Combination Cancer Therapy

Abstract

The present invention relates to a combination of a specific binding molecule that binds to human ANXA1 and a second active agent for use in the treatment of cancer. The second active agent includes thymidylate synthase inhibitors, nucleobase analogs, checkpoint inhibitors, proteasome inhibitors, taxanes, platinum-based chemotherapy agents, and nucleoside analogs. Suitable cancers for treatment include pancreatic cancer, colon cancer, breast cancer, lung cancer, myeloma, and mantle cell lymphoma. Also provided are related kits, products, and uses.

Description

[Technical field]

[0001] Field The present invention relates to the treatment of cancer with specific binding molecules to Annexin A1 (ANXA1) in combination with certain other therapeutic agents.

[0002] background Cancer is a group of diseases characterized by abnormal cell proliferation. The abnormal cell proliferation associated with cancer is characterized by the formation of a tumor (a solid mass of cells formed due to abnormal cell proliferation), but not always (especially in the case of blood cancers). In 2010, cancer was the single most common cause of death worldwide (about 8 million deaths) (Lozano et al., Lancet 380: 2095-2128, 2012). Furthermore, the rate of cancer is expected to increase with the aging of the global population. Thus, new and improved cancer treatments are urgently needed.

[0003] Moreover, many cancer deaths are due to cancers that have become resistant to chemotherapy drugs. The mechanisms by which cancers acquire drug resistance are reviewed in Housman et al. (Cancers 6: 1769-1792, 2014). As described in detail there, cancers can acquire drug resistance through a variety of mechanisms, including inactivating or metabolizing (or preventing metabolic activation of) drugs, mutating or modifying the drug target, and efflux of drugs via ABC transporters. Such mechanisms can lead to cancers becoming multidrug resistant (MDR). The development of resistance to drug-based therapies is a significant challenge in oncology today. Thus, new treatment options are needed for cancers that are inherently or have acquired resistance to conventional chemotherapy.

[0004] The present invention provides a new treatment option for cancer, specifically, a new treatment method that combines specific binding molecules (such as antibodies) against Annexin A1 (ANXA1) with certain specific partner drugs. As shown in the following examples, the combinations provided herein are particularly effective in treating cancer or certain types of cancer.

[0005] The amino acid sequence of full-length human ANXA1 is shown in SEQ ID NO: 17. ANXA1 is a member of the annexin protein family. Most of the proteins in this family, including ANXA1, are characterized by the presence of a "core" region that contains four homologous repeat domains. Each repeat domain binds at least one Ca 2+ It contains a binding site. Each member of the family is distinguished by a unique N-terminal region. ANXA1 is a monomeric amphipathic protein that is primarily located in the cytoplasm of cells in which it is expressed. However, ANXA1 can also be transported and localized to the cell surface (D'Acquisto et al., Br. J. Pharmacol. 155: 152-169, 2008).

[0006] ANXA1 is known to play a role in regulating the immune system. It is involved in maintaining the homeostasis of various cell types in both the innate and adaptive immune systems. For example, ANXA1 has been shown to regulate the homeostasis of cells of the innate immune system, such as neutrophils and macrophages, and also plays a role in T cells by regulating the strength of T cell receptor (TCR) signaling (D'Acquisto et al., Blood 109: 1095-1102, 2007). Inhibiting the role of ANXA1 in the adaptive immune system by using neutralizing antibodies against ANXA1 has been shown to be effective in treating various T cell-mediated diseases, including autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (WO2010 / 064012, WO2011 / 154705).

[0007] Antibodies against ANXA1 have also been shown to be useful in treating certain psychiatric conditions, particularly anxiety, obsessive-compulsive disorder (OCD) and related diseases (WO2013 / 088111), although the mechanism by which this occurs is unknown.

[0008] A number of monoclonal antibodies that recognize human ANXA1 are disclosed in WO2018 / 146230. As detailed in WO2020 / 030827, these antibodies were found to bind to human ANXA1 in a discontinuous epitope comprising amino acids 197-206, 220-224, and 227-237 (i.e., in an epitope comprising amino acids 197-206, 220-224, and 227-237 of SEQ ID NO: 17). The antibodies disclosed in WO2018 / 146230 have particularly advantageous properties in that they can bind to human ANXA1 with very high affinity. The antibodies disclosed in WO2018 / 146230 were subsequently shown to have potent anti-cancer activity (WO2020 / 030827). As shown in WO2020 / 030827, these antibodies demonstrated anti-proliferative effects against multiple cancer cell lines and also demonstrated therapeutic efficacy in a mouse model of triple-negative breast cancer.

[0009] The inventors have now found that combining treatment with the anti-ANXA1 antibodies disclosed in WO2018 / 146230 with certain other specific therapeutic agents results in an unexpectedly enhanced anti-cancer effect.

[0010] Summary of the Invention The present invention therefore provides in a first aspect a specific binding molecule that binds human ANXA1 and a second active agent for use in the treatment of cancer in a subject, comprising: (i) the specific binding molecule comprises complementarity determining regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3, each of said CDRs having the amino acid sequence VLCDR1 has the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8, or a modified sequence thereof containing conservative amino acid substitutions at positions 9 and / or 11; VLCDR2 has the sequence shown in SEQ ID NO:2, VLCDR3 has the sequence shown in SEQ ID NO:3, VHCDR1 has the sequence shown in SEQ ID NO:4, VHCDR2 has the sequence shown in SEQ ID NO:5, VHCDR3 has the sequence shown in SEQ ID NO:6, (ii) the second active agent is selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction of PD-1 with PD-L1, and a proteasome inhibitor. In other words, the present invention provides a specific binding molecule as defined herein for use in the treatment of cancer in a subject, wherein said specific binding molecule and a second active agent as defined herein are administered to said subject, i.e. wherein said treatment comprises a combination of said specific binding molecule and said second active agent.

[0011] In a second aspect, the present invention provides a specific binding molecule that binds human ANXA1 and a second active agent for use in treating breast cancer in a subject, said specific binding molecule being as defined above in relation to the first aspect, and said second active agent being selected from taxanes and platinum-based chemotherapeutic agents. In other words, the present invention provides a specific binding molecule as defined herein for use in treating breast cancer in a subject, wherein said specific binding molecule and a second active agent as defined herein are administered to said subject, i.e. said treatment is a combination of said specific binding molecule and said second active agent.

[0012] In a third aspect, the present invention provides a specific binding molecule that binds to human ANXA1 and a second active agent for use in treating pancreatic cancer in a subject, wherein the specific binding molecule is as defined above for the first aspect, and the second active agent is a nucleoside analogue.In other words, the present invention provides a specific binding molecule as defined herein for use in treating pancreatic cancer in a subject, wherein the treatment comprises administering the specific binding molecule and the second active agent as defined herein to the subject, i.e., the treatment comprises combining the specific binding molecule and the second active agent.

[0013] Relatedly, the invention provides in a fourth aspect a method of treating cancer in a subject comprising administering to the subject a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule is as defined above in relation to the first aspect and the second active agent is selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor.

[0014] In a fifth aspect, the present invention provides a method for treating breast cancer in a subject, comprising administering to the subject a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule is as defined above in relation to the first aspect, and the second active agent is selected from taxanes and platinum-based chemotherapeutic agents.

[0015] In a sixth aspect, the present invention provides a method for treating pancreatic cancer in a subject comprising administering to the subject a specific binding molecule that binds to human ANXA1 and a nucleoside analogue, wherein the specific binding molecule is as defined above in relation to the first aspect.

[0016] Relatedly, the present invention provides in a seventh aspect the use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating cancer, said specific binding molecule being as defined above in relation to the first aspect, and said treating cancer comprising administering to a subject the medicament and a second active agent, said second active agent being selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor.

[0017] In an eighth aspect, the present invention provides the use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating breast cancer, wherein the specific binding molecule is as defined above in relation to the first aspect, and the treatment of breast cancer comprises administering the medicament and a second active agent to a subject, and the second active agent is selected from taxanes and platinum-based chemotherapeutic agents.

[0018] In a ninth aspect, the present invention provides the use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating pancreatic cancer, wherein the specific binding molecule is as defined above in relation to the first aspect, and the treatment of pancreatic cancer comprises administering the medicament and a nucleoside analogue to a subject.

[0019] In a tenth aspect, the present invention provides a pharmaceutical composition comprising a specific binding molecule that binds to human ANXA1, a second active agent, and one or more pharma- ceutically acceptable diluents, carriers, or excipients, A pharmaceutical composition is provided, wherein said specific binding molecule and said second active agent are as defined above in relation to the first aspect.

[0020] In an eleventh aspect, the present invention provides a kit comprising a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule and the second active agent are as defined above in relation to the first aspect.

[0021] The present invention provides in a twelfth aspect a product comprising a specific binding molecule that binds to human ANXA1 as defined above in relation to the first aspect and a second active agent for separate, simultaneous or sequential use in the treatment of cancer in a subject, wherein said second active agent is selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor.

[0022] In a thirteenth aspect, the present invention provides a product comprising a specific binding molecule that binds to human ANXA1 as defined above in relation to the first aspect and a second active agent for separate, simultaneous or sequential use in the treatment of breast cancer in a subject, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents.

[0023] In a fourteenth aspect, the present invention provides a product comprising a specific binding molecule that binds to human ANXA1 and a nucleoside analogue as defined above in relation to the first aspect for separate, simultaneous or sequential use in the treatment of pancreatic cancer in a subject.

[0024] As discussed below, a third active agent may be used in each of the above aspects of the invention.

[0025] Description of the Invention The present invention provides new combinations that are effective in treating cancer. These combinations can help to increase the efficacy of the components compared to the use of each component separately. As an example, one of the components can enhance the effect of an agent that is otherwise less effective. This is particularly useful in treating drug-resistant cancers, for example, to provide new treatments and enhance the efficacy of agents to which cancers have acquired resistance. Furthermore, in view of the enhanced effect achieved with these combinations, the present invention allows the use of lower levels of the components (e.g., the second or third active agent). In a preferred embodiment, the combination exhibits a synergistic effect, i.e., an effect that is better than additive. In particular in such cases, it is possible to use less of one or both of the components (e.g., the second or third active agent) and to enhance the effect of one component (e.g., the second or third active agent) by using the combination. A specific binding molecule can be a component of a combination that enhances the activity of a second active agent (or a third active agent), and a second active agent (or a third active agent) can be a component of a combination that enhances the activity of a specific binding molecule.

[0026] As stated above, the present invention provides (in part) a specific binding molecule that binds to human ANXA1 for use in treating cancer (or a particular cancer) in a subject. A "specific binding molecule" as defined herein is a molecule that specifically binds to a specific molecular partner (in this case, human ANXA1). A molecule that specifically binds to human ANXA1 is a molecule that binds to human ANXA1 with a higher affinity than its binding affinity to other molecules, or at least most other molecules. Thus, for example, when a specific binding molecule that binds to human ANXA1 is contacted with a lysate of human cells, the specific binding molecule binds primarily to ANXA1. In particular, the specific binding molecule binds to a sequence or configuration present in human ANXA1. When the specific binding molecule is an antibody, said sequence or configuration is the epitope to which the specific binding molecule binds. The ANXA1 epitopes to which the specific binding molecules for use according to the present invention bind are detailed above.

[0027] A specific binding molecule for use as described herein may not necessarily bind only to human ANXA1, but may cross-react with some other undefined target molecules or may exhibit some degree of non-specific binding when contacted with a mixture of multiple molecules (e.g., cell lysates, etc.). For example, a specific binding molecule may exhibit some degree of cross-reactivity with other members of the human annexin family and / or with ANXA1 proteins from other animals. In any case, a specific binding molecule for use according to the present invention exhibits specificity for ANXA1. A person skilled in the art would be able to readily determine whether a specific binding molecule exhibits specificity for ANXA1 using standard methods in the art, such as ELISA, Western blot, surface plasmon resonance (SPR), etc. In certain embodiments, a specific binding molecule for use as described herein exhibits a K of less than 20 nM, less than 15 nM, or less than 10 nM. D (dissociation constant) of human ANXA1. In a preferred embodiment, the specific binding molecules for use herein have a K Dand binds to human ANXA1.

[0028] K for binding of specific binding molecules to ANXA1 D is preferably Ca 2+ The binding conditions are measured under conditions in which ions are present at a concentration of at least 1 mM, HEPES is optionally present at a concentration of 10-20 mM, and the pH is 7-8, preferably at physiological levels of 7.2 to 7.5. NaCl may be present, for example at a concentration of 100-250 mM, and a low concentration of a surfactant (e.g., polysorbate 20) may be present. Such a low concentration may be, for example, 0.01-0.5% v / v. The K of the interaction between a specific binding molecule and its ligand D There are many methods known in the art that can calculate . Known techniques include SPR (e.g., Biacore) and polarization-modulated oblique incidence reflectance difference (OI-RD).

[0029] As mentioned above, a molecule that "binds to human ANXA1" exhibits specificity for the human ANXA1 molecule. There are three isoforms of human ANXA1 that are derived by translation of four alternatively spliced ​​ANXA1·mRNAs. The full-length human ANXA1 protein is derived from translation of the ANXA1-002 or ANXA1-003 transcript, the amino acid sequence of which is shown in SEQ ID NO:17, as mentioned above. The ANXA1-004 and ANXA1-006 transcripts encode fragments of the full-length human ANXA1 protein, the amino acid sequences of which are shown in SEQ ID NO:18 and SEQ ID NO:19, respectively.

[0030] Specific binding molecules for use in accordance with the present invention bind to full-length human ANXA1 (i.e., ANXA1 of SEQ ID NO: 17, the 346 amino acid protein encoded by the ANXA1-002 or ANXA1-003 transcript). Specific binding molecules may also bind to specific fragments, portions, or variants of full-length ANXA1, such as the fragment encoded by the ANXA1-004 or ANXA1-006 transcript.

[0031] As discussed below, antibodies (and CDR-containing molecules) form suitable specific binding molecules for use in accordance with the present invention.

[0032] As mentioned above, a number of monoclonal antibodies that recognize human ANXA1 are disclosed in WO2018 / 146230. One antibody disclosed in WO2018 / 146230 has the following CDR sequence: VLCDR1: RSSQSLENSNAKTYLN (SEQ ID NO: 1), VLCDR2: GVSNRFS (SEQ ID NO: 2), VLCDR3: LQVTHVPYT (SEQ ID NO: 3), VHCDR1: GYTFTNYWIG (SEQ ID NO: 4), VHCDR2: DIYPGGDYTNYNEKFKG (SEQ ID NO: 5), and VHCDR3:ARWGLGYYFDY (sequence number 6). Another antibody disclosed in WO2018 / 146230 has the following CDR sequences: VLCDR1: RSSQSLENSNGKTYLN (SEQ ID NO: 7), VLCDR2: GVSNRFS (SEQ ID NO: 2), VLCDR3: LQVTHVPYT (SEQ ID NO: 3), VHCDR1: GYTFTNYWIG (SEQ ID NO: 4), VHCDR2: DIYPGGDYTNYNEKFKG (SEQ ID NO: 5), and VHCDR3:ARWGLGYYFDY (sequence number 6). Another antibody disclosed in WO2018 / 146230 has the following CDR sequences: VLCDR1: RSSQSLENTNGKTYLN (SEQ ID NO: 8), VLCDR2: GVSNRFS (SEQ ID NO: 2), VLCDR3: LQVTHVPYT (SEQ ID NO: 3), VHCDR1: GYTFTNYWIG (SEQ ID NO: 4), VHCDR2: DIYPGGDYTNYNEKFKG (SEQ ID NO: 5), and VHCDR3:ARWGLGYYFDY (sequence number 6). (Following standard nomenclature, VLCDR1, VLCDR2, and VLCDR3 represent CDR1, CDR2, and CDR3, respectively, of the antibody light chain, and VHCDR1, VHCDR2, and VHCDR3 represent CDR1, CDR2, and CDR3, respectively, of the antibody heavy chain.)

[0033] Thus, the CDR sequences of the antibodies disclosed in WO2018 / 146230 are identical, except for the VLCDR1 sequence, which is the wild-type VLCDR1 sequence found in the murine antibody MDX-001, constructed from minor mRNA sequences obtained from the hybridoma deposited at the European Collection of Cell Cultures (ECACC) under accession number 10060301.

[0034] MDX-001 antibody was humanized and surprisingly found that modifying the VLCDR1 sequence in these humanized antibodies resulted in enhanced antibodies. Substitution of the glycine residue at position 11 of SEQ ID NO:7 improves the stability and function of the antibody. Without being bound by theory, it is believed that this is achieved by removing a post-translational modification site in the CDR. Specifically, it is believed that substitution of this glycine residue removes a deamidation site from the protein. The VLCDR1 sequence shown in SEQ ID NO:7 contains a sequence motif of Ser-Asn-Gly. This sequence motif is associated with deamidation of Asn residues, converting asparagine residues to aspartic acid or isoaspartic acid, which may affect antibody stability and target binding. Substitution of any one residue within the Ser-Asn-Gly motif is believed to remove the deamidation site.

[0035] As described in detail in WO2018 / 146230, an antibody in which the glycine residue at position 11 of SEQ ID NO:7 (the glycine residue located within the deamidation site described above) is substituted with alanine has improved binding to the target (ANXA1) compared to the native MDX-001 antibody. The amino acid sequence of VLCDR1 in which the glycine at position 11 is substituted with alanine is: [ka] (The residues in bold are the alanines introduced by the substitutions described above), which is the sequence shown in SEQ ID NO:1. Furthermore, a humanized antibody comprising a VLCDR1 modified at position 9 by a serine to threonine substitution was also found to have improved binding to ANXA1 compared to MDX-001. The amino acid sequence of VLCDR1 with a serine to threonine substitution at position 9 is: [ka] (the residue in bold is the threonine introduced by the substitution above), which is the sequence shown in SEQ ID NO:8.

[0036] A specific binding molecule for use according to the present invention comprises the CDR sequence of any of the above three antibodies disclosed in WO2018 / 146230, or a particular variant thereof. In particular, as described above, the VLCDR1 of each antibody disclosed in WO2018 / 146230 has been found to tolerate at least conservative amino acid substitutions at positions 9 and 11 of the VLCDR1. Thus, a specific binding molecule for use according to the present invention comprises a CDR having the following amino acid sequence: VLCDR1 has the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8, or a modified sequence thereof containing conservative amino acid substitutions at positions 9 and / or 11; VLCDR2 has the sequence shown in SEQ ID NO:2, VLCDR3 has the sequence shown in SEQ ID NO:3, VHCDR1 has the sequence shown in SEQ ID NO:4, VHCDR2 has the sequence shown in SEQ ID NO:5, and VHCDR3 has the sequence shown in SEQ ID NO:6.

[0037] The term "conservative amino acid substitution" as used herein refers to an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Since amino acids with similar side chains tend to have similar properties, conservative substitution of an amino acid important in the structure or function of a polypeptide can be expected to have less effect on the structure / function of the polypeptide than non-conservative amino acid substitution at the same position. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine, etc.), acidic side chains (e.g., aspartic acid, glutamic acid, etc.), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, etc.), non-polar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine, etc.). Thus, conservative amino acid substitutions may be considered as substitutions that replace a particular amino acid residue with another amino acid from the same family.

[0038] Thus, in a particular embodiment, a specific binding molecule for use according to the invention comprises a VLCDR1 which is a variant of SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8 comprising a conservative amino acid substitution at position 9 relative to the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8. In another embodiment, a specific binding molecule for use according to the invention comprises a VLCDR1 which is a variant of SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8 comprising a conservative amino acid substitution at position 11 relative to the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8. In another embodiment, a specific binding molecule for use according to the invention comprises a VLCDR1 which is a variant of SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8 comprising a conservative amino acid substitution at both positions 9 and 11 relative to the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8.

[0039] In a preferred embodiment, a) a conservative amino acid substitution at position 9 relative to SEQ ID NO:7 or the sequence shown in SEQ ID NO:1 (when the amino acid at that position is serine) is asparagine, glutamine, threonine, or tyrosine; b) a conservative amino acid substitution at position 9 relative to the sequence set forth in SEQ ID NO:8 (when the amino acid at that position is threonine) is asparagine, glutamine, serine, or tyrosine; c) a conservative amino acid substitution at position 11 relative to the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 (when the amino acid at that position is glycine) is cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; d) A conservative amino acid substitution at position 11 relative to the sequence set forth in SEQ ID NO:1 (when the amino acid at that position is alanine) is glycine, cysteine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan.

[0040] In a preferred embodiment, a specific binding molecule for use according to the present invention comprises a CDR having the following amino acid sequence: VLCDR1 has the sequence shown in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8, VLCDR2 has the sequence shown in SEQ ID NO:2, VLCDR3 has the sequence shown in SEQ ID NO:3, VHCDR1 has the sequence shown in SEQ ID NO:4, VHCDR2 has the sequence shown in SEQ ID NO:5, and VHCDR3 has the sequence shown in SEQ ID NO:6.

[0041] Most preferably, the specific binding molecule for use in accordance with the present invention comprises a CDR having the following amino acid sequence: VLCDR1 has the sequence shown in SEQ ID NO:1, VLCDR2 has the sequence shown in SEQ ID NO:2, VLCDR3 has the sequence shown in SEQ ID NO:3, VHCDR1 has the sequence shown in SEQ ID NO:4, VHCDR2 has the sequence shown in SEQ ID NO:5, and VHCDR3 has the sequence shown in SEQ ID NO:6.

[0042] As mentioned above, the specific binding molecule for use according to the present invention comprises six CDRs consisting of a polypeptide sequence. As used herein, "protein" and "polypeptide" are interchangeable and both refer to a sequence consisting of two or more amino acids linked by one or more peptide bonds. Thus, the specific binding molecule may be a polypeptide. Alternatively, the specific binding molecule may comprise one or more polypeptides comprising the CDR sequences. Preferably, the specific binding molecule for use according to the present invention is an antibody or an antibody fragment.

[0043] The amino acids that make up the sequence of the CDR may include amino acids that are not naturally occurring amino acids but are modified versions of naturally occurring amino acids. These non-naturally occurring amino acids may be used to generate the CDRs described herein without reducing sequence identity, provided that they do not change the sequence or affect the specificity, i.e., are considered to provide the amino acids of the CDR. For example, amino acid derivatives such as methylated amino acids may be used. In one embodiment, the specific binding molecules for use according to the invention are not naturally occurring molecules, i.e., are not molecules found in nature.

[0044] The specific binding molecules for use according to the invention may be synthesized by any method known in the art. In particular, the specific binding molecules may be synthesized using a protein expression system, such as a cell expression system using prokaryotic cells (e.g., bacterial cells) or eukaryotic cells (e.g., yeast cells, fungal cells, insect cells, or mammalian cells). Another protein expression system includes an in vitro cell-free expression system, in which a base sequence encoding the specific binding molecule is transcribed into mRNA in vitro, and the mRNA is translated into a protein. Kits for cell-free expression systems are widely available, for example, from Thermo Fisher Scientific (USA). Alternatively, the specific binding molecules may be chemically synthesized in a non-biological system. Liquid or solid phase synthesis may be used to generate polypeptides that may form or be contained in the specific binding molecules for use according to the invention. A person skilled in the art can easily produce the specific binding molecules using appropriate techniques common in the art. In particular, the specific binding molecules may be expressed recombinantly in mammalian cells, such as CHO cells.

[0045] The specific binding molecule for use according to the present invention may be isolated (i.e., purified) if necessary. In this specification, "isolated" means that the specific binding molecule is the major component (i.e., the majority of the components) of the solution or the like containing it. In particular, when the specific binding molecule is initially produced in a mixture or mixed solution, the isolation of the specific binding molecule means that it has been separated or purified therefrom. Thus, for example, when the specific binding molecule is a polypeptide and the polypeptide is produced using a protein expression system as described above, the specific binding molecule is isolated so that it is the most abundant polypeptide in the solution or composition in which it is present, preferably so that it constitutes the majority of the polypeptides in the solution or composition, and is enriched over other polypeptides and biomolecules present in the original production medium. In particular, the specific binding molecule for use according to the present invention is isolated so that it is the major (majority) specific binding molecule in the solution or composition. In preferred aspects, the specific binding molecule is present in the solution or composition with a purity of at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or at least 99% w / w, when assessed relative to the abundance of other components in the solution or composition, particularly other polypeptide components.

[0046] When the specific binding molecule is, for example, a protein produced in a protein expression system, the solution of the specific binding molecule may be analyzed by quantitative proteomics to confirm whether the specific binding molecule for use according to the present invention is the most predominant and therefore isolated.For example, 2D gel electrophoresis and / or mass spectrometry may be used.Such isolated molecules may be present in a formulation or composition as described below.

[0047] The specific binding molecules of the present invention may be isolated using any method known in the art. For example, the specific binding molecules may be produced with affinity tags, such as polyhistidine tags, strep tags, FLAG tags, or HA tags, so that the molecules can be isolated by affinity chromatography using appropriate binding partners. For example, polyhistidine-tagged molecules can be isolated by affinity chromatography using Ni 2+ ions. In embodiments where the specific binding molecule is an antibody, the specific binding molecule may be isolated by affinity chromatography using one or more antibody binding proteins, such as Protein G, Protein A, Protein A / G, or Protein L. Alternatively, the specific binding molecule may be isolated, for example, by size exclusion or ion exchange chromatography. In contrast, specific binding molecules produced by chemical synthesis (i.e., non-biological methods) are more likely to be produced in isolated form. Thus, when a specific binding molecule for use according to the invention is synthesized by a method that produces an isolated molecule, no specific purification or isolation step is required for the specific binding molecule to be considered isolated.

[0048] Modifications to the amino acid sequences of the CDRs shown in SEQ ID NOs: 1-8 may be made using any suitable technique, such as site-directed mutagenesis of the encoding DNA sequence or solid phase synthesis.

[0049] The specific binding molecules for use according to the invention may contain, in addition to the CDRs described above, linker moieties or framework sequences that allow for the proper presentation of the CDRs. Additional sequences may be present that may conveniently confer additional properties, such as peptide sequences that allow for the isolation or identification of molecules that contain the CDRs as described above. In such cases, fusion proteins may be produced.

[0050] As mentioned above, the specific binding molecule for use in the present invention is preferably an antibody or an antibody fragment. The term "antibody" as used herein refers to antibodies that contain all the characteristics of natural immunoglobulins (as known in the art, see, for example, the description in WO2020 / 030827, which is incorporated herein by reference), as well as naturally occurring antibody variants (or variants that contain all the characteristics of natural immunoglobulins) that retain the CDRs but are presented in a different framework and function similarly, i.e., retain specificity for the antigen, as described below. Thus, antibodies include functional equivalents or functional homologs in which the naturally occurring domains are partially or entirely replaced by similarly functioning natural or non-natural equivalents or homologs.

[0051] When the specific binding molecule for use according to the invention is an antibody, it is preferably a monoclonal antibody. By "monoclonal antibody" is meant an antibody preparation consisting of a single antibody species. That is, all of the antibodies in the preparation have identical amino acid sequences, contain the same CDRs, and therefore bind to the same epitope on the target antigen ("target antigen" means an antigen that contains the epitope to which a particular antibody binds, i.e., the target antigen of an anti-Anx-A1 antibody is Anx-A1) to exert the same effect. In other words, the antibody for use according to the invention is preferably not part of a polyclonal antibody mixture.

[0052] As is well known in the art, in antibodies, CDR sequences are located in the variable domains of the heavy and light chains. The CDR sequences are present within the framework of the polypeptide, thereby positioning the CDRs appropriately for antigen binding. Thus, the remainder of the variable domain (i.e., the part of the variable domain sequence that does not form part of any CDR) constitutes the framework region. The mature variable domain N-terminus forms framework region 1 (FR1), the polypeptide sequence between CDR1 and CDR2 forms FR2, the polypeptide sequence between CDR2 and CDR3 forms FR3, and the polypeptide sequence linking CDR3 to the constant domain forms FR4. In an antibody or fragment thereof for use according to the invention, the amino acid sequence of the framework region of the variable region may be any suitable amino acid sequence such that the antibody or fragment thereof binds to human ANXA1 via its CDR. The constant region may be the constant region of any mammalian, preferably human, antibody isotype.

[0053] In certain embodiments of the invention, the specific binding molecule may be a multispecific monoclonal antibody, such as a bispecific monoclonal antibody. A multispecific binding molecule comprises regions or domains (antigen binding regions) that bind to at least two different molecular binding partners, e.g., bind to two or more different antigens or epitopes. In the case of a bispecific antibody, the antibody comprises two heavy chains and two light chains in a standard configuration, except that the variable domains of each of the two heavy chains and the two light chains are different, thereby forming two different antigen binding regions. In a multispecific (e.g., bispecific) binding molecule for use according to the invention, such as a multispecific monoclonal antibody, one of the antigen binding regions has the CDR sequence of a specific binding molecule for use according to the invention as defined herein, and thus binds to ANXA1. The other antigen binding region of the multispecific binding molecule for use according to the invention is different from the antigen binding region formed by the CDRs for use according to the invention, e.g., has a CDR sequence different from that defined herein for the specific binding molecule for use according to the invention. For example, in a bispecific antibody, the further (e.g., second) antigen-binding region of the specific binding molecule may bind to ANXA1, but at a different epitope than the first antigen-binding region (having the CDRs of the specific binding molecule for use according to the invention) that binds to ANXA1. Alternatively, the further (e.g., second) antigen-binding region may bind to a further (e.g., second) different antigen that is not ANXA1. In another embodiment, two or more antigen-binding regions in a specific binding molecule, such as an antibody, may each bind to the same antigen, i.e., providing a multivalent (e.g., bivalent) molecule.

[0054] The specific binding molecule may be an antibody fragment or a synthetic construct capable of binding to human ANXA1. Thus, an antibody fragment for use according to the invention comprises an antigen-binding domain (i.e., the antigen-binding domain of the antibody from which the antibody fragment originates), i.e., an antigen-binding fragment of an antibody. Antibody fragments are described in Rodrigo et al., Antibodies, Vol. 4(3), p. 259-277, 2015. Antibody fragments for use according to the invention are preferably monoclonal (i.e., not part of a polyclonal antibody fragment mixture). Antibody fragments include, for example, Fab fragments, F(ab')2 fragments, Fab' fragments, and Fv fragments. Fab fragments are described in Roitt et al., Immunology second edition (1989), Churchill Livingstone, London. Fab fragments consist of the antigen-binding domain of an antibody. That is, an individual antibody may be considered to contain two Fab fragments, each consisting of a light chain and the N-terminal portion of a heavy chain bound to it. Thus, a Fab fragment contains the entire light chain and the V of the heavy chain to which it binds. H Domain and C H 1 domain. The Fab fragment can be obtained by digesting an antibody with papain.

[0055] The F(ab')2 fragment consists of two Fab fragments of an antibody and the hinge region of the heavy domain, with a disulfide bond linking the two heavy chains. In other words, the F(ab')2 fragment can be considered as two Fab fragments covalently linked together. The F(ab')2 fragment can be obtained by digesting an antibody with pepsin. The F(ab')2 fragment can be reduced to obtain two Fab' fragments. These can be considered as Fab fragments with an additional sulfhydryl group that can serve to bind the fragment to other molecules.

[0056] Fv fragments consist of only the variable domains of the light and heavy chains. They are not covalently linked, but are only weakly held together by non-covalent interactions. Fv fragments can be modified to produce synthetic constructs known as single-chain Fv (scFv) molecules. Typically, such modifications are performed by engineering antibody genes such that a single polypeptide is expressed as a V H Domains and V L The scFv fragment can be produced recombinantly by producing a fusion protein containing both the V and V domains. H Area and V L The peptide linker may be comprised of 1-20 amino acids, for example 1, 2, 3, or 4 amino acids, or 5, 10, or 15 amino acids, or any other number conveniently in the range of 1-20. The peptide linker may be formed from commonly convenient amino acid residues such as glycine and / or serine. An example of a suitable linker is Gly4Ser. Multimers of such linkers may be used, such as dimers, trimers, tetramers, or pentamers ((Gly4Ser)2, (Gly4Ser)3, (Gly4Ser)4, or (Gly4Ser)5, etc.). However, the presence of a linker is not essential, and V may be used as a linker. L The domains are bounded by peptide bonds. H In the present specification, an scFv is defined as an antibody fragment.

[0057] The specific binding molecule may be an analog of an scFv. For example, scFvs may be linked to other specific binding molecules (e.g., other scFvs, Fab antibody fragments, and chimeric IgG antibodies (e.g., with human frameworks)). scFvs may be linked to other scFvs to form multimers that are multispecific binding proteins, such as dimers, trimers, or tetramers. Bispecific scFvs may be referred to as diabodies, trispecific scFvs as triabodies, and tetraspecific scFvs as tetrabodies. In other embodiments, scFvs for use according to the invention may be linked to other identical scFv molecules, thereby forming monospecific but multivalent multimers, such as bivalent dimers or trivalent trimers.

[0058] Synthetic constructs that can be used include CDR peptides. These are synthetic peptides that contain antigen-binding determinants. Peptide mimetics can also be used. These molecules are usually conformationally restricted organic rings that mimic the CDR loop structure and have side chains that interact with the antigen.

[0059] As noted above, specific binding molecules for use according to the invention comprise the amino acid sequence set out in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:8 (or variants thereof) and CDRs having the amino acid sequences set out in SEQ ID NOs:2-6. As detailed below, these are derived or modified from the murine antibody MDX-001. However, it is preferred that antibodies or fragments thereof for use according to the invention are humanized.

[0060] The antibodies or antibody fragments for use according to the invention may be human / mouse chimeric antibodies, preferably humanized. This is particularly the case for monoclonal antibodies and antibody fragments thereof. If the molecule is to be used as a human therapeutic, a humanized or chimeric antibody or antibody fragment thereof is desirable. Therapeutic treatment of humans with non-human antibodies (e.g., mouse antibodies) may be ineffective for several reasons, including the short half-life of antibodies in vivo, the low recognition rate of non-human heavy chain constant regions by Fc receptors on human immune effector cells, resulting in weak effector functions mediated by the xenogeneic heavy chain constant regions, sensitization of patients to the antibodies and (for mouse antibodies) the development of a human anti-mouse antibody (HAMA) response, and neutralization of mouse antibodies by HAMA, resulting in loss of therapeutic efficacy.

[0061] A chimeric antibody is an antibody that has a variable region derived from one species and a constant region derived from another species. Thus, an antibody or antibody fragment for use according to the invention may be a chimeric antibody or chimeric antibody fragment comprising a murine variable domain and a human constant domain.

[0062] Antibodies for use according to the invention, including chimeric antibodies, may have a constant region of any antibody isotype (in particular any human antibody isotype) and may have a constant region of any subclass within each isotype. For example, the antibody may be of isotype IgA, IgD, IgE, IgG, or IgM (i.e., a chimeric antibody may contain the heavy chain α, δ, ε, γ, or μ constant domains), but preferably, the antibody for use according to the invention is of the IgG isotype. The light chain of an antibody (e.g., a chimeric antibody) for use according to the invention may be a κ light chain or a λ light chain, and in particular may contain the constant region of a human λ light chain or may contain the constant region of a human κ light chain. Similarly, a chimeric antibody fragment is an antibody fragment that contains a constant domain (e.g., a Fab fragment, a Fab' fragment, or a F(ab')2 fragment). The constant domain of a chimeric antibody fragment for use according to the invention may be a constant domain as described above for chimeric monoclonal antibodies.

[0063] Chimeric antibodies may be produced using any suitable method, such as recombinant DNA techniques, in which a DNA sequence for a murine variable domain is fused to a DNA sequence for a human constant domain to encode the chimeric antibody. Chimeric antibody fragments may be obtained by using recombinant DNA techniques to produce DNA sequences encoding such polypeptides, or by processing a chimeric antibody for use according to the invention to produce the desired fragments as described above. Chimeric antibodies are expected to overcome the short in vivo half-life and weak effector function problems associated with the use of heterologous antibodies, such as murine antibodies, in human therapy, and may reduce the likelihood of sensitization and HAMA in patients. However, due to the presence of murine sequences in the variable domains, sensitization and HAMA in patients may still occur when chimeric antibodies are administered to human patients.

[0064] Therefore, the antibodies or antibody fragments for use according to the invention are preferably fully humanized. Humanized antibodies are antibodies derived from other species, such as mice, in which the constant domains of the antibody chains have been replaced with human constant domains, and the amino acid sequences of the variable regions have also been modified to replace the heterologous (e.g. mouse) framework sequences with human framework sequences, such that the only non-human sequences in the antibody are preferably CDR sequences. Humanized antibodies overcome all the problems associated with the therapeutic use of non-human antibodies in humans, including avoiding or minimizing the possibility of patient sensitization and HAMA.

[0065] Usually, antibody humanization is performed by a process known as CDR grafting, although other methods in the art may be used. Antibody grafting is well described in Williams, DG et al., Antibody Engineering, Vol. 1, eds. R. Kontermann and S. Dubel, Chapter 21, pp. 319-339. In this process, a chimeric antibody is first generated, as described above. Thus, in antibody humanization, the non-human constant domain is first replaced with a human constant domain, resulting in a chimeric antibody comprising a human constant domain and a non-human variable domain.

[0066] The subsequent humanization of the heterologous (e.g., mouse) variable domains involves inserting the mouse CDRs from each immunoglobulin chain into the FRs of the most suitable human variable domain. This is done by aligning the mouse variable domains with a database of known human variable domains (e.g., IMGT or Kabat). Suitable human framework regions are identified from the most well-aligned variable domains, such as those with high sequence identity between the human and mouse framework regions, those with the same length of CDRs, and those with the most similar structure (based on homology modeling). The mouse CDR sequences are then grafted into the appropriate positions of the first human framework sequences using recombinant DNA technology, and then humanized antibodies are produced and tested for binding to the target antigen. Those skilled in the art know and understand the antibody humanization process and can perform this method without further instruction. Antibody humanization services are also provided by many commercial companies, such as GenScript (USA / China) and MRC Technology (UK). Humanized antibody fragments can be readily derived from humanized antibodies, as described above.

[0067] Thus, an antibody or antibody fragment for use according to the invention may be from any species, for example a murine antibody or antibody fragment. However, it is preferred that the antibody or antibody fragment is a chimeric antibody or antibody fragment thereof, i.e. only the variable domains of the antibody or antibody fragment are of non-human origin, and all the constant domains are of human origin. Optimally, the antibody or antibody fragment for use according to the invention is a humanized antibody or antibody fragment thereof.

[0068] A humanized version of MDX-001 has been developed by the present inventors as detailed in WO2018 / 146230. A humanized light chain variable domain has been developed comprising the amino acid sequence set forth in SEQ ID NO:9 (known as the L1M2 variable region) and the amino acid sequence set forth in SEQ ID NO:10 (known as the L2M2 variable region), and the CDRs as described above. In certain embodiments, an antibody or fragment thereof for use according to the present invention comprises a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10, or an amino acid sequence having at least 70% sequence identity thereto (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and wherein the CDR sequences VLCDR1-3 are as defined above.

[0069] Humanized heavy chain variable domains have been developed with the amino acid sequence set forth in SEQ ID NO: 11 (known as the H4 variable region) and the amino acid sequence set forth in SEQ ID NO: 12 (known as the H2 variable region). In certain embodiments, an antibody or fragment thereof for use according to the invention comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence which has at least 70% sequence identity thereto (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and wherein the CDR sequences are as defined above.

[0070] Preferably, the antibody or fragment thereof for use according to the present invention comprises: (i) a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to said amino acid sequence, and wherein the CDR sequences VLCDR1 to 3 are as defined above; and (ii) a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, or an amino acid sequence having at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto, and wherein the CDR sequences are as defined above.

[0071] In a particular embodiment, the specific binding molecule for use according to the invention is a monoclonal antibody of the IgG1 isotype and comprises a light chain of the Kappa subtype. The L1M2 light chain is of the Kappa subtype and has the amino acid sequence shown in SEQ ID NO: 13. The H4 heavy chain has the amino acid sequence shown in SEQ ID NO: 14. In a particular embodiment, the specific binding molecule for use according to the invention is an L1M2H4 antibody comprising an L1M2 light chain and an H4 heavy chain (this antibody is also referred to as MDX-124). Thus, the specific binding molecule for use according to the invention may be a monoclonal antibody comprising or consisting of: i) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to said amino acid sequence, and wherein the CDR sequences VLCDR1 to 3 are as defined above, and ii) A heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence which has at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to this amino acid sequence, and wherein the CDR sequences VH CDR1-3 are as defined above.

[0072] Similarly, the L2M2 light chain is a kappa subtype light chain and has the amino acid sequence set forth in SEQ ID NO: 15. The H2 heavy chain has the amino acid sequence set forth in SEQ ID NO: 16. In a particular embodiment, a specific binding molecule for use according to the invention is an L2M2H2 antibody comprising an L2M2 light chain and an H2 heavy chain (this antibody is also referred to as MDX-222). Thus, a specific binding molecule for use according to the invention may be a monoclonal antibody comprising: i) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to said amino acid sequence, and wherein the CDR sequences VLCDR1 to 3 are as defined above, and ii) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence which has at least 70% (preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to this amino acid sequence, and wherein the CDR sequences VH CDR1 to VH CDR3 are as defined above.

[0073] In another embodiment, an L1M2 light chain may pair with an H2 heavy chain, and an L2M2 light chain may pair with an H4 heavy chain.

[0074] As is known to those skilled in the art, antibody chains are naturally produced with a signal sequence. The signal sequence of an antibody is an amino acid sequence located at the N-terminus of the light and heavy chains, i.e., the N-terminus of the variable region. The signal sequence allows the antibody chain to be transported from the cell in which it is produced. When produced in a cell expression system, the light and heavy chains having the amino acid sequences of SEQ ID NOs: 13 to 16 may be encoded with a signal sequence. The signal sequences of the L1M2 light chain and the L2M2 light chain are shown in SEQ ID NO: 20, and the signal sequences of the H2 heavy chain and the H4 heavy chain are shown in SEQ ID NO: 21. Thus, when synthesized with a signal sequence, the L1M2 chain may be synthesized with the amino acid sequence shown in SEQ ID NO: 22, the H4 chain may be synthesized with the amino acid sequence shown in SEQ ID NO: 23, the L2M2 chain may be synthesized with the amino acid sequence shown in SEQ ID NO: 24, and the H2 chain may be synthesized with the amino acid sequence shown in SEQ ID NO: 25. A nucleotide sequence encoding such a sequence can be easily derived by a person skilled in the art, but examples of sequences encoding the antibody chains of SEQ ID NOs: 22 to 25 and suitable for use in their synthesis include the nucleotide sequences shown in SEQ ID NOs: 26 to 29, respectively.

[0075] Sequence identity may be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs for pairwise or multiple alignment of sequences are useful, for example EMBOSS Needle or EMBOSS stretcher (both Rice P. et al., Trends Genet. 16, (6) pp. 276-277, 2000) may be used for pairwise sequence alignment, and Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. 32(5):1792-1797, 2004) may be used for multiple sequence alignment, although other suitable programs may be used. Whether pairwise or multiple alignment, it must be performed globally (i.e., across the entire reference sequence) rather than locally.

[0076] Sequence alignments and percent identity calculations may be performed, for example, using standard parameters for Clustal Omega, i.e., the matrix is ​​Gonnet, the gap opening penalty is 6, and the gap extension penalty is 1. Alternatively, standard parameters for EMBOSS needle may be used, i.e., the matrix is ​​BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5. Other suitable parameters may be used instead.

[0077] In the present application, in case of discrepancies between sequence identity values ​​obtained with different methods, the value obtained by performing a global pairwise alignment using the EMBOSS needle with default parameters shall be considered valid.

[0078] As mentioned above, the present invention provides specific binding molecules (as defined above) for use in combination with various second active agents, both in the treatment of cancers in general and in the treatment of various specific cancers. In some cases, additional active agents, such as a third active agent as described below, may be used. Alternatively, the treatment may be performed without a third active agent, and in particular, with only the specific binding molecule and the second active agent. The specific binding molecule and the second active agent (and optionally the third active agent, if present) act as active therapeutic agents. Any type of cancer may be treated according to the present invention, such as carcinoma (including adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, etc.), sarcoma, leukemia, lymphoma, etc. Cancers that may be treated include melanoma, lung cancer, colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, skin cancer, lymphoma (particularly Hodgkin's lymphoma or mantle cell myeloma), bladder cancer, kidney cancer, mesothelioma, liver cancer, and myeloma.

[0079] According to the present invention, any stage (i.e., grade) of cancer may be treated, such as stage I, stage II, stage III, and stage IV cancer. Both metastatic and localized (i.e., non-metastatic) cancers may be treated. In a preferred embodiment, the cancer treated by the present invention is drug resistant, for example multi-drug resistant (MDR). Drug resistant cancer refers to a cancer that is resistant to one chemotherapeutic drug. The drug to which the cancer is resistant may be a second or third active drug. MDR cancer refers to a cancer that is resistant to multiple chemotherapeutic drugs, particularly to multiple families of chemotherapeutic drugs. MDR cancer may be resistant to two, three, four, five or more different chemotherapeutic drugs or families (classes) of chemotherapeutic drugs. The term "MDR cancer" is well known in the art and is used in this context according to its meaning in the art. MDR cancer may be resistant to all known chemotherapeutic drugs. Multidrug resistance may be mediated by expression of one or more of the ABC transporters multidrug resistance protein (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), all three of which have broad substrate specificity and can efflux many different classes of chemotherapeutic agents from cells that express them.

[0080] In a first aspect of the invention, provided herein is a specific binding molecule as defined above and a second active agent for use in treating cancer in a subject, wherein the second active agent is a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, or a proteasome inhibitor. That is, the invention provides a specific binding molecule as defined above in combination with a second active agent for the treatment of cancer, wherein the second active agent is a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, or a proteasome inhibitor.

[0081] The nucleobase analogue may be any nucleobase analogue suitable for use in cancer treatment.As used herein, a nucleobase analogue is a compound that can replace a natural nucleobase in a nucleic acid molecule, for example, a compound that can base pair with the same partner base as the parent nucleobase of which it is an analogue.The nucleobase analogue may be any analogue of cytosine, guanine, adenine, thymine, or uracil that has a cytotoxic effect on cancer cells and / or is suitable for use in chemotherapy.In certain embodiments, the nucleobase analogue is a pyrimidine analogue, preferably a uracil analogue.5-fluorouracil is a nucleobase analogue chemotherapeutic agent that can be used according to the present invention.

[0082] Thymidylate synthase inhibitors inhibit the enzyme thymidylate synthase. Thymidylate synthase catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a nucleotide used in DNA synthesis. Thus, inhibition of thymidylate synthase inhibits dTMP production and DNA synthesis. Thus, thymidylate synthase inhibitors are drugs that inhibit the production of dTMP by thymidylate synthase. Such inhibitors may have any mode of action, for example, competitive or non-competitive. Any thymidylate synthase inhibitor that is suitable for use in chemotherapy can be used according to the present invention. Several thymidylate synthase inhibitors are known in the art (for example, the above-mentioned nucleobase analog 5FU is a thymidylate synthase inhibitor). Thymidylate synthase inhibitors can also be identified using known techniques for measuring thymidylate synthase activity, such as the tritium-labeled 5-fluoro-dUMP binding assay (see, e.g., Takezawa et al., British Journal of Cancer 103:354-361, 2010).

[0083] Most preferably, the nucleobase analog or thymidylate synthase inhibitor is 5-fluorouracil (5FU), the structure of which is shown in Formula I below. Formula I (5-Fluorouracil) [ka]

[0084] Another exemplary thymidylate synthase inhibitor that can be used according to the present invention is capecitabine, which is converted to 5FU in the body (i.e., is a 5FU prodrug) and therefore has the same mechanism of action as 5FU. The structure of capecitabine is shown below in Formula II. Formula II (Capecitabine) [ka]

[0085] When the specific binding molecule is used in combination with a nucleobase analog or a thymidylate synthase inhibitor (e.g., 5FU), the agent can be used to treat any cancer. For example, the combination can be used to treat colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, or skin cancer. In a preferred embodiment, the combination is used to treat pancreatic cancer or colon cancer. That is, in a preferred embodiment, the present invention provides a specific binding molecule as defined above and 5FU for use in treating pancreatic cancer or colon cancer. In another preferred embodiment, the present invention provides a specific binding molecule as defined above and capecitabine for use in treating pancreatic cancer.

[0086] Thus, in one preferred embodiment, the present invention provides a specific binding molecule as defined above and 5FU for use in the treatment of pancreatic cancer. In another preferred embodiment, the present invention provides a specific binding molecule as defined above and 5FU for use in the treatment of colon cancer.

[0087] The pancreatic cancer treated according to the present invention can be any pancreatic cancer. In certain embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0088] As shown in the Examples below, when used to treat pancreatic cancer cell lines in vitro, the combination of a specific binding molecule for use according to the invention with 5FU exhibited significant synergy in the anti-proliferative effect on the cell lines, demonstrating the unexpected advantage of combining these two agents for cancer treatment.

[0089] Checkpoint inhibitors are molecules that block the activity of immune checkpoints. These inhibitors are applied as anti-cancer drugs by activating the patient's immune system to attack cancer cells. Immune checkpoints suppress the immune system by preventing the death of healthy cells and autoimmunity. Immune checkpoints act as a "brake" on the immune system by preventing T cell activation. Checkpoint proteins are expressed on the surface of immune cells and bind to checkpoint ligands on the surface of target cells or antigen-presenting cells. As a result, immune cell activity is suppressed.

[0090] PD-1 (programmed cell death protein 1) is an example of an immune checkpoint. PD-1 is expressed by T cells and binds to PD-L1 (programmed death ligand 1) and PD-L2 expressed on the surface of cells such as target cells, lymphocytes, and antigen-presenting cells. Activation of PD-1 by binding to PD-L1 or PD-L2 inhibits T cell activation and proliferation. Thus, upregulation of PD-L1 and / or PD-L2 by cancer cells serves as a defense mechanism to prevent the destruction of cancer cells by T cells. Upregulation of PD-L1 and / or PD-L2 by healthy cells in the vicinity of the tumor exerts a similar dampening effect on the immune response.

[0091] The inventors have found that the combination of a specific binding molecule as defined herein with a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 provides unexpected beneficial effects in the treatment of cancer cells. A checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 may be a molecule that blocks the interaction to inhibit PD-1 and prevent its activation, thereby preventing downregulation of the immune response to cancer. A checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 binds to one of these proteins and prevents the interaction between the two proteins. Thus, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 may bind to PD-1 or to PD-L1. In a preferred embodiment, the checkpoint inhibitor binds to PD-1 or to PD-L1. In particular, such a checkpoint inhibitor may bind to the PD-L1 binding site of PD-1 or to the PD-1 binding site of PD-L1. To block the interaction of PD-1 with both PD-L1 and PD-L2, it may be advantageous to use checkpoint inhibitors that bind to PD-1 and block the interaction of PD-1 with its ligands.

[0092] In certain embodiments of the invention, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 is an antibody (preferably a monoclonal antibody, or a derivative or fragment thereof) that binds to PD-1. In other embodiments, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 is an antibody (preferably a monoclonal antibody, or a derivative or fragment thereof) that binds to PD-L1. Many such antibodies are known in the art, for example, the human anti-PD1 monoclonal IgG4 antibody nivolumab (Bristol-Myers Squibb); the humanized IgG4 anti-PD-1 antibody pembrolizumab (Merck); the human IgG4 anti-PD-1 antibody cemiplimab (Regeneron / Sanofi); the humanized anti-PD-L1 antibody atezolizumab (Genentech); and the human anti-PD-L1 antibody durvalumab (Medimmune / Astrazeneca), have all received regulatory approval and may be used in accordance with the present invention. Many other such antibodies are in development / testing that may also be used in accordance with the present invention, including the humanized anti-PD-1 antibody tislelizumab (BeiGene) and the fully human anti-PD-L1 antibody avelumab (Pfizer / Merck).

[0093] When the specific binding molecule is used in combination with a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, the agent can be used to treat any cancer.For example, this combination can be used to treat melanoma, lung cancer, breast cancer, lymphoma (particularly Hodgkin's lymphoma), gastric cancer, bladder cancer, esophageal cancer, kidney cancer, mesothelioma, colon cancer, or liver cancer.Alternatively, this combination can be used to treat cancer with mismatch repair deficiency or microsatellite instability and / or high tumor mutation burden (TMB-H).

[0094] Microsatellites (also called "short tandem repeats") are DNA sequences consisting of repeated sequences of units that are scattered throughout the genome, including both coding and noncoding regions. Individual microsatellites generally consist of 10-60 repeats of a repeat unit of 1-6 base pairs in length. Due to the repetitive nature of microsatellites, DNA polymerase is much more prone to make mistakes in these regions than in other regions of the genome. In cells with a functional mismatch repair (MMR) system, the MMR mechanism "proofreads" newly synthesized DNA strands and corrects errors made by the polymerase. Cancer cells with defective MMR mechanisms are unable to correct these errors and therefore experience a 100- to 1000-fold increase in point mutations in microsatellites. This increased mutation rate in microsatellites is known as microsatellite instability (MSI) (Dudley et al., Clin Cancer Res 22(4): 813-820, 2016). "Microsatellite instability high" (MSI-H) cancers are cancers that exhibit MSI. "Mismatch repair deficient" cancers are cancers in which the MMR mechanism is not functional.

[0095] TMB-H cancers are defined as tumors with 10 mutations / megabase or more. There is a significant, but not perfect, correlation between TMB-H and MSI-H tumors. That is, most, but not all, TMB-H tumors are also MSI-H, and most, but not all, MSI-H tumors are also TMB-H. Thus, the cancers treated according to this embodiment of the invention may be either MSI-H but not TMB-H cancers, TMB-H but not MSI-H cancers, or MSI-H and TMB-H cancers.

[0096] Preferably, the specific binding molecule as defined above is combined with a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 to treat breast or lung cancer. Thus, in a preferred embodiment, the present invention provides a specific binding molecule as defined above and a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 for use in the treatment of breast cancer. In another preferred embodiment, the present invention provides a specific binding molecule as defined above and a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 for use in the treatment of lung cancer.

[0097] The breast cancer treated according to this aspect of the invention may be any type of breast cancer, but in certain embodiments, the breast cancer is a triple-negative breast cancer (i.e., breast cancer that lacks expression of estrogen receptors, progesterone receptors, and the hormone epidermal growth factor receptor HER2). Alternatively, the breast cancer treated according to this aspect of the invention may be a hormone receptor-positive breast cancer, i.e., breast cancer that expresses one or more of the estrogen receptor, progesterone receptor, and HER2.

[0098] Similarly, the lung cancer treated according to this aspect of the invention may be any type of lung cancer, and in particular non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

[0099] As shown in the Examples below, when used to treat mouse models of lung and breast cancer, the combination of a specific binding molecule for use according to the invention with a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 exhibited significantly enhanced anti-cancer effects, demonstrating the unexpected advantage of combining these two agents for the treatment of cancer.

[0100] The proteasome inhibitor may be any proteasome inhibitor suitable for use in cancer therapy. Proteasomes are protein complexes present in cells that degrade damaged (e.g., misfolded) or unnecessary proteins by proteolysis after tagging with ubiquitin. The primary proteasome in mammals is the cytoplasmic 26S proteasome, which contains one 20S protein subunit (core particle) and two 19S regulatory cap subunits. The core particle consists of an α subunit (structural) and a β subunit (catalytic). Clinical and preclinical data support the role of the proteasome in maintaining the immortal phenotype of myeloma cells. Proteasome inhibition has been implicated in preventing the degradation of proapoptotic factors and inducing programmed cell death in tumor cells.

[0101] As used herein, a proteasome inhibitor is one that partially or completely inhibits the activity of the proteasome and has a cytotoxic effect on cancer cells, particularly multiple myeloma cancer cells. A preferred inhibitor inhibits the 26S proteasome by binding to the catalytic site of the 26S proteasome, but may exert inhibition by any mode of action, such as competitive or non-competitive, and the inhibition may be reversible or irreversible. Preferably, the inhibitor inhibits the proteasome subunit beta type-5 (PSMB5).

[0102] Preferred proteasome inhibitors are peptide analogues. Preferred inhibitors are bortezomib, ixazomib, and carfilzomib.

[0103] The structures of bortezomib, ixazomib, and carfilzomib are shown below in Formulas III-V. Formula III (Bortezomib) [ka] Formula IV (Ixazomib) [ka] Formula V (Carfilzomib) [ka]

[0104] When the specific binding molecule is used in combination with a proteasome inhibitor (e.g., bortezomib, ixazomib, or carfilzomib), the agent can be used to treat any cancer. For example, this combination can be used to treat melanoma, lung cancer, colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, skin cancer, lymphoma (particularly Hodgkin's lymphoma or mantle cell myeloma), bladder cancer, kidney cancer, mesothelioma, liver cancer, and myeloma. In a preferred embodiment, this combination is used to treat myeloma (also called multiple myeloma) or mantle cell lymphoma. That is, in a preferred embodiment, the present invention provides a specific binding molecule as defined above for use in the treatment of myeloma or mantle cell lymphoma, and bortezomib, ixazomib, or carfilzomib (particularly bortezomib for use in the treatment of myeloma).

[0105] Thus, in a preferred embodiment, the invention provides a specific binding molecule as defined above and bortezomib, ixazomib, or carfilzomib for use in the treatment of myeloma or mantle cell lymphoma.

[0106] As shown in the Examples below, when used to treat myeloma cell lines in vitro, the combination of a specific binding molecule for use according to the invention with bortezomib exhibited significantly improved anti-proliferative effects on the cell lines, demonstrating the unexpected advantage of combining these two agents for cancer treatment. In particular, the specific binding molecule was shown to enhance the effect of bortezomib.

[0107] In further embodiments, a third active agent may be used in cancer treatment. The third active agent may be selected from the second active agents described herein for this or other embodiments of the invention (i.e., two second active agents may be used), or another therapeutic molecule may be used. In some aspects of the invention, additional active agents may be used, but in some aspects of the invention, only the specific binding molecule and the second active agent (and optionally the third active agent) are used.

[0108] In a second aspect of the invention, provided herein is a specific binding molecule as defined above and a second active agent for use in treating breast cancer in a subject, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. That is, the invention provides a specific binding molecule as defined above in combination with a second active agent for the treatment of breast cancer, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents.

[0109] The breast cancer treated according to this aspect of the invention may be any breast cancer. In one embodiment, the breast cancer is triple negative breast cancer. In another embodiment, the breast cancer is hormone receptor positive breast cancer.

[0110] As mentioned above, in this aspect of the invention, the specific binding molecule may be used in combination with a taxane. Functionally, taxanes affect cell proliferation by binding and stabilizing microtubules, leading to cell cycle arrest and apoptosis. Taxanes are classified as diterpenes and contain a taxadiene core. Any taxane that has a cytotoxic effect on cancer cells and / or is suitable for use in chemotherapy may be used, such as paclitaxel, docetaxel, or cabazitaxel. In a preferred embodiment, the taxane is paclitaxel. The structure of paclitaxel is shown in Formula VI below. Formula VI (Paclitaxel) [ka]

[0111] Paclitaxel can be provided in various formulations.For example, it can be provided in the form of paclitaxel protein-bound formulation, for example, it is provided bound to albumin, as in the case of nab-paclitaxel (albumin-bound nanoparticle formulation of paclitaxel).It is considered that reference to paclitaxel includes such alternative formulations of paclitaxel.Similar considerations apply to other active agents described herein.

[0112] As mentioned above, in this aspect of the invention, the specific binding molecule may alternatively be used in combination with a platinum-based chemotherapeutic agent (i.e., a chemotherapeutic agent containing a platinum ion or a platinum atom, particularly as a platinum coordination compound). Platinum-based chemotherapeutic agents are sometimes referred to as platinum-based antineoplastic agents or platins. All platinum-based chemotherapeutic agents essentially work in the same way, reacting with the N-7 position of guanine residues to form DNA interstrand, intrastrand, and DNA-protein crosslinks. The crosslinks inhibit DNA synthesis and / or repair, and initiate apoptosis (Shen et al., Pharmacol. Rev. 64: 706-721, 2012). Any platinum-based chemotherapeutic agent may be used, such as, for example, cisplatin, oxaliplatin, nedaplatin, or carboplatin. In a preferred embodiment, the platinum-based chemotherapeutic agent is cisplatin. The structure of cisplatin is shown below in Formula VII. Formula VII (Cisplatin) [ka]

[0113] Thus, in a preferred embodiment, the second aspect of the invention provides a specific binding molecule as defined above and a second active agent for use in treating breast cancer in a subject, the second active agent being selected from paclitaxel and cisplatin (i.e., the second active agent is paclitaxel or cisplatin). In a particular embodiment, the invention provides a specific binding molecule as defined above and paclitaxel for use in treating breast cancer in a subject. In another embodiment, the invention provides a specific binding molecule as defined above and cisplatin for use in treating breast cancer in a subject.

[0114] As shown in the examples below, when used to treat breast cancer cell lines in vitro (particularly triple-negative breast cancer lines), the combination of a specific binding molecule for use according to the invention with cisplatin (or paclitaxel) exhibits synergistic anti-proliferative effects on the cell lines, demonstrating the unexpected advantage of combining these two agents for the treatment of breast cancer.

[0115] In further embodiments, a third active agent may be used in breast cancer treatment. The third active agent may be selected from the second active agents described herein for this or other embodiments of the invention (i.e., two second active agents may be used), or another therapeutic molecule may be used. In some aspects of the invention, additional active agents may be used, but in some aspects of the invention, only the specific binding molecule and the second active agent (and optionally the third active agent) are used.

[0116] In the third aspect of the present invention, the present invention provides a specific binding molecule as defined above and a second active agent for use in treating pancreatic cancer in a subject, wherein the second active agent is a nucleoside analogue.That is, the present invention provides a specific binding molecule as defined above in combination with a second active agent for treating pancreatic cancer, wherein the second active agent is a nucleoside analogue.

[0117] As known to those skilled in the art, a nucleoside consists of a nucleic acid base linked to a five-carbon sugar (ribose or 2'-deoxyribose). Nucleosides differ from nucleotides in that nucleotides additionally contain at least one phosphate group attached to the sugar moiety.

[0118] The nucleoside analogue may be an analogue of any nucleoside, i.e., an analogue of adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. Nucleoside analogues as used herein are compounds that can replace natural nucleosides in nucleic acid molecules, e.g., compounds that can base pair with the same partner base as the parent nucleoside of which they are analogues. Nucleoside analogues for use according to the invention have cytotoxic and / or chemotherapeutic effects, regardless of the natural nucleoside from which they are derived. That is, they are preferably used in cancer treatment. That is, they are chemotherapeutic nucleoside analogues. In a preferred embodiment, the nucleoside analogue is an analogue of cytidine and / or deoxycytidine. Most preferably, the nucleoside analogue is gemcitabine. The structure of gemcitabine is shown below in Formula VIII. Formula VIII (Gemcitabine) [ka]

[0119] As shown in the Examples below, when used to treat pancreatic cancer cell lines in vitro, the combination of a specific binding molecule for use according to the invention with gemcitabine exhibited a significantly enhanced anti-proliferative effect on the cell lines, demonstrating the unexpected advantage of combining these two agents for the treatment of pancreatic cancer.

[0120] In further embodiments, a third active agent may be used to treat pancreatic cancer. The third active agent may be selected from the second active agents described herein for this or other embodiments of the present invention (i.e., two second active agents may be used), or another therapeutic molecule may be used. In some aspects of the present invention, additional active agents may be used, but in some aspects of the present invention, only the specific binding molecule and the second active agent (and possibly the third active agent) are used.

[0121] In a preferred embodiment, the third active agent is a taxane, preferably paclitaxel.

[0122] As shown in the following examples, when used to treat pancreatic cancer in mouse models, the combination of the specific binding molecule for use according to the present invention with gemcitabine and paclitaxel shows significantly enhanced anti-proliferative effect on tumors, demonstrating the unexpected advantage of combining these three drugs for treating pancreatic cancer.Therefore, in a preferred embodiment, gemcitabine and paclitaxel are used to treat pancreatic cancer.

[0123] Preferred combinations for the treatment of cancer are as described in the Examples.

[0124] In all of the above aspects of the invention, the cancer treated according to the invention may express ANXA1 (meaning that cells in the cancer express ANXA1, e.g., on the surface of the cells). It is straightforward for one of skill in the art to determine whether a cancer expresses ANXA1. ANXA1 expression may be analyzed in a cancer biopsy sample, e.g., at the protein level by immunohistochemical analysis of the sample. The sample may be immunostained with an anti-ANXA1 antibody (such as the antibody described above) to detect ANXA1 expression, according to standard procedures in the art. Both intracellular and extracellular ANXA1 may be detected by permeabilizing the sample (e.g., with a detergent, as is standard in the art).

[0125] Alternatively, the expression of ANXA1 can be analyzed at the nucleic acid level, for example by quantitative PCR (qPCR). mRNA can be extracted from tissue samples and reverse transcribed into DNA using standard procedures in the art. ANXA1 expression level can then be determined by quantitative amplification of target ANXA1 sequence. Suitable qPCR techniques, such as TaqMan, are well known in the art.

[0126] In certain embodiments, the cancer overexpresses ANXA1. "Overexpressing ANXA1" means that the cancer expresses ANXA1 at a higher level than healthy tissue from the same origin. That is, the cancer cells express ANXA1 at a higher level than healthy (i.e., non-cancerous) cells from the same origin. "Same origin" means the same tissue. For example, if a pancreatic ductal adenocarcinoma expresses ANXA1 at a higher level than healthy pancreatic ductal tissue, it would be considered to overexpress ANXA1. To determine whether a cancer tissue overexpresses ANXA1, a quantitative comparison of ANXA1 expression in at least two different tissues (cancer tissue and healthy control tissue) is required. Any suitable technique may be used to make this comparison, but qPCR would be the most suitable. It would be easy for a person skilled in the art to determine whether a cancer overexpresses ANXA1. In certain embodiments, the difference in the expression level of ANXA1 between a cancer overexpressing ANXA1 and a healthy tissue is statistically significant. In other embodiments, expression of ANXA1 in cancer tissue is increased by at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to corresponding healthy tissue.

[0127] ANXA1-expressing cancers treated according to the present invention may express ANXA1 on their surface (i.e., ANXA1 may be expressed on the surface of the cells of the cancer). ANXA1 expression on the surface of cancer cells means that the cells express ANXA1 and that the expressed ANXA1 is transported and localized on the cell surface. ANXA1 expression on the cell surface can be identified immunohistochemically, as described above. In particular, to analyze ANXA1 expression on the cell surface, immunohistochemical analysis is performed without cell permeabilization. This means that the antibody used to detect ANXA1 cannot enter the interior of the cell, and only extracellular (e.g., surface-localized) protein is detected. Since transported ANXA1 is usually attached to the cell surface (rather than released into plasma or other extracellular space), ANXA1 detected by immunohistochemistry of non-permeabilized cells may be considered to be surface-localized ANXA1. Nevertheless, tissues may be washed before staining to remove free extracellular material, such as proteins, according to standard protocols.

[0128] One or more (preferably all) of the specific binding molecule, the second active agent, and the third active agent (if used) may be in free form (i.e., not bound or associated with another molecule, such as a carrier). Thus, in preferred embodiments, no carrier is used for one or more (preferably all) of the specific binding molecule, the second active agent, and the third active agent.

[0129] Alternatively, one or more of the specific binding molecule, the second active agent, and the third active agent (if used) may be bound or associated with a carrier. The carrier may be a particle, a vesicle, or any other solid support (e.g., a scaffold). In a preferred embodiment, when a carrier is used, the carrier is not a solid support. That is, the specific binding molecule and / or the second active agent (and / or the third active agent (if present)) are associated with the carrier but are not bound. In this embodiment, the carrier can be used, for example, to package one or more of the specific binding molecule, the second active agent, and the third active agent without binding them to the molecule. By way of example, in one embodiment, the carrier can encapsulate one or more of the specific binding molecule, the second active agent, and the third active agent, for example, the carrier can be a free-floating lipid vesicle, for example, a liposome.

[0130] As yet another alternative, where a carrier is used that binds to or associates with a specific binding molecule, a second active agent, and / or a third active agent, the carrier used is proteinaceous.

[0131] When a carrier is used, the carrier may be bound to one or more of the specific binding molecule, the second active agent, and / or the third active agent. However, when a carrier is used, it is preferred that the carrier is bound to only one of the specific binding molecule, the second active agent, and / or the third active agent. In that case, different carriers may be used for different molecules / agents, and / or one or more of the molecules / agents may be in free form.

[0132] As described herein, the specific binding molecule and the second active agent (and the third active agent, if present) may be administered separately (e.g., in separate compositions), sequentially, or simultaneously. In the latter case, the different molecules / agents may be provided in combination (i.e., in one composition). In all cases, as described above, the molecules / agents may be provided to be administered with a carrier or without a carrier. When a carrier is used, it is preferred that only one of the specific binding molecule, the second active agent, and / or the third active agent is present on each carrier. That is, the other molecule / agent is in free form when provided in the same composition. Alternatively, separate carriers may be used for the different molecules / agents. Thus, by way of example, the specific binding molecule may be provided with a first carrier, and separately, the second active agent may be provided with a second carrier, and the third active agent (if present) may be provided in free form. However, in a preferred embodiment, all agents are provided in free form.

[0133] The specific binding molecule, the second active agent, and optionally the third (or further) active agent, if present, may each be administered to the subject to be treated in the form of a pharmaceutical composition. Such a composition may contain one or more pharma- ceutically acceptable diluents, carriers, or excipients. As used herein, "pharma- ceutically acceptable" refers to an ingredient that is compatible with the other ingredients of the composition and physiologically acceptable to the recipient. The type and dosage of the composition and carrier or excipient material may be selected in the usual manner, depending on preference, the desired route of administration, etc. The dosage may also be determined in the usual manner and may depend on the type of molecule, the age of the patient, the mode of administration, etc. As further described below, the specific binding molecule and the second active agent (and optionally the third active agent) may be administered in the same pharmaceutical composition or in separate pharmaceutical compositions.

[0134] The pharmaceutical composition may be prepared by any suitable means for administration to a subject. Such administration may be, for example, oral, rectal, nasal, topical, vaginal, or parenteral. Oral administration herein includes buccal and sublingual administration. Topical administration herein includes transdermal administration. Parenteral administration as defined herein includes subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal administration.

[0135] The pharmaceutical compositions disclosed herein may be in the form of a solution or syrup, a solid composition such as a powder, granules, tablets, or capsules, a cream, an ointment, and other compositions commonly used in the art. Suitable pharma-ceutically acceptable diluents, carriers, and excipients for use in such compositions are well known in the art. For example, suitable excipients include lactose, corn starch or its derivatives, stearic acid or its salts, vegetable oils, waxes, fats, and polyols. Suitable carriers or diluents include carboxymethylcellulose (CMC), methylcellulose, hydroxypropylmethylcellulose (HPMC), dextrose, trehalose, liposomes, polyvinyl alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, gelatin, oils and fats, alcohols, surfactants, and emulsifiers such as polysorbates. Stabilizing agents, wetting agents, emulsifying agents, sweeteners, and the like may also be used.

[0136] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: sterile diluents such as water for injection, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can act as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0137] Thus, the pharmaceutical composition for use according to the invention may be administered in an appropriate manner. The dosage and frequency of administration will depend on factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials. The specific binding molecule and / or second active agent (and optionally the third active agent) for use according to the invention may be conveniently provided to the subject in a daily, weekly or monthly administration, or in intermediate frequency administration, for example, administration may be performed every 2, 3, 4, 5 or 6 days, or every 2, 3, 4, 5 or 6 weeks, or every 2, 3, 4, 5 or 6 months, or once a year or twice a year. Administration may be performed for a total of at least 2 weeks, preferably at least 2 months, for example 3 to 24 months. Administration may be in amounts of 100 ng / kg to 5 g / kg of body weight, for example, in amounts of 10 μg / kg to 1 g / kg, or in amounts of 1 mg / kg to 100 mg / kg. A dose is considered to be the application of the specific binding molecule or second active agent (or third active agent) at one time or over a continuous period of time, for example, as a single bolus or continuously over a discrete period of time.

[0138] When using a second active agent (or a third active agent) that is already approved, the agent may be conveniently used at the approved dosage. For example, 5FU may be administered at 400 mg / m on day 1. 2 as an intravenous bolus, then 2400-3000 mg / m2 every 2 weeks 2as a continuous intravenous infusion over 46 hours. For an adult weighing 70 kg, this corresponds to approximately 11 mg / kg (bolus) and 68-85 mg / kg for each infusion (considered to be a single dose). Pembrolizumab may be administered as an intravenous infusion of 200 mg every 3 weeks or 400 mg every 6 weeks (corresponding to approximately 6-11 mg / kg for adults). Bortezomib may be administered intravenously or subcutaneously at a single dose of 1-5 mg to adults twice weekly for at least 2 weeks, and once weekly in subsequent cycles. Ixazomib may be administered orally at a single dose of 1-5 mg to adults once weekly for 4-week cycles. Carfilzomib may be administered intravenously at a single dose of 10-100 mg to adults twice weekly for 3 weeks in the first cycle. Approved dosages of other existing therapies are well known in the art. Alternatively, the combination of a second active agent (and optionally a third active agent) with a specific binding molecule for ANXA1 may allow the second active agent (and / or optionally a third active agent) to be used at a lower dosage than currently approved for use, especially in cases where synergistic effects are observed between the two components. For example, the second active agent (and / or optionally a third active agent) can be used at a dosage that is up to 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more lower than currently approved dosages. A skilled clinician will be able to calculate the appropriate dosage for a patient based on all relevant factors, such as age, height, weight, and the condition being treated.

[0139] The specific binding molecule and the second active agent may be provided in the amounts described above, for example in amounts conventionally used or in reduced amounts. Advantageously, the specific binding molecule and the second active agent are used in a molar ratio of 2000:1 to 1:2000.

[0140] Preferably, the specific binding molecule and the second active agent (or pharmaceutical composition containing them) (and optionally the third active agent) for use according to the present invention are administered to a subject in need thereof in a therapeutically effective amount. By "therapeutically effective amount" is meant an amount sufficient to have an effect on the subject's condition. Whether an amount is sufficient to have an effect on the subject's condition may be determined by a physician / veterinarian.

[0141] The specific binding molecule as defined above and the second active agent (and optionally the third active agent) may be administered to a subject separately, simultaneously or sequentially. By "separate" administration herein is meant that the specific binding molecule and the second active agent (and optionally the third active agent) are administered to a subject simultaneously, or at least substantially simultaneously, but by different administration routes. By "simultaneous" administration herein is meant that the specific binding molecule and the second active agent (and optionally the third active agent) are administered to a subject simultaneously, or at least substantially simultaneously, by the same administration route. By "sequential" administration herein is meant that the specific binding molecule and the second active agent (and optionally the third active agent) are administered to a subject at different times. In particular, the administration of the specific binding molecule is completed before the administration of the second active agent (and optionally the third active agent) is started (or the administration of the specific binding molecule is completed before the administration of the second active agent (and optionally the third active agent) is started). Sequential administration may occur when the administration of the two agents is separated by 10 minutes to 30 days, for example, 1 hour to 96 hours (or 2 weeks). When administered sequentially to a subject, the two agents may be administered by the same or different routes of administration.

[0142] The specific binding molecules for use according to the present invention may also be administered to a subject in combination with radiation therapy and / or surgery.

[0143] As detailed above, the present invention is for use in the treatment of cancer in a subject. The treatment may be curative (or may be intended to be curative), but may also be palliative (i.e., merely limiting, relieving or ameliorating the symptoms of cancer, or prolonging life). Preferably, the treatment shrinks the tumor or slows or reduces its rate of growth. It is preferred that the tumor size is reduced by at least 10%, preferably at least 20%, 30% or 50% (e.g., by 30%, 50%, 75% or 100%). The same is preferred for the level of growth reduction.

[0144] The subject to be treated in the present invention may be any mammal, for example, a domestic animal such as a cow, horse, sheep, pig, or goat, a pet animal such as a rabbit, cat, or dog, or a primate such as a monkey, chimpanzee, gorilla, or human. Most preferably, the subject is a human. The subject may be any animal, preferably a human, suffering from or suspected of suffering from cancer. Thus, the subject is an individual in need of treatment for cancer, or a particular cancer, as set forth in the various aspects of the present invention detailed above.

[0145] As detailed above, the first aspect of the invention provides a specific binding molecule that binds human ANXA1 as defined above and a second active agent (and optionally a third active agent) for use in treating cancer in a subject, the second active agent being selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor. This aspect of the invention can be considered as a method of treating cancer in a subject, comprising administering to the subject a specific binding molecule that binds human ANXA1 and a second active agent (and optionally a third active agent), the specific binding molecule being as defined above, and the second active agent being selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor. Such a method thus constitutes a fourth aspect of the invention. All features of this fourth aspect of the invention may be as defined above with respect to the first aspect.

[0146] Similarly, as mentioned above, the second aspect of the invention provides a specific binding molecule that binds human ANXA1 and a second active agent (and optionally a third active agent) for use in treating breast cancer in a subject, the specific binding molecule being as defined above and the second active agent being selected from taxanes and platinum-based chemotherapeutic agents. This aspect of the invention can alternatively be considered as providing a method of treating breast cancer in a subject, comprising administering to the subject a specific binding molecule that binds human ANXA1 and a second active agent (and optionally a third active agent), the specific binding molecule being as defined above in relation to the first aspect and the second active agent being selected from taxanes and platinum-based chemotherapeutic agents. Such a method thus constitutes a fifth aspect of the invention. All the features of this fifth aspect of the invention may be as defined above in relation to the second aspect.

[0147] Similarly, as mentioned above, the third aspect of the present invention provides a specific binding molecule that binds to human ANXA1 and a second active agent (and optionally a third active agent) for use in treating pancreatic cancer in a subject, the specific binding molecule being as defined above and the second active agent being a nucleoside analogue. This aspect of the present invention can alternatively be considered as providing a method of treating pancreatic cancer in a subject, comprising administering to the subject a specific binding molecule that binds to human ANXA1 and a nucleoside analogue (and optionally a third active agent), the specific binding molecule being as defined above. Such a method thus constitutes the sixth aspect of the present invention. All the features of this sixth aspect of the present invention may be as defined above for the third aspect.

[0148] The first aspect of the present invention may alternatively be considered as providing a use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating cancer, the specific binding molecule being as defined above, and the treatment of cancer comprising administering the medicament and a second active agent (and optionally a third active agent) to a subject, the second active agent being selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1, and a proteasome inhibitor. Such a use thus constitutes the seventh aspect of the present invention. All the features of this seventh aspect of the present invention may be as defined above for the first aspect. As an alternative to this aspect, a second active agent (and optionally a third active agent) may be used to manufacture a medicament, and the treatment comprises administering the medicament and a specific binding molecule as defined above.

[0149] The second aspect of the present invention may alternatively be considered as providing a use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating breast cancer, the specific binding molecule being as defined above, and the treatment of breast cancer comprising administering the medicament and a second active agent (and optionally a third active agent) to a subject, the second active agent being selected from taxanes and platinum-based chemotherapeutic agents. Such a use thus constitutes an eighth aspect of the present invention. All the features of this eighth aspect of the present invention may be as defined above for the second aspect. As an alternative to this aspect, a second active agent (and optionally a third active agent) may be used to manufacture a medicament, and the treatment comprises administering the medicament and a specific binding molecule as defined above.

[0150] The third aspect of the present invention may alternatively be considered as providing a use of a specific binding molecule that binds to human ANXA1 in the manufacture of a medicament for treating pancreatic cancer, the specific binding molecule being as defined above, and the treatment of pancreatic cancer comprising administering the medicament and a nucleoside analogue (and optionally a third active agent) to a subject. Such a use thus constitutes the ninth aspect of the present invention. All the features of this ninth aspect of the present invention may be as defined above for the third aspect. As an alternative to this aspect, a second active agent (and optionally a third active agent) may be used to manufacture the medicament, and the treatment comprises administering the medicament and a specific binding molecule as defined above.

[0151] In the seventh, eighth and ninth aspects of the invention, in line with the above teachings, the medicament produced may contain both a specific binding molecule that binds to human ANXA1 and a second active agent (and optionally a third active agent), or may contain only one of either the specific binding molecule that binds to human ANXA1 or the second active agent (and optionally a third active agent), in which case the two (or three) agents are administered to the subject as separate medicaments.

[0152] In a tenth aspect, the present invention provides a pharmaceutical composition comprising a specific binding molecule that binds to human ANXA1 as described above, a second active agent (and optionally a third active agent) as defined in the first aspect of the invention, and one or more pharma- ceutically acceptable diluents, carriers, or excipients. The pharmaceutical composition and the pharma-ceutically acceptable diluents, carriers, or excipients are described above, all of which teachings are applicable to the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be used to treat cancer, particularly cancer as described above with respect to the first aspect of the present invention.

[0153] In an eleventh aspect, the present invention provides a kit comprising a specific binding molecule that binds to human ANXA1 as defined above, and a second active agent (and optionally a third active agent) as defined in relation to the first aspect of the present invention. The specific binding molecule and the second active agent (and optionally a third active agent) may be provided as separate components, for example in separate compositions, and they may be provided together in a single container or in separate containers. Alternatively, the specific binding molecule and the second active agent (and optionally a third active agent) may be provided in a single composition in a single container. Each therapeutic agent may be provided in any suitable form, for example as an aqueous solution or as a lyophilisate.

[0154] In a twelfth aspect, the present invention provides a product comprising a specific binding molecule that binds to a defined human ANXA1 and a second active agent (and optionally a third active agent) for separate, simultaneous or sequential use in the treatment of cancer in a subject, wherein the second active agent is selected from a thymidylate synthase inhibitor, a nucleobase analogue, a checkpoint inhibitor that blocks the interaction of PD-1 with PD-L1, and a proteasome inhibitor. The features of the product and its use of the twelfth aspect may be as defined above in relation to the first aspect.

[0155] In a thirteenth aspect, the present invention provides a product comprising a specific binding molecule that binds to human ANXA1 as defined above and a second active agent (and optionally a third active agent) for separate, simultaneous or sequential use in the treatment of breast cancer in a subject, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. The characteristics of the product of the thirteenth aspect and its use may be as defined above for the second aspect.

[0156] In a fourteenth aspect, the present invention provides a product comprising a specific binding molecule that binds to human ANXA1 as defined above in relation to the first aspect and a nucleoside analogue (and optionally a third active agent) for separate, simultaneous or sequential use in the treatment of pancreatic cancer in a subject. The characteristics of the product of the fourteenth aspect and its use may be as defined above in relation to the third aspect.

[0157] In an article of manufacture for use according to the invention, the specific binding molecule and the second active agent (and optionally the third active agent) may be provided as separate components, e.g., in separate compositions, which may be provided together in a single container or in separate containers. Alternatively, the specific binding molecule and the second active agent (and optionally the third active agent) may be provided in a single composition in a single container. Each therapeutic agent may be provided in any suitable form, e.g., as an aqueous solution or as a lyophilisate.

[0158] All documents cited in this application are hereby incorporated by reference in their entirety.

[0159] The invention will be further understood by reference to the following non-limiting examples. [Brief description of the drawings]

[0160] [Figure 1]Figure 1 shows that application of antibody MDX-124 to pancreatic cancer cell lines MIA PaCa-2 (A) and PANC-1 (B) significantly inhibited cancer cell proliferation in vitro, both when the antibody was used as a single agent and when combined with chemotherapy with 5FU. The antibody was applied to the cells in the concentration range of 0-10 μM, and 5FU was applied at its IC50, with ****p<0.0001, ***p<0.001, and **p<0.01 (MDX-124 vs. MDX-124+5FU IC50), or ○p<0.05 and ○○p<0.01 (MDX-124 vs. IgG isotype control). [Diagram 2] Figure 2 shows that application of antibody MDX-124 to the pancreatic cancer cell line PANC-1 significantly inhibited cancer cell growth in vitro both when the antibody was used as a single agent and when combined with chemotherapy with gemcitabine. The antibody was applied to the cells in a concentration range of 0-10 μM, and gemcitabine was applied at its IC50, with ****p<0.0001, ***p<0.001, and **p<0.01 (MDX-124 vs. MDX-124 + gemcitabine IC50), or ○○○○p<0.0001 (MDX-124 vs. IgG isotype control). [Diagram 3] Figure 3 shows that application of antibody MDX-124 to the breast cancer cell line HCC1806 significantly inhibited cancer cell proliferation in vitro, both when the antibody was used as a single agent and when combined with chemotherapy with cisplatin. The antibody was applied to the cells at a concentration range of 0-10 μM, and cisplatin was applied at its IC50, with ****p<0.0001 (MDX-124 vs. MDX-124 + cisplatin IC50), or ○p<0.05, ○○p<0.01, and ○○○p<0.001 (MDX-124 vs. IgG isotype control). This figure is representative of two independent experiments. [Figure 4]Figure 4 shows that application of antibody MDX-124 to the breast cancer cell line HCC1806 significantly inhibited cancer cell proliferation in vitro, both when the antibody was used as a single agent and when combined with chemotherapy with paclitaxel. The antibody was applied to the cells in a concentration range of 0-10 μM, and paclitaxel was applied at its IC20, with ****p<0.0001 (MDX-124 vs. MDX-124 + paclitaxel IC20), or ○○○p<0.001 and ○○○p<0.0001 (MDX-124 vs. IgG isotype control). [Diagram 5] Figure 5 shows tumor volume in the EMT6 mouse model of breast cancer. Mice were inoculated with cancer cells and then administered either vehicle control (PBS), MDX-001 (10 mg / kg, QW), anti-PD-1 antibody (10 mg / kg, BIW), or a combination of the MDX-001 and anti-PD-1 regimens (n=10 per group). Tumor volume was calculated at each time point shown. As shown, the combination therapy group showed the best results in terms of tumor growth inhibition. [Figure 6] Figure 6 shows the EMT6 tumor volume for individual mice analyzed in Figure 5. Results are shown comparing vehicle-treated mice with anti-PD-1 antibody (A) or with MDX-001 and anti-PD-1 antibody combination therapy (B). As shown, more mice treated with MDX-001 and anti-PD-1 antibody showed tumor regression than those treated with anti-PD-1 monotherapy. [Figure 7] Figure 7 shows the mean tumor volume in the LL / 2 mouse model of lung cancer. Mice were inoculated with cancer cells and then administered either vehicle control (PBS), MDX-001 (10 mg / kg, QW), anti-PD-1 antibody (10 mg / kg, BIW), or a combination of the MDX-001 and anti-PD-1 regimens (n=10 per group). Tumor volumes were calculated at the indicated time points. As shown, neither the MDX-001 nor the anti-PD-1 antibody showed efficacy against tumors when administered alone, but a significant anti-tumor effect was observed when administered in combination. [Figure 8]Figure 8 shows the mean tumor volume in the Pan02 mouse model of pancreatic cancer. Mice were inoculated with cancer cells and then administered either gemcitabine (80 mg / kg, Q3D x 4) and nab-paclitaxel (Abraxane, 30 mg / kg, Q3D x 4) (n=50) or MDX-124 (10 mg / kg, twice weekly) with gemcitabine (80 mg / kg, Q3D x 4) and nab-paclitaxel (Abraxane, 30 mg / kg, Q3D x 4) (n=30). Tumor volumes were calculated at each time point shown and presented as mean tumor volume ± SEM. [Figure 9] Figure 9 shows the effect of MDX-124 + / - bortezomib on apoptosis in multiple myeloma cell lines. (A) H929, (B) JJN3, and (C) U266 human myeloma cell lines were treated with MDX-124 (20 μM), bortezomib (20 nM), or a combination of MDX-124 and bortezomib. All data are presented as the mean ± SD of three independent experiments, each performed in duplicate. Statistical analysis was performed using two-way ANOVA with Tukey correction for multiple comparisons. [Figure 10] FIG. 10 shows the effect of MDX-124 and bortezomib on the expression of p-STAT3 and p-BCL2 in multiple myeloma cell lines. Aliquots of each human multiple myeloma cell line were treated with either MDX-124 (20 μM), bortezomib (20 nM), or a combination of MDX-124 and bortezomib for 4 hours and then stained with Alexa488-labeled p-STAT3 (Tyr705) and PE-labeled p-BCL2 (pS70) antibodies. The mean fluorescence intensity values ​​for (A) p-BCL2 and (B) p-STAT3 after each treatment group were compared to untreated control cells. All data are presented as the mean ± SD of three independent experiments, each performed in duplicate. Statistical analysis was performed using two-way ANOVA with Tukey correction for multiple comparisons. [Figure 11]Figure 11 shows the effect of MDX-124 and bortezomib on intracellular IL-6 production in multiple myeloma cell lines. Aliquots of each human multiple myeloma cell line were treated with either MDX-124 (20 μM), bortezomib (20 nM), or a combination of MDX-124 and bortezomib for 24 hours, after which intracellular IL-6 was analyzed in fixed and permeabilized cells. The mean fluorescence intensity values ​​for IL-6 for each treatment group were compared to untreated control cells. All data are presented as the mean ± SD of three independent experiments, each performed in duplicate. Statistical analysis was performed using two-way ANOVA with Tukey correction for multiple comparisons.

[0161] Working Example Example 1 - In vitro testing of anti-ANXA1 antibody combination therapy against cancer cell lines Combination of MDX-124 and 5FU against pancreatic cancer cell lines MTT cell proliferation assays were performed on pancreatic cancer cell lines MIA-PaCa-2 and PANC-1. The cell lines were obtained from Public Health England Culture Collections. MIA-PaCa-2 is a human pancreatic cancer cell line, and PANC-1 is a human pancreatic epithelioid carcinoma cell line. MIA PaCa-2 and PANC-1 cells were cultured in DMEM containing 10% FBS, 1% penicillin / streptomycin, and 1% L-glutamine at 37°C in an atmosphere containing 5% CO2. MDX-124, described in the description of the invention above, is a humanized IgG1 antibody against ANXA1 having a light chain of SEQ ID NO:13 and a heavy chain of SEQ ID NO:14. To measure the metabolic activity of cells, cell proliferation was measured using the MTT colorimetric assay. In this assay, NADPH-dependent cellular oxidoreductase reduces a yellow tetrazolium dye, or MTT, to an insoluble purple formazan product, which is quantified by measuring the absorbance at 500–600 nm using a spectrophotometer. The amount of formazan is proportional to the level of cell proliferation, with rapidly dividing cells reducing higher levels of MTT. Assays were performed in triplicate. Cells were seeded in a final volume of 100 μL. MIA PaCa-2 and PANC-1 cells were seeded at 1 × 10 per well. 4 The cells were seeded at a density of 100×. Cells were then cultured for 24 hours before assay, after which cell proliferation was measured. For proliferation assays, cells were cultured for 72 hours in the presence of IgG isotype negative control (Thermo Fisher Scientific, USA, Cat. No. 31154) at concentrations ranging from 2.5 to 10 μM, in the presence of MDX-124 (2.5 to 10 μM), or in the presence of a combination of MDX-124 (2.5 to 10 μM) and 100 μM (for MIA PaCa-2 cells) 5FU or 1 mM (for PANC-1 cells) 5FU. The antiproliferative effect of each treatment was measured as the percentage response compared to untreated control cells. In both cell lines, 5FU had an IC 50 Used in IC 50 The concentrations represent the concentrations at which a substance exerts half of its maximal inhibitory effect. IC for each cell line 50 The IC was calculated by treating cancer cells with a 10-fold dilution series of 5FU ranging from 1 nM to 10 mM. Using the MTT assay, the survival rate of cancer cells after 72 hours of incubation with each concentration of 5FU was calculated. This was repeated eight times, and the average concentration at which 50% of the cells became non-viable was calculated as the IC 50 was considered a value. As expected, MDX-124 alone showed relatively strong antiproliferative effects on both cell lines, especially MIA-PaCa-2. Nevertheless, 5FU (its IC 50When combined with 5FU (at 100 mg / kg / day), cancer cell viability for both cell lines was significantly reduced compared to either treatment alone (Figure 1). The combination of MDX-124 and 5FU reduced cancer cell viability by 99.8% in the MIA PaCa-2 cell line and by 91.2% in the PANC-1 cell line. Analysis of the results by unpaired t-test using the "SynergyFinder" software (Ianevski et al., Nucleic Acids Research 48(W1): W488-W493, 2020) showed that MDX-124 had strong synergistic activity when combined with 5FU.

[0162] MDX-124 in combination with gemcitabine against pancreatic cancer cell lines MTT cell proliferation assays were performed on the pancreatic cancer cell line PANC-1 as described above. Cell proliferation was measured using the MTT colorimetric assay as described above, using the same IgG isotype control. The IC of gemcitabine on PANC-1 cells in the presence of MDX-124 (2.5–10 μM) or in the presence of MDX-124 (2.5–10 μM) was 50 Cells were cultured in the presence of combination with 20 μM gemcitabine, IC 50 was calculated as described above using a series of gemcitabine dilutions ranging from 0.1 nM to 100 μM, and the assay was repeated three times to determine the mean concentration at which 50% of the cells were non-viable (i.e., IC 50 ) was obtained. As expected, MDX-124 alone showed a relatively strong antiproliferative effect on the cell line. 50 When combined with rituximab (at 0.01 mg / kg / day), cancer cell viability was significantly decreased compared to either treatment alone (Figure 2). Results were analyzed by unpaired t-test.

[0163] Combination of MDX-124 and cisplatin against breast cancer cell lines MTT cell proliferation assay was performed on triple-negative breast cancer cell line HCC1806. The cell line was obtained from ATCC. HCC1806 cells were cultured in DMEM containing 10% FBS, 1% penicillin / streptomycin, and 1% L-glutamine at 37°C in an atmosphere containing 5% CO2. Cell proliferation was measured using the MTT colorimetric assay as described above, using the same IgG isotype control. IC values ​​for HCC1806 cells in the presence of MDX-124 (2.5–10 μM) or in the presence of MDX-124 (2.5–10 μM) were 50 Cells were cultured in the presence of 0.65 μM cisplatin in combination with IC 50 was calculated as described above using a series of cisplatin dilutions ranging from 0.1 nM to 100 μM, and the assay was repeated four times to determine the mean concentration at which 50% of the cells became non-viable (i.e., IC 50 ) was obtained. As expected, MDX-124 alone had an antiproliferative effect on the cell lines, which was substantially enhanced by combination with cisplatin (Figure 3). Analysis of the results by unpaired t-test using the "SynergyFinder" software (see above) showed that MDX-124 had strong synergistic activity when combined with cisplatin.

[0164] Combination of MDX-124 and paclitaxel against breast cancer cell lines MTT cell proliferation assay was performed on the triple-negative breast cancer cell line HCC1806. Cell proliferation was measured using the MTT colorimetric assay as described above, using the same IgG isotype control. IC values ​​for HCC1806 cells in the presence of MDX-124 (2.5–10 μM) or in the presence of MDX-124 (2.5–10 μM) were 20 Cells were cultured in the presence of a combination of 0.4 nM paclitaxel, IC 20was calculated as described above using a series of paclitaxel dilutions ranging from 0 to 10 nM, and the assay was repeated twice to determine the mean concentration at which 20% of cells were nonviable (i.e., IC 20 ) was obtained. As expected, MDX-124 alone had an antiproliferative effect on the cell lines, which was substantially enhanced by combination with paclitaxel (Figure 4). Analysis of the results by unpaired t-test using the "SynergyFinder" software (see above) showed that MDX-124 had strong synergistic activity when combined with paclitaxel.

[0165] Example 2 - Testing combination treatments of anti-ANXA1 antibodies in in vivo models of cancer Breast Cancer Models 5 × 10 5 EMT6 triple-negative breast cancer cells were inoculated subcutaneously. 3 When tumor volume reached 100 μg / mL (measured by calipers), mice (n=10 per group) were administered either vehicle control (PBS), MDX-001 (10 mg / kg, once per week), anti-PD-1 antibody (10 mg / kg, twice per week), or a combination regimen of MDX-001 (10 mg / kg, once per week) and anti-PD-1 antibody (10 mg / kg, twice per week). Tumor volumes were measured twice per week for 3 weeks. The anti-PD-1 antibody used was the murine antibody RMP-1-14 (Yamazaki et al., Journal of Immunology 175(3): 1586-1592, 2005), PBS was administered intraperitoneally, and the antibody was administered intravenously. As detailed above, MDX-001 is an anti-ANXA1 antibody and is the parent of MDX-124. MDX-001 has a light chain as shown in SEQ ID NO:30 and a heavy chain as shown in SEQ ID NO:31. Compared to the vehicle control, MDX-001 monotherapy did not significantly affect tumor growth, but tumor growth was significantly slower when mice were administered anti-PD-1 monotherapy. When anti-PD-1 antibody was combined with MDX-001, the mean tumor volume was reduced by an additional 15% compared to anti-PD-1 monotherapy (Figure 5). Of note, 30% of mice treated with MDX-001 and anti-PD-1 combination therapy showed evidence of tumor regression at day 21 compared to day 18, compared to only 10% of mice receiving anti-PD-1 therapy alone (Figure 6). No weight loss was observed in any treatment group, and no side effects were observed with treatment with MDX-001, anti-PD-1, or combination therapy.

[0166] Lung cancer model 3 × 10 5 LL / 2 lung cancer cells were inoculated subcutaneously. 3 When tumor volume reached 100 μg / mL (measured by calipers), mice (n=10 per group) were treated with either vehicle control (PBS), MDX-124 (10 mg / kg once per week), anti-PD-1 (10 mg / kg twice per week), or a combination regimen of MDX-124 (10 mg / kg once per week) and anti-PD-1 (10 mg / kg twice per week). Drugs were administered as described above. Tumor volumes were measured on days 2, 5, 8, 12, and 15, at which point vehicle and monotherapy groups were terminated. Combination groups were measured again on day 19 before being terminated. Results were analyzed using a two-way repeated measures ANOVA / mixed effects model. No significant differences were observed between the vehicle group and MD-124 monotherapy, or between the vehicle group and anti-PD-1 monotherapy. However, the MDX-124 / anti-PD-1 combination group showed significantly slower tumor growth than the vehicle control (P = 0.022), MDX-124 alone (P = 0.0003), or anti-PD-1 alone (P = 0.037), indicating a synergistic effect of the combination of MDX-124 and anti-PD-1 therapy. Mice in the combination group survived longer and remained in the study until day 19, whereas the other groups were terminated on day 15 (Figure 7). No weight loss was detected in this study.

[0167] Example 3 - Testing combination treatments of anti-ANXA1 antibodies in an in vivo model of pancreatic cancer Pancreatic Cancer Models 5 × 10 6 Pan02 pancreatic cancer cells were inoculated subcutaneously, and tumors were 100 mm 3 Once mice reached a median age of 10 days (measured by calipers), they were randomly assigned to each treatment group. During the first 13-day treatment period, mice received either gemcitabine (Hospira Inc., Lake Forest, IL) (80 mg / kg, Q3D x 4) and nab-paclitaxel (Abraxane, Celgene Corp., Summit, NJ; Celgene Europe, Germany) (30 mg / kg, Q3D x 4) (n = 50), or a combination regimen of MDX-124 (10 mg / kg, twice weekly) with gemcitabine (80 mg / kg, Q3D x 4) and nab-paclitaxel (30 mg / kg, Q3D x 4) (n = 30). Drugs were administered as described above. A vehicle control was also performed using saline (BIW, 10 mL / kg). Tumor volumes were measured on days 3, 7, 10, and 13. On day 13, the group receiving only gemcitabine and nab-paclitaxel had a mean tumor volume of 106.7 mm 3 The mean tumor volume in the group receiving the combination regimen of MDX-124 with gemcitabine and nab-paclitaxel was 92.6 mm compared with mice treated with vehicle control (data not shown). 3 Thus, the addition of MDX-124 to gemcitabine and nab-paclitaxel increased the mean tumor growth inhibition compared to gemcitabine and nab-paclitaxel alone in Pan02 mice (FIG. 8).

[0168] Example 4 - In vitro testing of combination treatments of anti-ANXA1 antibodies against multiple myeloma cell lines Several assays were used to evaluate the anticancer activity of MDX-124 in combination with bortezomib against a panel of human multiple myeloma cancer cell lines.

[0169] Materials and Methods Apoptosis assay The effects of MDX-124, bortezomib, and the combination of both drugs on apoptosis were assessed using Annexin V and 7-AAD labeling. Human multiple myeloma cell lines (H929, JJN3, and U266, all obtained from DSMZ, Leibniz Institute, German Collection of Microorganisms and Cell Cultures, GmbH, Germany) were incubated for 48 h with either MDX-124 (20 μM), bortezomib (20 nM, Cell Signaling Technology, MA, USA), or the combination of both drugs (fixed molar ratio of 1000:1) in 1 mL of RPMI medium supplemented with 10% FCS. The percentage of apoptosis above untreated control cell levels for each treatment group was quantified using flow cytometry.

[0170] Expression of apoptosis-related proteins The effects of MDX-124, bortezomib, and the combination of both drugs on the expression of two apoptosis-related proteins, p-BCL2 and p-STAT3, were assessed using flow cytometry. Aliquots of each human multiple myeloma cell line were treated with either MDX-124 (20 μM), bortezomib (20 nM), or the combination of MDX-124 and bortezomib for 4 hours and then stained with Alexa488-labeled p-STAT3 (Tyr705, BD Biosciences, catalog number 557814) and PE-labeled p-BCL2 (pS70, BD Biosciences, catalog number 562532) antibodies. Mean fluorescence intensity values ​​for each treatment group were compared to untreated control cells.

[0171] IL-6 expression The effect of each agent alone and in combination on interleukin 6 (IL-6) expression was assessed using flow cytometry. Aliquots of each human multiple myeloma cell line were treated with either MDX-124 (20 μM), bortezomib (20 nM), or a combination of MDX-124 and bortezomib for 24 hours, and intracellular IL-6 was analyzed in fixed and permeabilized cells. The mean fluorescence intensity values ​​for IL-6 for each treatment group were compared to untreated control cells.

[0172] result Apoptosis assay Although MDX-124 alone affected apoptosis, bortezomib alone significantly induced apoptosis when compared to untreated control cells (Figure 9). However, the addition of MDX-124 to bortezomib promoted apoptosis compared to bortezomib alone in all cell lines tested (Figure 9).

[0173] Expression of apoptosis-related proteins Overexpression of STAT3 in multiple myeloma is associated with poor prognosis and has been hypothesized to be involved in microenvironment-dependent therapeutic resistance. In addition to its growth-promoting role, STAT3 upregulates antiapoptotic proteins and causes dysregulation of microRNAs in multiple myeloma (Chong et al., 2019, Cancers, Vol. 11(5), 731). The BCL2 protein is an attractive target because it is an oncogene that promotes cell survival and is often upregulated in multiple myeloma (Gupta et al., 2021, Blood Lymphat. Cancer, Vol. 11, 11-24). Both MDX-124 and bortezomib alone reduced p-BCL2 or p-STAT3 expression in the multiple myeloma cell lines tested (Figure 10). However, the combination of MDX-124 and bortezomib reduced p-BCL2 and p-STAT3 more than either treatment alone in all cell lines tested (Figure 10).

[0174] IL-6 expression IL-6 is not only a growth factor but also a survival factor for multiple myeloma and inhibits apoptosis of myeloma cells. Inhibition of IL-6 activity has been implicated in tumor regression (Harmer et al., 2019, Front. Endocrinol., Vol. 9, doi: 10.3389 / fendo.2018.00788). Although intracellular IL-6 expression was modestly reduced by both MDX-124 and bortezomib, the combination of MDX-124 and bortezomib reduced it more than either treatment alone in all cell lines tested (Figure 11). Overall, these data suggest that the addition of MDX-124 to bortezomib enhances the anticancer effect compared with either agent alone in multiple myeloma cell lines.

[0175] Sequence Listing The sequences provided in the sequence listing are shown in the following table: [Table 1]

Claims

1. A pharmaceutical composition for use in combination with a second active agent for the treatment of cancer in a target, comprising a specific binding molecule that binds to human ANXA1, (i) The specific binding molecule comprises complementarity-determining regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3, each having the following amino acid sequences: VLCDR1 has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 8, or a modified sequence thereof containing a conservative amino acid substitution at position 9 and / or position 11. VLCDR2 has the sequence shown in Sequence ID No. 2, VLCDR3 has the sequence shown in Sequence ID 3, VHCDR1 has the sequence shown in Sequence ID No. 4, VHCDR2 has the sequence shown in Sequence ID No. 5, and VHCDR3 has the sequence shown in Sequence ID 6, and also, (ii) The second active agent is selected from thymidylate synthase inhibitors, nucleic acid base analogs, checkpoint inhibitors that block the interaction between PD-1 and PD-L1, and proteasome inhibitors. If necessary, the treatment of the cancer is a pharmaceutical composition in which a third active agent is used in combination with the specific binding molecule and the second active agent.

2. The pharmaceutical composition according to claim 1, wherein the second active agent is 5 FU.

3. The pharmaceutical composition according to claim 2, wherein the cancer is pancreatic cancer or colorectal cancer.

4. The pharmaceutical composition according to claim 1, wherein the second active agent is an antibody that binds to PD-1 or an antibody that binds to PD-L1.

5. The pharmaceutical composition according to claim 4, wherein the antibody that binds to PD-1 is nivolumab, pembrolizumab, semiprimab, or tislerizumab, or the antibody that binds to PD-L1 is atezolizumab, durvalumab, or avelumab.

6. The pharmaceutical composition according to claim 4, wherein the cancer is breast cancer or lung cancer.

7. The pharmaceutical composition according to claim 6, wherein the breast cancer is triple-negative breast cancer.

8. The pharmaceutical composition according to claim 1, wherein the second active agent is bortezomib, ixazomib, or carfilzomib.

9. The pharmaceutical composition according to claim 8, wherein the cancer is myeloma or mantle cell lymphoma.

10. A pharmaceutical composition for use in combination with a second active agent for the treatment of breast cancer in a subject, comprising a specific binding molecule that binds to human ANXA1, The specific binding molecule is as defined in claim 1, and the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. Preferably, the second active agent is selected from paclitaxel and cisplatin. If necessary, the treatment of breast cancer is a pharmaceutical composition in which a third active agent is used in combination with the specific binding molecule and the second active agent.

11. A pharmaceutical composition for use in combination with a second active agent for the treatment of pancreatic cancer in a subject, comprising a specific binding molecule that binds to human ANXA1, The specific binding molecule is as defined in claim 1, and the second active agent is a nucleoside analog, preferably gemcitabine. If necessary, the treatment of pancreatic cancer is a pharmaceutical composition in which a third active agent is used in combination with the specific binding molecule and the second active agent.

12. The pharmaceutical composition according to claim 11, wherein the second active agent is gemcitabine and the third active agent is paclitaxel.

13. Each of the aforementioned specific binding molecules has the following amino acid sequence, i.e., VLCDR1 has the sequence shown in Sequence ID No. 1, VLCDR2 has the sequence shown in Sequence ID No. 2, VLCDR3 has the sequence shown in Sequence ID 3, VHCDR1 has the sequence shown in Sequence ID No. 4, VHCDR2 has the sequence shown in Sequence ID No. 5, and The pharmaceutical composition according to any one of claims 1 or 10 to 12, wherein VHCDR3 has the sequence shown in Sequence ID No.

6.

14. The pharmaceutical composition according to claim 1 or any one of claims 10 to 12, wherein the specific binding molecule is an antibody or a fragment thereof.

15. (i) The antibody or fragment thereof is humanized, and / or (ii) The antibody is a monoclonal antibody, or the antibody fragment is a Fab antibody fragment, a Fab' antibody fragment, or F(ab') 2 Antibody fragments, or scFv molecules, The pharmaceutical composition according to claim 14.

16. The antibody or fragment thereof i) A light chain variable region comprising an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence that has at least 70% sequence identity to said sequence, ii) A heavy chain variable region comprising an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that has at least 70% sequence identity to said sequence, The pharmaceutical composition according to claim 15, comprising:

17. The aforementioned specific binding molecule i) A light chain comprising an amino acid sequence that includes the amino acid sequence shown in Sequence ID No. 13, or an amino acid sequence that has at least 70% sequence identity to said sequence, ii) A heavy chain containing the amino acid sequence shown in Sequence ID No. 14, or an amino acid sequence having at least 70% sequence identity to said sequence, The pharmaceutical composition according to claim 16, wherein the monoclonal antibody contains a monoclonal antibody.

18. The aforementioned specific binding molecule i) A light chain containing the amino acid sequence shown in Sequence ID No. 15, or a light chain containing an amino acid sequence having at least 70% sequence identity to said sequence, ii) A heavy chain containing the amino acid sequence shown in Sequence ID No. 16, or a sequence having at least 70% sequence identity to said sequence, The pharmaceutical composition according to claim 16, wherein the monoclonal antibody contains a monoclonal antibody.

19. (i) The cancer expresses ANXA1, (ii) The specific binding molecule, the second active agent, and optionally the third active agent, if present, are administered to the subject separately, simultaneously, or sequentially, and / or (iii) The subject is a human, A pharmaceutical composition according to claim 1 or any one of claims 10 to 12.

20. The use of a specific binding molecule that binds to human ANXA1 in the manufacture of a pharmaceutical for the treatment of cancer, wherein the specific binding molecule is as defined in claim 1, and the treatment of cancer comprises administering the pharmaceutical and a second active agent to a target, wherein the second active agent is selected from thymidylate synthase inhibitors, nucleoside analogs, checkpoint inhibitors that block the interaction between PD-1 and PD-L1, and proteasome inhibitors. Preferably, the cancer expresses ANXA1, and the specific binding molecule, the second active agent, and optionally the third active agent, if present, are administered to the subject separately, simultaneously, or sequentially, and / or the subject is human.

21. (i) The second active agent is 5FU and the cancer is pancreatic cancer or colorectal cancer, or (ii) The second active agent is an antibody that binds to PD-1 or an antibody that binds to PD-L1, preferably the antibody that binds to PD-1 is nivolumab, pembrolizumab, semiprimab, or tislerizumab, or the antibody that binds to PD-L1 is atezolizumab, durvalumab, or avelumab and the cancer is breast cancer or lung cancer. (iii) The second active agent is bortezomib, ixazomib, or carfilzomib, and the cancer is myeloma or mantle cell lymphoma, or (iv) A third active agent is used in the treatment of the aforementioned cancer. The use described in claim 20.

22. The use of a specific binding molecule that binds to human ANXA1 in the manufacture of a pharmaceutical for the treatment of breast cancer, wherein the specific binding molecule is as defined in claim 1, and the treatment of breast cancer comprises administering the pharmaceutical and a second active agent to a target, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. Preferably, the second active agent is selected from paclitaxel and cisplatin, the cancer expresses ANXA1, and the specific binding molecule, the second active agent, and optionally the third active agent, if present, are administered to the subject separately, simultaneously, or sequentially, the subject being human, and / or the third active agent is used to treat the breast cancer.

23. The use of a specific binding molecule that binds to human ANXA1 in the manufacture of a pharmaceutical product for the treatment of pancreatic cancer, wherein the specific binding molecule is as defined in claim 1, and the treatment of the pancreatic cancer comprises administering the pharmaceutical product and a nucleoside analog to the target. Preferably, the nucleoside analog is gemcitabine, the cancer expresses ANXA1, and the specific binding molecule and the second active agent, and optionally the third active agent, if present, are administered to the subject separately, simultaneously, or sequentially, the subject being human, and / or the third active agent is used to treat the breast cancer.

24. The use according to claim 23, wherein a third active agent is used in the treatment of the pancreatic cancer, the second active agent is gemcitabine, and the third active agent is paclitaxel.

25. A pharmaceutical composition comprising a specific binding molecule that binds to human ANXA1, a second active agent, optionally a third active agent, and one or more pharmaceutically acceptable diluents, carriers, or excipients, A pharmaceutical composition wherein the specific binding molecule is as defined in claim 1, the second active agent is as defined in claim 1, preferably 5FU, and the third active agent, if present, is preferably paclitaxel.

26. A kit comprising a specific binding molecule that binds to human ANXA1, a second active agent, and optionally a third active agent, wherein the specific binding molecule is as defined in claim 1, the second active agent is as defined in claim 1, and the third active agent, if present, is preferably paclitaxel.

27. A product comprising a specific binding molecule that binds to human ANXA1 as defined in claim 1, a second active agent, and optionally a third active agent, for use separately, simultaneously, or sequentially in the treatment of cancer in a subject, wherein the second active agent is selected from thymidylate synthase inhibitors, nucleoside base analogs, checkpoint inhibitors that block the interaction between PD-1 and PD-L1, and proteasome inhibitors.

28. (i) The second active agent is 5FU and the cancer is pancreatic cancer or colorectal cancer, or (ii) The second active agent is an antibody that binds to PD-1 or an antibody that binds to PD-L1, preferably the antibody that binds to PD-1 is nivolumab, pembrolizumab, semiprimab, or tislerizumab, or the antibody that binds to PD-L1 is atezolizumab, durvalumab, or avelumab and the cancer is breast cancer or lung cancer, preferably the breast cancer is triple-negative breast cancer, or (iii) The product according to claim 27, wherein the second active agent is bortezomib, ixazomib, or carfilzomib, and the cancer is myeloma or mantle cell lymphoma.

29. A product comprising a specific binding molecule that binds to human ANXA1 as defined in claim 1, a second active agent, and optionally a third active agent, for use separately, simultaneously, or sequentially in the treatment of breast cancer in a subject, wherein the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. Preferably, the second active agent is selected from paclitaxel and cisplatin in the product.

30. A product comprising a specific binding molecule that binds to human ANXA1 as defined in claim 1, a nucleoside analog, and optionally a third active agent, for use separately, simultaneously, or sequentially in the treatment of pancreatic cancer in a subject, Preferably, the nucleoside analog is gemcitabine and / or the third active agent is paclitaxel, in the product.

31. A pharmaceutical composition for use in the treatment of cancer in a subject, comprising a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule is as defined in Claim 1, and the second active agent is selected from thymidylate synthase inhibitors, nucleic acid base analogs, checkpoint inhibitors that block the interaction between PD-1 and PD-L1, and proteasome inhibitors. A pharmaceutical composition comprising, if necessary, a third active agent.

32. A pharmaceutical composition for use in the treatment of breast cancer in a subject, comprising a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule is as defined in Claim 1, and the second active agent is selected from taxanes and platinum-based chemotherapeutic agents. Preferably, the second active agent is selected from paclitaxel and cisplatin, and the pharmaceutical composition optionally comprises a third active agent.

33. A pharmaceutical composition for use in the treatment of pancreatic cancer in a subject, comprising a specific binding molecule that binds to human ANXA1 and a second active agent, wherein the specific binding molecule is as defined in Claim 1, and the second active agent is a nucleoside analog, preferably gemcitabine. The pharmaceutical composition optionally comprises a third active agent, preferably paclitaxel.

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