Antibody against fibroblast growth factor receptor 2b and use thereof

Abstract

The present invention relates to an antibody targeting fibroblast growth factor receptor 2b (FGFR2b). The present invention further relates to a detection system comprising such antibodies for diagnosing diseases and / or disorders associated with abnormal expression of FGFR2b.

Description

Antibodies against fibroblast growth factor receptor 2b and uses thereof TECHNICAL FIELD

[0001] The present invention relates to antibodies targeting fibroblast growth factor receptor 2b (FGFR2b), and detection systems comprising such antibodies, for the diagnosis of diseases and / or disorders associated with abnormal expression of FGFR2b. BACKGROUND

[0002] The fibroblast growth factor receptor (FGFR) family of tyrosine kinases (RTKs) consists of fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3 and FGFR4 and encompasses high affinity receptors for up to 18 different FGF ligands. Fibroblast growth factor receptors are transmembrane tyrosine kinases, comprising an extracellular region, a transmembrane region and an intracellular kinase region. FGFR signaling drives downstream pathways, including the mitogen-activated protein kinase (MAPK) and AKT pathways, which are critical for cell proliferation, differentiation, survival and migration. Binding of FGF to the receptor induces dimerization of the FGF:FGFR complex. Dimerization leads to kinase activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of FGFR, and activation of downstream signaling of the phosphoinositide 3-kinase (PI3K)-AKT and MAPK extracellular signal-regulated kinase (ERK) pathways.

[0003] Aberrant expression (e.g., overexpression) of FGFR2 has been reported in various cancers. FGFR2 overexpression affects signaling without altering the endogenous kinase activity of the receptor. FGFR2 protein is overexpressed in about 3% of breast cancers, including triple-negative breast cancers, and about 10% of gastric / esophageal cancers. FGFR2 overexpression has also been found in other cancers, including colon cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, glioma and head and neck cancer. In addition, mutations in the FGFR2 gene have been reported in about 12% of endometrial cancers. FGFR2 overexpression has been associated with poor survival rates in patients with gastric cancer.

[0004] The extracellular region of FGFR2 comprises three immunoglobulin-like domains, IgI, IgII and IgIII, of which IgIII is the region where the extracellular ligand binds primarily, encoded by exons 7, 8, 9. Alternative splicing of exons 7-9 of the FGFR2 gene results in alternative splice isoforms of the IgIII loop, FGFRIIIb or FGFRIIIc. Normally, FGFR2-IIIb (also known as FGFR2(beta)IIIb, FGFR2b) is expressed mainly in epithelial cells and is able to bind FGF1 and KGF family of cell growth factors (FGF7, FGF10, FGF22) with high affinity; FGFR2-IIIc (also known as FGFR2(beta)IIIc, FGFR2c) is expressed mainly in mesenchymal cells and is able to bind FGF1 and FGF2 with high affinity. FGFR2IIIb and FGFR2IIIc bind different extracellular ligands.

[0005] Immunohistochemistry (IHC) is a routine technique used in medical diagnostics to determine the presence of a protein in a tissue. Prior to performing IHC analysis, tumor tissue is typically formalin-fixed and paraffin-embedded. Typically, therapeutic antibodies targeting FGFR2b do not efficiently detect endogenously expressed FGFR2b in tumor tissue, and thus, diagnostic FGFR2b antibodies should be able to efficiently detect expressed FGFR2b in tissue samples. However, currently known anti-FGFR2b antibodies do not specifically and efficiently detect endogenous FGFR2b in formalin-fixed paraffin-embedded tissue samples. Therefore, there remains a need in the art for detection and diagnostic means that can accurately and reliably detect the expression and distribution of the FGFR2b target antigen in pathological tissue of a patient or suspected patient. Molecular typing is of prognostic significance for cancer diagnosis and prognosis, but current molecular typing is not fine enough to guide gastric cancer targeted therapy. The present application solves this problem to some extent. SUMMARY

[0006] To meet the above needs, the present application provides an antibody HDM-45 which binds to FGFR2b. HDM-45 is suitable for clinical pathological detection and can be used on tissue samples in diagnostic methods obtained from individuals, particularly from patients suspected of suffering from a neoplastic disease associated with and / or identifiable by abnormal FGFR2b expression. The antibody can also be used as a diagnostic tool for detecting FGFR2b in laboratory animals. As shown in the examples of the present application, the antibody of the present application has the characteristics of high specificity and high sensitivity. The application of the antibody of the present application can effectively detect tumors with abnormal expression of FGFGR2b but not FGFGR2c in immunohistochemical (IHC) detection, thereby allowing diagnosis of the relevant specific cancer / tumor, identifying FGFGR2b-positive cancer patients suitable for receiving anti-FGFGR2b drug treatment, and predicting the treatment responsiveness of patients to anti-FGFGR2b drugs and evaluating prognosis.

[0007] The present application further provides a polynucleotide encoding the antibody, a vector comprising the polynucleotide, a host cell expressing the polynucleotide, and a diagnostic composition comprising the same, and a kit comprising the antibody of the present application as a diagnostic tool and other components including but not limited to at least one of a secondary antibody, an enzyme, a buffer, an instruction manual, a calibration tool, a control, etc.

[0008] In one aspect, the present application provides an antibody or an antigen-binding fragment thereof which specifically binds to FGFR2b, wherein the antibody binds to cell surface FGFR2b.

[0009] The present application provides an antibody or an antigen-binding fragment thereof which specifically binds to FGFR2b, wherein the antibody contains or consists of a VH region having at least 90% identity to the sequence set forth in SEQ ID NO: 1, and / or the antibody contains or consists of a VL region having at least 90% identity to the sequence set forth in SEQ ID NO: 2.

[0010] The present application provides an antibody or an antigen-binding fragment thereof which specifically binds to FGFR2b, wherein the antibody contains a heavy chain variable region containing HCDR1 of SEQ ID NO: 3; and / or the antibody contains HCDR2 of SEQ ID NO: 4; and / or the antibody contains HCDR3 of SEQ ID NO: 5.

[0011] The antibody or antigen-binding fragment thereof that specifically binds to FGFR2b provided herein, wherein the antibody comprises a light chain variable region comprising a LCDR1 of the sequence of SEQ ID NO: 6; and / or, the antibody comprises a LCDR2 of the sequence of SEQ ID NO: 7; and / or, the antibody comprises a LCDR3 of the sequence of SEQ ID NO: 8.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VH region as set forth in SEQ ID NO: 1 comprising a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 4, and a HCDR3 as set forth in SEQ ID NO: 5.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VL region as set forth in SEQ ID NO: 2 comprising a LCDR1 as set forth in SEQ ID NO: 6, a LCDR2 as set forth in SEQ ID NO: 7, and a LCDR3 as set forth in SEQ ID NO: 8.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VH region as set forth in SEQ ID NO: 1 comprising a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 4, and a HCDR3 as set forth in SEQ ID NO: 5; and a VL region as set forth in SEQ ID NO: 2 comprising a LCDR1 as set forth in SEQ ID NO: 6, a LCDR2 as set forth in SEQ ID NO: 7, and a LCDR3 as set forth in SEQ ID NO: 8.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b provided herein is a monoclonal antibody.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b provided herein specifically binds to human FGFR2b in an immunohistochemistry assay, to cells expressing FGFR2b in a fluorescence activated cell sorting assay, to fixed and permeabilized cells expressing FGFR2b, optionally, it does not bind to huFGFR2c.

[0017] In some embodiments, the antibody provided herein is an IgG, IgD, IgA, IgE, or IgM antibody, preferably an IgG antibody, such as an IgGl, IgG2, IgG3, or IgG4 antibody.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof specifically binding to FGFR2b according to the present application comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence as set forth in SEQ ID NO: 9, or comprises a sequence at least 90% identical to SEQ ID NO: 9, or consists of SEQ ID NO: 9; and the light chain comprises a sequence as set forth in SEQ ID NO: 10, or comprises a sequence at least 90% identical to SEQ ID NO: 10, or consists of SEQ ID NO: 10.

[0019] As a facet of the present application, the present application further relates to a polynucleotide encoding any of the aforesaid antibodies.

[0020] As a facet of the present application, the present application further relates to a vector comprising the aforesaid polynucleotide.

[0021] As a facet of the present application, the present application further relates to a host cell transformed or transfected with the aforesaid polynucleotide or vector.

[0022] In some embodiments, the present application further relates to a method for producing the aforesaid antibody, comprising culturing the aforesaid host cell under conditions permitting expression of the antibody and recovering the antibody produced from the culture.

[0023] As a facet of the present application, the present application further relates to a composition comprising the aforesaid antibody or the aforesaid polynucleotide.

[0024] As a facet of the present application, the present application further relates to a detection system comprising the aforesaid antibody or the antibody produced by the aforesaid method.

[0025] As a facet of the present application, the present application further provides a detection method, comprising contacting a sample with any of the aforesaid antibodies to detect whether the sample comprises FGFR2b, optionally, to detect the expression level of FGFR2b in the sample.

[0026] As a facet of the present application, the present application further relates to a diagnostic method, comprising detecting whether a sample from a subject comprises FGFR2b using the aforesaid antibody or the aforesaid detection system, thereby diagnosing whether the subject is suffering from a disease associated with abnormal expression of FGFR2b.

[0027] In some embodiments, the present application further relates to use of the aforesaid antibody or the antibody produced by the aforesaid method or the aforesaid detection system in the preparation of a diagnostic reagent for a diagnostic method.

[0028] As one aspect of the application, the present application further relates to a method of detecting a neoplastic growth. In some embodiments, the present application also relates to one or more uses of the aforementioned antibody, or of the antibody produced by the aforementioned method, or of the aforementioned detection system, in a method of detecting a neoplastic growth.

[0029] In some embodiments, the method of detecting a neoplastic growth comprises determining the amount of FGFR2b expression in a sample from a patient suspected of having a cancer and a negative control, and optionally a positive control.

[0030] In some embodiments, the method of detecting a neoplastic growth comprises comparing the amount of FGFR2b expression between the samples, optionally wherein the amount of expression in the negative control and / or the positive control can be derived from stored data in at least one positive control and / or negative control obtained in the method of detecting a neoplastic growth.

[0031] In some embodiments, the method of detecting a neoplastic growth comprises determining the amount of FGFR2b expression, further optionally wherein the amount of expression in the negative control sample and / or the positive control sample can be derived from stored data comprising the average amount of expression in a plurality of negative control samples and / or a plurality of positive control samples obtained in the method of detecting a neoplastic growth.

[0032] In some embodiments, the method of detecting a neoplastic growth comprises determining the amount of FGFR2b expression, wherein the sample is a solid tissue sample and / or a liquid tissue sample.

[0033] In some embodiments, the present application provides a method for identifying a cell expressing FGFR2b, the method comprising contacting the cell with an antibody of the present application and detecting specific binding of the antibody to the cell.

[0034] In some embodiments, the present application provides a method for identifying the presence of FGFR2b in a tissue sample, the method comprising contacting the tissue sample with an antibody of the present application and detecting specific binding of the antibody to the tissue, identifying the tissue as expressing FGFR2b.

[0035] In some embodiments, the present application provides a method for identifying a subject having or at risk of having a disease associated with aberrant expression of FGFR2b, the method comprising contacting a tissue sample of the subject with an antibody of the present application and detecting specific binding of the antibody to the tissue, thereby identifying whether the subject has or is at risk of having a disease associated with aberrant expression of FGFR2b.

[0036] In some embodiments, the present application relates to a method for detecting and / or quantifying FGFR2b expression in a sample, the method comprising the steps of:

[0037] (a) using an antibody according to any one of the preceding antibodies of the application or produced according to the preceding method of the application, or using a detection system according to the preceding of the application, for determining the amount of FGFR2b expression in a sample;

[0038] (b) comparing the amount of FGFR2b expression determined in step (a) to (i) a predefined value for the amount of FGFR2b expression, (ii) the amount of FGFR2b expression determined in a control sample, or (iii) the amount of FGFR2b expression determined in a sample obtained from the same source or subject at a previous time point.

[0039] In some embodiments, the application relates to a method for diagnosing a neoplastic disease associated with FGFR2b expression or increased FGFR2b expression, the method comprising the steps of:

[0040] (a) using an antibody according to any one of the preceding antibodies of the application or produced according to the preceding method of the application, or using a detection system according to the preceding of the application, for determining the amount of FGFR2b expression in a sample;

[0041] (b) comparing the amount of FGFR2b expression determined in step (a) to (i) a predefined threshold value for the amount of FGFR2b expression indicative of the absence of such disease, or (ii) the amount of FGFR2b expression determined in a negative control sample representative of the absence of such neoplastic disease, wherein a higher amount of FGFR2b expression determined in step (a) compared to the predefined threshold value of (i) or the amount of FGFR2b expression determined in the negative control sample of (ii) is indicative of the presence of a disease associated with FGFR2b expression or increased FGFR2b expression.

[0042] In some embodiments, the application relates to a method for monitoring the progression of a disease associated with FGFR2b expression or with increased FGFR2b expression or for monitoring the response to a treatment of a disease associated with FGFR2b expression or with increased FGFR2b expression, the method comprising the steps of:

[0043] (a) using an antibody according to any one of the preceding antibodies of the application or produced according to the preceding method of the application, or using a detection system according to the preceding of the application, for determining the amount of FGFR2b expression in a sample obtained from a subject diagnosed with such disease at a first time point;

[0044] (b) using an antibody according to any one of the preceding antibodies of the application or produced according to the preceding method of the application, or using a detection system according to the preceding of the application, for determining the amount of FGFR2b expression in a sample obtained from the subject at a second time point or after treatment; and

[0045] (c) comparing the FGFR2b expression level determined in step (a) with the FGFR2b expression level determined in step (b);

[0046] wherein a higher FGFR2b expression level determined in step (b) compared to the FGFR2b expression level determined in step (a) is indicative of the disease progressing, and / or wherein a lower FGFR2b expression level determined in step (b) compared to the FGFR2b expression level determined in step (a) is indicative of the disease entering remission or the disease responding to the treatment.

[0047] In some embodiments, the aforementioned diagnostic methods and uses of the present application, the sample used is a biological sample, preferably a human biological sample, such as a tissue sample or a sample comprising cultured cells.

[0048] In some embodiments, the aforementioned diagnostic methods and uses of the present application, the sample used is obtained from a human subject, preferably a human subject suspected of having or having a disease associated with FGFR2b expression or increased FGFR2b expression level, or a subject who has received a treatment for a disease associated with FGFR2b expression or increased FGFR2b expression level.

[0049] In some embodiments, the aforementioned diagnostic methods and uses of the present application, wherein the disease is selected from the group consisting of lung cancer, lung squamous carcinoma, lung adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, intrahepatic cholangiocellular carcinoma, bladder cancer, colorectal cancer, prostate cancer, cervical cancer, colon cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, renal cell carcinoma, glioma, head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, oral mucosa cancer, and urothelial cancer; preferably the disease is gastric cancer, breast cancer, lung cancer.

[0050] In some embodiments, any of the foregoing antibodies of the application and any of the foregoing diagnostic methods and uses of the application are used in an immunohistochemical method (IHC method) for detecting FGFR2b expression, which IHC method comprises the step of providing a tissue sample (including liquid biopsy material, such as cells obtained from an individual or animal or from a cell culture). In some embodiments, the sample material is typically provided on a support, e.g. a carrier such as a glass or plastic slide, and optionally, the tissue can be fixed using a fixation medium known in the art, such as paraffin, ethanol, or acetone, or any other suitable medium ensuring fixation of the tissue or cells on the support. In some embodiments, the method further comprises the step of preparing the sample, e.g. deparaffinization, with the aim of allowing access of the antibodies described in the application to the target FGFR2b of interest. In some embodiments, the method further comprises the step of incubating the tissue sample with a culture medium comprising a sufficient amount of the antibodies disclosed in the application to allow binding of these antibodies to FGFR2b present in the tissue sample. After a generally known and reasonable incubation time, the reaction of the tissue sample with the antibodies is generally stopped by washing the support and tissue sample in a medium, such as PBS. To check the influence of the incubation medium, fixation medium, incubation time and temperature, tests are generally performed using positive control material known to express the target FGFR2b and a negative control of a sample known not to express FGFR2b. The antibodies are added to the positive control material in a titration assay, whereby the concentration can be determined that prevents the antibodies of the application from specifically and / or selectively detecting the protein FGFR2b. Determining the correct amount of antibodies to perform proper positive and negative controls is known to the skilled person in the art of immunocytochemistry and in particular immunohistochemistry. Once the appropriate controls are available, the test material can be analyzed, e.g. material obtained from a patient suspected of having a disease that can be characterized by pathologically enhanced / distributed FGFR2b expression, such as a cancer. As discussed further below, the antibodies can carry a detectable label, or they can be recognized by another conjugate carrying such a label. The terminology for these techniques is referred to as direct and indirect immunocytochemistry / immunohistochemistry detection. Once the required incubation steps and washing steps have been performed on the tissue sample and controls, the tissue can be prepared for analysis, e.g. using an immunofluorescence-based detection system. The intensity and amount of detectable label can be determined and compared to the positive and negative controls and / or to known standard values. Based thereon, the scientist is able to draw a conclusion as to whether the sample is FGFR2b expression positive or not.

[0051] As one aspect of the application, the application also provides a kit comprising an antibody specifically binding FGFR2b as previously described in the application, or a composition thereof.

[0052] In some embodiments, detecting the amount of FGFR2b in a sample means contacting the sample with an FGFR2b antibody of the present application.

[0053] In some embodiments, detecting the amount of FGFR2b in a sample means determining the presence or absence of FGFR2b in the sample by binding of an FGFR2b antibody of the present application.

[0054] In some embodiments, detecting the amount of FGFR2b in a sample means determining the level or amount of FGFR2b expressed in the sample by binding of an FGFR2b antibody of the present application.

[0055] The present application provides a first monoclonal antibody (or an antigen-binding fragment thereof) of a detection system:

[0056] (a) a VH region as depicted in SEQ ID NO: 1 comprising a HCDR1 as depicted in SEQ ID NO: 3, a HCDR2 as depicted in SEQ ID NO: 4 and a HCDR3 as depicted in SEQ ID NO: 5; and, a VL region as depicted in SEQ ID NO: 2 comprising a LCDR1 as depicted in SEQ ID NO: 6, a LCDR2 as depicted in SEQ ID NO: 7 and a LCDR3 as depicted in SEQ ID NO: 8;

[0057] (b) a monoclonal antibody that binds to the same FGFR2b protein as the antibody of (a) or competes for binding to FGFR2b with the antibody of (a).

[0058] The present application also provides a monoclonal antibody of a detection system:

[0059] (a) comprising a VH region as depicted in SEQ ID NO: 1 ;

[0060] (b) comprising a VL region as depicted in SEQ ID NO: 2;

[0061] (c) comprising a VH region as depicted in SEQ ID NO: 1 and comprising a VL region as depicted in SEQ ID NO: 2;

[0062] (d) a monoclonal antibody that binds to the same FGFR2b protein as the antibody of c) or competes for binding to FGFR2b with the antibody of c); or

[0063] The present application also provides the use of an antibody of the present application for or the use of a detection system of the present application for:

[0064] 1. detecting FGFR2b in a sample;

[0065] 2. quantifying FGFR2b in a sample;

[0066] 3. diagnosing a disease associated with increased FGFR2b;

[0067] 4. stratifying a patient diagnosed with a disease associated with increased FGFR2b;

[0068] 5. monitoring the progression of a disease associated with increased FGFR2b; or

[0069] 6. monitoring the response to treatment of a disease associated with increased FGFR2b.

[0070] The present application also provides a method for detecting and / or quantifying FGFR2b in a sample, the method comprising the steps of:

[0071] (a) determining the FGFR2b content in the sample using an antibody of the present application or using a detection system of the present application; and

[0072] (b) comparing the FGFR2b content determined in step (a) to (i) a predefined value for the FGFR2b content, (ii) the FGFR2b content determined in a control sample, or (iii) the FGFR2b content determined in a sample obtained from the same source or subject at a previous time point.

[0073] The present application provides a method for diagnosing a disease associated with increased FGFR2b, the method comprising the steps of:

[0074] (a) determining the FGFR2b content in the sample using an antibody of the present application or using a detection system of the present application; and

[0075] (b) comparing the FGFR2b content determined in step (a) to (i) a predefined threshold value for the FGFR2b content indicative of the absence of such a disease, or (ii) the FGFR2b content determined in a control sample representative of the absence of such a disease, wherein a higher FGFR2b content determined in step (a) compared to the predefined threshold value of (i) or the FGFR2b content determined in the control sample of (ii) is indicative of the presence of a disease associated with increased FGFR2b.

[0076] The present application also provides a method for monitoring the progression of a disease associated with increased FGFR2b or for monitoring the response to treatment of a disease associated with increased FGFR2b, the method comprising the steps of:

[0077] (a) determining the FGFR2b content in a biological sample obtained from a subject diagnosed with such a disease at a first time point using an antibody of the present application or using a detection system of the present application;

[0078] (b) determining the FGFR2b content in a biological sample obtained from the subject at a second (later) time point or after treatment using an antibody of the application, or using a detection system of the application; and

[0079] (c) comparing the FGFR2b content determined in step (a) with the FGFR2b content determined in step (b);

[0080] wherein a higher FGFR2b content determined in step (b) compared to the FGFR2b content determined in step (a) is indicative of the disease progressing, and / or wherein a lower FGFR2b content determined in step (b) compared to the FGFR2b content determined in step (a) is indicative of the disease going into remission or the disease responding to the treatment.

[0081] The antibody or antigen-binding fragment thereof of the application that specifically binds FGFR2b comprises a VH region having at least 75% or 80% homology, preferably at least 85%, 90%, 91 %, 92%, 93%, 94% homology, more preferably 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 1.

[0082] The VH of the FGFR2b antibody or antigen-binding fragment thereof of the application comprises a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 4 and a HCDR3 as set forth in SEQ ID NO: 5.

[0083] The antibody or antigen-binding fragment thereof of the application that specifically binds FGFR2b comprises a VL region having at least 75% or 80% homology, preferably at least 85%, 90%, 91 %, 92%, 93%, 94% homology, more preferably 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 2.

[0084] The VL of the FGFR2b antibody or antigen-binding fragment thereof of the application comprises a LCDR1 as set forth in SEQ ID NO: 6, a LCDR2 as set forth in SEQ ID NO: 7 and a LCDR3 as set forth in SEQ ID NO: 8.

[0085] The antibody or antigen-binding fragment thereof according to the present application that specifically binds to FGFR2b comprises a VH region having at least 75% or 80% homology, preferably at least 85%, 90%, 91%, 92%, 93%, 94% homology, more preferably 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 1, which comprises a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 4 and a HCDR3 as set forth in SEQ ID NO: 5.

[0086] The antibody or antigen-binding fragment thereof according to the present application that specifically binds to FGFR2b comprises a VL region having at least 75% or 80% homology, preferably at least 85%, 90%, 91%, 92%, 93%, 94% homology, more preferably 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 2, which comprises a LCDR1 as set forth in SEQ ID NO: 6, a LCDR2 as set forth in SEQ ID NO: 7 and a LCDR3 as set forth in SEQ ID NO: 8.

[0087] The antibody or antigen-binding fragment thereof according to the present application that specifically binds to FGFR2b comprises

[0088] (a) a VH region as set forth in SEQ ID NO: 1;

[0089] (b) a VL region as set forth in SEQ ID NO: 2;

[0090] (c) a VH region as set forth in SEQ ID NO: 1 and a VL region as set forth in SEQ ID NO: 2;

[0091] (d) a monoclonal antibody that binds to the same FGFR2b protein as the antibody of c) or competes for binding to FGFR2b with the antibody of c).

[0092] In some embodiments, the competition for binding to FGFR2b by the antibody or antigen-binding fragment thereof according to the present application is defined as a competition that occurs between at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the two test antibodies.

[0093] The present application provides a composition comprising the antibody or antigen-binding fragment thereof according to the present application that specifically binds to FGFR2b.

[0094] The present application provides a detection system comprising:

[0095] (a) a first monoclonal antibody that binds to FGFR2b according to the present application, and / or

[0096] (b) a second monoclonal antibody that binds to the same FGFR2b as the antibody of (a) or competes for binding to FGFR2b with the antibody of (a);

[0097] wherein the binding of the first monoclonal antibody to FGFR2b occurs in the presence of the second monoclonal antibody that binds to FGFR2b, and / or wherein the binding of the second monoclonal antibody to FGFR2b occurs in the presence of the first monoclonal antibody that binds to FGFR2b.

[0098] In some embodiments, the detection system according to the present application, the first monoclonal antibody and / or the second monoclonal antibody comprises a mouse VH region and / or a mouse VL region.

[0099] In some embodiments, the detection system according to the present application, wherein the affinity (KD) of the first monoclonal antibody and / or the second monoclonal antibody for FGFR2b is about < 10 -7 M, < 10 -8 M, < 10 -9 M, or < 10 -10 M.

[0100] In some embodiments, the detection system according to the present application, wherein the first monoclonal antibody comprises:

[0101] (a) a VH region as set forth in SEQ ID NO: 1 comprising a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 4 and a HCDR3 as set forth in SEQ ID NO: 5; and, a VL region as set forth in SEQ ID NO: 2 comprising a LCDR1 as set forth in SEQ ID NO: 6, a LCDR2 as set forth in SEQ ID NO: 7 and a LCDR3 as set forth in SEQ ID NO: 8;

[0102] (b) a monoclonal antibody that binds to the same FGFR2b protein as the antibody of (a) or competes for binding to FGFR2b with the antibody of (a).

[0103] The present application provides a diagnostic kit comprising a FGFR2b antibody or antigen binding fragment thereof according to the present application.

[0104] The present application also provides a kit for a single dose administration unit. The kit of the present application can also contain a first container comprising a dried / lyophilized FGFR2b antibody or composition of the present application and a second container comprising an aqueous formulation.

[0105] Definitions of Terms

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the following terms are defined below.

[0107] The term "about" when used in connection with a numerical value means encompassing numerical values within a range having a lower limit that is 10% (preferably 5%) less than the specified numerical value and an upper limit that is 10% (preferably 5%) greater than the specified numerical value.

[0108] The term "and / or" when used in connection with a list of items means that any one of the items in the list or a combination of any two or more of the items in the list can be employed.

[0109] As used herein, the term "comprising" or "including," or "having" means including, but not limited to, the recited elements, integers, or steps, etc. In this connection, the term "comprising" or "including" or "having" also covers the case where the recited elements, integers, or steps, etc. are comprised or included in the present application, unless otherwise indicated. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to cover an antibody variable region consisting of the particular sequence.

[0110] The present application provides an antibody (or a portion or derivative thereof) that binds to FGFR2b. Whenever the term "antibody" (i.e., an antibody that binds to FGFR2b) is used in the present application, it encompasses "antibody fragments". The definition and description of "antibody" (e.g., a monoclonal antibody that binds to FGFR2b) of the present application provided below applies analogously to any chemically or enzymatically modified antibody, e.g., an antibody carrying a label such as a fluorescent, radioactive, luminescent or chromogenic label or an enzyme capable of producing a detectable signal.

[0111] An "antibody" (sometimes also referred to as an immunoglobulin) is a polypeptide that immunospecifically binds to its target, comprising at least a light or heavy immunoglobulin variable region. Antibodies recognize unique targets, known as antigens, through their variable regions. An "antibody" can be of any immunoglobulin isotype, including IgG (including IgGl, IgG2, IgG3, and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM, and IgE. The term "antibody" can include, for example, monoclonal antibodies, chimeric antibodies, recombinant antibodies, de-immunized antibodies, affinity matured antibodies, humanized antibodies, and human antibodies, as well as antibodies from other species such as rodents, rabbits, mice, rats, hamsters, goats, and the like. Antibodies can be derived from only a single source, or can be "chimeric," that is, different portions of the antibody (such as the CDRs, framework regions, variable regions, constant regions) can be derived from two different antibodies. The definition of "antibody" according to the present application includes full-length antibodies, but also camelid antibodies and other immunoglobulins generated by biotechnological or protein engineering methods or processes. Antibodies can also be produced in hybridomas.

[0112] A "full antibody" (used interchangeably herein with "full-length antibody", "complete antibody" and "intact antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable region is the domain of the heavy or light chain of an antibody that is involved in binding the antibody to its antigen. The constant region does not participate in the binding of the antibody to the antigen, but exhibits various effector functions. The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be assigned to one of five different types, depending on the amino acid sequence of its constant region: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called a, d, e, g, and m, respectively.

[0113] The term "isotype" refers to the antibody class that is determined by the heavy chain constant region. For example, an antibody according to the present application can be an IgA (e.g., IgAl or IgA2), IgGl, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibody. In some embodiments, an antibody according to the present application is an IgGl antibody. More preferably, an IgGl antibody according to the present application has a non-human (e.g., murine-derived) IgGl constant region.

[0114] The Fc region of an antibody is the "tail" region of a classical antibody that interacts with cell surface receptors and some proteins of the complement system known as Fc receptors. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains (CH2 and CH3) of the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4) in each polypeptide chain. The Fc region also contains a portion of the so-called "hinge" region that is held together by one or more disulfide bonds and non-covalent interactions. The Fc region of naturally occurring IgG has highly conserved N-glycosylation sites. The glycosylation of the Fc fragment is required for Fc receptor-mediated activity.

[0115] The monoclonal antibodies of the present invention can be IgG, IgD, IgE, IgM or IgA antibodies. In some embodiments, the monoclonal antibodies are IgG antibodies, such as IgG1, IgG2, IgG3 or IgG4 antibodies. The isotype and subclass of the antibodies can be those of mouse, rabbit, hamster, etc. (e.g., mouse IgG, mouse IgG1, etc.).

[0116] In the present invention, the term "variable region" refers to those parts of an antibody or immunoglobulin domain that exhibit its sequence variability and are involved in determining the specificity and binding affinity of a particular antibody (i.e., "one or more variable regions"). Typically, the pairing of the heavy chain variable region (VH) and the light chain variable region (VL) together forms a single antigen binding site. Variability is not evenly distributed throughout the variable region of an antibody; it is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable region are called "framework" (FR) regions and provide a scaffold for the six CDRs in three-dimensional space to form an antigen binding surface. The variable regions of naturally occurring antibody heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4) that primarily adopt a β-sheet configuration. Together with the CDRs, they form the following sequence within the variable heavy or light chain: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable regions in each chain are held together in close proximity by framework regions and, often together with the hypervariable regions from another chain, contribute to the formation of the antigen-binding site.

[0117] The term "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs in a variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures.

[0118] It should be noted that the boundaries of CDRs of a variable region of the same antibody can vary slightly based on the assignment system used. That is, the CDR sequences of the same antibody variable region defined under different assignment systems can vary slightly. Thus, where an antibody is defined in terms of specific CDR sequences as defined herein, the scope of the antibody also encompasses antibodies whose variable region sequences contain the specific CDR sequences recited but whose CDR boundaries differ from the specific CDR boundaries defined herein due to the application of different schemes (e.g., different assignment systems or combinations).

[0119] Unless otherwise indicated, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above.

[0120] CDRs can also be determined based on having the same Kabat or AbM numbering position as a reference CDR sequence, such as any of the CDRs exemplified herein. In some embodiments, the CDRs of the antibodies of the application are determined according to the Kabat or AbM numbering scheme.

[0121] The term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications that can be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (or epitopes), each monoclonal antibody is directed against a single antigenic site or determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, so that they are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0122] For preparation of the monoclonal antibodies, any technique which provides the antibodies produced by the continuous cell line culture can be used. For example, the monoclonal antibodies or binding domains to be used can be prepared by the hybridoma method first described by Koehler et al. Nature, 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Examples of additional techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.

[0123] Standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE) analysis can be used to screen the hybridomas to identify one or more hybridomas that produce an antibody or binding domain that immunospecifically binds to the specified antigen.

[0124] Exemplary methods of making antibodies or antigen-binding fragments thereof include screening protein expression libraries, such as phage display or ribosome display libraries. In addition to using display libraries, a non-human animal, such as a rodent (such as a mouse, hamster, rabbit, or rat), can be immunized with the relevant antigen.

[0125] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, e.g., humanized, de-immunized, chimerized, etc. Examples of modified antibodies, constructs or binding domains include humanized variants of non-human antibodies / antibody constructs, "affinity matured" antibodies, constructs or binding domains, and antibody variants or mutants with one or more altered effector functions.

[0126] The present application also contemplates amino acid sequence modifications of the antibodies described herein. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by peptide synthesis or by introducing appropriate nucleotide changes into the nucleic acid molecule encoding the antibody. All of the following described amino acid sequence modifications are intended to result in antibodies that retain the biological activity required of the unmodified parent molecule (bind to FGFR2b).

[0127] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (lie or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), with modified, synthetic, or rare amino acids being used as desired.

[0128] Amino acid modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequence of the monoclonal antibody or binding domain. Any combination of deletion, insertion, and / or substitution is made to arrive at the final monoclonal antibody or binding domain, provided that the final antibody possesses the desired characteristics, e.g., biological activity of the unmodified parent molecule (such as binding to FGFR2b). The amino acid changes also can alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

[0129] For example, 1, 2, 3, 4, 5, or 6 amino acids can be inserted, deleted, and / or substituted in each CDR (depending on their respective lengths), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be inserted, deleted, and / or substituted in each FR. Amino acid sequence insertions also include N-terminal and / or C-terminal additions of from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides of, for example, more than 10, such as one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0130] Amino acid modifications (particularly for amino acid substitutions) are most interesting at positions which are known or suspected to be involved in binding of the antibody to its antigen. Such positions include those in the hypervariable regions, particularly in the respective CDRs of the heavy and / or light chains, but the application also contemplates FR changes in the heavy and / or light chains. Substitutions can be conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted in the CDRs, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be substituted in the framework regions (FRs), depending on the length of the CDR or FR, respectively. For example, if a CDR sequence encompasses 6 amino acids, then one, two, or three of these amino acids are envisaged to be substituted. Similarly, if a CDR sequence encompasses 15 amino acids, then one, two, three, four, five, or six of these amino acids are envisaged to be substituted. Generally, if amino acids are substituted in one or more or all CDRs of the heavy and / or light chain / variable region, then the "substituted" sequence obtained when is envisaged to have at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identity / homology to the "original" or "parental" CDR sequence.

[0131] “Sequence identity,”“sequence homology” are used interchangeably herein to refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis in a comparison window. The percent “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity. The optimal alignment of sequences for determining percent sequence identity can be achieved by using a variety of algorithms known in the art, such as those of Needleman et al. (1970) J. Mol. Biol. 48:443-453; or Smith et al. (1990) J. Mol. Biol. 215:403-410; or by using the computer software of Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; or by using the computer software of Altschul et al. (1990) J. Mol. Biol. 215:403-410. Appropriate parameters for alignment are known to those skilled in the art.

[0132] A conservative substitution (also referred to as a conservative mutation or conservative substitution) is an amino acid substitution that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, size). Conservative substitutions in a protein generally have less impact on protein function than non-conservative substitutions.

[0133] The term“antibody derivative” according to the present application can also comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab’, F(ab’)2, or“r IgG” (a“half-antibody” consisting of a heavy chain and a light chain). Antibody fragments can be produced by enzymatic or chemical cleavage of intact antibodies. The antibody construct according to the present application can also comprise modified fragments of antibodies, also referred to as antibody variants or antibody derivatives.

[0134] The terms "antigen binding fragment", "binding domain" or "domain that binds to" are used interchangeably herein in reference to antibodies of the present application and refer to a portion of an antibody that specifically binds to / interacts with / recognizes a protein / epitope on a target or antigen (here: FGFR2b) that does not possess the entire structure of the whole antibody, but only comprises a part or a local variant of the whole antibody, which local or local variant possesses the ability to bind to the antigen. "Antigen binding fragments" or "antibody fragments" herein include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single domain antibodies. The structure and function of the binding domain is based on the structure and / or function of an antibody. The "binding domain" or "domain that binds to" can comprise the minimum structural requirements of an antibody that allow for immunospecific target binding. This minimum structural requirement of a binding domain can be defined, for example, by the presence of at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs. The "domain that binds to" (or "binding domain") typically can comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily have to comprise both, but can comprise only one of VH or VL.

[0135] The terms "binds to" or "binds to specifically or immunospecifically" or "recognizes" or "reacts with" mean that according to the present application, an antibody or binding domain interacts with a given protein / epitope on a target molecule (antigen) (FGFR2b) or interacts (immuno-)specifically. This interaction or binding occurs more frequently, more rapidly, with longer duration, with greater affinity, or some combination thereof, in the protein / epitope on the particular target than in alternative substances (non-target molecules). However, due to sequence similarity between homologous proteins in different species, an antibody or binding domain that binds to its target (such as a human target) immunospecifically can cross-react with a homologous target molecule from a different species (such as from a non-human primate, such as a cynomolgus monkey). Thus, the term "specific / immunospecific binding" can include binding of an antibody or binding domain to a protein / epitope or structurally related protein / epitope in more than one species.

[0136] In the context of the present application, the term "epitope" refers to a portion or region of an antigen that is recognized / immunospecifically recognized by the binding domain, antibody or derivative thereof. The "epitope" is antigenic and thus the term epitope is sometimes also referred to as "antigenic structure" or "antigenic determinant". The portion of the binding domain, antibody or antibody construct that binds to the epitope is referred to as the paratope. It is believed that specific binding is achieved by specific motifs in the amino acid sequences of the binding domain, antibody or antibody construct and the antigen. Thus, binding is achieved as a result of their primary, secondary and / or tertiary structure and potential secondary modifications of said structures. The specific interaction of the paratope with its antigenic determinant can result in a simple binding of said site to the antigen. In some cases, the specific interaction can alternatively or additionally result in the initiation of a signal, for example due to inducing a change in the conformation of the antigen, oligomerization of the antigen, etc.

[0137] The interaction between the monoclonal antibody and the protein / epitope of the target antigen means that the variable region exhibits a clear or significant affinity for the epitope / target antigen (FGFR2b) and generally does not exhibit a significant affinity for proteins or antigens other than the target antigen. "Significant affinity" includes an affinity of about < 10 -6 M. Preferably, the binding affinity is about < 10 -7 M, < 10 -8 M, < 10 -9 M, or < 10 -10 M. Thus, the affinity (dissociation constant, KD) of the monoclonal antibody (or antibody construct) of the present application for FGFR2b is about < 10 -7 M, < 10 -8 M, < 10 -9 M, or < 10 -10 M. These values are preferably measured in a surface plasmon resonance assay, such as a Biacore assay.

[0138] Whether an antibody reacts or binds (immuno-)specifically with a target is determined, for example, by comparing the affinity of the antibody to its desired target protein or antigen with the affinity of the antibody to a non-target protein or antigen (here: a protein other than FGFR2b). Preferably, the antibodies of the present application do not significantly bind to a protein or antigen other than FGFR2b, unless any other binding domain(s) directed to another target is intentionally introduced into the antibody / antibody construct of the present application. The term "do not significantly bind" means that the monoclonal antibody (or antibody construct) of the present application does not bind to a protein or antigen other than FGFR2b. Thus, the antibody construct exhibits < 30%, preferably < 20%, more preferably < 10%, particularly preferably < 9%, < 8%, < 7%, < 6%, < 5%, < 4%, < 3%, < 2%, or < 1% reactivity with a protein or antigen other than FGFR2b.

[0139] The antibodies of the present application can be "in vitro generated antibodies" and / or "recombinant antibodies". In the present application, the term "in vitro generated" refers to antibodies according to the above definition, wherein all or part of the variable region is generated in a non-immune cell.

[0140] The antibodies of the present application can be "isolated" or "substantially pure" antibodies. "Isolated" or "substantially pure" when used to describe antibodies of the present application means that the antibodies have been identified, separated and / or recovered from a component of their production environment.

[0141] In some embodiments, the antibodies are derived from mice. The term "antibody" includes antibodies and binding domains having antibody-derived regions, such as variable regions and constant regions or domains that substantially correspond to germline immunoglobulin sequences known in the art, respectively.

[0142] Whether an antibody competes with another given antibody for binding to an antigen can be measured in a competition assay, such as a competitive ELISA.

[0143] Covalent modifications of the antibodies of the present application are also included within the scope of the present application, and are typically, but not always, made post-translationally.

[0144] The "sample" of the present application can be a biological sample. In some embodiments, the sample is a human sample, e.g. a human biological sample. The biological sample can be a (human) serum sample, a plasma sample, a blood sample, a bone marrow sample or a tissue sample. The sample can also be a supernatant obtained from a cell culture of (human) bone marrow mononuclear cells or (human) peripheral blood mononuclear cells. The sample can be obtained from a subject, e.g. a human subject suspected of having or having (been diagnosed with) a disease associated with increased FGFR2b, or a subject who has received a treatment for a disease associated with increased FGFR2b.

[0145] According to the present application, the monoclonal antibody is conjugated to a detectable label. In some embodiments, covalent modification of the monoclonal antibody of the present application includes the addition of one or more labels, such as detection labels. Labels or label groups can be conjugated to the antibody via spacers of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to practice the present application.

[0146] The term "label" or "label group" refers to any detectable marker.

[0147] The term "FGFR2b positive cell" refers to a cell that expresses FGFR2b on the cell surface, such as a cultured cell, a tumor cell that overexpresses FGFR2b. The level of FGFR2b expression on the cell surface can be determined by using the anti-FGFR2b antibody of the present application, as well as any conventional method known in the art for determining the level of cell surface antigen expression (e.g., immunohistochemical staining method, FACS detection method, or immunofluorescent staining method). It is known that FGFR2b has a significantly higher expression level on various tumor tissues / cells than on normal tissues / cells.

[0148] The term "FGFR2b positive tissue" refers to a tissue that aberrantly expresses FGFR2b, such as a tumor tissue. The level of FGFR2b expression on the tissue can be determined by using the anti-FGFR2b antibody of the present application, as well as any conventional method known in the art for determining the level of tissue surface antigen expression (e.g., immunohistochemical staining method, FACS detection method, or immunofluorescent staining method). It is known that FGFR2b has a significantly higher expression level on various tumor tissues / cells than on normal tissues / cells.

[0149] The terms "disease and / or disorder associated with aberrant expression of FGFR2b", "disease associated with increased FGFR2b", and "disease and / or disorder associated with increased expression of FGFR2b" can be used interchangeably in the present application to refer to a disease state that is initiated or driven by aberrant expression (e.g., overexpression or aberrant activation) of fibroblast growth factor receptor 2b (FGFR2b). In a particular embodiment of the present application, the term "disease and / or disorder associated with aberrant expression of FGFR2b" refers to a cancer or a tumor associated with aberrant expression of FGFR2b.

[0150] The present application further provides a polynucleotide / nucleic acid molecule encoding an antibody of the present application. Nucleic acid molecules are biopolymers constructed from nucleotides. Polynucleotides are biopolymers constructed from 13 or more nucleotide monomers covalently bonded in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotide / nucleic acid molecules with different biological functions. Nucleotides are organic molecules that act as monomers or subunits of nucleic acid molecules like DNA or RNA. The nucleic acid molecule or polynucleotide of the present application can be double-stranded or single-stranded, linear or circular. It is envisaged that the nucleic acid molecule or polynucleotide is comprised in a vector. Such a vector is comprised in a host cell. The host cell is capable of expressing the antibody, e.g. after being transformed or transfected with a vector or polynucleotide / nucleic acid molecule of the present application. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0151] In some embodiments, the polynucleotide / nucleic acid molecule of the present application encoding an antibody of the present application is in the form of one single molecule or in the form of two or more separate molecules. If the antibody construct of the present application is single-chained, the polynucleotide / nucleic acid molecule encoding such a construct will most likely also be in the form of one single molecule. In case the different components of the antibody (such as heavy and light chains) are located on separate polypeptide chains, the polynucleotide / nucleic acid molecule will most likely be in the form of two (or more) separate molecules in this case.

[0152] The present application also provides a vector comprising a polynucleotide / nucleic acid molecule of the present application. Vectors are nucleic acid molecules used as vehicles to transfer (foreign) genetic material into cells, usually for replication and / or expression. The term "vector" encompasses, but is not limited to, plasmids, viruses, cosmids and artificial chromosomes. Some vectors are designed specifically for cloning (cloning vectors), others for protein expression (expression vectors). So-called transcription vectors are mainly used for amplification of their insert.

[0153] The term "host cell" refers to a cell that is transformed or transfected with a polynucleotide / nucleic acid molecule of the present application or a vector of the present application. As used herein, the term "host cell" or "recipient cell" is intended to include any single cell or cell culture that can be or has been a recipient of a vector, foreign nucleic acid molecule and / or polynucleotide encoding an antibody of the present application, and / or the antibody itself. The corresponding material is introduced into the cell by transformation, transfection or the like. The term "host cell" is also intended to include progeny or potential progeny of the single cell or cell culture that originally is or has been a recipient of a vector, foreign nucleic acid molecule and / or polynucleotide encoding an antibody of the present application, and / or the antibody itself.

[0154] The present application provides a method for producing an antibody of the present application, said method comprising culturing a host cell of the present application under conditions allowing expression of an antibody of the present application and recovering the produced antibody from the culture.

[0155] The term "culturing" refers to the in vitro maintenance, differentiation, growth, proliferation and / or propagation of cells under suitable conditions in a culture medium. Cells are grown and maintained in a cell growth medium at an appropriate temperature and gas mixture. Culture conditions vary greatly for each cell type. Typical growth conditions are a temperature of about 37°C, a CO2 concentration of about 5% and a humidity of about 95%. The formulation of the growth medium can vary, for example, in pH, in the concentration of carbon source such as glucose, in the nature and concentration of growth factors, and in the presence of other nutrients such as amino acids or vitamins. Growth factors used to supplement the culture medium are usually derived from serum of animal blood, such as fetal bovine serum (FBS), fetal calf serum (FCS), horse serum and pig serum. Cells can be grown in suspension or as adherent cultures. There also exist cell lines that have been modified to be able to survive in suspension culture, so they can be grown at higher density than would be allowed in adherent conditions.

[0156] The term "expression" includes any step involved in the production of an antibody of the application, including but not limited to transcription, post-transcriptional modification, translation, folding, post-translational modification, targeting to a particular subcellular or extracellular location, and secretion.

[0157] The term "recovery" refers to a series of processes aimed at isolating an antibody from a cell culture. The "recovery" or "purification" process can separate protein and non-protein parts of the cell culture and eventually isolate the desired antibody from all other polypeptides and proteins. The separation steps usually exploit differences in protein size, physico-chemical properties, binding affinities and biological activities. Preparative purification aims at producing relatively large amounts of purified protein for subsequent use, while analytical purification produces relatively small amounts of protein for various research or analytical purposes. The antibodies of the application prepared from host cells can be recovered or purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.

[0158] The present application provides a composition comprising an antibody of the present application or comprising an antibody produced according to the method of the present application. The composition is preferably a diagnostic composition. The term "diagnostic composition" as used in the present application relates to a composition suitable for use in a diagnostic kit or detection system. A possible diagnostic composition of the present application comprises one or more antibodies of the present application, preferably in an amount useful for detecting FGFR2b in a sample.

[0159] The present application provides a detection system comprising: a) a first monoclonal antibody (or a derivative thereof) that binds to FGFR2b, and / or b) a second monoclonal antibody (or a derivative thereof) that optionally binds to FGFR2b, wherein the binding of the first monoclonal antibody (or a derivative thereof) to FGFR2b occurs in the presence of the second monoclonal antibody (or a derivative thereof) that binds to FGFR2b.

[0160] A "detection system" is a kit or tool (or diagnostic kit / tool) containing reagents for performing an analytical assay. In the present invention, the assay detects and / or quantifies the presence of FGFR2b in a sample (typically a tissue sample). The detection system contains an antibody that binds to FGFR2b. Typically, the detection system involves the use of a solid support (such as a microtiter plate or a membrane) that acts as a surface to immobilize the antigen to be detected (for example in the case of a "direct ELISA") or a (monoclonal) antibody that binds to FGFR2b ("capture antibody") or a "secondary antibody" (for example, an anti-Fc antibody) that binds to the antibody that binds to FGFR2b (capture antibody). Typically, this immobilization occurs non-specifically (by adsorption onto the surface) or specifically (by capture by an antibody, for example a secondary antibody). In addition, the detection system can contain a (monoclonal) detection antibody that binds to FGFR2b (optionally coupled to an enzyme, detectable label or reporter group), and optionally a secondary antibody (for example, an anti-Fc antibody) that binds to the detection antibody and is coupled to an enzyme, detectable label or reporter group.

[0161] The detection system can be an ELISA assay, which can be used for the purposes of the present invention. ELISA is used to detect an antigen in a sample or to quantify an unknown amount of an antigen. The steps can include: providing a surface to which a known amount of "capture antibody" is bound. This binding can occur directly by adsorption of the capture antibody onto the surface, or by a secondary antibody that is adsorbed onto the surface and binds to the capture antibody. Any non-specific binding sites on the surface are blocked. The sample containing the antigen is applied to the surface, and the antigen is captured (bound) by the antibody. The plate is washed to remove unbound antigen. A "detection antibody" is added and the "detection antibody" binds to the antigen. The detection antibody can be coupled (for example, covalently linked) to an enzyme, detectable label or reporter group. If this is not the case, then a secondary antibody is applied that is coupled to an enzyme, detectable label or reporter group and binds to the detection antibody. The plate is washed to remove any unbound antibody. A chemical substrate is added that is converted to a detectable form, such as a light signal or an electrochemical signal. The absorbance or fluorescence or electrochemical signal of the plate wells or surface is measured to determine the presence and / or amount of antigen.

[0162] Traditional ELISA typically involves a chromogenic reporter molecule and substrate that produces an observable color change to indicate the presence of the antigen. Newer technologies similar to ELISA use fluorescent, electrochemiluminescent and quantitative PCR reporter molecules to produce a quantifiable signal.

[0163] The detection system can be used in either a qualitative or quantitative format. The qualitative result provides a simple positive or negative result (yes or no) for the sample. In the quantitative format, the optical density (OD) or electrochemical signal of the sample is compared to a standard curve.

[0164] The term "diagnosis" or "medical diagnosis" is the process of determining which disease or condition can explain a subject's symptoms and signs. Typically, one or more diagnostic procedures, such as diagnostic tests or medical tests, are performed in this process.

[0165] The term "monitoring" refers to the observation of a disease, condition, or one or more medical parameters over time.

[0166] The term "improvement" refers to any improvement in the disease state of a patient having a disease as specified herein by the administration of an antibody to a subject in need thereof. Such improvement can also be seen as a slowing down or a stop of the progression of the disease in the patient.

[0167] The term "prevention" means avoiding the onset or recurrence of a patient having a tumor or cancer or metastatic cancer as specified herein by the administration of an antibody to a subject in need thereof.

[0168] The term "disease" refers to any condition that would benefit from treatment with an antibody construct or pharmaceutical composition described herein.

[0169] The terms "tumor" and "cancer" are used interchangeably herein to refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. The term encompasses both primary forms and metastatic forms of tumors. The term also encompasses all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0170] The terms "subject," "individual," or "patient" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human. In one embodiment, subjects who have already suffered from a disease as well as subjects who are to be prevented from a disease are included.

[0171] The term "amount" or "content" of (FGFR2b) can be used interchangeably with the term "level" or "concentration" of (FGFR2b). A "predefined value" of the FGFR2b content can be a "domain value" that has been determined beforehand. The value may, for example, indicate that a certain FGFR2b content in a sample is indicative of a disease associated with increased FGFR2b or an FGFR2b positive tumor.

[0172] The term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. The pharmaceutical composition comprises preferably a therapeutically effective amount of one or more antibodies of the present application. The composition can be administered to a subject in a suitable dose.

[0173] The terms "biological sample," "sample," "tissue sample," and the like are used interchangeably herein to refer to a collection of cells, tissues, or body fluids obtained from an individual or subject. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate sample; blood or any blood component; a body fluid such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during gestation or development. The tissue sample can contain compounds not naturally associated with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, tumor cells in circulation, serum, plasma, plasma proteins in circulation, ascites, primary cell cultures or cell lines derived from a tumor or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor tissue sections or frozen tumor samples.

[0174] The terms "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refer to a sample, cell, tissue, or standard used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased portion of the same subject or individual, is a healthy and / or non-diseased tissue or cell. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy tissue or cell of an individual that is not the subject.

[0175] The term "effective amount" refers to the amount or dose of an antibody or composition of the present application, which elicits the desired effect in a patient after administration to the patient in a single dose or multiple doses. The effective amount can be readily determined by the attending physician, as one of ordinary skill in the art, by taking into consideration a variety of factors, such as species of mammal; body mass, age and general health condition of the mammal; the particular disease involved; the extent or severity of the disease; the individual patient's response; the particular antibody involved; the mode of administration; the bioavailability of the formulation administered; the dosing regimen selected; and any concomitant therapy being administered.

[0176] The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody fragment or composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody moiety to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system predictive of efficacy in humans.

[0177] The term "kit" means two or more components (one of which corresponds to an FGFR2b antibody or composition of the application) packaged together in a container, receptacle, or other packaging. A kit can be described as a set of products and / or implements sufficient to achieve a certain goal, which can be sold as a single unit. A kit can include one or more containers of any appropriate shape, size, and material containing a dose suitable for administration of an antibody or pharmaceutical composition of the application. A kit can additionally contain instructions for use, means for administering an antibody of the application, means for reconstituting an antibody of the application, and / or means for diluting an antibody of the application. BRIEF DESCRIPTION OF DRAWINGS

[0178] Figure 1 shows the specificity of the antibodies of the application for FGFR2b by immunohistochemistry (IHC). Figure 1A shows that the HDM-45 antibody of the application specifically binds CHOK1 cells overexpressing FGFR2b, Figure IB shows that the HDM-45 antibody of the application does not recognize CHOK1 cells overexpressing FGFR2c, and Figure 1C shows that the isotype negative control antibody does not recognize CHOK1 cells overexpressing FGFR2b.

[0179] Figure 2 shows the recognition of the FGFR2b positive gastric cancer cell line KATO-III by the anti-FGFR2b antibodies of the application and commercially available FGFR2b antibodies by immunohistochemistry (IHC) (all at an antibody concentration of 1 μg / ml). Figure 2A shows specific staining of the gastric cancer cell line KATO-III by the HDM-45 antibody of the application, Figure 2B shows specific staining of the gastric cancer cell line KATO-III by the commercially available antibody SDT-423-18, Figure 2C shows specific staining of the gastric cancer cell line KATO-III by the commercially available antibody SDT-423-2, and Figure 2D shows that the isotype negative control antibody IgGl does not stain KATO-III.

[0180] Figure 3 shows the recognition of the negative gastric cancer cell line SNU-5, which does not express FGFR2b, by the anti-FGFR2b antibodies of the present application and a commercially available FGFR2b antibody, as tested by immunohistochemistry (IHC) (antibody concentration 1 μg / ml). Figure 3A shows staining of the gastric cancer cell line SNU-5 by the HDM-45 antibody of the present application, Figure 3B shows staining of the gastric cancer cell line SNU-5 by the commercially available antibody SDT-423-18, Figure 3C shows staining of the gastric cancer cell line SNU-5 by the commercially available antibody SDT-423-2, and Figure 3D shows staining of SNU-5 by an isotype negative control antibody IgGl.

[0181] Figure 4 shows the staining sensitivity results of the same gastric cancer serial sections by the anti-FGFR2b antibodies of the present application and a commercially available FGFR2b antibody, as tested by immunohistochemistry (IHC) (antibody concentration 1 μg / ml). Figure 4A shows staining results of the gastric cancer sections by the HDM-45 antibody of the present application, and Figure 4B shows staining results of the gastric cancer sections by the commercially available FGFR2b antibody SDT-423-18.

[0182] Figure 5 shows the staining results of tonsil (negative tissue that does not express FGFR2b) sections by the anti-FGFR2b antibody HDM-45 of the present application, as tested by immunohistochemistry (IHC) (antibody concentration 1 μg / ml).

[0183] Figure 6 shows the staining results of triple negative breast cancer sections by the anti-FGFR2b antibody HDM-45 of the present application, as tested by immunohistochemistry (IHC) (antibody concentration 1 μg / ml).

[0184] Figure 7 shows the results of flow cytometric sorting assays for the binding of the anti-FGFR2b antibody HDM-45 of the present application to FGFR2b and FGFG2c. DETAILED DESCRIPTION

[0185] In some embodiments, the present application provides a method of making an anti-FGFR2b antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-FGFR2b antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-FGFR2b antibody, and optionally isolating the anti-FGFR2b antibody. In a certain embodiment, the method further comprises recovering the anti-FGFR2b antibody from the host cell (or host cell culture medium).

[0186] For recombinant production of the anti-FGFR2b antibodies of the application, the nucleic acid encoding the heavy and light chains of the anti-FGFR2b antibodies of the application is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes that are specific for the nucleic acid encoding the anti-FGFR2b antibodies of the application.

[0187] The anti-FGFR2b antibodies of the application prepared as described herein can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. The purity of the anti-FGFR2b antibodies of the application can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0188] Aspects of the application will be further detailed in the following subsections.

[0189] I. Antibodies of the application

[0190] Through screening, the present application obtains a monoclonal antibody that recognizes the cell membrane surface protein FGFR2b, and its performance is detected and verified on various cell lines and different types of tumor tissue samples. The results show that the anti-FGFR2b antibody of the present application has high specificity, high accuracy and high sensitivity characteristics that meet the expected detection / diagnosis purposes. The antibody of the present application can be reliably used by pathologists to detect FGFR2b expression in human cancer tissues, and can also assist medical personnel in diagnosing abnormalities.

[0191] In a first aspect, the present application provides an anti-FGFR2b antibody or antigen binding fragment thereof.

[0192] In one embodiment, the present application provides an antibody or antigen binding fragment thereof that binds FGFR2b, comprising three complementarity determining regions of a light chain variable region, LCDR1, LCDR2 and LCDR3, and three complementarity determining regions of a heavy chain variable region, HCDR1, HCDR2 and HCDR3, wherein:

[0193] 1) the CDRs as determined according to the definition of Kabat:

[0194] HCDR1 as shown in SEQ ID NO: 3,

[0195] HCDR2 as shown in SEQ ID NO: 4,

[0196] HCDR3 as shown in SEQ ID NO: 5,

[0197] LCDR1 as shown in SEQ ID NO: 6,

[0198] LCDR2 as shown in SEQ ID NO: 7, and

[0199] LCDR3 as shown in SEQ ID NO: 8;

[0200] or

[0201] 2) CDRs determined according to the AbM definition:

[0202] HCDR1 as shown in SEQ ID NO: 11,

[0203] HCDR2 as shown in SEQ ID NO: 12,

[0204] HCDR3 as shown in SEQ ID NO: 13,

[0205] LCDR1 as shown in SEQ ID NO: 14,

[0206] LCDR2 as shown in SEQ ID NO: 15, and

[0207] LCDR3 as shown in SEQ ID NO: 16;

[0208] or

[0209] 3) CDRs determined according to the IMGT definition:

[0210] HCDR1 as shown in SEQ ID NO: 17,

[0211] HCDR2 as shown in SEQ ID NO: 18,

[0212] HCDR3 as shown in SEQ ID NO: 19,

[0213] LCDR1 as shown in SEQ ID NO: 20,

[0214] LCDR2 as shown in SEQ ID NO: 21, and

[0215] LCDR3 as shown in SEQ ID NO: 22;

[0216] or

[0217] 4) CDRs determined according to the Chothia definition:

[0218] HCDR1 as shown in SEQ ID NO: 23,

[0219] HCDR2 as set forth in SEQ ID NO: 24,

[0220] HCDR3 as set forth in SEQ ID NO: 25,

[0221] LCDR1 as set forth in SEQ ID NO: 26,

[0222] LCDR2 as set forth in SEQ ID NO: 27, and

[0223] LCDR3 as set forth in SEQ ID NO: 28.

[0224] In one embodiment, the present application provides an anti-FGFR2b antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consists of SEQ ID NO: 1. In another embodiment, the present application provides an anti-FGFR2b antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consists of SEQ ID NO: 2.

[0225] In some preferred embodiments, the present application provides an anti-FGFR2b antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consists of SEQ ID NO: 1, and wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consists of SEQ ID NO: 2. Preferably, the antibody or antigen-binding fragment comprises a heavy chain variable region of SEQ ID No: 1 and a light chain variable region of SEQ ID No: 2.

[0226] In some embodiments, the antibody according to the present application can comprise a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprised in the antibody of the present application can be of any isotype or subtype, e.g. a heavy chain constant region of an IgGl, IgG2, IgG3 or IgG4 isotype, preferably an IgGl heavy chain constant region. The light chain constant region comprised in the antibody of the present application can be a kappa light chain constant region or a lambda light chain constant region.

[0227] In some embodiments, the antibody according to the application is a full-length antibody consisting of two heavy chains and two light chains.

[0228] In some preferred embodiments, the application provides an anti-FGFR2b antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consisting of SEQ ID NO: 9, and wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or consisting of SEQ ID NO: 10. Preferably, the antibody or antigen-binding fragment comprises a heavy chain of SEQ ID No: 9 and a light chain of SEQ ID No: 10.

[0229] II. Polynucleotides, vectors and hosts and methods of antibody production

[0230] In a second aspect, the application provides a nucleic acid encoding an anti-FGFR2b antibody or fragment thereof according to the first aspect of the application; also provided are vectors comprising the nucleic acid; and host cells comprising the nucleic acid or the vector; and methods of producing the antibody or fragment thereof.

[0231] In some embodiments, the application provides nucleic acid molecules comprising a polynucleotide encoding a heavy chain VH or light chain VL sequence of an anti-FGFR2b antibody of the first aspect. In some embodiments, the application provides polynucleotides encoding a heavy chain and / or a light chain of an anti-FGFR2b antibody of the first aspect. As will be apparent to those skilled in the art, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences due to codon degeneracy.

[0232] In one embodiment, the application provides one or more vectors comprising a nucleic acid of the application, including cloning vectors and expression vectors. In one embodiment, the vector is an expression vector, e.g., a eukaryotic expression vector.

[0233] In one embodiment, the present application provides a host cell comprising a vector of the present application. Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. In one embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for making an antibody or antigen-binding fragment thereof. Examples of useful mammalian host cell lines include COS-7 monkey kidney line transformed with SV40 (COS-7); human embryonic kidney line (293 HEK or 293 cells); Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NS0 and Sp2 / 0.

[0234] In one embodiment, the present application provides a method of making an anti-FGFR2b antibody or fragment thereof of the present application. In one embodiment, the method of the present application comprises culturing a host cell comprising a nucleic acid encoding an antibody of the present application under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0235] Antibodies of the present application prepared as described herein can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. The purity of an antibody of the present application can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0236] III. Detection and diagnostic methods and uses and kits

[0237] 1. Detection methods and detection uses

[0238] In some embodiments, the present application provides a method for detecting the presence or absence of FGFR2b in a sample. In this context, the term "detecting" includes quantitative, semi-quantitative, or qualitative detection. Exemplary detection methods include, but are not limited to, immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry (e.g., FACS), ELISA assays, Western blot assays, immunofluorescence (IF) assays, and / or co-immunoprecipitation (Co-IP) detection.

[0239] In some embodiments, the detection method of the present application comprises:

[0240] (a) contacting a biological sample with an antibody or antigen-binding fragment thereof of the first aspect; and

[0241] (b) detecting the presence or absence of a complex formed by said antibody or antigen binding fragment thereof and FGFR2b, wherein the presence of a complex indicates the presence of FGFR2b in the sample;

[0242] Optionally, the antibody is directly or indirectly detectably labeled.

[0243] In some embodiments, there is provided the use of an antibody or antigen binding fragment thereof according to the application in a detection method.

[0244] The biological sample suitable for use in the methods of the application is not particularly limited and includes, but is not limited to, a cell sample (e.g., cultured cells, cell lines, cell samples from a subject), a tissue sample (e.g., tissue sections, pathology sections, and tissue microarrays). In some embodiments, the biological sample comprises cells or tissues. In other embodiments, the biological sample is from a hyperproliferative or cancerous lesion. In some preferred embodiments, the biological sample is a tissue section (e.g., a paraffin section or a frozen section), and more preferably the biological sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

[0245] The anti-FGFR2b antibody used in the methods of the application can be directly or indirectly detectably labeled, depending on the detection format chosen. In the context of the present application, an antibody is "directly labeled" if the antibody is directly linked to a label; correspondingly, an antibody is "indirectly labeled" if the antibody is linked to a label via one or more bridging reagents, e.g., a primary antibody can be indirectly detectably labeled via a secondary antibody. Labels useful in the present application include, but are not limited to, fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as enzymes or ligands.

[0246] In some embodiments, the detection of the complex of step (b) is performed by immunohistochemistry (IHC) according to the methods of the application. Immunohistochemistry (IHC) is one of the label-based protein detection techniques. The principle of this technique is based on the specific binding of a particular antibody to a matching specific antigen in situ in a sample (tissue or cells). The antigen-antibody complex after binding can then be visualized by a range of detection techniques, allowing the display and recording of the high-resolution distribution and localization of specific cellular components in the cellular and appropriate histological context. Labels useful for IHC visualization include, e.g., enzymatic labels (such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) and their corresponding chromogenic substrates) and fluorescent labels (such as fluorescent dyes or fluorophores). Depending on the label and the sample, the detection of the complex can be performed by image analysis, microscopic observation, etc., and can be qualitative, semi-quantitative, and quantitative assessment. The biological sample suitable for use in IHC is not particularly limited and includes, e.g., paraffin sections and frozen sections.

[0247] In some embodiments of the methods of the application, antigen-antibody complex detection is performed using IHC. Depending on the biological sample applied, the skilled person can select an appropriate known IHC procedure. As one specific example of an IHC procedure applied to paraffin sections, the IHC comprises: adding an anti-FGFR2b antibody; a negative control antibody; or a positive control antibody diluted to a working concentration to a paraffin section (e.g. a FFPE section) after deparaffinization and hydration; and visualizing the antigen bound to the antibody. In some embodiments, the visualizing comprises using a label, e.g. an enzymatic label or a fluorescent label, to directly or indirectly label the antibody according to the application.

[0248] 2. Diagnostic methods and diagnostic uses

[0249] In clinical pathology diagnosis, it is of great importance to be able to perform accurate and sensitive immunohistochemical (IHC) analysis of the target FGFR2b antigen using routinely processed tissue sections for reliably discriminating true target antigen positive cases and for determining whether a patient is suitable for receiving an immunotherapy directed against the target antigen. Furthermore, a validated IHC analysis on paraffin-embedded (e.g. FFPE) samples also contributes to the prediction of clinical responsiveness of a patient to an immunotherapy.

[0250] In some embodiments, the application provides a method for diagnosing whether a subject is afflicted with a disease associated with abnormal expression of FGFR2b, comprising: detecting FGFR2b and / or the amount of expression thereof in a biological sample from the subject, optionally comparing the detected presence or amount of expression of FGFR2b to the presence or amount of expression of FGFR2b in a reference sample, to determine whether there is abnormal expression (e.g. overexpression) of FGFR2b and / or the amount of expression in the biological sample. In some embodiments, the presence or amount of expression of FGFR2b in the reference sample can be (i) a pre-defined value for the amount of FGFR2b expression; (ii) the amount of FGFR2b expression determined in a healthy individual control sample, or (iii) the amount of FGFR2b expression determined in a sample obtained from the same source or subject at a previous time point.

[0251] In some embodiments, the presence and / or the amount of expression of FGFR2b in a patient biological sample that is elevated relative to the presence or amount of expression of FGFR2b in a reference sample is indicative of the presence of a disease associated with abnormal expression of FGFR2b. In one embodiment, the disease is a FGFR2b-positive tumor.

[0252] In some embodiments, there is provided use of an anti-FGFR2b antibody or antigen-binding fragment thereof of the application in the manufacture of a product for diagnosing whether a subject is afflicted with a disease associated with abnormal expression of FGFR2b.

[0253] In some embodiments, the present application provides a method of monitoring the progression of a disease associated with abnormal expression of FGFR2b comprising: detecting the presence and / or amount of FGFR2b in biological samples taken from a subject at different time points using the detection method of the present application and comparing the detected presence or amount of FGFR2b in samples obtained at different time points. In some specific embodiments, the method comprises:

[0254] (a) detecting the amount of FGFR2b in a biological sample obtained from a subject at a first time point using the detection method of the present application;

[0255] (b) detecting the amount of FGFR2b in a biological sample obtained from a subject at a second (later) time point or after treatment using the detection method of the present application; and

[0256] (c) comparing the amount of FGFR2b determined in step (a) with the amount of FGFR2b determined in step (b);

[0257] wherein a higher amount of FGFR2b determined in step (b) compared to the amount of FGFR2b determined in step (a) is indicative of the disease progressing and / or wherein a lower amount of FGFR2b determined in step (b) compared to the amount of FGFR2b determined in step (a) is indicative of the disease going into remission or the disease responding to the treatment.

[0258] In some embodiments, the patient is a patient who has been treated with an anti-neoplastic agent including but not limited to a metabolic inhibitor, an antibiotic anticancer agent, a plant alkaloid-based anticancer agent, a topoisomerase inhibitor, an anti-neoplastic alkylating agent, a monoclonal antibody, an ADC, and the like.

[0259] In some embodiments, there is provided the use of an anti-FGFR2b antibody or antigen binding fragment thereof of the present application in the manufacture of a product for monitoring the progression of a disease associated with abnormal expression of FGFR2b.

[0260] In some embodiments, the present application provides a method of determining whether a patient is suitable for treatment with an anti-FGFR2b drug, comprising: detecting the presence and / or amount of FGFR2b in a biological sample from the patient using a detection method according to the present application, and optionally comparing the detected presence or amount of FGFR2b to a reference amount of FGFR2b, wherein a higher amount of FGFR2b in the patient sample than the reference amount of FGFR2b indicates that the patient is suitable for treatment with an anti-FGFR2b drug. In some embodiments, the reference amount of FGFR2b is (i) a predefined threshold of FGFR2b content indicative of the absence of such a disease, or (ii) a FGFR2b content determined in a control sample representative of the absence of such a disease.

[0261] In some embodiments, there is provided use of an anti-FGFR2b antibody or antigen binding fragment thereof according to the present application in the manufacture of a product for determining whether a patient is suitable for treatment with an anti-FGFR2b drug.

[0262] In some embodiments, the present application provides a method of determining whether a patient is benefiting from treatment with an anti-FGFR2b drug, comprising:

[0263] (a) detecting the amount of FGFR2b in a biological sample obtained from the subject at a first time point using a detection method according to the present application;

[0264] (b) detecting the amount of FGFR2b in a biological sample obtained from the subject at a second (later) time point using a detection method according to the present application; and

[0265] (c) comparing the amount of FGFR2b determined in step (a) to the amount of FGFR2b determined in step (b);

[0266] wherein a lower amount of FGFR2b determined in step (b) than in step (a) indicates that the patient is benefiting from treatment with an anti-FGFR2b drug.

[0267] In some embodiments, the anti-FGFR2b drug is an anti-FGFR2b antibody.

[0268] In some embodiments, there is provided use of an anti-FGFR2b antibody or antigen binding fragment thereof according to the present application in the manufacture of a product for determining whether a subject is benefiting from treatment with an anti-FGFR2b drug.

[0269] In some embodiments, the present application provides a method of predicting the responsiveness of a subject to treatment with an anti-FGFR2b drug, comprising:

[0270] (a) determining the FGFR2b content in a biological sample obtained from the subject at a first time point using the detection method of the application;

[0271] (b) determining the FGFR2b content in a biological sample obtained from the subject at a second (later) time point using the detection method of the application; and

[0272] (c) comparing the FGFR2b content determined in step (a) with the FGFR2b content determined in step (b);

[0273] wherein if the FGFR2b content determined in step (b) is lower than the FGFR2b content determined in step (a), it is indicative that the subject is responsive to treatment with an anti-FGFR2b class of drugs.

[0274] In some embodiments, the anti-FGFR2b class of drugs is an anti-FGFR2b antibody.

[0275] In some embodiments, there is provided the use of an anti-FGFR2b antibody or antigen binding fragment thereof of the application in the manufacture of a product for predicting the responsiveness of a subject to treatment with an anti-FGFR2b class of drugs.

[0276] In some embodiments of the above diagnostic, prognostic, determining and predicting methods according to the application, the method comprises:

[0277] 1) contacting a biological sample from the subject with the anti-FGFR2b antibody or antigen binding fragment thereof;

[0278] 2) detecting the binding of the antibody or antigen binding fragment to the biological sample; and

[0279] 3) optionally, comparing the presence or amount of FGFR2b in the biological sample from the subject to the presence or amount of FGFR2b in a reference sample.

[0280] Preferably, the detection is by immunohistochemistry (IHC) staining.

[0281] In some embodiments, the biological sample is a tissue section (e.g. a paraffin section or a frozen section), more preferably the sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

[0282] In some embodiments, the biological sample is a pre-cancer sample, a primary cancer sample, or a sample obtained after the cancer has metastasized. In some embodiments, the biological sample is obtained prior to treatment with a therapeutic agent.

[0283] 3. Kits

[0284] In some embodiments, the present application provides a kit useful for the detection or diagnostic methods of the present application. In some embodiments, a kit according to the present application comprises an anti-FGFR2b antibody of the present application, and optionally a package insert instructing the use of the kit.

[0285] In some embodiments, a kit according to the present application further comprises reagents for detecting the anti-FGFR2b antibody of the present application. In some embodiments, a kit according to the present application further comprises a secondary antibody detection system. In some embodiments, the secondary antibody detection system comprises a secondary antibody with a detectable label. In other embodiments, the secondary antibody detection system comprises: an enzyme-labeled secondary antibody; a chromogenic substrate for the enzyme.

[0286] In some embodiments, the present application also provides the use of an antibody of the present application or a kit of the present application in the manufacture of a product for use in the above-mentioned methods.

[0287] Examples

[0288] The following examples further illustrate the present application, however, it is to be understood that the examples are described by way of illustration and not by way of limitation and that modifications can be made by those skilled in the art. Where specific conditions are not specified in the examples, they are carried out under conventional conditions or as recommended by the manufacturer. Where the manufacturer of a reagent or instrument is not named, conventional products available from commercial vendors have been employed. The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0289] Immunohistochemistry (IHC) is a technique that uses color development of an antibody bound to an antigen to localize and qualitatively and quantitatively determine a target antigen in cells of a tissue. IHC has been widely used in the diagnosis of tumor pathology. By examining the distribution, localization, and abundance of a tumor biomarker on a clinicopathological sample, IHC can provide information on the stage and grade of a tumor, whether the tumor is benign or malignant, and can be used to predict the clinical responsiveness of a patient to a biomarker-targeted therapy. A key element in such a pathological diagnosis is an IHC antibody that has been validated to reliably detect biomarker-positive cases.

[0290] When preparing a sample for immunohistochemical analysis, some epitopes of an antigen expressed on the membrane surface of the sample can be masked or destroyed, such that some antibodies of the antigen can no longer recognize the epitopes. In addition, even if the epitopes are present, different antibodies of the antigen can exhibit significantly different detection performance, influenced by factors such as the location of the epitopes and the membrane surface binding activity. Therefore, a monoclonal antibody for IHC often needs to be specially screened and validated.

[0291] Example 1: Preparation of Anti-FGFR2b Antibodies

[0292] SJL mice (5-6 weeks old female, Shanghai Slac Laboratory Animal Limited Liability Company) were immunized by intraperitoneal injection of fusion protein FGFR2(beta) IIIb-Fc (KACTUS, Cat# FGR-MM1BB) and anti-CD25 antibody or anti-CD40 antibody every other week, the first immunization was anti-CD25 antibody, 100 μg per animal, then anti-CD40 antibody, 2 doses, 50 μg per animal. The antigen was suspended in MPL / TDM (Sigma-Aldrich). Three days after the last injection, popliteal lymphocytes were extracted and fused with Sp2 / 0-Ag14 mouse myeloma cells (ATCC, Cat# CRL-1581) at a ratio of 1:1 using the Hybrimune electrofusion system (Cyto Pulse Sciences). After 24 hours, the hybridoma was selected by adding 2x HAT (Sigma). Ten days after fusion, the hybridoma culture supernatant was collected and screened using ELISA to detect the ability of the antibody in the supernatant to bind to FGFR2 IIIb-his and FGFR2 IIIc-his, and hybridomas that only bind to FGFR2 IIIb-his and not to FGFR2 IIIc-his were selected. The selected hybridomas were then cloned twice using the limiting dilution technique. After sequencing the subcloned hybridomas, a number of recombinant monoclonal anti-FGFR2b mouse antibodies were obtained, including mAb HDM-45, the sequence of HDM-45 is as recited in the sequence listing.

[0293] Control antibodies SDT-423-2 and SDT-423-18 are commercially purchased antibodies.

[0294] Example 2: Verification of the specificity of anti-FGFR2b antibody HDM-45

[0295] This example evaluates the binding of HDM-45 antibody to FGFR2b and FGFR2c. First, FGFR2b-CHOK1 and FGFR2c-CHOK1 overexpression stable transfection cell lines were prepared. Nucleic acid molecules encoding human FGFR2b (P21802-3) and FGFR2c (P21802-1) were cloned into a lentiviral vector, respectively, and stable expression cell lines were obtained by lentiviral transfection.

[0296] 2.1 Experimental materials

[0297] 2.1.1. Experimental reagents

[0298] FACS buffer: lx PBS + 1% FBS

[0299] 2.1.2. Experimental instruments

[0300] Bench top centrifuge: Eppendorf 5810R

[0301] FACS instrument: BD FACSCanto II

[0302] 2.2 Virus packaging, transfection of target cells

[0303] 1) 2.2E7 cells in logarithmic growth phase of 293T were inoculated into a T75 culture flask treated with poly-D-lysine (PDL);

[0304] 2) The lentiviral transfection system was configured and the 293T cells were transfected, and then cultured in an incubator;

[0305] 3) The supernatant of lentiviral particles was collected and stored in a 4-degree refrigerator;

[0306] 4) The collected virus supernatant was centrifuged at 3750 rpm for 15 min at 4 degrees, and then filtered with a 250 ml, 0.45 um filter. Then, according to the total amount of the supernatant, the corresponding virus concentration column was selected to concentrate the virus;

[0307] 5) CHOK1 was used as the host target cell, and the cells were plated 18-24 hours before transfection. The cell culture medium was replaced with CHOK1 complete culture medium containing 5 ug / ml polybrene, and 80 ul / well of virus was added to the well. After 6 hours, the liquid was replaced with complete culture medium;

[0308] 6) Repeated infection and FACS detection of virus infection effect.

[0309] 2.3 Screening of monoclonal cells:

[0310] 1) After determining the success of virus infection, the cell culture medium was replaced with new complete culture medium containing 8 ug / mL puromycin;

[0311] 2) Using a sorting instrument, a single cell infected with virus was inoculated into a 96-well plate at 1 cell / well to obtain a monoclonal cell.

[0312] 3) After the number of monoclonal cells increased, FACS was used to detect the expression amount of the stable cell strain, and CHOK1 cells stably overexpressing FGFR2b and FGFR2c were obtained, respectively, for subsequent experiments.

[0313] The FACS detection method is as follows:

[0314] 1) After cell counting, 2E5 cells were added to 100 μL / well in round-bottom 96-well plates, and washed once with FACS buffer;

[0315] 2) Primary antibody (5 μg / mL) was added at 100 μL / well, and incubated at 4°C for 1 hour, and washed three times with FACS buffer;

[0316] 3) Secondary antibody (1:1000) was added at 100 μL / well, and incubated at 4°C for 1 hour, and washed three times with FACS buffer;

[0317] 4) Resuspended with 80 μL 1xPBS, and detected with a FACS instrument.

[0318] 2.4 The binding specificity of the antibody of the present application to CHOK1 cells overexpressing FGFR2b was detected by immunohistochemistry (IHC) method, and staining with an isotype negative control antibody was used as a negative control.

[0319] IHC experimental method:

[0320] 1) Section preparation: The CHOK1 cells overexpressing FGFR2b obtained above were fixed in 10 ml of 4% formaldehyde (PBS solution) at 4°C overnight, and then incubated in fresh 4% formaldehyde at 65°C for 3 minutes. 4% agarose (1:1) in 1x Tris-acetate-EDTA (TAE) buffer was added to the cell suspension, transferred to a 24-well plate, and incubated at 4°C overnight, followed by fixation in 4% formaldehyde at 4°C overnight. The agarose cell block was embedded in paraffin to prepare a wax block for sectioning.

[0321] 2) Sectioning: The wax block was sectioned according to the conventional method, and the sections were baked at 60°C for 30 min.

[0322] 3) Staining: The baked sections were placed in a Leica Bond III full-automatic immunohistochemical staining machine for staining;

[0323] The staining procedure was as follows:

[0324] After the staining was completed, the slides were removed and sequentially placed in 95% ethanol, anhydrous ethanol I, anhydrous ethanol II, xylene I, and xylene II for 5 min each.

[0325] 3) Mounting: An appropriate amount of neutral resin was dropped onto the tissue sections using a dropper, and then covered with a cover glass gently, avoiding the generation of air bubbles during the process. The mounted sections were placed in a fume hood for natural solidification of the neutral resin.

[0326] 4) Reading: The staining results were observed under a microscope and photographed.

[0327] The results are shown in Figure 1. Figure 1A shows that HDM-45 antibody specifically recognizes FGFR2b, Figure 1B shows that HDM-45 antibody does not recognize FGFR2c (antibody concentration 1 μg / ml), and Figure 1C shows the staining of cells with isotype negative control antibody IgGl. The results show that HDM-45 antibody specifically recognizes FGFR2b, but not FGFR2c.

[0328] Example 3: IHC assay of HDM-45 to identify positive cells expressing FGFR2b

[0329] To determine whether anti-FGFR2b antibody HDM-45 can specifically recognize FGFR2b expressed by cancer cells, an immunohistochemistry assay was performed on gastric cancer cell KATO-III (origin: ATCC, HTB-103, P22) according to the method of Example 2, using HDM-45 at an antibody concentration of 1 μg / ml, commercial FGFR2b antibodies SDT-423-18 and SDT-423-2 at 1 μg / ml. The results are shown in Figure 2.

[0330] Figure 2A shows that HDM-45 antibody can specifically stain gastric cancer cell line KATO-III, indicating that it specifically recognizes endogenous FGFR2b expressed in the gastric cancer cell line, Figure 2B shows staining of gastric cancer cell line KATO-III with commercial antibody SDT-423-18, Figure 2C shows staining of gastric cancer cell line KATO-III with commercial antibody SDT-423-2, and Figure 2D shows staining of KATO-III with isotype negative control. The results show that both HDM-45 and commercial FGFR2b antibodies can stain positive gastric cancer cells expressing FGFR2b, but HDM-45 stains more uniformly with a clearer cell membrane staining, indicating that the antibody of the application can more specifically recognize FGFR2b on the cell membrane of gastric cancer cells and can perform more fine staining of cancer tissue expressing FGFR2b.

[0331] Example 4: IHC assay of HDM-45 to identify negative cells expressing FGFR2b

[0332] To determine the staining of FGFR2b negative cells with anti-FGFR2b antibody HDM-45, an immunohistochemistry assay was performed on FGFR2b negative gastric cancer cell SNU-5 (origin: ATCC, CRL-5973, P5) according to the method of Example 2, using HDM-45 at an antibody concentration of 1 μg / ml, commercial FGFR2b antibodies SDT-423-18 and SDT-423-2 at 1 μg / ml.

[0333] Figure 3A shows staining of HDM-45 on gastric cancer cell line SNU-5, Figure 3B shows staining of commercial antibody SDT-423-18 on gastric cancer cell line SNU-5, Figure 3C shows staining of commercial antibody SDT-423-2 on gastric cancer cell line SNU-5, and Figure 3D shows SNU-5 staining of isotype negative control. The results show that HDM-45 does not stain FGFR2b negative cell SNU-5, while SDT-423-18 and SDT-423-2 both have some non-specific binding to SNU-5. Thus, the HDM-45 antibody obtained in the present application has more specific binding to FGFR2b, and can more sensitively distinguish between positive cells expressing FGFR2b and negative cells not expressing FGFR2b.

[0334] Example 5: Determination of sensitivity of HDM-45 in specific recognition of gastric cancer tissue

[0335] Using the immunohistochemistry method disclosed in Example 2, the cell material was replaced with gastric cancer tissue expressing FGFR2b (source: Guangzhou Anbiping) for routine section preparation. Then the gastric cancer tissue section expressing FGFR2b was detected using HDM-45. The results showed that HDM-45 can sensitively bind to the gastric cancer tissue expressing FGFR2b. Figure 4A shows the staining results of HDM-45 antibody on the gastric cancer section, and Figure 4B shows the staining results of commercial FGFR2b antibody SDT-423-18 on the gastric cancer section. The results show that HDM-45 has higher sensitivity than commercial antibody SDT-423-18 in staining of the consecutive sections of the same gastric cancer.

[0336] The above determination was repeated using HDM-45 in FGFR2b negative tissue (e.g. tonsil), and Figure 5 shows that HDM-45 does not stain the tonsil section (source: Guangzhou Anbiping).

[0337] The results show that HDM-45 can specifically recognize the gastric cancer tissue expressing FGFR2b with higher sensitivity than known antibodies, and does not have non-specific recognition of negative tissue. Therefore, FGFR2b in gastric cancer can be detected with high sensitivity, and the FGFR2b expression level of the corresponding cancer tissue can be determined, thereby identifying patients suitable for FGFR2b targeted therapy.

[0338] In addition, in view of the higher sensitivity of the HDM-45 antibody of the present application to cancer tissue expressing FGFR2b than known antibodies, it can be more sensitively used to diagnose whether a subject is suffering from the corresponding cancer, thereby improving the accuracy of diagnosis. The high sensitivity of HDM-45 can also be used for earlier diagnosis of cancer, thereby discovering cancer earlier to provide a more favorable therapeutic window period for the corresponding treatment.

[0339] Example 6: HDM-45 for immunohistochemical detection of triple negative breast cancer

[0340] Using similar immunohistochemical method as in Example 5, HDM-45 was used to detect triple negative breast cancer tissue sections (source: Guangzhou Anbiping). The results are shown in Figure 6, indicating that HDM-45 has clear membrane positive staining on triple negative breast cancer tissue sections. The results show that HDM-45 can specifically recognize FGFR2b in breast cancer, and can be used to determine the level of FGFR2b expression, thereby identifying patients suitable for FGFR2b targeted therapy.

[0341] Example 7: HDM-45 specificity verification

[0342] This example uses flow cytometry sorting (FACS) method to evaluate the binding of HDM-45 to FGFR2b and FGFG2c.

[0343] The FACS detection method is disclosed in Example 2, wherein the primary antibody is an anti-FGFR2b antibody, including HDM-45, control FPA144, Aprutumab and isotype negative control.

[0344] The preparation of control antibody FPA144 is referred to WO2015017600, which is incorporated herein by reference in its entirety. FPA144 is a clinical phase III antibody drug, which only recognizes FGFR2b and does not recognize FGFR2c.

[0345] The preparation of control antibody Aprutumab is referred to WO2013076186, which is incorporated herein by reference in its entirety. Aprutumab is a human FGFR2 monoclonal antibody, which can bind to FGFR2b and FGFR2c.

[0346] The results of flow cytometry sorting method are shown in Figures 7A and 7B, indicating that HDM-45 antibody only specifically recognizes FGFR2b and does not recognize FGFR2c. Therefore, the anti-HDM-45 antibody provided in the present application can effectively subtype cancers expressing different molecules (FGFR2b, but not FGFR2c), thereby guiding subsequent targeted therapy and prognosis.

[0347] Sequence Listing

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Fibroblast Growth Factor Receptor 2b (FGFR2b), comprising (i) HCDR1, HCDR2 and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 1, and (ii) LCDR1, LCDR2 and LCDR3 of a light chain variable region as set forth in SEQ ID NO: 2, preferably wherein the CDRs are defined according to IMGT, Kabat, Chothia or Contact, or any combination thereof.

2. The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 1, comprising: 1) CDRs determined according to the Kabat definition: HCDR1 as set forth in SEQ ID NO: 3, HCDR2 as set forth in SEQ ID NO: 4, HCDR3 as set forth in SEQ ID NO: 5, LCDR1 as set forth in SEQ ID NO: 6, LCDR2 as set forth in SEQ ID NO: 7, and LCDR3 as set forth in SEQ ID NO: 8; or 2) CDRs determined according to the AbM definition: HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 12, HCDR3 as set forth in SEQ ID NO: 13, LCDR1 as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16; or 3) CDRs determined according to the IMGT definition: HCDR1 as set forth in SEQ ID NO: 17, HCDR2 as set forth in SEQ ID NO: 18, HCDR3 as set forth in SEQ ID NO: 19, LCDR1 as set forth in SEQ ID NO: 20, LCDR2 as set forth in SEQ ID NO: 21, and LCDR3 as set forth in SEQ ID NO: 22; or 4) CDRs determined according to the Chothia definition: HCDR1 as set forth in SEQ ID NO: 23, HCDR2 as set forth in SEQ ID NO: 24, HCDR3 as set forth in SEQ ID NO: 25, LCDR1 as set forth in SEQ ID NO: 26, LCDR2 as set forth in SEQ ID NO: 27, and LCDR3 as set forth in SEQ ID NO:

28.

3. The anti-FGFR2b antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or consisting of SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or consisting of SEQ ID NO:

2.

4. The anti-FGFR2b antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, The antigen binding fragment is an antibody fragment selected from the group consisting of a Fab, Fab', Fab'-SH, Fv, single chain antibody, scFv, scFab, disulfide bonded scFv, disulfide bonded scFab, (Fab')2 fragment or a linear antibody.

5. The anti-FGFR2b antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain and a light chain, wherein: The heavy chain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or consists of SEQ ID NO: 9; and the light chain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or consists of SEQ ID NO:

10.

6. An isolated nucleic acid encoding the anti-FGFR2b antibody or antigen binding fragment thereof of any of the preceding claims.

7. A vector comprising the nucleic acid of claim 6, preferably the vector is an expression vector.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7, preferably the host cell is a mammalian cell.

9. A method of producing an anti-FGFR2b antibody or antigen binding fragment thereof, the method comprising culturing the host cell of claim 8 under conditions suitable for the expression of the nucleic acid encoding the antibody or antigen binding fragment thereof of any of the preceding claims 1-5, optionally the method further comprises recovering the anti-FGFR2b antibody or antigen binding fragment thereof from the host cell.

10. A kit comprising the anti-FGFR2b antibody or antigen binding fragment thereof according to any of claims 1-5, or produced by the method of claim 9, or the nucleic acid of claim 6, or the vector of claim 7, or the host cell of claim 8, optionally the kit further comprises a secondary antibody detection system, optionally the secondary antibody detection system comprises a secondary antibody labeled with an enzyme, optionally the secondary antibody detection system further comprises components to detect the secondary antibody.

11. A composition comprising the antibody or antigen binding fragment thereof of any of claims 1-5 or produced by the method of claim 9, or the nucleic acid of claim 6, or the vector of claim 7, or the host cell of claim 8.

12. Use of an anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or of a kit according to claim 10, or of a composition according to claim 11, for the manufacture of a product for diagnosing whether a subject is afflicted with a disease associated with an abnormal expression of FGFR2b, or for monitoring the progression of a disease associated with an abnormal expression of FGFR2b, or for determining whether a patient is suitable for receiving an anti-FGFR2b drug treatment, or for determining whether a patient would benefit from an anti-FGFR2b drug treatment, or for predicting the responsiveness of a subject to an anti-FGFR2b drug treatment.

13. Use according to claim 12, wherein said diagnosis, or monitoring of the progression of the disease, or determining whether a patient is suitable for receiving a treatment, or determining whether a patient would benefit from an anti-FGFR2b drug treatment, or predicting the responsiveness of a treatment comprises: 1) contacting a biological sample from said subject with an anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; 2) detecting whether a complex is formed between said antibody or antigen-binding fragment and said biological sample; 3) determining or assessing the presence or the amount of FGFR2b in the biological sample; optionally, comparing the presence or the amount of FGFR2b in the biological sample from said subject with the presence or the amount of FGFR2b in a reference sample; wherein the presence or the amount of FGFR2b in said reference sample is: (i) a predefined threshold of FGFR2b amount indicative of the absence of the disease, (ii) the FGFR2b amount determined in a control sample from a healthy individual, or (iii) the FGFR2b amount determined in a sample from the same patient collected at a second time point; preferably, said detection is performed by immunohistochemistry (IHC) staining; more preferably, said biological sample is a tissue section (e.g. a paraffin section or a frozen section), more preferably said sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

14. A method for detecting the presence or the absence of FGFR2b in a biological sample and / or for quantifying the amount of FGFR2b in a sample, said method comprising: (a) contacting a biological sample with an anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or with an anti-FGFR2b antibody or antigen-binding fragment thereof comprised in a kit according to claim 10 or 11 ; and (b) detecting whether a complex is formed between said antibody or antigen-binding fragment and FGFR2b, and / or determining the amount of FGFR2b in the sample; optionally, said antibody is directly or indirectly labeled with a detectable label.

15. The method according to claim 14, wherein said biological sample is a solid tissue sample and / or a liquid tissue sample, preferably selected from the group consisting of: a cell sample, a tissue sample (e.g. a tissue section and a tissue microarray), preferably said sample is a tissue section (e.g. a paraffin section or a frozen section), more preferably said sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

16. The method according to any one of claims 14-15, wherein the detection of the complex is performed by immunohistochemical (IHC) staining.

17. A method of diagnosing whether a subject suffers from a disease associated with abnormal expression of FGFR2b, comprising: (a) detecting FGFR2b and / or the amount of FGFR2b expression in a biological sample from the subject using an antibody or antigen-binding fragment thereof according to any one of claims 1-5 of the present application, or using a kit according to claim 10 of the present application, or using a composition according to claim 11 of the present application; (b) comparing the amount of FGFR2b expression determined in step (a) to: (i) a predefined threshold of FGFR2b expression indicative of the absence of such a disease, or (ii) the amount of FGFR2b expression determined in a control sample of a healthy individual, wherein a higher amount of FGFR2b expression determined in step (a) compared to the predefined threshold of (i) or the amount of FGFR2b expression determined in the control sample of (ii) is indicative of the subject suffering from a disease associated with abnormal expression of FGFR2b.

18. A method of monitoring the progression of a disease associated with abnormal expression of FGFR2b in a subject, or a method of determining whether a subject is suitable for treatment with an anti-FGFR2b drug, or a method of determining whether a subject will benefit from treatment with an anti-FGFR2b drug, or a method of predicting the responsiveness of a subject to treatment with an anti-FGFR2b drug, comprising: (a) detecting the amount of FGFR2b expression in a sample obtained from the subject at a first time point using an antibody or antigen-binding fragment thereof according to any one of claims 1-5 of the present application, or using a kit according to claim 10 of the present application, or using a composition according to claim 11 of the present application; (b) detecting the amount of FGFR2b expression in a sample obtained from the subject at a second time point or after treatment; and (c) comparing the amount of FGFR2b determined in step (a) to the amount of FGFR2b determined in step (b), wherein if the amount of FGFR2b determined in step (b) is lower than the amount of FGFR2b determined in step (a), it is indicative that the progression of the disease associated with abnormal expression of FGFR2b in the subject is controlled or alleviated, or that the subject is suitable for treatment with an anti-FGFR2b drug, or that the subject will benefit from treatment with an anti-FGFR2b drug, or that the subject is responsive to treatment with an anti-FGFR2b drug.

19. The method of claim 17 or 18, wherein the sample is a solid tissue sample and / or a liquid tissue sample, preferably selected from the group consisting of a cell sample, a tissue sample (e.g. a tissue section and a tissue microarray), preferably the sample is a tissue section (e.g. a paraffin section or a frozen section), more preferably the sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

20. The method according to any one of claims 17 to 19, wherein the disease associated with abnormal expression of FGFR2b is a disease associated with increased amount of FGFR2b expression, preferably a cancer or tumor associated with increased amount of FGFR2b expression.

21. The method according to claim 20, wherein the disease is selected from the group consisting of lung cancer, lung squamous carcinoma, lung adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, intrahepatic cholangiocellular carcinoma, bladder cancer, colorectal cancer, prostate cancer, cervical cancer, large intestine cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, renal cell carcinoma, glioma, head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, oral mucosa cancer, and urothelial cancer; preferably the disease is gastric cancer, breast cancer, lung cancer.

22. The method according to any one of claims 17 to 21, wherein the detecting is performed by immunohistochemistry (IHC) staining.

23. The method according to claim 16 or 22, wherein the immunohistochemistry (IHC) staining method comprises: (i) obtaining a paraffin section; (ii) deparaffinization and hydration; (iii) antigen retrieval; (iv) contacting the section with the anti-FGFR2b antibody; and (v) detecting the binding of the anti-FGFR2b antibody to the antigen FGFR2b, optionally, using a secondary antibody recognizing the anti-FGFR2b antibody, optionally, the secondary antibody is an enzyme-labeled secondary antibody.

24. The method according to claim 23, wherein the step (iv) comprises incubating the antibody at a concentration of 1-10 pg / ml for 10-30 minutes at about 15-30 °C, preferably, at a concentration of 1-5 pg / ml, especially about 1 pg / ml, for 10-20 minutes at 20-26 °C. ​

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