peritoneal cavity of 6-8 week old BALB/c mice, and the ascites was collected after culturing for a period of time. The antibody samples were analyzed by SDS-PAGE protein electrophoresis to check the purification effect. The results are shown in Figure 1, where NR represents non-reducing conditions; R represents reducing conditions. As can be seen from Figure 1, high-purity anti-LIF antibodies were obtained.

Example 3. Anti-LIF Antibody Humanization

Since the mouse-derived anti-LIF antibody obtained in the above example has a heterologous reaction to the human body, it is possible to induce the human anti-mouse antibody effect (Human anti-mouse antibodies, HAMA reaction). In order to reduce the immunogenicity of the antibody and improve its performance in human applications, the inventors engineered the antibody.

First, the B cell epitope immunogenicity was evaluated on the variable region sequences of the light and heavy chains of the obtained murine anti-LIF antibody, and the immunogenicity of the sequences was analyzed by software to obtain the B cell immunogenicity score of the antibody sequence, and Targets to be engineered to reduce or remove immunogenicity were initially selected based on this score.

Then, the T-cell epitope analysis of MHC I and MHCII was performed on the variable region sequences of the light and heavy chains of the obtained murine anti-LIF antibody. The evaluation criteria for strong epitopes were that the predicted IC50 value of the MHCII epitope was less than 50, and the predicted IC50 of the MHCII epitope was less than 50. The value is less than 100. The number, intensity and strong epitope information of T cell epitopes of the antibody light and heavy chains are thus obtained.

According to the above analysis results, the framework region of the mouse-derived anti-LIF antibody sequence was de-immunized and designed to be humanized to obtain a humanized antibody, the sequence of which is shown in the following table:

Table IV: Heavy and Light Chain Variable Region Sequences of Humanized Anti-LIF Antibodies:

Table V: Heavy and Light Chain Sequences of Humanized Anti-LIF Antibodies:

The obtained humanized anti-LIF antibody was correspondingly expressed according to the method of Example 2 and purified using the method described in Example 2.

Example 4. Blockade of LIF activity by anti-LIF antibodies and their humanized antibodies

M1 cells (ATCC TIB-192 cells, mouse leukemia cells) that express high LIF receptors were diluted to a final concentration of 20,000 cells/mL in DMEM medium containing 10% fetal bovine serum, and added to 50 mL of M1 cell suspension. LIF protein at a concentration of 2.5ng/mL, and mix well. The obtained M1 cell mixture was divided into equal parts and mixed with the anti-LIF antibody (50ng/mL) of the present application. The negative control was an equal volume of PBS solution, and then inoculated into 96-well plates, and DMEM was added to each well for culture. 100 μL of base, incubate for 24 hours, and then use CCK-8 detection kit to measure the absorbance at 630 nm and 450 nm of each well according to the method recommended by the manufacturer to detect the binding of anti-LIF antibody and its humanized antibody to LIF, that is, to LIF blocking of activity. The experiment was repeated three times.

Since the dehydrogenase activity in M1 cells is positively correlated with the amount of LIF added, blocking LIF can reduce the dehydrogenase activity in M1 cells, so it can be used to evaluate the blocking of LIF activity by anti-LIF antibodies. Relative to the control, a greater down-regulation of absorbance values indicates a stronger blockade of LIF activity.

The results showed that both anti-LIF antibody and its humanized antibody could block the function of LIF factor.

Example 5. Affinity of anti-LIF antibodies and their humanized antibodies to LIF

Antibody affinity for antigen was tested by surface plasmon resonance (SPR). Biacore 8K (GE Healthcare Life Sciences, GE) was used, and the corresponding affinity test was performed according to the manufacturer's instructions.

Chip preparation

First, the CM5 chip was activated with 400 mM EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 100 mM NHS (N-hydroxysuccinimide) at a flow rate of 10 μL/min Surface 420s. Then, 25 μg/mL mouse anti-human IgG (Fc) antibody prepared in immobilization reagent (10 mM sodium acetate, pH 5.0) was injected into the experimental channel (FC2) at a flow rate of 10 μL/min for about 420 s, and 50 μL mouse anti-human IgG(Fc) antibody was added to 950μL of immobilization reagent for immobilization of eight channels, and the immobilization amount was about 9000 to 14000RU. Finally, the chip was blocked with 1 M ethanolamine at 10 μL/min for 420 s.

capture ligand

The anti-LIF antibody and its humanized antibody were diluted to 5 μg/mL with running reagent, and were sequentially injected into the experimental channel (FC2) for about 200 RU at a flow rate of 10 μL/min. The reference channel (FC1) does not require ligand capture.

Analyte Multicycle Analysis

Recombinant human leukemia inhibitory factor protein was prepared with running reagent (10 mM N-(2-hydroxyethyl) piperazine-N-2 sulfonic acid (HEPES), 150 mM sodium chloride, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20 (Tween-20, pH 7.4) was diluted to obtain solutions with concentrations of 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, and 0 nM, respectively. The diluted recombinant human leukemia inhibitory factor protein was sequentially injected into the experimental channel and the reference channel at a flow rate of 50 μL/min, the binding time was 150s, and the dissociation time was 600s. Both binding and dissociation steps are performed in the running reagent. After analyzing each concentration, the chip needed to be regenerated with 3M magnesium chloride at a flow rate of 20 μL/min.

KD values for each antibody were calculated using Biacore 8K analysis software. The reference channel (FC1) is used for background subtraction.

The affinity results of the anti-LIF antibody and its humanized antibody of the present application are shown in Table 1:

Table 1. Affinities of anti-LIF antibodies and their humanized antibodies

Ligand	ka(1/Ms)	kd(1/s)	KD(M)	Rmax(RU)
Mouse anti-LIF antibody	1.38E+06	1.88E-07	1.37E-13	54.9
Humanized anti-LIF antibody	1.73E+06	7.79E-09	4.51E-15	57.5

It can be seen that the mouse-derived anti-LIF antibody and its corresponding humanized antibody obtained in this application can bind to the recombinant human leukemia inhibitory factor protein with high affinity, and the affinity of the mouse-derived anti-LIF antibody is 1.37×10 ^-13 mol/L ; The affinity of the humanized anti-LIF antibody was 4.51×10 ⁻¹⁵ mol/L. After humanization, the humanized antibody obtained in the present application can bind to human recombinant leukemia inhibitory factor with higher affinity.

Example 6. Immunogenicity evaluation of the applicant's anti-LIF antibody

The mouse-derived anti-LIF antibody and its humanized antibody were divided into two groups to immunize the mice respectively. The immunogens were injected into the limbs and abdominal cavity of the mice respectively, and a total of one primary immunization and four booster immunizations were performed within 14 days. , the immunization time points are the 0th day, the 3rd day, the 7th day, the 10th day and the 12th day, the total immunization dose is about 70ug/mouse; The 14-day and 21-day mouse serum was used to evaluate the titer level of ADA (Anti-drug antibody, ADA) in mouse serum by ELISA method.

Serum anti-drug antibody titers were assessed using ELISA. Specifically, humanized anti-LIF antibody and mouse anti-LIF antibody were diluted with PBS solution to 1 μg/mL and coated with ELISA plate, 100 μL per well, incubated overnight at 4°C or incubated at 37°C for 2 hr; then PBS solution Plates were washed 3 times and blocked with 5% milk-PBS for 1 hr at room temperature; then washed once with PBS solution; at the same time, the plates were serially diluted in 5% milk-PBS buffer containing 10 μg/mL of human total IgG and collected at different time points The mouse tail blood serum (1:500, 1:1000, 1:5000, 1:10000, 1:50000) was left at room temperature for 1 hr; then the pre-diluted tail blood serum of each gradient was added to the ELISA plate respectively , 100 μL per well, incubate for 1 hr at room temperature; then wash the plate 3 times with PBS solution and pat dry, add 1:2000 diluted HRP-labeled goat anti-mouse IgG (Fc) secondary antibody, and react at room temperature for 1 hr; wash the plate with PBS solution for 5 After several times, pat dry, add equal volumes of solution A and solution B (HRP substrate chromogenic solution, purchased from Beijing Qinbang Biotechnology Co., Ltd.), and react at room temperature for 20 min in the dark; then add 50 μL of stop solution and mix well Then read the absorbance values at OD450 and OD630 on the microplate reader, and the experiment was repeated three times. And calculate the difference between the absorbance value at OD450 minus the absorbance value at OD630.

According to the obtained data, the abscissa is the blood sampling time, and the ordinate is the difference of absorbance at 450nm and 630nm. As shown in Figure 2, the humanized anti-LIF antibodies disclosed in the present application are similar in immunogenicity to their murine antibodies in mice.

In addition, for the sera of mice in each group on the 21st day after immunization, the applicant also used ELISA method to study the ADA titers of the sera at different dilutions, and the specific experimental steps were the same as above. As shown in Figure 3, 21 days after the injection of the antibody, the humanized anti-LIF antibody obtained in the present application was similar in immunogenicity to the parental murine antibody.

As can be seen, the present application obtained anti-LIF humanized antibodies with reduced immunogenicity for human subjects.

Example 7. Anti-LIF antibodies inhibit tumor growth in a mouse model of pancreatic cancer

To test the ability of the applicant's personalized antibodies and their parental murine antibodies to inhibit LIF-positive cancers in vivo, the antibodies were tested in a mouse model of pancreatic cancer. This experiment was performed in accordance with national and international guidelines for the care and use of laboratory animals and was approved by the local ethics committee.

In this example, human pancreatic cancer PANC-1 (purchased from the National Biomedical Experimental Cell Resource Bank) cells were used to prepare a pancreatic cancer-bearing mouse model, and the cells were inoculated into the right axilla of nude mice (Balb/c) at a rate of 5.0×10 ⁶ cells per mouse. Subcutaneously, 10 days later, they were randomly divided into 4 groups (6 mice in each group): control group (IgG), gemcitabine (Gemcitabine hydrochloride for injection, Shandong Qilu Pharmaceutical) group (Gem), mouse LIF antibody group, mouse LIF antibody group In combination with gemcitabine. The mice in each group were administered by intraperitoneal injection, wherein the mice in each group were given the corresponding LIF antibody once every 1 day at a dose of 25 mg/kg; for the combination group and the gemcitabine group alone, gemcitabine was administered at a dose of 80 mg/kg. , once every 3 days. The experiment was terminated after 18 consecutive days of dosing.

During the experiment, the tumor volume was measured with a vernier caliper every two days, and the tumor volume (TV) (unit: mm ³ ) was calculated according to the formula (length×width ² )/2.

The experimental results of the tumor tissues of each group of mice are shown in Figure 4. Compared with the control group, the transplanted tumor volume and tumor weight of the anti-LIF antibody group, gemcitabine group and combination group were significantly lower, and the combination of Gem and anti-LIF antibody group had more significant tumor inhibition, and Gem and LIF antibody alone Significantly lower than the administration group. It is suggested that anti-LIF antibody can significantly inhibit the growth of subcutaneous transplanted tumor, reduce tumor volume and weight, and the inhibitory effect is more significant after combining with Gem (Figure 4). The experimental results show that the single administration of the anti-LIF antibody of the present application can inhibit the growth of pancreatic cancer tumors and reduce the volume and weight of the tumor. The combination of the anti-LIF antibody of the present application and gemcitabine has the greatest inhibitory effect on tumor volume.

The applicant expects that the applicant's humanized anti-LIF antibody also has a corresponding inhibitory effect on pancreatic cancer, and the humanized anti-LIF antibody is used to verify in vivo pancreatic cancer-bearing mice.

Specifically, human pancreatic cancer PANC-1 cells were subcutaneously inoculated into the right axilla of nude mice (Balb/c) at a dose of 5.0×10 ⁶ cells/mouse to prepare a pancreatic cancer-bearing mouse model. After 10 days, they were randomly divided into 4 groups: Control group (IgG), gemcitabine group (Gem), humanized anti-LIF antibody group, humanized anti-LIF antibody combined with gemcitabine group. The mice in each group were administered by intraperitoneal injection, wherein the mice in each group were given the corresponding LIF antibody once every 1 day at a dose of 25 mg/kg; for the combination group and the gemcitabine group alone, gemcitabine was administered at a dose of 80 mg/kg. , once every 3 days. The experiment was terminated after 18 consecutive days of dosing.

During the experiment, the tumor volume was measured with a vernier caliper every two days, and the tumor volume (unit: mm ³ ) was calculated according to the formula (length×width ² )/2.

The experimental results show that the humanized anti-LIF antibody can significantly inhibit the growth of tumor compared with the control group, and even reduce the volume and weight of the tumor. The combination of Gem and humanized anti-LIF antibody has a more significant inhibitory effect on the tumor.

Applicants then further validated the tumor suppressive effect of the humanized anti-LIF antibody in NSG mice.

Specifically, female NSG mice (18-20g/mice, SPF grade) were obtained from Speifu Biotechnology Co., Ltd., and human pancreatic cancer PANC1 cells with a density of 5×10 ⁸ cells/mL were collected, and 0.2 mL was inoculated into NSG The mice were subcutaneously placed on the dorsum of the armpits, and when the tumors grew to a size of 1000 mm ³ , they were taken out aseptically, divided into equal-sized tumor masses, and then evenly inoculated into the dorsum of the armpits of other NSG mice. On the 6th day, the animals were randomly divided into groups (control group, gemcitabine group, anti-LIF group, gemcitabine + anti-LIF combination group), and the intraperitoneal injection was started (recorded as day 0), among which the control antibody (30 mg/kg), human Anti-LIF antibody (30 mg/kg) was administered every other day, and gemcitabine (50 mg/kg) was administered twice a week for the combination group and the gemcitabine alone group. NSG mice were sacrificed by dislocation on day 11, and tumor tissue was dissected, weighed and photographed. Tumor volume was calculated by weighing twice weekly and measuring tumor length and width with vernier calipers. And the tumor inhibition rate was calculated according to the following formula:

Inhibition rate (%)=(1-T/C)×100, T is the TV or tumor weight of the treatment group, and C is the TV or tumor weight of the negative control group.

The applicant found that the antibody of the present application and its drug combination with gemcitabine still had excellent antitumor activity in NSG mice. The results are shown in Figure 4d-f, compared with the control group, the anti-LIF antibody alone group and the combination group with gemcitabine significantly reduced the tumor volume and tumor weight, and the combination group of gemcitabine and anti-LIF antibody inhibited the tumor. effect is more significant. It is worth noting that 1 animal died in both the gemcitabine experimental group and the antibody and gemcitabine combination group, indicating that gemcitabine has certain side effects.

It can be seen that the anti-LIF antibody obtained in the present application has excellent effects of inhibiting tumor growth and reducing tumor volume and weight.

Example 8. LIF antibody combined with PD-1 antibody inhibits tumor growth in a mouse model of pancreatic cancer

In order to test the ability of the applicant's antibody and its parent murine antibody in combination with PD-1 antibody to inhibit LIF-positive cancer in vivo, the effect of the combination of the antibody was tested in a mouse model of pancreatic cancer. This experiment was performed in accordance with national and international guidelines for the care and use of laboratory animals and was approved by the local ethics committee.

In this example, mouse pancreatic cancer Pan02 cells (purchased from the National Biomedical Experimental Cell Resource Bank) were used to prepare a pancreatic cancer-bearing mouse model, and 4.0×10 ⁶ cells per mouse were subcutaneously inoculated into the right armpit of C57 mice, and randomized 5 days later. Divided into 4 groups (8 mice in each group): control group (IgG), PD-1 antibody group (anti-mouse PD-1 antibody purchased from BioX cell, anti-mouse PD-1 (Cat. No.: CD279)), mouse anti-LIF antibody group , mouse anti-LIF antibody and PD-1 antibody combination group. The mice in each group were administered by intraperitoneal injection, wherein the mice in each group were given the corresponding LIF antibody once every other day at a dose of 25 mg/kg; for the combination group and the PD-1 antibody group alone, they were given weekly The PD-1 antibody was administered twice at a dose of 200 μg/only. The experiment was terminated after 16 consecutive days of dosing.

During the experiment, the tumor volume was measured with a vernier caliper every two days, and the tumor volume (unit: mm ³ ) was calculated according to the formula (length*width ² )/2.

The experimental results are shown in Figure 5. The results showed that in the Pan02 mouse pancreatic cancer xenograft model, the tumor volume of mice in the immunotherapy group with anti-LIF antibody and anti-PD-1 antibody alone was significantly lower than that in the control group (Figure 5a and Figure 5b). , the tumor weight of the mice treated with single antibody was also significantly smaller than that of the mice in the control group (Fig. 5c), that is, the growth of the transplanted tumor was delayed, and the size and weight of the transplanted tumor were even reduced. The combination therapy group achieved the greatest tumor volume inhibition and greatest tumor volume reduction.

The experimental results show that the single administration of the anti-LIF antibody of the present application can inhibit the growth of pancreatic cancer in tumor-bearing mice, and the combination of the anti-LIF antibody of the present application and the PD-1 antibody has the best inhibitory effect on tumor volume.

The applicant expects that the applicant's humanized anti-LIF antibody also has a corresponding inhibitory effect on pancreatic cancer, and the humanized anti-LIF antibody is used to verify in vivo pancreatic cancer-bearing mice.

Specifically, mouse pancreatic cancer Pan02 cells were inoculated subcutaneously into the right armpit of C57 mice at 4.0×10 ⁶ cells/mouse to prepare a pancreatic cancer-bearing mouse model, and 5 days later, they were randomly divided into 4 groups: control group (IgG) , PD-1 antibody group (the antibody was purchased from BioX cell, anti-mouse PD-1 (Cat. No.: CD279)), humanized anti-LIF antibody group, humanized anti-LIF antibody combined with PD-1 antibody group. The mice in each group were administered by intraperitoneal injection, wherein the mice in each group were given the corresponding LIF antibody once every other day at a dose of 25 mg/kg; for the combination group and the PD-1 antibody group alone, they were given weekly The PD-1 antibody was administered twice at a dose of 200 μg/only. The experiment was terminated after 16 consecutive days of dosing.

During the experiment, the tumor volume was measured with a vernier caliper every two days, and the tumor volume was calculated according to the following formula: V=(length*width2)/2 (unit: mm3). The tumor inhibition rate was calculated according to the following formula: tumor inhibition rate (%)=(1-T/C)×100, T is the TV or tumor weight of the treatment group, and C is the TV or tumor weight of the negative control group.

The experimental results show that the humanized anti-LIF antibody can significantly inhibit tumor growth compared with the control group, and even reduce the volume and weight of the tumor. The combination of humanized anti-LIF antibody and anti-PD-1 antibody inhibits tumor. effect is more significant. For example, as shown in Figure 6, compared with the control group, the tumor weight and tumor volume of mice in the immunotherapy group treated with anti-LIF antibody and anti-PD-1 antibody alone were significantly reduced (Figures 6a and 6b), and the tumor inhibition rate The anti-tumor effect of the antibody combination group (combo group) was more significant, showing stronger tumor-suppressing activity, and the tumor-inhibiting rate could reach 77.16%. Figure 6c shows tumor growth curves of mice in each treatment group.

Example 9. Further optimization of humanized anti-LIF antibodies

The inventors modified the humanized anti-LIF antibody obtained in Example 3 to further optimize its performance, thus obtained a humanized antibody with the following sequence, and named it humanized anti-LIF antibody- 1.

Table VI: Heavy and Light Chain Variable Region Sequences of Humanized Anti-LIF Antibody-1:

Table VII: Heavy and Light Chain Sequences of Humanized Anti-LIF Antibody-1:

According to the method of Example 2, the newly obtained humanized anti-LIF antibody-1 was correspondingly expressed and purified, and compared with the humanized anti-LIF antibody obtained in Example 3 in terms of protein expression. Briefly, according to conventional methods, plasmids containing humanized anti-LIF antibody and humanized anti-LIF antibody-1 sequences were respectively transfected into CHO-K1 cells, cultured and expressed the target protein, and the supernatant was harvested. The target protein was purified by MabSelect PrisMA affinity chromatography, and the expression levels of the two antibody molecules were calculated.

The results showed that the expression level of humanized anti-LIF antibody was 40-45 mg/L, while the expression level of humanized anti-LIF antibody-1 was 100-110 mg/L. It can be seen that the humanized anti-LIF antibody- 1 has higher expression levels than its parental antibody, which is advantageous in production and applications requiring large quantities of antibody.

Afterwards, we verified that the humanized anti-LIF antibody-1 still has an excellent ability to bind to the antigen by detecting the affinity with the antigen LIF.

Using the parent antibody obtained in Example 3 as a control, the method described in Example 5 was used for affinity detection. The results are shown in Table 2. The affinity of humanized anti-LIF antibody-1 was 1.26×10 ^-13 mol/L . It can be seen that after transformation and optimization, the newly obtained humanized anti-LIF antibody-1 still retains the excellent ability to bind to the antigen.

Table 2. Affinities of Humanized Anti-LIF Antibody and Humanized Anti-LIF Antibody-1

Ligand	ka(1/Ms)	kd(1/s)	KD(M)	Rmax(RU)
Humanized anti-LIF antibody	7.37E+05	4.85E-08	6.59E-14	53.0
Humanized anti-LIF antibody-1	1.31E+06	1.66E-07	1.26E-13	47.6

Next, we further tested the ability of humanized anti-LIF antibody-1 to kill pancreatic cancer cells, and compared it with the humanized anti-LIF antibody obtained in Example 3 and the murine anti-LIF antibody obtained in Example 1. Performance was compared with an irrelevant antibody as a negative control. For the ability to inhibit human pancreatic cancer cell lines and pancreatic cancer patient-derived organoids at the in vitro level, all experiments below were repeated three times.

First, a horizontal cell proliferation assay was performed using the pancreatic cancer cell line PANC-1. Single cell suspensions of PANC-1 cells were prepared according to conventional methods, and cell counts were performed. 1000 cells per well (100 μL complete medium) were inoculated into a 96-well plate. After the cells adhered, the medium in each well was replaced with the corresponding 100 μL containing 3 μg/mL of various anti-LIF antibodies or control antibodies. The cells were cultured in a cell culture incubator, and the cell proliferation was detected with CCK8 after 3 days. The experimental results are shown in Figure 7a. Compared with the control group, both the humanized anti-LIF antibody-1 group and the humanized anti-LIF antibody group significantly reduced the proliferation ability of tumor cells, among which the humanized anti-LIF antibody group The tumor cell inhibitory effect of the antibody-1 group was more significant.

Second, the pancreatic cancer cell lines PANC-1 and SW1990 (purchased from the National Biomedical Experiment Cell Resource Bank) were used to detect the proliferation of tumor cell-derived organoids (CDO) at the level. Single cell suspensions of PANC-1 cells and SW1990 were prepared according to conventional methods, and cell counts were performed. 3000 cells per well (resuspended in 40 μL matrigel) were seeded into a 96-well plate and left to stand for 10 minutes in a 37° C., 5% CO ₂ cell incubator. The medium in each well was replaced with the corresponding 100 μL cell complete medium (containing 1% penicillin and 10% fetal bovine serum) containing 3 μg/mL of various anti-LIF antibodies or control antibodies, respectively. After 7 days, the organoid proliferation was detected by cellTiter. The experimental results are shown in Figures 7b and 7c. Compared with the control group, both the humanized anti-LIF antibody-1 group and the humanized anti-LIF antibody group significantly reduced the proliferation ability of tumor organoids, and the humanized anti-LIF antibody group significantly reduced the proliferation ability of tumor organoids. The -LIF antibody-1 group had a more significant inhibitory effect on tumor organoids.

Finally, proliferation assays were performed at the level of pancreatic cancer patient-derived organoids (PDO). This experiment was approved by the Ethics Committee of Cancer Hospital, Chinese Academy of Medical Sciences. Informed consent of the pancreatic cancer patients has been obtained as required by the ethics committee. All experiments were performed in accordance with relevant guidelines and regulations. Routinely, when the PDO has grown to an appropriate level, it is digested into individual organoids with Tryp LE, and the cells are resuspended in an appropriate volume of medium for cell counting. 3000 cells per well (resuspended in 40 μL matrigel) were seeded into a 96-well plate and left to stand for 10 minutes in a 37° C., 5% CO ₂ cell incubator. The medium in each well was replaced with the corresponding 100 μL pancreatic cancer organoid medium containing 3 μg/mL of various anti-LIF antibodies or control antibodies, respectively, and the culture was continued in a cell incubator. CellTiter was used to detect cell proliferation after 7 days. The experimental results are shown in Figure 7d. Compared with the control group, the humanized anti-LIF antibody-1 group and the humanized anti-LIF antibody group significantly reduced the proliferation ability of tumor organoids.

The above experimental results show that both the humanized anti-LIF antibody-1 and humanized anti-LIF antibody of the applicant can significantly inhibit the proliferation of pancreatic cancer tumors, and the humanized anti-LIF antibody-1 group has an inhibitory effect on tumors. more pronounced.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (15)

1. Anti-human leukemia inhibitory factor antibody or antigen-binding fragment thereof comprising 3 CDRs of the variable region of the heavy chain as shown in SEQ ID NO:7, and 3 CDRs of the variable region of the light chain as shown in SEQ ID NO:8 CDRs.
2. An anti-human leukemia inhibitory factor antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

   (a) CDRs comprising the following heavy chain variable regions:

   CDR1 comprising the sequence of SEQ ID NO: 1;

   CDR2 comprising the sequence of SEQ ID NO:2;

   CDR3 comprising the sequence of SEQ ID NO: 3, and

   (b) CDRs comprising the following light chain variable regions:

   CDR1, it comprises the sequence of SEQ ID NO:4;

   CDR2, it comprises the sequence of SEQ ID NO:5;

   CDR3, which comprises the sequence of SEQ ID NO:6.
3. The anti-human leukemia inhibitory factor antibody of any one of claims 1-2, comprising the following heavy chain variable region and light chain variable region:

   (1) a heavy chain variable region sequence comprising the heavy chain CDR of claim 1 or 2 and having at least 80% identity to SEQ ID NO: 7, 11 or 15, and comprising the heavy chain variable region sequence of claim 1 or 2 A light chain variable region sequence that is at least 80% identical to SEQ ID NO: 8, 12 or 16 in the light chain CDRs; or

   (2) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 7, or a heavy chain variable region comprising at least 80% identity with SEQ ID NO: 7 and comprising the heavy chain CDRs described in claim 1 or 2 A variable region, and a light chain variable region comprising the sequence shown in SEQ ID NO: 8, or a light chain comprising at least 80% identity with SEQ ID NO: 8 and comprising the light chain CDRs of claim 1 or 2 variable region; or

   (3) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 11, or a heavy chain variable region comprising at least 80% identity with SEQ ID NO: 11 and comprising the heavy chain CDRs described in claim 1 or 2 A variable region, and a light chain variable region comprising the sequence shown in SEQ ID NO: 12, or a light chain comprising at least 80% identity with SEQ ID NO: 12 and comprising the light chain CDRs of claim 1 or 2 variable region; or

   (4) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 15, or a heavy chain variable region comprising at least 80% identity with SEQ ID NO: 15 and comprising the heavy chain CDRs described in claim 1 or 2 A variable region, and a light chain variable region comprising the sequence shown in SEQ ID NO: 16, or a light chain comprising at least 80% identity with SEQ ID NO: 16 and comprising the light chain CDRs of claim 1 or 2 variable region.
4. The anti-human leukemia inhibitory factor antibody of any one of claims 1-3, comprising the following heavy chain and light chain:

   (1) a heavy chain variable region sequence comprising the heavy chain CDR of claim 1 or 2 and having at least 80% identity to SEQ ID NO: 9, 13 or 17, and comprising the heavy chain variable region sequence of claim 1 or 2 A light chain variable region sequence that is at least 80% identical to SEQ ID NO: 10, 14 or 18 in the light chain CDRs; or

   (2) a heavy chain comprising the sequence shown in SEQ ID NO: 9, or a heavy chain having at least 80% identity with SEQ ID NO: 9 and comprising the CDRs of the heavy chain according to claim 1 or 2, and comprising SEQ ID NO: 9 A light chain of the sequence shown in ID NO: 10, or a light chain comprising at least 80% identity to SEQ ID NO: 10 and comprising the light chain CDRs of claim 1 or 2; or

   (3) a heavy chain comprising the sequence shown in SEQ ID NO: 13, or a heavy chain having at least 80% identity with SEQ ID NO: 13 and comprising the CDRs of the heavy chain according to claim 1 or 2, and comprising SEQ ID NO: 13 A light chain of the sequence shown in ID NO: 14, or a light chain comprising at least 80% identity to SEQ ID NO: 14 and comprising the light chain CDRs of claim 1 or 2; or

   (4) a heavy chain comprising the sequence shown in SEQ ID NO: 17, or a heavy chain having at least 80% identity with SEQ ID NO: 17 and comprising the CDRs of the heavy chain according to claim 1 or 2, and comprising SEQ ID NO: 17 A light chain of the sequence shown in ID NO: 18, or a light chain comprising at least 80% identity to SEQ ID NO: 18 and comprising the light chain CDRs of claim 1 or 2.
5. The anti-human leukemia inhibitory factor antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragment.
6. A nucleic acid molecule encoding the anti-human leukemia inhibitory factor antibody or antigen-binding fragment thereof of any one of claims 1 to 5.
7. A vector comprising the nucleic acid molecule of claim 6, preferably the vector is an expression vector.
8. The host cell comprising the vector of claim 7 or the nucleic acid molecule of claim 6, preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from Escherichia coli cells, yeast cells, Mammalian cells or other cells suitable for the production of antibodies or antigen-binding fragments thereof, wherein the mammalian cells are, for example, CHO cells, HEK293 cells or COS cells.
9. A method of producing an antibody or antigen-binding fragment thereof that binds to human leukemia inhibitory factor, comprising culturing the nucleic acid of claim 8 under conditions suitable for expressing a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 The host cell, optionally isolating the antibody or antigen-binding fragment thereof, and optionally collecting the antibody or antigen-binding fragment thereof produced.
10. The antibody or antigen-binding fragment thereof produced by the method of claim 9.
11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5 or 10 and a pharmaceutically acceptable carrier.
12. The pharmaceutical composition of claim 11, further comprising an additional therapeutic agent, for example, said therapeutic agent is gemcitabine and its salts, anti-PD-1 antibody, optionally said gemcitabine is gemcitabine hydrochloride, said anti-PD- 1 The antibody is for example selected from nivolumab, pembrolizumab, Lambrolizumab, Pidilizumab and other anti-PD-1 antibodies.
13. A method for treating a disease associated with abnormal expression of human leukemia inhibitory factor, comprising adding an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-5 or 10 or any one of claims 11-12. One of the pharmaceutical compositions is administered to a subject in need thereof.
14. The method of claim 13, wherein the antibody or antigen-binding fragment thereof or the pharmaceutical composition is used in combination with another therapeutic agent, eg, the therapeutic agent is gemcitabine and its salts, an anti-PD-1 antibody , optionally, in combination with gemcitabine hydrochloride or an anti-PD-1 antibody.
15. The method of claim 13 or 14, wherein the disease associated with abnormal expression of human leukemia inhibitory factor is a malignant tumor, such as ovarian cancer, lung cancer, colorectal cancer, brain glioma, leukemia, rectal cancer, bladder cancer, Breast cancer, pancreatic cancer, preferably, the cancer is pancreatic cancer.

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