FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
ANTIBODIES AGAINST LIPOCALIN-2 AND USES THEREOF
APPLICANT(S):
(1) Name: ADVANCED CENTRE FOR TREATMENT RESEARCH AND
EDUCATION IN CANCER, TATA MEMORIAL CENTRE
Address: ACTREC, Tata Memorial Centre, Kharghar Node, Navi Mumbai,
Maharashtra-410210
Nationality: Indian
(2) Name: MAZUMDAR SHAW MEDICAL FOUNDATION
Address: 8th Floor, Mazumdar Shaw Medical Center, Narayana Health City, Bommasandra, Bangalore, Karnataka - 560099, India Nationality: Indian
(3) Name: BEYOND ANTIBODY LLP
Address: S005-Krishna Greens, Krishna Temple Road, Dodda Bomasandra, Bangalore, Karnataka-560097, India Nationality: Indian
(4) Name: DEPARTMENT OF BIOTECHNOLOGY
Address: Block 2, 8th Floor, CGO Complex Lodhi Road, New Delhi, Delhi -110003
India
Nationality: Indian
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF INVENTION
The present invention pertains to the field of monoclonal antibodies. More particularly, the invention relates to monoclonal antibodies for targeting cells overexpressing Lipocalin-2 (LCN2).
BACKGROUND OF THE INVENTION
Lipocalin-2 (hereinafter, LCN2), also known as NGAL (neutrophil gelatinase associated lipocalin) is a secreted glycoprotein and is required to maintain the integrity of the gastro-intestinal mucosa. It has been reported that LCN2 is over-expressed in many tumour types and is an indication for the presence of various types of malignancies. An increase in LCN2 expression has been observed in colorectal carcinomas, breast cancer, pancreatic cancer etc.
Plaque protein plakophilin3 (PKP3) is another protein which is responsible for desmosome formation and cell-cell adhesion. It has been reported that the loss of PKP3 leads to decrease in cell-cell adhesion that is followed by increased tumor formation and metastasis.
Though LCN2 and PKP3 had been studied individually, the correlation between the expression of each protein was not known. Further, it was not known whether LCN2 and PKP3expression levels correlate with the resistance to radio or chemotherapy.
The current therapeutic modalities for targeting malignant cells include neo-adjuvant chemo radiotherapy (NACTRT) followed by surgery. However, there are serious limitations to this form of therapeutic modality as 20% of the patients fail to respond to neo-adjuvant chemo radiotherapy (NACTRT) across all histological subtypes. Therefore, identification of potential therapeutic targets that contribute to the resistance to chemo radiotherapy would help improve patient outcomes. Further, limitations of current therapeutic modalities include reduced action of non-specific targeted drugs.
An ideal solution to overcome these limitations is identification of specific biomarkers and development of antibodies specific to the market so that cancer progression can be stopped at the root level.
The inventors have decoded the correlation between PKP3 and LCN2 expression levels and have established LCN2 as potential biomarker. Further, the inventors have addressed the above issues by developing extremely potent monoclonal antibodies which can target LCN2. Apart from other advantages, the developed antibodies help inhibiting chemo-resistance, inhibiting tumour growth, regressing tumour growth and sensitizes the tumors to chemotherapeutics and radiotherapy. Consequently, the invention provides for a novel therapeutic which can be used for treating cancer.

The present invention thus overcomes the problems of the prior art to solve a long-standing problem of providing monoclonal antibodies which can target LCN2 and inhibit LCN2-mediated tumour progression. Further, the antibodies can also reverse the LCN2 mediated resistance to chemo and radio therapy and can serve as a potent therapeutic agent in multiple tumour types.
SUMMARY OF THE INVENTION
Technical Problem
The technical problem to be solved in this invention is to provide monoclonal antibodies which can target LCN2 and inhibit LCN2-mediated tumour progression. Solution to the problem
The problem has been solved by developing monoclonal antibodies which can bind to LCN2 and based on the following complementarity determining regions (CDRs) in the light chain and heavy chain:

SEQUENCE NAME SEQUENCE SEQUENCES OR CONSERVATIVE
ID NO VARIANTS
Light Chain CDR 1 (LCDR1) 3 KASQDINKYIA
Light Chain CDR 2 (LCDR2) 4 YTSTLQP
Light Chain CDR 3 (LCDR3) 5 LQYDNLYT
Heavy Chain 1CDR 1 6 GGSISSYYWS
(H1CDR1)
Heavy Chain 1 CDR 2 7 RIYTSGSTNYNPSLKS
(H1CDR2)
Heavy Chain 1 CDR 3 8 DAVGGRDY
(H1CDR3)
Heavy Chain 2 CDR 1 9 GGSISSSSYYWG
(H2CDR1)
Heavy Chain 2 CDR 2 10 SIYYSGSTYYNPSLKS
(H2CDR2)
Heavy Chain 2 CDR 3 11 NPTRYSSSPFDYYYYYMDV
(H2CDR3)
Table 1: Complementarity determining regions of monoclonal antibodies

Overview of the present invention
In one embodiment, the invention provides monoclonal antibodies or fragment thereof that binds to LCN2 protein, comprising a heavy chain variable domain and a light chain variable domain. The light chain variable domain comprises at least one light chain complementarity determining region (LCDRs) LCDR1 (SEQ ID NO: 3), LCDR2 (SEQ ID NO: 4) and LCDR3 (SEQ ID NO: 5) or conservative variants of the LCDRs. The heavy chain variable domain comprises at least one heavy chain complementarity determining region (HCDRs) H1CDR1 (SEQ ID NO: 6), H1CDR2 (SEQ ID NO: 7), H1CDR3 (SEQ ID NO: 8), H2CDR1 (SEQ ID NO: 9), H2CDR2 (SEQ ID NO: 10) and H2CDR3 (SEQ ID NO: 11) or conservative variants of the HCDRs.
In another embodiment, the invention provides monoclonal antibodies or fragment thereof having the light chain complementarity determining regions (LCDRs) and the heavy chain complementarity determining regions (HCDRs) grafted onto human framework and constant regions.
In another embodiment, the monoclonal antibodies or fragment thereof further comprises the following light chain variable domain framework regions (LCFRs): LCFR1 (SEQ ID NO: 12), LCFR2 (SEQ ID NO: 13), LCFR3 (SEQ ID NO: 14) and LCFR4 (SEQ ID NO: 15) or conservative variants of the LCFRs.
In another embodiment, the monoclonal antibodies or fragment thereof further comprises the following heavy chain variable domain framework regions (HCFRs): HC1FR1 (SEQ ID NO: 16), HC1FR2 (SEQ ID NO: 17), HC1FR3 (SEQ ID NO: 18), HC1FR4 (SEQ ID NO: 19), HC2FR1 (SEQ ID NO: 20), HC2FR2 (SEQ ID NO: 21), HC2FR3 (SEQ ID NO: 22) and HC2FR4 (SEQ ID NO: 23) or conservative variants of the HCFRs.
In another embodiment, the invention provides monoclonal antibodies or fragment thereof wherein the variable region of light chain comprises the amino acid sequence of SEQ ID NO: 24 or conservative variants thereof. In another embodiment, the invention provides monoclonal antibodies wherein the variable region of heavy chain comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26 or conservative variants thereof.
In yet another embodiment, the invention provides a composition comprising a therapeutically effective amount of the monoclonal antibody or fragment thereof, or any combination or mutants of the fragment thereof of the present invention. In another embodiment, the composition further comprises one or more pharmaceutically acceptable carrier, chemotherapeutic agent or excipients.

The invention also provides for nucleic acids encoding the monoclonal antibodies of the present invention, expression vectors comprising the nucleic acid and host cells comprising the expression vector.
The invention also provides methods for producing the monoclonal antibodies of the present invention comprising culturing the host cell expressing the monoclonal antibodies in a culture medium under conditions sufficient to produce the monoclonal antibody.
The invention also provides methods of treating a subject exhibiting LCN2 overexpression disorder by administering to the subject a therapeutically effective amount of the composition comprising the monoclonal antibodies of the present invention.
The invention also provides the structural basis of making any new construct (combination of nucleotide sequence) which will bind to LCN2 and neutralize it.
DESCRIPTION OF THE DRAWINGS
Figure 1 depicts that PKP3 expression was decreased and LCN2 expression was increased in human colorectal cancer (CRC). mRNA prepared from CRC tumour samples and adjacent normal controls was used as a template in real time PCR reactions to determine the levels of PKP3 (Figure 1A) and LCN2 (Figure 1B) and the fold change plotted on the Y-axis. The levels of PKP3 are decreased in tumour samples as compared to the normal controls.
Figure 2 depicts that PKP3 protein levels are decreased and LCN2 protein levels increased in colorectal cancer (CRC) clinical specimens. Figure 2A depicts protein extracts prepared from adjacent normal and tumor tissues being resolved on SDS-PAGE gels followed by Western blotting with anti-LCN2 antibodies. A ponceau stain of the blot serves as a loading control. Figure 2B depicts immunohistochemistry (IHC) analysis of PKP3 levels in adjacent normal and matched tumor tissues. Most samples show a decrease in PKP3 signal.
Figure 3 depicts that LCN2 levels are increased in multiple tumor types.
Figure 4 depicts that LCN2 expression level is elevated in Triple Negative Breast Cancer (TNBC) and is poorly correlated with survival in HNSCC. A. The TCGA data set was analysed for the expression of LCN2 in TNBC vs other tumour types. LCN2 levels are significantly increased (p = 8.12xe-17) in triple negative patients as compared to the other tumour types. B. Kaplan-Meier survival analysis of patients divided into LCN2-high and LCN2-low expression groups, based on median expression level. Log-rank test shows statistically significant difference (p < 0.05) between the two survival graphs.
Figure 5 depicts LCN2 is required for the increased resistance to radiation and 5-fluorouracil (5-FU) upon PKP3 loss. The vector control (vec), the PKP3 knockdown clones (shpkp3.1 and shpkp3.2) and the PKP3 and LCN2 double knockdown clones (shpkp3.2 +

shLCN2-1 and shpkp3.2 + shLCN2-2) were treated with various doses of γ-radiation (A) or 5-FU (B) and clonogenic assays performed. At 14 days post treatment the colonies were stained with crystal violet and counted, and the survival fraction plotted on the Y-axis. The PKP3 loss leads to an increase in resistance to radiation and 5-FU which disappears upon LCN2 loss.
Figure 6 depicts that PKP3 loss leads to radio resistance in vivo. 2 x 106 of the HCT116 derived vector control (vec) or PKP3 knockdown clone (shpkp3.2) were injected sub-cutaneously on the thigh of nude mice (A). Tumor formation and volume were monitored every 2-3 days. Once the tumors reached a certain size, (100 mm3) one set of tumors was irradiated with 4Gy of radiation twice a week, for a total dose of 24Gy. Tumor volume was determined using Vernier caliper using the formula: (0.5 x LV2) where L is the largest dimension and V its perpendicular dimension. A picture of the un-irradiated and irradiated tumors is shown (B) and mean tumor volume and SEM plotted on the Y-axis (n=6) (C). p values were obtained using a student’s t test. Note that the PKP3 knockdown cells show no decrease in tumor formation post radiation.
Figure 7 depicts that PKP3 loss leads to 5-FU resistance in vivo. Immunocompromised mice were injected sub-cutaneously in the dorsal flank with 1 x 106 cells of the HCT116 derived vector control (vec) or PKP3 knockdown clone (shpkp3.2) (A). Once the tumors reached a certain size (30-50 mm3) mice were either injected with the vehicle control (PBS) or 30mg/kg 5-FU (IP) thrice a week for 2 weeks. Tumor volume was monitored using a vernier-calipers using the formula (0.5 x LV2) where L is the largest dimension and V its perpendicular dimension. The mean tumor value and SEM are plotted on the Y-axis. p values were determined using a Student’s t-test. ns=not significant.
Figure 8 depicts that LCN2 is required for increase in resistance to 5-FU. 1000 cells of PKP3+LCN2 double knockdown and the vector control were seeded in 35mm dishes. 24 hours later, the cells were treated with either the vehicle control, recombinant LCN2 (R-LCN2) or heat inactivated recombinant LCN2 (HI-LCN2) for 12 hours followed by treatment with 3µM 5FU for 48 hours. Post treatment the media was changed and 14 days post treatment with 5FU colonies were counted. The survival fraction in plotted on the Y-axis. The mean and SEM of three independent experiments are plotted and p values were generated using a Students t-test.
Figure 9 depicts that PKP3 loss leads to an increase in autophagy upon irradiation. The HCT116 derived vector control (vec), PKP3 knockdown (shpkp3.2), the PKP3 vector clone (shPKP3.2 + vec) and the PKP3 LCN2 double knockdown (shpkp3.2 + shlcn2.1) cells were unirradiated (A) or irradiated (B-C) followed by transmission electron microscopy to detect autophagosomes. Note that the number of autophagosomes (red arrows) increases post

radiation in the cells with PKP3 knockdown (shpkp3.2 and shpkp3.2 + vec) in comparison to the vector control and the double knockdown cells. D. The indicated cell types were irradiated, and Western blots performed using the indicated antibodies. Actin served as a loading control. E. Unirradiated or irradiated cells at various time points post radiation were stained with antibodies to LC3B followed by immunofluorescence analysis and the number of LC3B foci (mean and SEM) plotted on the Y-axis at different time points post radiation. Where indicated p values were determined using a Student’s t-test.
Figure 10 depicts that PKP3 loss leads to an increase in autophagy upon treatment with 5-FU. The HCT116 derived vector control (vec), PKP3 knockdown (shpkp3.2), the PKP3 vector clone (shPKP3.2 + vec) and the PKP3 LCN2 double knockdown (shpkp3.2 + shlcn2.1) cells were untreated (vehicle control) or treated with 5-FU for various lengths of time and stained with antibodies to LC3B followed by immunofluorescence analysis. The number of LC3B foci (mean and standard deviation) plotted on the Y-axis at different time points post treatment. Where indicated p values were determined using a Student’s t-test.
Figure 11 depicts that PKP3 loss leads to a decrease in ROS or efficient ROS clearance in irradiated cells. The HCT116 derived vector control (vec), PKP3 knockdown (shpkp3.2), the PKP3 vector clone (shPKP3.2 + vec) and the PKP3 LCN2 double knockdown (shpkp3.2 + shlcn2.1) cells were unirradiated and stained with dyes to detect total ROS (A) or mitochondrial ROS (B), followed by fluorescence microscopy. Note that the levels of ROS increase post-irradiation in all cell types but decrease dramatically at later time points in the cells with PKP3 knockdown (shpkp3.2 and shpkp3.2 + vec) in comparison to the vector control and the double knockdown cells. The mean intensity and SEM are plotted on the Y-axis and where indicated p values were determined using a Student’s t-test.
Figure 12 depicts that PKP3 loss leads to a decrease in ROS or efficient ROS clearance upon treatment with 5-FU. The HCT116 derived vector control (vec), PKP3 knockdown (shpkp3.2), the PKP3 vector clone (shPKP3.2 + vec) and the PKP3 LCN2 double knockdown (shpkp3.2 + shlcn2.1) cells were treated with 5FU and stained with dyes to detect total ROS followed by fluorescence microscopy. Note that the levels of ROS increase post treatment in all cell types but decrease dramatically at later time points in the cells with PKP3 knockdown (shpkp3.2 and shpkp3.2 + vec) in comparison to the vector control and the double knockdown cells. The mean intensity and SEM are plotted on the Y-axis and where indicated p values were determined using a Student’s t-test.
Figure 13 depicts that LCN2 levels affect the level of cellular iron concentration in HCT116 cells. The HCT116 derived vector control (vec), PKP3 knockdown (shpkp3.2), the

PKP3 vector clone (shPKP3.2 + vec) and the PKP3 LCN2 double knockdown (shpkp3.2 + shlcn2.1) cells were unirradiated or irradiated with 4Gy radiation (A) or treated with the vehicle control or 5FU (B). At different time points, the cells were harvested and the levels of total iron, Fe2+ or Fe3+ determined in three independent experiments and the mean and SEM plotted on the Y-axis. C-D. Catechol levels in the indicated cell lines were determined by LC/MS-MS ESI Q-TRAP post radiation (C) and post treatment with 5-FU (D). Where indicated p values were determined using a Student’s t-test.
Figure 14 depicts the process of generation of monoclonal antibodies to LCN2 and that one of the purified antibodies neutralized VEGF-165 induced proliferation of hTERT-RPE1 cell lines. A. Full length LCN2 was cloned into pET28b (+) digested with NdeI and XhoI so that it would be in frame with the His tag. B. Bacterial protein extracts that were either un-induced or induced with 0.5mM IPTG were resolved on a 15% SDS-PAGE gel followed by silver staining. The supernatant and pellet fractions show the soluble and insoluble fractions respectively. The recombinant LCN2 was purified on Ni/NTA beads and the purified eluted protein and the unbound fractions loaded as indicated followed by Silver staining. Note that the purified protein is present as a single band at the right MW. MW markers are indicated. The purified protein was resolved on 15%SDS-PaGE gels transferred to PVDF and Western blots performed with the indicated antibodies. C. A sandwich ELISA was performed to determine the affinity of clone 3D12B2 to LCN2. Note that clone 6 forms a complex with LCN2 as expected. D. hTERT RPE1 cells were seeded in a 96 well dish in the presence of recombinant LCN2 or LCN2 + clone 3D12B2 and proliferation measured using the CCK8 kit from Sigma as per the manufacturer’s instructions. Note that LCN2 addition resulted in an increase in proliferation that was significantly suppressed by addition of clone 6. p values were obtained using a Student’s t-test.
Figure 15 depicts that treatment with the antibodies generated by the 3D12B2 clone sensitizes the PKP3 knockdown clones to 5-FU in comparison to non-specific mouse IgG. A. The vector control (vec) and the PKP3 knockdown clones (shpkp3.1 and shpkp3.2) were treated with 5-FU or the vehicle control in the presence of non-specific mouse IgG or Clone 3D12B2 or the vehicle control and survival fractions determined. Note that treatment with Clone 3D12B2 sensitizes the PKP3 knockdown clones to 5-FU in comparison to the non-specific mouse IgG. B. Immunocompromised mice were injected sub-cutaneously in the dorsal flank with 1 x 106 cells of the HCT116 derived PKP3 knockdown clone (shpkp3.2). Once the tumors reached a certain size (100 mm3) mice were either injected with the vehicle control (PBS) or 30mg/kg 5-FU (IP) every alternate day for 2 weeks in the presence of either the

vehicle control, non-specific mouse IgG or Clone 3D12B2 (100µg IV). The arrows indicate the time of injection. Tumor volume was monitored using a vernier-calipers using the formula (0.5 x LV2) where L is the largest dimension and V its perpendicular dimension. The mean tumor value and SEM are plotted on the Y-axis. p values were determined using a Student’s t-test. ns=not significant. Note that treatment with Clone 3D12B2 results in an inhibition of tumor growth and treatment with both Clone 3D12B2 and 5FU results in a regression in tumor growth.
Figure 16 depicts that the antibodies sensitizes tumour cells to radiation and inhibits tumour progression in comparison to non-specific IgG. Immunocompromised mice were injected sub-cutaneously in the dorsal flank with 2 x 106 cells of the HCT116 derived PKP3 knockdown clone (shpkp3.2). Once the tumors reached a certain size (50-100 mm3) tumors were irradiated with 4Gy for a total dose of 24Gy, in the presence of either the non-specific mouse IgG or Clone 3D12B2 (100µg IV). The arrows indicate the time of injection of antibody. Tumor volume was monitored using a vernier-calipers using the formula (0.5 x LV2) where L is the largest dimension and V its perpendicular dimension. The mean tumor value and standard deviation are plotted on the Y-axis. p values were determined using a Student’s t-test. ns=not significant. Note that treatment with Clone 3D12B2 results in an inhibition of tumor growth and treatment with both Clone 3D12B2 and radiation results in a regression in tumor growth.
Figure 17 depicts that LCN2 expression is sufficient to induce invasion and chemoresistance. A. HCT116 derived vector control and PKP3 knockdown clones were untreated (UT) or pre-treated with either non-specific mouse IgG or Clone 3D12B2 for 12 hours and then seeded in a Bowden's chamber coated with matrigel to determine invasive potential. The number of cells observed in ten random fields of the membrane for each clone was determined. The mean and standard deviation of three independent experiments is plotted. B. 300 mg of acetone precipitated cell supernatants or 75 mg of protein extracts were prepared from the HCT116 derived vector control (vector) or LCN2 over-expressing clones (LCN2.1 and LCN2.3) were resolved on a 12% gel followed by Western blotting with the indicated antibodies. A Ponceau stain or a Western blot of b-actin served as loading controls for the cell supernatant and the whole cell extracts respectively. C. The HCT116 derived vector control and LCN2 over-expressing clones were treated with 5FU for 48 hours and clonogenic assays performed. The graph is an average of three independent experiments and the mean and standard deviation plotted. The Y-axis represents the survival fraction and the X-axis the concentration of 5FU. D. The HCT116 derived vector control and LCN2 over-expressing clones were untreated (UT) or pre-treated with either non-specific mouse IgG or Clone 3D12B2

for 12 hours and then seeded in a Bowden's chamber coated with matrigel to determine invasive potential. The number of cells observed in ten random fields of the membrane for each clone was determined. The mean and SEM of three independent experiments is plotted. Where indicated p values were determined using a Student’s t-test.
Figure 18-19 depict the plasmid map of pFUSE-CH-Ig-hg-ABH vectors used for cloning the heavy chains of the partially humanized monoclonal antibody.
Figure 20 depicts the plasmid map of pFUSE-CL-Ig-hk-ABL vectors used for cloning the light chains of the partially humanized monoclonal antibody.
Figure 21-23 depicts the plasmid map of pCDNA3 puro used for generating single-chain variable fragment (scFv) antibodies.
BRIEF DESCRIPTION OF SEQUENCE LISTING
SEQ ID NO: 1 represents cDNA sequence of LCN2 protein.
SEQ ID NO: 2 represents amino acid sequence of LCN2 protein.
SEQ ID NO: 3 represents amino acid sequence of Light Chain Complementarity Determining Region 1 (LCDR1).
SEQ ID NO: 4 represents amino acid sequence of Light Chain Complementarity Determining Region 2 (LCDR2).
SEQ ID NO: 5 represents amino acid sequence of Light Chain Complementarity Determining Region 3 (LCDR3).
SEQ ID NO: 6 represents amino acid sequence of Heavy Chain 1 Complementarity Determining Region 1 (H1CDR1).
SEQ ID NO: 7 represents amino acid sequence of Heavy Chain 1 Complementarity Determining Region 2 (H1CDR2).
SEQ ID NO: 8 represents amino acid sequence of Heavy Chain 1 Complementarity Determining Region 3 (H1CDR3).
SEQ ID NO: 9 represents amino acid sequence of Heavy Chain 2 Complementarity Determining Region 1 (H2CDR1).
SEQ ID NO: 10 represents amino acid sequence of Heavy Chain 2 Complementarity Determining Region 2 (H2CDR2).
SEQ ID NO: 11 represents amino acid sequence of Heavy Chain 2 Complementarity Determining Region 3 (H2CDR3).
SEQ ID NO: 12 represents amino acid sequence of Light Chain Framework Region 1 (LCFR1).

SEQ ID NO: 13 represents amino acid sequence of Light Chain Framework Region 2 (LCFR2).
SEQ ID NO: 14 represents amino acid sequence of Light Chain Framework Region 3 (LCFR3).
SEQ ID NO: 15 represents amino acid sequence of Light Chain Framework Region 4 (LCFR4).
SEQ ID NO: 16 represents amino acid sequence of Heavy Chain 1 Framework Region
1 (HC1FR1).
SEQ ID NO: 17 represents amino acid sequence of Heavy Chain 1 Framework Region
2 (HC1FR2).
SEQ ID NO: 18 represents amino acid sequence of Heavy Chain 1 Framework Region
3 (HC1FR3).
SEQ ID NO: 19 represents amino acid sequence of Heavy Chain 1 Framework Region
4 (HC1FR4).
SEQ ID NO: 20 represents amino acid sequence of Heavy Chain 2 Framework Region
1 (HC2FR1).
SEQ ID NO: 21 represents amino acid sequence of Heavy Chain 2 Framework Region
2 (HC2FR2).
SEQ ID NO: 22 represents amino acid sequence of Heavy Chain 2 Framework Region
3 (HC2FR3).
SEQ ID NO: 23 represents amino acid sequence of Heavy Chain 2 Framework Region
4 (HC2FR4).
SEQ ID NO: 24 represents amino acid sequence of variable region of the Light Chain (VL).
SEQ ID NO: 25 represents amino acid sequence of variable region of the Heavy Chain
1 (VH1).
SEQ ID NO: 26 represents amino acid sequence of variable region of the Heavy Chain
2 (VH2).
SEQ ID NO: 27 represents nucleotide sequence of Light Chain Complementarity Determining Region 1 (LCDR1).
SEQ ID NO: 28 represents nucleotide sequence of Light Chain Complementarity Determining Region 2 (LCDR2).
SEQ ID NO: 29 represents nucleotide sequence of Light Chain Complementarity Determining Region 3 (LCDR3).

SEQ ID NO: 30 represents nucleotide sequence of Heavy Chain 1 Complementarity Determining Region 1 (H1CDR1).
SEQ ID NO: 31 represents nucleotide sequence of Heavy Chain 1 Complementarity Determining Region 2 (H1CDR2).
SEQ ID NO: 32 represents nucleotide sequence of Heavy Chain 1 Complementarity Determining Region 3 (H1CDR3).
SEQ ID NO: 33 represents nucleotide sequence of Heavy Chain 2 Complementarity Determining Region 1 (H2CDR1).
SEQ ID NO: 34 represents nucleotide sequence of Heavy Chain 2 Complementarity Determining Region 2 (H2CDR2).
SEQ ID NO: 35 represents nucleotide sequence of Heavy Chain 2 Complementarity Determining Region 3 (H2CDR3).
SEQ ID NO: 36 represents nucleotide sequence of Light Chain Framework Region 1 (LCFR1).
SEQ ID NO: 37 represents nucleotide sequence of Light Chain Framework Region 2 (LCFR2).
SEQ ID NO: 38 represents nucleotide sequence of Light Chain Framework Region 3 (LCFR3).
SEQ ID NO: 39 represents nucleotide sequence of Light Chain Framework Region 4 (LCFR4).
SEQ ID NO: 40 represents nucleotide sequence of Heavy Chain 1 Framework Region
1 (HC1FR1).
SEQ ID NO: 41 represents nucleotide sequence of Heavy Chain 1 Framework Region
2 (HC1FR2).
SEQ ID NO: 42 represents nucleotide sequence of Heavy Chain 1 Framework Region
3 (HC1FR3).
SEQ ID NO: 43 represents nucleotide sequence of Heavy Chain 1 Framework Region
4 (HC1FR4).
SEQ ID NO: 44 represents nucleotide sequence of Heavy Chain 2 Framework Region
1 (HC2FR1).
SEQ ID NO: 45 represents nucleotide sequence of Heavy Chain 2 Framework Region
2 (HC2FR2).
SEQ ID NO: 46 represents nucleotide sequence of Heavy Chain 2 Framework Region
3 (HC2FR3).

SEQ ID NO: 47 represents nucleotide sequence of Heavy Chain 2 Framework Region 4 (HC2FR4).
SEQ ID NO: 48 represents nucleotide sequence of variable region of the Light Chain (VL).
SEQ ID NO: 49 represents nucleotide sequence of variable region of the Heavy Chain
1 (VH1).
SEQ ID NO: 50 represents nucleotide sequence of variable region of the Heavy Chain
2 (VH2).
SEQ ID NO: 51 and SEQ ID NO: 52 represents the forward and reverse primers used for amplification of Variable Immunoglobin chains (IgV).
SEQ ID NO: 53 represents amino acid sequence of variable region of the Light Chain (VL).
SEQ ID NO: 54 represents amino acid sequence of variable region of the Heavy Chain 1 (VH1)
SEQ ID NO: 55 represents the amino acid sequence of variable region of the Heavy Chain 2 (VH2).
SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61,SEQ ID NO: 62 and SEQ ID NO: 63 represents the nucleotide sequences of the primers used for amplifying the variable regions of the heavy (VHC) and light (VLC) chains and the IL2 signal sequence.
SEQ ID NO: 64 and SEQ ID NO: 65 represents the amino acid sequence of the scFv fragments of the anti-LCN2 antibodies.
SEQ ID NO: 66 and SEQ ID NO: 67 represents the nucleotide sequence of the scFv fragments of the anti-LCN2 antibodies.
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any antibody, compositions or methods similar or equivalent to those described herein can also be used in the practice or testing of the embodiments of the present invention, representative illustrative methods and compositions are now described.
Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and

are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.
It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Antibody means a polypeptide comprising at least a “light chain” and a “heavy chain”. Each heavy chain and light chain are comprised of a variable region and a constant region (the regions are also known as “domains”). The variable regions of heavy and light chains recognize and binds to an epitope of an antigen, such as LCN2, or a fragment thereof. The variable domains of heavy chain (VH) and the variable domains of light chain (VL) are responsible for binding the antigen recognized by the antibody. The variable regions of heavy and light chains can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs) or “constant regions”.
Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

The term “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and/or heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies. The term also includes humanized antibodies (also called a “fully human” antibody) which includes human framework regions (also known as constant regions) and at least one of the CDRs.
The term “humanized antibody” or “humanized monoclonal antibody” is a monoclonal antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins have to be constructed by means of genetic engineering.
The term “partially humanized” antibody means an antibody that contains heavy and light chain variable regions of, e.g., murine origin, joined onto human heavy and light chain constant regions.
The term “single chain variable fragment (scFv)” refers to a fusion of the variable regions of the heavy and light chains of immunoglobulin, linked together with a short linker. Single chain antibodies can be single chain composite polypeptides having antigen-binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of immunoglobulin light and heavy chain (linked VH-VL or single chain Fv (scFv)).
The term “expression vector” refers to a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation or transfection into the host.
The term “host cell” includes an individual cell or cell culture, which can be, or has been, a recipient for the subject of expression constructs. Host cells include progeny of a single host cell. Host cell can be any expression host including prokaryotic cell such as but not limited to Escherichia coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.

The term “therapeutically effective amount” refers to the amount of monoclonal antibody composition which is effective to achieve an intended purpose without undesirable side effects (such as toxicity, irritation or allergic response). Although individual needs may vary, optimal ranges for effective amounts of formulations can be readily determined by one of ordinary skill in the art.
The term “chemotherapeutic agent” refers to any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer etc.
The term “conservative variant” as used herein refers to proteins with amino acid substitutions that do not substantially decrease the binding affinity of an antibody for an antigen. For example, a human antibody that specifically binds LCN2 can include at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and specifically bind the LCN2 polypeptide. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody retains binding affinity for LCN2. Non-conservative substitutions are those that reduce an activity or binding to LCN2.
The phrase "pharmaceutically acceptable carrier and excipient" as used herein means a pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material. Each carrier or excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

Amino acids
Group 1 Alanine (A), Serine (S), Threonine (T), Glycine (G), Proline (P)
Group 2 Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q)
Group 3 Arginine (R), Lysine (K), Histidine (H)
Group 4 Isoleucine (I), Leucine (L), Methionine (M), Valine (V)
Group 5 Phenylalanine (F), Tyrosine (Y), Tryptophan (W)
Group 6 Cysteine (C)
Table 2: Amino acid substitution table

DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses monoclonal antibodies which can efficiently and effectively target LCN2 protein. Further, the invention relates to compositions comprising therapeutically effective amount of the monoclonal antibody, methods for producing the monoclonal antibody and methods of treating a subject exhibiting LCN2 overexpression disorder by administering the monoclonal antibodies.
The inventors have contemplated a unique approach by identifying LCN2 as a potential therapeutic target and then developed monoclonal antibodies for targeting LCN2 overexpressing cells.
For the first time, the inventors have been able to generate monoclonal antibodies which are highly effective for treatment of cancers in which LCN2 overexpression takes place.
The effectiveness of the monoclonal antibodies is demonstrated by the following:
1. Ability of antibodies in inhibiting chemo-resistance (Example 13)
2. Ability of antibodies in inhibiting tumor progression and causing regression in tumour growth (Example 14)
3. Ability in suppression in invasive and migration of the malignant cells (Example 15)
4. Sensitization of tumors to chemotherapeutics and radiotherapy (Example 15)
The enzyme can facilitate highly effective treatment for a large number of cancer types and has huge therapeutic potential.
Before the antibodies and methods of the present disclosure are described in greater detail, it is to be understood that the invention is not limited to particular embodiments and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.
In one embodiment, the present invention provides monoclonal antibodies, wherein the antibody contains at least one Complementarity Determining Regions (CDRs) set forth by the following sequences:

SEQUENCE NAME SEQUENCE ID NO SEQUENCE
Light Chain CDR 1 (LCDR1) 3 KASQDINKYIA
Light Chain CDR 2 (LCDR2) 4 YTSTLQP
Light Chain CDR 3 (LCDR3) 5 LQYDNLYT

Heavy Chain 1 CDR 1 6 GGSISSYYWS
(H1CDR1)
Heavy Chain 1 CDR 2 7 RIYTSGSTNYNPSLKS
(H1CDR2)
Heavy Chain 1 CDR 3 8 DAVGGRDY
(H1CDR3)
Heavy Chain 2 CDR 1 9 GGSISSSSYYWG
(H2CDR1)
Heavy Chain 2 CDR 2 10 SIYYSGSTYYNPSLKS
(H2CDR2)
Heavy Chain 2 CDR 3 11 NPTRYSSSPFDYYYYYMDV
(H2CDR3)
Table 3: Amino acid sequences of Complementarity Determining Region
In another embodiment, the monoclonal antibody of the present invention comprises at least one or at least two Light Chain Complementarity Determining Region (LCDR) selected from LCDR1, LCDR2 and LCDR3.
In yet another embodiment, the monoclonal antibody of the present invention comprises at least one or at least two Heavy Chain Complementarity Determining Region (HCDR) selected from H1CDR1, H1CDR2 and H1CDR3.
In yet another embodiment, the monoclonal antibody of the present invention comprises at least one or at least two Heavy Chain Complementarity Determining Region (HCDR) selected from H2CDR1, H2CDR2 and H2CDR3.
In another embodiment, the complementarity determining regions (CDRs)of antibodies of the present invention are conservative variants of the CDRs described herein.
In another embodiment, the nucleic acid sequences encoding the Complementarity Determining Regions (CDRs) are shown in the following table:

SEQUENCE NAME SEQUENCE ID NO SEQUENCE
Light Chain CDR 1 (LCDR1) 27 AAAGCGAGCCAGGACATCAACAAG TACATTGCG
Light Chain CDR 2 (LCDR2) 28 TACACCAGCACCCTGCAACCG
Light Chain CDR 3 (LCDR3) 29 CTGCAATACGATAACCTGTATACC

Heavy Chain 1 CDR 1 30 GGTGGCAGCATCAGCAGCTACTATT
(H1CDR1) GGAGC
Heavy Chain 1 CDR 2 31 CGTATTTACACCAGCGGCAGCACCA
(H1CDR2) ACTATAACCCGAGCCTGAAGAGC
Heavy Chain 1 CDR 3 32 GACGCGGTGGGTGGCCGTGATTAC
(H1CDR3)
Heavy Chain 2 CDR 1 33 GGTGGTAGCATCAGCAGCAGCAGCT
(H2CDR1) ACTATTGGGGT
Heavy Chain 2 CDR 2 34 AGCATTTACTATAGCGGCAGCACCT
(H2CDR2) ACTATAACCCGAGCCTGAAGAGC
Heavy Chain 2 CDR 3 35 AACCCGACCCGTTACAGCAGCAGCC
(H2CDR3) CGTTTGACTACTATTACTATTACATG GATGTG
Table 4: Nucleotide sequences of Complementarity Determining Region
In another embodiment, the nucleic acid encoding the Complementarity Determining Regions (CDRs)may contains the preferred codons for enhanced expression a host cell.
In another embodiment, the framework region of the light chain is lambda (λ) or kappa (k) types.
In another embodiment, the framework region of the light chain contains regions from both lambda (λ) and kappa (k) types.
In another embodiment, the framework region of the heavy chain is selected from a group comprising IgM, IgD, IgG, IgA and IgE.
In yet another embodiment, the framework region of the heavy chain contains regions from one or more of IgM, IgD, IgG, IgA and IgE.
In another embodiment, the present invention provides monoclonal antibodies, wherein the sequences of the Light Chain Framework Regions (LCFRs) and Heavy Chain Framework Regions (HCFRs) are following:

SEQUENCE NAME SEQUENC E ID NO SEQUENCE
Light Chain FR 1 (LCFR1) 12 DIVLTQSPSSLSASLGGKVTITC
Light Chain FR 2 (LCFR2) 13 WYQHKPGKGPRLLIH
Light Chain FR 3 (LCFR3) 14 GIPSRFSGSGSGNDYSFSISNLEPEDIAT YYC

Light Chain 1 FR 4 (LCFR4) 15 FGGGTKLEIKRA
Heavy Chain 1 FR 1 (HC1FR1) 16 QMQLQESGPGLVKPSETLSLTCTVS
Heavy Chain 1 FR 2 (HC1FR2) 17 WIRQPTGKGLEWIG
Heavy Chain 1 FR 3 (HC1FR3) 18 RVTMSVDTSKNQFSLNLSFVTAADTAV YYCAR
Heavy Chain 1 FR 4 (HC1FR4) 19 WGQGTLVTVSS
Heavy Chain 2 FR 1 (HC2FR1) 20 QIQLQESGPGLVKPSETLSLTCTVS
Heavy Chain 2 FR 2 (HC2FR2) 21 WIRQPPGKGLEWIG
Heavy Chain 2 FR 3 (HC2FR3) 22 RVTIGIDTSKRQFSLELSSVTAADTAMY YCAR
Heavy Chain 2 FR 4 (HC2FR4) 23 WGKGTTVTVSS
Table 5: Amino acid sequences of Framework Regions
In another embodiment, the monoclonal antibody of the present invention comprises at least one, or at least two, or at least three Light Chain Framework Region (LCFR) selected from LCFR1, LCFR2, LCFR3and LCFR4.
In another embodiment, the monoclonal antibody of the present invention comprises at least one, or at least two, or at least three Heavy Chain Framework Region (HC1FR) selected from HC1FR1, HC1FR2, HC1FR3 and HC1FR4.
In another embodiment, the monoclonal antibody of the present invention comprises at least one, or at least two, or at least three Heavy Chain Framework Region (HC2FR) selected from HC2FR1, HC2FR2, HC2FR3 and HC2FR4.
In another embodiment, the variable region of the Light Chain (VL) of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 25.
In another embodiment, the variable region of the Heavy Chain (VH1) of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 26.
In another embodiment, the variable region of the Heavy Chain (VH2) of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 27.
In another embodiment, the nucleic acid sequences encoding the Light Chain Framework Regions (LCFRs) and Heavy Chain Framework Regions (HCFRs) are following:

SEQUENCE NAME SEQUENCE ID NO SEQUENCE

Light Chain FR 1 (LCFR1) 36 GATATCGTTCTGACCCAAAGCCCGA
GCAGCCTGAGCGCGAGCCTGGGTGG
CAAGGTTACCATTACCTGC
Light Chain FR 2 (LCFR2) 37 TGGTATCAACACAAGCCGGGTAAAG GTCCGCGTCTGCTGATCCAC
Light Chain FR 3 (LCFR3) 38 GGTATTCCGAGCCGTTTCAGCGGCA GCGGTAGCGGTAACGATTATAGCTT TAGCATCAGCAACCTGGAGCCGGAA GACATTGCGACCTACTATTGC
Light Chain FR 4 (LCFR4) 39 TTCGGTGGTGGCACCAAGCTGGAGA TCAAACGTGCC
Heavy Chain 1 FR 1 40 CAGATGCAACTGCAAGAAAGCGGTC
(HC1FR1) CGGGCCTGGTGAAGCCGAGCGAAA CCCTGAGCCTGACCTGCACCGTTAG C
Heavy Chain 1 FR 2 41 TGGATTCGTCAACCGACCGGTAAAG
(HC1FR2) GCCTGGAGTGGATCGGT
Heavy Chain 1 FR 3 42 CGTGTGACCATGAGCGTTGACACCA
(HC1FR3) GCAAAAACCAATTCAGCCTGAACCT GAGCTTTGTGACCGCGGCGGATACC GCGGTTTATTACTGCGCGCGT
Heavy Chain 2 FR 4 43 TGGGGTCAGGGCACCCTGGTGACCG
(HC2FR4) TTAGCAGC
Heavy Chain 2 FR 1 44 CAGATCCAACTGCAAGAAAGCGGTC
(HC2FR1) CGGGCCTGGTGAAGCCGAGCGAAA CCCTGAGCCTGACCTGCACCGTTAG C
Heavy Chain 2 FR 2 45 TGGATTCGTCAACCGCCGGGTAAAG
(HC2FR2) GCCTGGAGTGGATCGGT
Heavy Chain 2 FR 3 46 CGTGTGACCATCGGCATTGACACCA
(HC2FR3) GCAAACGTCAGTTCAGCCTGGAACT GAGCAGCGTTACCGCGGCGGATACC GCGATGTACTATTGCGCGCGT

Heavy Chain 2 FR 4 47 TGGGGCAAGGGCACCACCGTGACCG
(HC2FR4) TTAGCAGC
Table 6: Nucleotide sequences of Framework Regions
In another embodiment, the nucleic acid encoding the Light Chain Framework Regions (LCFRs) and Heavy Chain Framework Regions (HCFRs) may contains the preferred codons for enhanced expression a host cell.
In another embodiment, the variable region of the Light Chain (VL) of the monoclonal antibody comprises the nucleotide sequence of SEQ ID NO: 48.
In another embodiment, the variable region of the Heavy Chain (VH1) of the monoclonal antibody comprises the nucleotide sequence of SEQ ID NO: 49.
In another embodiment, the variable region of the Heavy Chain (VH2) of the monoclonal antibody comprises the nucleotide sequence of SEQ ID NO: 50.
In another embodiment, the monoclonal antibody is a humanized monoclonal antibody comprising the humanized light chain and a humanized heavy chain immunoglobulin.
In another embodiment, the humanized monoclonal antibody binds to the LCN2 with similar affinity.
In another embodiment, the monoclonal antibody contains the same or conservative variants of the same complementarity determining regions.
In other embodiment, humanized monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering.
In another embodiment, the present invention provides for an expression vector for recombinant production of the monoclonal antibodies. Any suitable vector known to a person skilled in the art can be chosen.
In another embodiment, the present invention provides for a host cell for recombinant production of the monoclonal antibodies. Any suitable host cell known to a person skilled in the art can be chosen.
In another embodiment, the present invention provides for compositions comprising the monoclonal antibodies of the present invention.
In another embodiment, the compositions contain an effective concentration of monoclonal antibodies required for the treatment of cancer. The effective amount would relieve to some extent one or more of the symptoms of the disorder being treated.

In reference to the treatment of cancer, an effective concentration refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis emergence, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, and/or (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer. Therapeutic or pharmacological effectiveness of the doses and administration regimens may also be characterized as the ability to induce, enhance, maintain or prolong disease control and/or overall survival in patients with these specific tumors, which may be measured as prolongation of the time before disease progression".
The compositions may be administered together or independently of one another by any route known to a person skilled in the art.
In one embodiment, the compositions are particularly useful in treatment or alleviation of a disease or disorder in which LCN2 is overexpressed.
The composition of the present invention may further comprise a pharmaceutically acceptable carrier or excipient. The carriers include, but are not limited to, solid diluents or fillers, excipients, sterile aqueous media and various non-toxic organic solvents.
In another embodiment, the invention provides treatment of a subject exhibiting LCN2 overexpression disorder, comprising administering to the subject a therapeutically effective amount of the composition comprising the monoclonal antibodies. Repetition of the administration or dosing regimens may be conducted as necessary to achieve the desired reduction or diminution of cancer cells.
In a further embodiment, the disease or disorder is selected from the group comprising solid, diffused, liquid and resistant forms of cancer.
In a further embodiment, examples of cancer include, but are not limited to liver cancer, melanoma, lung cancer, ovarian cancer, breast cancer, colon cancer, rectal cancer, oropharynx cancer, hypopharynx cancer, esophageal cancer, stomach cancer, pancreas cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, female genital tract cancer, male genital tract cancer, endocrine gland cancer, skin cancer, hemangioma, sarcoma, brain tumor, nerve cancer, eye tumor, meninges cancer, solid tumors from hematopoietic malignancy and tumor metastases.
EXAMPLES
The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.

Example 1: Studies on regulation of Lipocalin-2 (LCN2) gene and plakophilin-3 (PKP3) gene in normal and colorectal cancer tissues
mRNA prepared from colorectal cancer (CRC) tumour samples and normal tissues were used as templates in RT-PCR reaction to determine the levels of LCN2 and PKP3.
It was found that PKP3 mRNA levels were lower in tumor samples as compared to normal tissue. The results are depicted in Figure 1A.
Further, it was found that LCN2 mRNA levels were higher in tumor tissue as compared to the normal tissue. The results are depicted in Figure 1B.
The results depict that an increase in LCN2 expression can drive tumor progression in vivo.
Protein extracts prepared from adjacent normal and tumor tissues were resolved on SDS-PAGE gels followed by Western blotting with anti-LCN2 antibodies. The results as depicted in Figure 2A clearly shows that LCN2 protein levels increase in the CRC tumor samples.
Further, immunohistochemistry (IHC) analysis of PKP3 levels in normal and tumor tissues clearly shows that PKP3 protein levels decrease in the CRC tumor samples as compared to normal samples. The results are depicted in Figure 2B.
Table 7: Summary of the data on PKP3 and LCN2 levels in colon tumour samples
The data summarized in Table 7depicts that LCN2 protein levels are elevated in 28/40 (70%) of the colon tumour samples as compared to the adjacent normal tissue and PKP3 protein levels are decreased in 10/22 (45%) of the tumour samples as compared to normal tissue. Most of the tumour samples with PKP3 levels lower than the normal tissue exhibited high LCN2 levels (9/10). It was found that when PKP3 levels in tumours are decreased as compared to normal tissue, 90% of these samples show elevated LCN2 levels.

Example 2: Studies on LCN2 regulation in multiple tumour types
In order to check whether, LCN2 expression levels increase in multiple tumour types as compared to normal samples, expression analysis was carried out across RNA panel of 95 tumor samples obtained from the ACTREC and TMH tissue repositories, India.
The expression was compared to normal RNA for each tumor type, which was procured from Agilent and was a pool of at least six healthy donors.
Figure 3 depicts the results of the experiments. Points denote fold changes of target LCN2 expression intensity in tumor tissue samples compared with normal tissue. Horizontal bars show the median value of fold changes in each tissue type. The q-RT PCR analysis confirmed that expression of LCN2 was significantly increased in invasive breast carcinoma (n=9), hepatocellular carcinoma (n=8), glioblastoma (n=8), stomach adenocarcinoma (n=8), testicular seminoma(n=4), colon adenocarcinoma (n=9), rectal adenocarcinoma (n=8) and penile squamous cell carcinoma (n=8) when compared with respective normal tissues (p = 0.0001, p = 0.006, p=0.001, p=0.0004, p=0.0001, p=0.001, p=0.0001 and p=0.03 respectively). In contrast, LCN2 expression was not significantly altered in of tongue squamous cell carcinoma (n=9), buccal squamous cell carcinoma (n=8), renal cell carcinoma (n=8) and ovarian serous adenocarcinoma (n=8) compared to the respective normal samples (p = 0.3, p = 0.83, p=0.42 and p=0.43 respectively).
The results depict that LCN2 expression is increased in multiple tumour types and can be as a potential target.
Studies were also conducted to check the expression of LCN2 in Triple Negative Breast Cancer (TNBC) samples (MCF-7, MCF10A and MDAMB231 cell lines maintained at ACTREC, Navi Mumbai, India). The Cancer Genome Atlas (TCGA) data sets were analysed for expression of LCN2 in in Triple Negative Breast Cancer (TNBC) samples as compared to other tumour types. It was found that LCN2 expression is significantly elevated in Triple Negative Breast Cancer (p=8.12 X e-17) as compared to other breast tumour types. The results are depicted in Figure 4A.
Further, studies were conducted to compare the RNA expression levels in Head and Neck Squamous cell carcinoma (HNSCC). The Cancer Genome Atlas (TCGA) shows lower expression of LCN2 in tumor samples in comparison to the normal samples present in the database.
The Kaplan-Meier survival analysis was conducted based on patients LCN2-high and LCN2-low expression groups, based on median expression levels. The log-rank test shows statistically significant difference (p<0.05) between the two survival graphs. The regression

analysis of survival data clearly depicts that in a pool of Head and Neck cancer patients, high expression of LCN2 leads poorer survival probability. The results are depicted in Figure 4B.
Example 3: Studies to establish that loss in PKP3 expression and increase in LCN2 expression is required for increasing the radio-resistance in all cell types
The inventors had previously demonstrated that loss in PKP3 expression leads to increase of radio-resistance in cells. The role of LCN2 in increasing the radio-resistance of cells was studied.
HCT116 (obtained from Johns Hopkins Medicine, USA) was used for the purposes of the experiments. Five different HCT116 derived knockdown clones and one vector control was used for the purposes of the experiment as given below:

Clone Name Description
vec Empty vector
shpkp3-1 PKP3 knockdown HCT116 clone
shpkp3-2 PKP3 knockdown HCT116 clone
shpkp3-2+vec Derived by transfecting empty vector in the shpkp3-2 clone
shpkp3-2 + shLCN2-1 PKP3 + LCN2 double knockdown clone
shpkp3-2 + shLCN2-2 PKP3 + LCN2 double knockdown clone
The clones were treated with various doses of γ-radiation to calculate survival fraction of the cells. Clonogenic assay was performed and the colonies were stained with crystal violet for counting at 14 days post-treatment. The survival fraction was plotted on the Y-axis and the radiation dosage (in Grays) was plotted on X-axis. Figure 5A depicts the results of the experiment. The experiments clearly depict that LCN2 is required for the increase in radio-resistance observed upon PKP3 loss.
Studies were conducted to determine whether the resistance to radiation were also exhibited in vivo.
2 X 106 of the HCT116 derived vector control (vec) or PKP3 knockdown clone (shpkp3-2) were injected subcutaneously on the thigh of nude mice. Tumor formation and volume were monitored every 2-3 days.
Once the tumours reached a certain size (about 100 mm3), one set of tumours were irradiated with 4Gy of radiation for a total dose of 24 gray. Only the thigh was irradiated to ensure that any toxic effects of radiation on the animal would not interfere with the interpretation of the experiment.

Tumour volume was determined using vernier callipers using the formula (0.5 X LV2) where L is the largest dimension and V is the perpendicular dimension.
Figure 6A depicts the different mice samples with and without radiation. Figure 6B depicts unirradiated and irradiated tumours. Figure 6C depicts the mean tumour volume and standard deviation plotted on Y-axis (n=6). p values were obtained using Student's t-test.
It was noted that the vector control clones showed decrease in tumour formation post few doses of radiation, while the PKP3 knockdown clones did not show decrease in tumour formation when treated with radiation and grew to the same degree as that observed in un-irradiated mice.
This clearly depicts that loss in expression of PKP3 leads to radioresistance in vivo.
Example 5: Studies to establish that loss in PKP3 expression and increase in LCN2 expression is required for increasing the resistance to chemotherapeutic agents
It was also studied whether the loss of PKP3 leads to resistance to the chemotherapeutic agent 5-fluorouracil (5FU) and whether the increase in radioresistance is also dependent upon LCN2 expression Experiments were performed with the following clones:

Clone Name Description
vec Empty vector
shpkp3-1 PKP3 knockdown HCT116 clone
shpkp3-2+vec Derived by transfecting empty vector in the shpkp3-2 clone
shpkp3-2 + shLCN2-1 PKP3 + LCN2 double knockdown clone
shpkp3-2 + shLCN2-2 PKP3 + LCN2 double knockdown clone
The clones were treated with various concentration of 5-fluorouracil to calculate survival fraction of the cells. Clonogenic assay was performed and the colonies were stained with crystal violet for counting at 14 days post-treatment. The survival fraction was plotted on the Y-axis and the concentration of 5-fluorouracil (in µM) was plotted on X-axis. Figure 5B depicts the results of the experiment. The experiments clearly depict that loss of PKP3 leads to resistance to the chemotherapeutic agent 5-fluorouracil (5FU) and this increase in resistance is directly proportional to LCN2 expression.
Studies were conducted to determine whether the resistance to chemotherapeutic agent 5-fluorouracil (5FU) were also exhibited in vivo.

Immunocompromised mice were injected subcutaneously in the dorsal flank with 1 X 106 cells of the HCT116 derived vector control (vec) or PKP3 knockdown clone (shpkp3-2). Tumor formation and volume were monitored.
Once the tumours reached a certain size (about 30-50 mm3), the mice were either injected with the vehicle control (PBS) or 30 mg/kg 5-FU (IP) every alternative day.
Tumour volume was determined using Vernier callipers using the formula (0.5 X LV2) where L is the largest dimension and V is the perpendicular dimension. The mean tumour volume and standard deviation plotted on Y-axis and the number of days is plotted on X-axis. p values were obtained using Student's t-test.
Figure 7A depicts the different mice samples with and without chemotherapeutic agent. Figure 7B depicts the mean tumour volume and standard deviation (p) plotted on Y-axis with time. N.S denotes that the standard deviations are not significant.
It was noted that the vector control clones showed decrease in tumour formation post administration with 5-FU, while the PKP3 knockdown clones did not show decrease in tumour formation when administered with 5-FU and grew to the same degree as that observed in un-irradiated mice.
This clearly depicts that loss in expression of PKP3 leads to resistance to chemotherapeutic agents in vivo.
Example 6: Studies to determine whether LCN2 leads to resistance to 5FU
Studies were conducted to determine whether restoration of LCN2 led to a reversal of the sensitivity observed in the PKP3 LCN2 double knockdown clones.
1000 cells of PKP3+LCN2 double knockdown and vector control were seeded in 35 mm dishes. 24 hours later, the cells were treated with either the vehicle control, recombinant LCN2 (R-LCN2) or heat inactivated LCN2 (HI-LCN2) for 12 hours followed by treatment with 3 µM 5-FU for 48 hours.
Post-treatment, the media was changed, and clonogenic assays were performed to count the colonies after 14 days post-treatment. The survival fraction was plotted on Y-axis. The mean and SEM of the three independent experiments are plotted and p values were generated using Student’s t-test. Figure 8 depicts the results of the experiments.
It was found that treatment with R-LCN2 restored resistance to 5FU in the double knockdown cells as compared to the vehicle control or HI-LCN2. These results depict that increase in LCN2 expression increases the resistance to 5FU.

Example 7: LCN2 expression stimulates autophagy
Studies were conducted to determine whether loss of PKP3 led to an increase in autophagy. HCT116 derived vector control (vec), PKP3 knockdown (shpkp3-2), PKP3 vector clone (shpkp3-2+vec) and PKP3 LCN2 double knockdown clone (shpkp3-2 + shLCN2-1) were irradiated and then processed for imaging using a Transmission Electron Microscope (TEM) to detect autophagosomes.
It was noted that the number of autophagosomes increased post radiation in the cells with PKP3 knockdown (shpkp3-2 and shpkp3-2+vec) in comparison to vector control and double knockdown clone (shpkp3-2 + shLCN2-1).
Figure 9A depicts that in the absence of irradiation, none of the cells showed an increase in the number of autophagic vacuoles. Figure 9B depicts that upon irradiation, the PKP3 knockdown clones show an increase in the number of autophagosomes as compared to the vector controls. Figure 9C depicts that loss of LCN2 in the PKP3 knockdown cells leads to a reduction in the number of autophagosomes as compared to the vector controls.
The above results were confirmed by monitoring the lipidation of LC3B protein which is an initial step in the formation of autophagosomes. It was confirmed that PKP3 loss led to the formation of more lipidated form of LC3B. The results were reversed in double knockdown clone (shpkp3-2 + shLCN2-1). Figure 9D depicts the Western blots performed to monitor the lipidation of LC3B with actin serving as a loading control.
Further studies were conducted to quantitate the number of autophagosomes formed. The cells were irradiated and stained with anti-LC3B antibodies, followed by immunofluorescence analysis. The number of LC3B foci (mean and SEM) were plotted on Y-axis at different points post radiation. p values were calculated using Student’s t-test.
The results are depicted in Figure 9E. It was clearly shown that the numbers of LC3B foci were increased in cells lacking PKP3 as compared to the vector control cells and cells lacking LCN2. These results depict that LCN2 expression stimulates autophagy in irradiated cells.
Similarly, studies were conducted to confirm whether PKP3 loss leads to an increase in autophagy upon treatment with 5-FU.
HCT116 derived vector control (vec), PKP3 knockdown (shpkp3-2), PKP3 vector clone (shpkp3-2+vec) and PKP3 LCN2 double knockdown clone (shpkp3-2 + shLCN2-1) were treated with 5-FU for various lengths of time and stained with antibodies to LC3B followed by immunofluorescence analysis.

The number of LC3B foci (mean and standard deviation) plotted on Y-axis at different time points post treatment. p values were calculated using Student’s t-test
The results are depicted in Figure 10. The results clearly depict that autophagy is enhanced upon PKP3 loss and that the increase in autophagy is dependent upon LCN2 expression.
Example 8: LCN2 expression stimulates response to oxidative stress
Studies were conducted to determine whether increase in LCN2 expression levels in the PKP3 knockdown cells leads to a decrease in reactive oxygen species (ROS) levels post radiation or treatment with 5-fluorouracil (5-FU).
HCT116 derived vector control (vec), PKP3 knockdown (shpkp3-2), PKP3 vector clone (shpkp3-2+vec) and PKP3 LCN2 double knockdown clone (shpkp3-2 + shLCN2-1) were irradiated and stained with dyes to detect total ROS or mitochondrial ROS, followed by fluorescence microscopy.
The cells were cultured in glass bottom dishes, irradiated and at the indicated time points, cells were treated with 5 μM Cell ROXTM Orange (dye), which fluoresces in the presence of reactive oxygen species (ROS) for 20 minutes at 37°C.
The total fluorescence was measured in three independent experiments. Figure 11A depicts that the levels of ROS increased in all cell types upon irradiation. However, the ROS levels decreased precipitously at 24 hours post radiation in the PKP3 knockdown cells (shpkp3-2 and shpkp3-2+vec). However, the ROS does not decrease in the vector control or the PKP3 LCN2 double knockdown cells.
To determine mitochondrial ROS levels in live cell assays post radiation, cells were stained with 100 nMMitoTracker Green (to identify mitochondria) and 5 μMMitoSOXTM Red (MitoSox Red dye specifically accumulates in mitochondria which upon oxidation by superoxide radicals shows red fluorescence) post irradiation for 20 minutes at 37 °C. The total fluorescence was measured in three independent experiments.
Figure 11B depicts that the levels of mitochondrial ROS increased in all cell types upon irradiation. However, the mitochondrial ROS levels decreased precipitously at 24 hours post radiation in the PKP3 knockdown cells (shpkp3-2 and shpkp3-2+vec).However, the ROS does not decrease in the vector control or the PKP3 LCN2 double knockdown cells.
To determine ROS levels in live cell assays post treatment with 5-FU, cells were treated with 5-FU and stained with dyes to detect total ROS followed by fluorescence microscopy. The total fluorescence was measured in two independent experiments.

Figure 12 depicts that the cells treated with 5FU showed an increase in ROS production that was rapidly decreased in cells expressing LCN2 as opposed to the vector control or the LCN2 double knockdown cells. The results depict that the chemo and radio resistance exhibited by the PKP3 knockdown cells is due to their ability to rapidly clear ROS.
Example 9: Increase in LCN2 levels upon PKP3 knockdown regulate iron metabolism
In mammals, LCN2 binds to iron in complex with a small molecule called catechol. LCN2 forms a complex with Fe3+ bound catechol in circulation and this complex is taken up in cells via endocytosis and the Fe3+ recycled in endosomes in a pH dependent manner. In the absence of LCN2, the catechol facilitates the conversion of Fe3+ to Fe2+ resulting in the generation of hydroxyl radicals.
Studies were conducted to check whether LCN2 inhibits the conversion of Fe3+ to Fe2+ by catechols.
Levels of total iron and the Fe3+ to Fe2+ forms in cells at different time points were determined post radiation using a colorimetric kit (Abcam catalogue no. ab83366). The kit measures the levels of total iron and ferrous iron and the levels of ferric iron are determined by subtracting the level of ferrous iron from total iron.
Figure 13A depicts that the levels of total iron or the Fe3+ to Fe2+ forms are unchanged in all the cell types tested. However, post-radiation the levels of total iron fall in the PKP3 knockdown cells as compared to the vector control and the double knockdown cells. In addition, while the levels of Fe2+ iron increase significantly in the vector control and the double knockdown cells, they do not increase to the same degree in the PKP3 knockdown cells.
Figure 13B depicts that the levels of total iron or the Fe3+ to Fe2+ forms are unchanged in all the cell types tested. However, post-treatment with 5-FU, the levels of total iron fall in the PKP3 knockdown cells as compared to the vector control and the double knockdown cells. In addition, while the levels of Fe2+ iron increase significantly in the vector control and the double knockdown cells, they do not increase to the same degree in the PKP3 knockdown cells.
These results depict that the increase in LCN2 levels upon PKP3 knockdown regulate iron metabolism, which correlates with the results regarding the reduction in ROS levels observed upon PKP3 loss.
Studies were conducted to determine if the change in ROS levels is accompanied by a change in catechol levels. For the studies, intracellular catechol levels were measured.
Cells were mechanically harvested and passed through a syringe to generate a single cell suspension. 2x106 cells were lysed by sonication and the supernatant processed for catechol extraction. The catechol was purified on a C18-E STRATSA cartridge with a capacity of 50mg

(Phenomenex, Torrance, CA, US) as per the manufacturer's protocol and the sample eluted using 1ml of Formic acid: methanol (5:95). The eluted sample was vacuum dried and reconstituted in 50μl of Acetonitrile: Methanol: Water (10:20:70). The supernatant was vacuum dried and reconstituted in 20μl of Acetonitrile: Methanol: Water (10:20:70) mix and used for further LC/MS-MS analysis.
Liquid chromatography was performed on a Shimanzu Nexera-X2 series equipped with a gradient pump with vacuum degasser, an auto sampler and a column oven. The chromatographic separation was accomplished by Gradient elution. Mass spectro-metric analysis was performed on a Triple Quadrupole LC/MS-MS Q-TRAP 4500 series mass spectrometer (AB SCIEX QTRAP 4500Canada) equipped with an Electro Spray Ionization (ESI) source.
MS analysis was performed in the targeted multiple reactions monitoring (MRM) negative mode. Data acquisition and processing was conducted using Analyst1.6.2 software. The mobile phase was - Solvent A -Water with 0.1% formic acid in H2O and solvent B– acetonitrile with 0.1% formic acid in acetonitrile (ACN).
Figure 13C depicts that the catechol levels are unchanged in all the cell types tested. However, post-radiation there was no appreciable change in catechol levels for any cell type.
Figure 13D depicts that the catechol levels are unchanged in all the cell types tested. However, post-treatment with 5-FU the there was no appreciable change in catechol levels for any cell type.
Figure 13C and 13D depicts that there was no appreciable change in catechol levels and therefore PKP3 expression loss is not correlated with the amount of catechol.
Example 10: Production of monoclonal antibody
Full length LCN2 cDNA (SEQ ID NO: 1) was amplified and codon optimized for bacterial expression was synthesized (by GenScript, USA) and cloned into pET-28b (+) (by GenScript, USA) digested with NdeI and XhoI sites as depicted in Figure 14A.
LCN2 protein (SEQ ID NO: 2) was expressed in and purified from E. coli Rosetta strain (maintained at IISc, Bangalore, India). The protein was purified using Ni/NTA beads followed by silver staining for purity check and was characterized via Western blotting using anti-His antibody and a polyclonal antibody to LCN2 as depicted in Figure 14B.
Balb/c mice (Vivo Sciences, Bangalore, India) were injected with 10µg of pure LCN2 protein with Freund’s complete adjuvant in 1:1 ratio followed by two boosters at a gap of 21 days each with Freund’s Incomplete Adjuvant in 1:1 ratio.

Finally, an intra-peritoneal injection of 50µg of pure LCN2 protein (5x of primary immunization) was given and the fusion was done within 5 days of final immunization.
For Hybridoma generation, the spleenocytes of the immunised mice were fused with mouse myeloma cells SP2/O (provided by Dr. Anjali Karande, IISc) with the help of PEG (Poly-ethylene-glycol) in a ratio of 7:1. The clones thus formed were selected using HAT (hypoxanthine-aminopterin-thymidine)-HT (Hypoxanthine-Thymidine) selection method.
Cells with secreting antibody were single-cell-cloned by dilution plating. The developed clones were checked using ELISA for antibody production. Selected monoclonal hybridomawere grown in T75 flasks and secreting antibodies were purified from cell supernatantswith Protein A beads.
One of the purified antibodies were shown to be neutralizing VEGF-165 induced proliferation of hTERT-RPE1 cell lines (obtained from ATCC, USA) at MSMF.
The monoclonal antibody was named as 3D12B2. The antibody was further characterized.
Example 11: Characterization of the monoclonal antibody
3D12B2 was characterized for its ability to form a complex with LCN2 as determined by Western blot as depicted in Figure 14B and a sandwich ELISA as depicted in Figure 14C.
To determine if it can neutralize LCN2, recombinant LCN2 (rLCN2) was added to hTERT RPE1 in the presence or absence of 3D12B2 and proliferation measured using the CCK8 kit (Sigma).
Figure 14D depicts that 3D12B2 inhibited the proliferation induced by rLCN2. These results suggested that 3D12B2 can inhibit LCN2 function.
Example 12: Structural characterization of the monoclonal antibody
The sequence of the monoclonal antibody was characterized in order to determine the Complementarity determining regions (Hypervariable regions) and the Framework Regions of the variable chains.
The sequences of the Complementarity Determining Regions (CDRs) are provided in the following table:

SEQUENCE NAME SEQUENCE ID NO SEQUENCE
Light Chain CDR 1 (LCDR1) 3 KASQDINKYIA
Light Chain CDR 2 (LCDR2) 4 YTSTLQP
Light Chain CDR 3 (LCDR3) 5 LQYDNLYT

Heavy Chain 1CDR 1
6 GGSISSYYWS
(H1CDR1)
Heavy Chain 1 CDR 2 7 RIYTSGSTNYNPSLKS
(H1CDR2)
Heavy Chain 1 CDR 3 8 DAVGGRDY
(H1CDR3)
Heavy Chain 2 CDR 1 9 GGSISSSSYYWG
(H2CDR1)
Heavy Chain 2 CDR 2 10 SIYYSGSTYYNPSLKS
(H2CDR2)
Heavy Chain 2 CDR 3 11 NPTRYSSSPFDYYYYYMDV
(H2CDR3)
Table 9: Amino acid sequences of sequences of the Complementarity Determining
Regions
The sequences of the Framework regions (FRs) are provided in the following table:

SEQUENCE NAME SEQUENCE ID NO SEQUENCE
Light Chain FR 1 (LCFR1) 12 DIVLTQSPSSLSASLGGKVTITC
Light Chain FR 2 (LCFR2) 13 WYQHKPGKGPRLLIH
Light Chain FR 3 (LCFR3) 14 GIPSRFSGSGSGNDYSFSISNLEPEDIATYYC
Light Chain 1 FR 4 (LCFR4) 15 FGGGTKLEIKRA
Heavy Chain 1 FR 1 (HC1FR1) 16 QMQLQESGPGLVKPSETLSLTCTVS
Heavy Chain 1 FR 2 (HC1FR2) 17 WIRQPTGKGLEWIG
Heavy Chain 1 FR 3 (HC1FR3) 18 RVTMSVDTSKNQFSLNLSFVTAADTAVYYCAR
Heavy Chain 1 FR 4 (HC1FR4) 19 WGQGTLVTVSS

Heavy Chain 2 FR 1 (HC2FR1) 20 QIQLQESGPGLVKPSETLSLTCTVS
Heavy Chain 2 FR 2 (HC2FR2) 21 WIRQPPGKGLEWIG
Heavy Chain 2 FR 3 (HC2FR3) 22 RVTIGIDTSKRQFSLELSSVTAADTAMYYCAR
Heavy Chain 2 FR 4 (HC2FR4) 23 WGKGTTVTVSS
Table 10: Amino acid sequences of sequences of the Framework Regions Example 13: Treatment with antibodies can inhibit chemoresistance
Studies were conducted to determine whether the monoclonal antibody (3D12B2) can inhibit the resistance exhibited upon LCN2 expression.
HCT116 derived vector control (vec), PKP3 knockdown clones (shpkp3-1 and shpkp3-2), were treated with 5-FU or the vehicle control, and the survival fractions were determined.
It was found that treatment with the antibodies generated by the 3D12B2 clone sensitizes the PKP3 knockdown clones to 5-FU in comparison to non-specific mouse IgG. The results are depicted in Figure 15A.
The study clearly depicts that treatment with 3D12B2 could inhibit chemo-resistance observed in the PKP3 knockdown cells. However, treatment with non-specific mouse IgG cannot inhibit chemo-resistance.
Example 14: Treatment with monoclonal antibody inhibited tumor progression
Studies were conducted to determine if treatment with 3D12B2 could lead to tumor regression in vivo.
Immunocompromised mice were injected subcutaneously in the dorsal flank with 1 X 106 cells of the HCT116 derived PKP3 knockdown clone (shpkp3-2). Tumor formation and volume were monitored.
Once the tumours reached a certain size (about 100 mm3), the mice were either injected with the vehicle control (PBS) or 30 mg/kg 5-FU (IP) every alternative day in the presence of the vehicle control (PBS), non-specific mouse IgG and monoclonal antibodies from Clone 3D12B2 (100 µg IV).
Tumour volume was determined using vernier callipers using the formula (0.5 X LV2) where L is the largest dimension and V is the perpendicular dimension. The mean tumour

volume and standard deviation plotted on Y-axis and the number of days is plotted on X-axis. p values were obtained using Student's t-test.
It was noted that tumours formed efficiently in mice injected with the PKP3 knockdown clones that have high LCN2 levels both in the presence or absence of 5-FU. Treatment with the non-specific mouse immunoglobulin did not affect tumour formation. Treatment with antibodies from Clone 3D12B2 results in inhibition of tumour growth. Further, treatment with antibodies from Clone 3D12B2 and 5-FU results in regression in tumour growth.
Figure 15B depicts the mean tumour volume and standard deviation (p) plotted on Y-axis with time. The arrows indicate the time of injection. N.S denotes that the standard deviations are not significant.
It was noted that the treatment with antibodies from Clone 3D12B2 results in inhibition of tumour growth. Further, treatment with antibodies from Clone 3D12B2 and 5-FU results in regression in tumour growth.
A similar study was conducted to check the effects of irradiation along with 3D12B2 antibodies.
Immunocompromised mice were injected subcutaneously in the dorsal flank with 2 X 106 cells of the HCT116 derived PKP3 knockdown clone (shpkp3-2). Tumor formation and volume were monitored.
Once the tumours reached a certain size (about 50-100 mm3), the tumours were irradiated with 4Gy of radiation for a total dose of 24 gray in the presence of non-specific mouse IgG and monoclonal antibodies from Clone 3D12B2 (100 µg IV).
Tumour volume was determined using vernier callipers using the formula (0.5 X LV2) where L is the largest dimension and V is the perpendicular dimension. The mean tumour volume and standard deviation plotted on Y-axis and the number of days is plotted on X-axis. p values were obtained using Student's t-test.
It was noted that treatment with the non-specific mouse immunoglobulin did not affect tumour formation. Treatment with antibodies from Clone 3D12B2 results in inhibition of tumour growth. Further, treatment with antibodies from Clone 3D12B2 and radiation results in regression in tumour growth.
Figure 16 depicts the mean tumour volume and standard deviation (p) plotted on Y-axis with time. The arrows indicate the time of injection. N.S denotes that the standard deviations are not significant. These results clearly depict that 3D12B2 antibodies could serve as a lead molecule for tumour therapy.

Example 15: Treatment with monoclonal antibody results in decrease in invasion
Studies were conducted to determine whether 3D12B2 can inhibit invasion. Invasion assays were performed in the vector control and PKP3 knockdown clones in the presence or absence of antibody.
HCT116 derived vector control and PKP3 knockdown clones (shpkp3-2) were untreated and pre-treated with either non-specific mouse IgG or antibodies from Clone 3D12B2 for 12 hours and then seeded in Bowden’s chamber coated with matrigel to determine invasive potential. The number of cells observed in in ten random fields of the membrane for each clone was determined. The mean and standard deviation of three independent experiments were plotted.
Figure 17A depicts the results. It can be clearly seen that antibodies from Clone 3D12B2 inhibited the increased invasion upon PKP3 loss. Thus, the antibodies from Clone 3D12B2 inhibits tumour progression by inhibiting invasion and migration.
Further studies were conducted to determine whether LCN2 was both necessary and sufficient for the increase in chemo resistance and invasion in these cells HCT116 cells were transfected with either the vector control or an over-expression clone of LCN2 followed by selection in G418.
300 mg of acetone precipitated cell supernatants or 75 mg of protein extracts were prepared from the HCT116 derived vector control (vector) or LCN2 over-expressing clones (LCN2.1 and LCN2.3) were resolved on a 12% gel followed by Western blotting with the indicated antibodies. A Ponceau stain or a Western blot of b-actin served as loading controls for the cell supernatant and the whole cell extracts respectively. The Western blot performed for LCN2 depicted that the selected clones over-expressed LCN2 in comparison to the vector control as shown in Figure 17B.
The HCT116 derived vector control and LCN2 over-expressing clones were treated with 5-FU for 48 hours and clonogenic assays performed. The graph is an average of three independent experiments and the mean and standard deviation plotted. The Y-axis represents the survival fraction and the X-axis the concentration of 5FU. The assays as shown in Figure 17C demonstrated that the two clones showed increased resistance to 5-FU.
The HCT116 derived vector control and LCN2 over-expressing clones were untreated (UT) or pre-treated with either non-specific mouse IgG or Clone 3DI2B2 for 12 hours and then seeded in a Bowden's chamber coated with matrigel to determine invasive potential. The number of cells observed in ten random fields of the membrane for each clone was determined.

The mean and SEM of three independent experiments is plotted. Where indicated p values were determined using Student’s t-test.
The results as depicted in Figure 17D clearly shows that the two clones had increased invasion as compared to the vector control. , Treating the cells with 3D12B2 antibodies but not the non-specific IgG resulted in a decrease in invasion indicating the therapeutic potential of the antibodies to target LCN2 expressing cells.
These above experiments exhibit that 3D12B2 antibodies can inhibit LCN2 function which can result in tumor regression due to a suppression of the invasive and migratory phenotype and could also sensitize the tumors to chemotherapeutics and radiotherapy.
Example 16: Generation of partially humanized monoclonal antibodies
Partially humanized monoclonal antibodies were generated. The CDR regions of the LCN2 antibody for both the heavy and light chains as described in Example 12 were cloned into pGEM-T Easy vector (Promega Corporation, USA).
For cloning, the total RNA was isolated from monoclonal antibody secreting hybridoma clone 3D12B2 using Trizol® reagent (Invitrogen). The mRNA was converted to first strand cDNA using SuperScript™ IV Reverse Transcriptase (Invitrogen™). The variable light chain was amplified using the primers VL FOR (forward primer) and VL REV (reverse primer) having the sequences in Table 11.

Primer Orientation Sequence (5’ to 3’) SEQ ID NO
VL FOR ↑ Fwd 5’-ATGGGATGGAGCTGGATC-3’ 51
VL REV ↓ Rev 5’-CTGGACAGGGATCCAGAGTTCCA-3’ 52
VH Kit from AbGenics Pvt. Ltd., Pune.
Table 11: Primers used for amplification of Variable Immunoglobin chains (IgV)
The variable light chain (VL) was amplified using Taq polymerase (SibEnzyme) and cloned in to pGEM-T Easy Vector (Promega) by standard PCR. The variable heavy chain (VH) was amplified using the kit from AbGenics Pvt. Ltd, Pune using Herculase II Fusion DNA Polymerases (Agilent) by Touchdown PCR.
The amplicon was A-tailed by Taq DNA Polymerase (New England Biolabs) followed by cloning into pGEM-T Easy Vector. Bidirectional sequencing of MCS yielded one productive VL and two productive VH. The inserts were sequenced, and the protein sequences are listed in Table 12 and in nucleotide sequences are listed in Table 13.

Insert Amino Acid Sequence SEQ
name ID NO
LCN2VL DIVLTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRL
LIHYTSTLQPGIPSRFSGSGSGNDYSFSISNLEPEDIATYYCLQYDNL
YTFGGGTKLEIKRA 53
LCN2VH QMQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPTGKGLE 54
1 WIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLNLSFVTAADTA VYYCARDAVGGRDYWGQGTLVTVSS
LCN2VH QIQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKG 55
2 LEWIGSIYYSGSTYYNPSLKSRVTIGIDTSKRQFSLELSSVTAADTA MYYCARNPTRYSSSPFDYYYYYMDVWGKGTTVTVSS
Table 12: Protein sequence of the variable region (IgV) of the anti LCN2 antibody for the Light (VL) chain and for the Heavy (HL) chain

Insert Nucleotide Sequence SEQ
name ID NO
LCN2VL GATATCGTTCTGACCCAAAGCCCGAGCAGCCTGAGCGCGAGCC
TGGGTGGCAAGGTTACCATTACCTGCAAAGCGAGCCAGGACAT
CAACAAGTACATTGCGTGGTATCAACACAAGCCGGGTAAAGGT
CCGCGTCTGCTGATCCACTACACCAGCACCCTGCAACCGGGTA
TTCCGAGCCGTTTCAGCGGCAGCGGTAGCGGTAACGATTATAG
CTTTAGCATCAGCAACCTGGAGCCGGAAGACATTGCGACCTAC
TATTGCCTGCAATACGATAACCTGTATACCTTCGGTGGTGGCAC
CAAGCTGGAGATCAAACGTGCC 48
LCN2VH CAGATGCAACTGCAAGAAAGCGGTCCGGGCCTGGTGAAGCCG 49
1 AGCGAAACCCTGAGCCTGACCTGCACCGTTAGCGGTGGCAGCA
TCAGCAGCTACTATTGGAGCTGGATTCGTCAACCGACCGGTAA
AGGCCTGGAGTGGATCGGTCGTATTTACACCAGCGGCAGCACC
AACTATAACCCGAGCCTGAAGAGCCGTGTGACCATGAGCGTTG
ACACCAGCAAAAACCAATTCAGCCTGAACCTGAGCTTTGTGAC
CGCGGCGGATACCGCGGTTTATTACTGCGCGCGTGACGCGGTG
GGTGGCCGTGATTACTGGGGTCAGGGCACCCTGGTGACCGTTA
GCAGC

LCN2VH CAGATCCAACTGCAAGAAAGCGGTCCGGGCCTGGTGAAGCCG 50
2 AGCGAAACCCTGAGCCTGACCTGCACCGTTAGCGGTGGTAGCA
TCAGCAGCAGCAGCTACTATTGGGGTTGGATTCGTCAACCGCC GGGTAAAGGCCTGGAGTGGATCGGTAGCATTTACTATAGCGGC AGCACCTACTATAACCCGAGCCTGAAGAGCCGTGTGACCATCG GCATTGACACCAGCAAACGTCAGTTCAGCCTGGAACTGAGCAG CGTTACCGCGGCGGATACCGCGATGTACTATTGCGCGCGTAAC CCGACCCGTTACAGCAGCAGCCCGTTTGACTACTATTACTATTA CATGGATGTGTGGGGCAAGGGCACCACCGTGACCGTTAGCAGC
Table 13: Nucleotide sequence of the variable region (IgV) of the anti LCN2 antibody for the Light (VL) chain and for the Heavy (HL) chain
These sequences and the IL-2 signal sequence were amplified using the primers in Table 14 and cloned into the pTZ57R/T (Fermentas) plasmid and sequenced to confirm that no mutations had been introduced during the amplification.
Name of the primer Sequence (5’to 3’) SEQ ID NO
VHC1 forward primer EcoRV AGATATCCAGATGCAGCTGCAGGAGTC 56
VHC1reverse primer NheI AGCTAGCCGAGGAGACGGTGACCAGG 57
VHC2 forward primer EcoRV AGATATCCAGATTCAGCTGCAGGAGTC 58
VHC2 reverse primer NheI AGCTAGCCGAGGAGACGGTGACAGTG 59
VLC forward primer NcoI ACCATGGGATATTGTGCTGACCCAGTCT 60
VLC reverse primer NcoI ACCATGGGGCCCGTTTTATTTCCAGCTT 61
IL2 signal sequence forward AACCGGTATGTACAGGATGCAACTCCTGT 62
AgeI primer
IL2 signal sequence reverse ACCATGGCGAATTCGTGACAAGTGCAAG 63
NcoI primer
Table 14: Primers used for amplifying the variable regions of the heavy (VHC) and light (VLC) chains and the IL2 signal sequence with the appropriate restriction sites
Heavy chain cloning into pFUSE-CH-Ig-hg-ABH vector:The VH1 and VH2 constructs cloned in pTZ57R/T were digested with EcoRV and NheI and cloned into pFUSE CH-Ig-hk-ABL (Invivogen). A map of the resulting plasmids is shown in Figures 18-19. This plasmid encodes a partially humanized fragment of the two different heavy chain sequences of the LCN2 antibody.

Light chain cloning into pFUSE-CL-Ig-hk-ABL vector:As a first step the IL-2 sequence cloned in pTZ57RTwas digested with AgeI and NcoI and cloned into pFUSE-CL-Ig-hk-ABL (Invivogen). Once this plasmid was generated, it and the VLC sequence cloned in pTZ57RT were digested with NcoI and subsequently ligated to generate a plasmid with a partially humanized light chain of the LCN2 antibody. A map of the resulting plasmid is shown in Figure 20.
Example 17: Generation of single-chain variable fragment (scFv) antibodies
Single-chain variable fragment (scFv) antibodies were generated. The obtained constructs were codon optimized for E. coli bacterial system by GenScript and were synthesized by Eurofins Genomics. The VL cDNA was synthesized within NcoI and NaeI restriction sites. The VH was synthesized within BamHI and XhoI restriction sites. The linker was chosen as the 15-mer (Gly4Ser)3 linker which exist within NaeI and BamHI sites.
The VL and VH were doubly digested with NcoI/NaeI and BamHI/XhoI and were subsequently cloned into pEX-A128 cloning vector in the presence of the (Gly4Ser)3 linker. The resulting full scFv constructs were double digested with NcoI/XhoI enzymes and were ligated into pET-28a(+) vector (Novagen) which was digested at the same sites. All cloning was performed in E. coli DH5α cells in the presence of respective antibiotic resistances. The amino acid sequences for these fragments are listed in Table 15.

Fragment name Sequence Type Sequence SEQ ID NO
DIVLTQSPSSLSASLGGKVTITCKASQDINKYIAW YQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGND
Amino acid YSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK RAGGGGSGGGGSGGGGSQMQLQESGPGLVKPSE TLSLTCTVSGGSISSYYWSWIRQPTGKGLEWIGRI 64
scFv1 YTSGSTNYNPSLKSRVTMSVDTSKNQFSLNLSFVT AADTAVYYCARDAVGGRDYWGQGTLVTVSS

GATATCGTTCTGACCCAAAGCCCGAGCAGCCTG
Nucleic Acid AGCGCGAGCCTGGGTGGCAAGGTTACCATTACC TGCAAAGCGAGCCAGGACATCAACAAGTACAT
TGCGTGGTATCAACACAAGCCGGGTAAAGGTCC GCGTCTGCTGATCCACTACACCAGCACCCTGCA 66

ACCGGGTATTCCGAGCCGTTTCAGCGGCAGCGG
TAGCGGTAACGATTATAGCTTTAGCATCAGCAA
CCTGGAGCCGGAAGACATTGCGACCTACTATTG
CCTGCAATACGATAACCTGTATACCTTCGGTGG
TGGCACCAAGCTGGAGATCAAACGTGCCGGCG
GTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGT
GGCGGATCCCAGATGCAACTGCAAGAAAGCGG
TCCGGGCCTGGTGAAGCCGAGCGAAACCCTGA
GCCTGACCTGCACCGTTAGCGGTGGCAGCATCA
GCAGCTACTATTGGAGCTGGATTCGTCAACCGA
CCGGTAAAGGCCTGGAGTGGATCGGTCGTATTT
ACACCAGCGGCAGCACCAACTATAACCCGAGC
CTGAAGAGCCGTGTGACCATGAGCGTTGACACC
AGCAAAAACCAATTCAGCCTGAACCTGAGCTTT
GTGACCGCGGCGGATACCGCGGTTTATTACTGC
GCGCGTGACGCGGTGGGTGGCCGTGATTACTGG
GGTCAGGGCACCCTGGTGACCGTTAGCAGC
DIVLTQSPSSLSASLGGKVTITCKASQDINKYIAW
YQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGND
YSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK
Amino RAGGGGSGGGGSGGGGSQIQLQESGPGLVKPSET 65
acid LSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSI

YYSGSTYYNPSLKSRVTIGIDTSKRQFSLELSSVTA ADTAMYYCARNPTRYSSSPFDYYYYYMDVWGK
scFv2 GTTVTVSS

GATATCGTTCTGACCCAAAGCCCGAGCAGCCTG
AGCGCGAGCCTGGGTGGCAAGGTTACCATTACC TGCAAAGCGAGCCAGGACATCAACAAGTACAT
Nucleic TGCGTGGTATCAACACAAGCCGGGTAAAGGTCC 67
Acid GCGTCTGCTGATCCACTACACCAGCACCCTGCA

ACCGGGTATTCCGAGCCGTTTCAGCGGCAGCGG TAGCGGTAACGATTATAGCTTTAGCATCAGCAA CCTGGAGCCGGAAGACATTGCGACCTACTATTG

CCTGCAATACGATAACCTGTATACCTTCGGTGG TGGCACCAAGCTGGAGATCAAACGTGCCGGCG GTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGT GGCGGATCCCAGATCCAACTGCAAGAAAGCGG TCCGGGCCTGGTGAAGCCGAGCGAAACCCTGA GCCTGACCTGCACCGTTAGCGGTGGTAGCATCA GCAGCAGCAGCTACTATTGGGGTTGGATTCGTC AACCGCCGGGTAAAGGCCTGGAGTGGATCGGT AGCATTTACTATAGCGGCAGCACCTACTATAAC CCGAGCCTGAAGAGCCGTGTGACCATCGGCATT GACACCAGCAAACGTCAGTTCAGCCTGGAACTG AGCAGCGTTACCGCGGCGGATACCGCGATGTAC TATTGCGCGCGTAACCCGACCCGTTACAGCAGC AGCCCGTTTGACTACTATTACTATTACATGGAT GTGTGGGGCAAGGGCACCACCGTGACCGTTAG
CAGC
Table 15: Amino acid sequence of the scFv fragments of the anti LCN2 antibody cloned
into pET28a (+)
Bacterial expression of anti-LCN2 scFvs: The pET-28a (+) vectors bearing the anti-LCN2 scFvs were transformed into Rosetta-gami2 (DE3) pLysS Cells via heat shock transformation. They were propagated in Kanamycin (50 mg/ml) and Chloramphenicol (50 mg/ml) and were optimally expressed using 0.5 mM IPTG inducer for 4 hours at 37°C. The expression of the respective constructs was confirmed by gel electrophoresis. The pET28a (+) clones were digested with BglII and XhoI and cloned into pCDNA3 puro (1) digested with BamHI and XhoI. The map of the resulting plasmid is shown in Figure 21-23.

We claim:
1. A monoclonal antibody or fragment thereof that binds to LCN2, comprising a heavy
chain variable domain and a light chain variable domain,
wherein the light chain variable domain comprises at least one light chain
complementarity determining regions (LCDRs) selected from LCDR1, LCDR2 and
LCDR3,
wherein the heavy chain variable domain comprises at least one heavy chain
complementarity determining regions (HCDRs) selected from H1CDR1, H1CDR2,
H1CDR3, H2CDR1, H2CDR2 and H2CDR3, and wherein:
a. the LCDR1 comprises the amino acid sequence set forth as SEQ ID NO: 3 or a
conservative variant thereof, the LCDR2 comprises the amino acid sequence set
forth as SEQ ID NO: 4 or a conservative variant thereof, and the LCDR3
comprises the amino acid sequence set forth as SEQ ID NO: 5 or a conservative
variant thereof; and
b. the H1CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 6 or
a conservative variant thereof, the H1CDR2 comprises the amino acid sequence
set forth as SEQ ID NO: 7 or a conservative variant thereof, and the H1CDR3
comprises the amino acid sequence set forth as SEQ ID NO: 8 or a conservative
variant thereof, the H2CDR1 comprises the amino acid sequence set forth as
SEQ ID NO: 9 or a conservative variant thereof, the H2CDR2 comprises the
amino acid sequence set forth as SEQ ID NO: 10 or a conservative variant
thereof, and the H2CDR3 comprises the amino acid sequence set forth as SEQ
ID NO: 11 or a conservative variant thereof.
2. The monoclonal antibody or fragment thereof as claimed in claim 1, wherein the antibody has the light chain complementarity determining regions (LCDRs) and the heavy chain complementarity determining regions (HCDRs) grafted onto human framework and constant regions.
3. The monoclonal antibody or fragment thereof as claimed in claim 1, wherein the antibody further comprises the following light chain variable domain framework regions (LCFRs):
a. a LCFR1 comprising the amino acid sequence set forth as SEQ ID NO: 12 or a conservative variant thereof;

b. a LCFR2 comprising the amino acid sequence set forth as SEQ ID NO: 13 or a
conservative variant thereof;
c. a LCFR3 comprising the amino acid sequence set forth as SEQ ID NO: 14 or a
conservative variant thereof; and
d. a LCFR4 comprising the amino acid sequence set forth as SEQ ID NO: 15 or a
conservative variant thereof;
4. The monoclonal antibody or fragment thereof as claimed in claim 1, wherein the
antibody further comprises at least one of the following heavy chain variable
domain framework regions (HCFRs):
a. a HC1FR1 comprising the amino acid sequence set forth as SEQ ID NO: 16 or
a conservative variant thereof;
b. a HC1FR2 comprising the amino acid sequence set forth as SEQ ID NO: 17 or
a conservative variant thereof;
c. a HC1FR3 comprising the amino acid sequence set forth as SEQ ID NO: 18 or
a conservative variant thereof; and
d. a HC1FR4 comprising the amino acid sequence set forth as SEQ ID NO: 19 or
a conservative variant thereof.
e. a HC2FR1 comprising the amino acid sequence set forth as SEQ ID NO: 20 or
a conservative variant thereof;
f. a HC2FR2 comprising the amino acid sequence set forth as SEQ ID NO: 21 or
a conservative variant thereof;
g. a HC2FR3 comprising the amino acid sequence set forth as SEQ ID NO: 22 or
a conservative variant thereof; and
h. a HC2FR4 comprising the amino acid sequence set forth as SEQ ID NO: 23 or a conservative variant thereof.
5. The monoclonal antibody or fragment thereof as claimed in claim 1, wherein the variable region of light chain comprises the amino acid sequence of SEQ ID NO: 24 or a conservative variant thereof.
6. The monoclonal antibody or fragment thereof as claimed in claim 1, wherein the variable region of heavy chain comprises the amino acid sequence selected from a group comprising SEQ ID NO: 25, SEQ ID NO: 26 or a conservative variant thereof.
7. The monoclonal antibody as claimed in claim 1, wherein the antibody is a partially humanized monoclonal antibody selected from a group comprising SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55 or a conservative variant thereof.

8. The monoclonal antibody as claimed in claim 1, wherein the antibody is a Single-chain variable fragment (scFv) antibody selected from a group comprising SEQ ID NO: 64 and SEQ ID NO: 65 or a conservative variant thereof.
9. A composition comprising a therapeutically effective amount of the monoclonal antibody or fragment thereof as claimed in claim 1.
10. The composition as claimed in claim 9, further comprising one or more pharmaceutically acceptable carrier, chemotherapeutic agent or excipients.
11. A nucleic acid encoding the monoclonal antibody as claimed in claim 1.
12. An expression vector comprising the nucleic acid as claimed in claim 11.
13. A host cell comprising the expression vector as claimed in claim 12.
14. A method of producing the monoclonal antibody as claimed in claim 1, comprising culturing the host cell as claimed in claim 13 in a culture medium under conditions sufficient to produce the monoclonal antibody.
15. A method of treating a subject exhibiting LCN2 overexpression disorder, comprising administering to the subject a therapeutically effective amount of the composition of claim 9.
16. The method as claimed in claim 15, wherein the LCN2 overexpression disorder is selected from a group comprising liver cancer, melanoma, lung cancer, ovarian cancer, breast cancer, colon cancer, rectal cancer, oropharynx cancer, hypopharynx cancer, esophageal cancer, stomach cancer, pancreas cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, female genital tract cancer, male genital tract cancer, endocrine gland cancer, skin cancer, hemangioma, sarcoma, brain tumor, nerve cancer, eye tumor, meninges cancer, solid tumors from hematopoietic malignancy and tumor metastases.
17. Monoclonal antibody or fragment thereof as claimed in claim 1 for use in treatment of a subject exhibiting LCN2 overexpression disorder, wherein the wherein the LCN2 overexpression disorder is selected from a group comprising liver cancer, melanoma, lung cancer, ovarian cancer, breast cancer, colon cancer, rectal cancer, oropharynx cancer, hypopharynx cancer, esophageal cancer, stomach cancer, pancreas cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, female genital tract cancer, male genital tract cancer, endocrine gland cancer, skin cancer, hemangioma, sarcoma, brain tumor, nerve cancer, eye tumor, meninges cancer, solid tumors from hematopoietic malignancy and tumor metastases.