HK1261816A1 - Administration of an anti-lgr5 monoclonal antibody - Google Patents

Description

Administration of anti-LGR5 monoclonal antibodies

RELATED APPLICATIONS

The present application claims the benefit OF U.S. provisional application No. 62/311,631 entitled "ADMINISTRATION OF AN ANTI-LGR5 MONOCLONAL ANTIBODY (ADMINISTRATION OF AN ANTI-LGR5 MONOCLONAL ANTIBODY)" filed on 2016, 3, 22, the contents OF which are expressly incorporated herein in their entirety by reference.

Technical Field

The present invention relates generally to the field of cancer biology. Some embodiments of the methods and compositions provided herein relate to the administration of humanized antibodies or antigen-binding fragments thereof that specifically bind leucine-rich repeat unit-rich G protein-coupled receptor 5(LGR5) to treat certain cancers.

Reference to sequence listing

This application is submitted concurrently with the sequence listing in electronic format. The sequence listing is provided in a file titled BIONO14WOSEQLISTING, which was generated at 3, 8 and 2017, and is about 40Kb in size. The information in the sequence listing in electronic format is incorporated by reference herein in its entirety.

Background

Leucine-rich repeat G-protein coupled receptor 5(LGR5), also known as GPR49/HG38/FEX, belongs to the G-protein coupled receptor (GPR) protein family of leucine-rich repeat G-protein coupled receptor (LGR)/receptor proteins that are structurally similar to glycoprotein hormone receptors. LGR is divided into three subgroups: (1) glycoprotein hormone receptors, including Thyroid Stimulating Hormone (TSH) receptor, Follicle Stimulating Hormone (FSH) receptor, and Luteinizing Hormone (LH) receptor; (2) relaxin receptors LGR7 and LGR 8; and (3) LRG4, LGR5, and LGR 6. LGR5 is expressed in a variety of tissues, including the intestine, skeletal muscle, placenta, brain, and spinal cord.

Summary of The Invention

Embodiments of the methods and compositions provided herein include methods of treating a human subject having metastatic colorectal cancer, comprising administering to a subject in need thereof an effective amount of a humanized monoclonal antibody that specifically binds leucine rich repeat G protein-coupled receptor 5(LGR5), wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg.

In some embodiments, the monoclonal antibody is administered in combination with folinic acid (folinic acid), fluorouracil, and irinotecan (irinotecan). In some embodiments, an initial dose of monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan. In some embodimentsInitial dose of irinotecan of about 180mg/m²About 90 minutes of administration; the initial dose of folinic acid is about 400mg/m²About 120 minutes, and is administered concurrently with an initial dose of irinotecan; initial dose of fluorouracil is about 400mg/m²Administered after administration of an initial dose of folinic acid; and folinic acid, fluorouracil and irinotecan are administered every 14 days.

Embodiments of the methods and compositions provided herein include methods of treating a subject having cancer comprising administering to a subject in need thereof an effective amount of a humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds leucine-rich repeat unit-rich G protein-coupled receptor 5(LGR5), wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg.

In some embodiments, the monoclonal antibody is administered in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of folinic acid, fluorouracil, irinotecan, gemcitabine (gemcitabine), and nanoparticle albumin-bound paclitaxel (ABRAXANE).

In some embodiments, an initial dose of the monoclonal antibody is administered prior to administration of the chemotherapeutic agent.

In some embodiments, the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.

In some embodiments, the initial dose of irinotecan is about 180mg/m²Administration was for about 90 minutes. In some embodiments, the initial dose of folinic acid is about 400mg/m²Administered for about 120 minutes and concurrently with the initial dose of irinotecan. In some embodiments, the initial dose of fluorouracilIs about 400mg/m²Administered after administration of the initial dose of folinic acid. In some embodiments, folinic acid, fluorouracil, and irinotecan are administered every 14 days.

In some embodiments, the monoclonal antibody is administered in combination with an additional therapeutic agent selected from bevacizumab (bevacizumab), aflibercept (aflibercept), cetuximab (cetuximab), and panitumumab (panitumumab).

In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is selected from colon cancer, colorectal cancer, pancreatic cancer, breast cancer, and lung cancer. In some embodiments, the cancer is selected from colon cancer containing a mutation in APC, colon cancer containing a mutation in KRAS, metastatic colorectal cancer, metastatic pancreatic cancer, triple negative breast cancer, and small cell lung cancer. In some embodiments, the cancer is metastatic colorectal cancer.

In some embodiments, the subject has a characteristic selected from the group consisting of: prior to administration of the monoclonal antibody, at least 1 line (line) of prior chemotherapy failed for metastatic disease; no known brain metastases; has an expected life of 12 weeks or more; (ii) has an absolute neutrophil count of greater than about 1500 cells/mL without growth factor support within 14 days prior to administration of the monoclonal antibody; (ii) has a platelet count of greater than 100,000 platelets/mL under transfusion-free conditions within 14 days prior to administration of the monoclonal antibody; has a hemoglobin content of greater than or equal to 9.0 g/dL; and serum albumin having greater than or equal to 3 g/dL.

In some embodiments, the subject is a mammal, e.g., a human.

Embodiments of the methods and compositions provided herein include a container comprising a pharmaceutical composition comprising an administered dose of a humanized monoclonal antibody that specifically binds leucine rich repeat G protein-coupled receptor 5(LGR5), and a suitable pharmaceutical carrier, wherein the dose of the monoclonal antibody is from about 2.5mg/kg to about 15 mg/kg. In some embodiments, the pharmaceutical composition is suitable for intravenous administration.

Embodiments of the methods and compositions provided herein include humanized monoclonal antibodies that specifically bind leucine rich repeat G-protein coupled receptor 5(LGR5) for the treatment of metastatic colorectal cancer, wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg. In some embodiments, the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan. In some embodiments, the initial dose of irinotecan is about 180mg/m²About 90 minutes of administration; the initial dose of folinic acid is about 400mg/m²(ii) is administered for about 120 minutes and concurrently with the initial dose of irinotecan; initial dose of fluorouracil is about 400mg/m²(ii) administered after administration of the initial dose of folinic acid; and folinic acid, fluorouracil and irinotecan are administered every 14 days.

Brief description of the drawings

FIG. 1 is a graph showing direct FACS binding of humanized monoclonal antibody 18G7H6A3 to human LGR5 (CHO).

Figure 2 is a graph showing the effect of FOLFIRI alone and in combination with 18G7H6a3 on CT3 CRC tumor volume.

FIG. 3 is a graph showing that 18G7H6A3 treatment significantly reduced MDA-MB-231-LM3 primary tumor volume.

Fig. 4 shows a graph of LGR5 upregulation resulting from FolFiri treatment of mice with CT1 or CT3 tumors.

Fig. 5 is a bar graph showing that chemotherapy resulted in upregulation (greater than 4-fold) of LGR5 in JH109 tumors.

Figure 6 is a graph showing that significant 18G7H6a3 activity was observed when administered in combination with chemotherapy (gemcitabine).

Fig. 7 is a dot plot showing that antibody 18G7H6a3 reduces the number of survival events for the CT1 cancer stem cell population.

Fig. 8 is a line graph showing that cells isolated from mice treated with the combination of anti-LGR5 antibody 18G7H6A3 and FOLFIRI have substantially reduced tumorigenicity compared to cells isolated from mice treated with FOLFIRI alone.

Fig. 9 is a line graph showing that reimplanted cells from the 18G7H6A3FOLFIRI combination have a significantly delayed time to progression.

Figure 10 is a line graph showing that significant humanized antibody 18G7H6a3 activity was observed when administered prophylactically in combination with chemotherapy (FOLFIRI).

figure 11 is a dot plot showing that antibody 18G7H6A3 is capable of inhibiting Wnt signaling in tumor cells in vivo as shown by phospho-Thr 41/Ser45- β -catenin immunoassay.

Fig. 12 is a bar graph showing that increasing the concentration of soluble antibody 18G7H6A3 did not affect the induction of TCF/LEF promoter-driven GFP expression by Wnt3a plus RSPO2 combination, indicating that anti-LGR5 antibody 18G7H6A3 did not block RSPO-driven TCF/LEF promoter activation. The positive control antibody C12 demonstrated the ability to inhibit Wnt3a/RSPO 2-driven activation of the TCF/LEF promoter.

Fig. 13 is a line graph showing that R-spondin does not block binding of antibody 18G7H6a3 to LGR 5.

Fig. 14 is a bar graph showing that binding of antibody 18G7H6a3 to LGR5 inhibits formation of a ternary complex.

Fig. 15 shows the expression level of LGR5 in the treated samples.

FIG. 16 shows the expression level of CTNNB1 and the expression level of p- β -catenin in treated samples.

FIG. 17 shows transcripts differentially expressed in different treatment samples.

Figure 18 shows genes differentially expressed in 18G7H6a3- (BNC101) treated tumors.

Figure 19 shows genes differentially expressed in FOLFIRI treated tumors.

Figure 20 shows genes differentially expressed in tumors treated in combination.

Figure 21 shows the levels of LGR5 in circulating HLA + cells.

Fig. 22A and 22B show the levels of LGR5 in circulating HLA + cells.

Fig. 23 is a graph showing animal survival for mice treated with gemcitabine/albumin-bound paclitaxel or with gemcitabine/albumin-bound paclitaxel and 18G7H6a 3.

Detailed Description

Some embodiments of the methods and compositions provided herein relate to the administration of humanized antibodies or antigen-binding fragments thereof that specifically bind leucine-rich repeat unit-rich G protein-coupled receptor 5(LGR5) to treat certain cancers. Embodiments of such humanized antibodies and antigen-binding fragments thereof are disclosed in PCT publication No. WO 2015/153916, published on 8/10/2017, which is incorporated herein by reference in its entirety.

LGR5 was identified by lineage tracing studies, and is a highly specific marker of normal stem cells and tumor initiating cells in the gut. Approximately 150 genes were previously identified, the expression of which was quenched after abrogation of Wnt expression. Comprehensive characterization of these "Wnt target genes" revealed that LGR5 was selectively expressed on 10-14 proliferating wedge cell populations at the crypt base (crypt-base). These crypt-based columnar cells have previously been suggested as candidate stem cell populations. In vivo lineage tracing using a heritable lacZ-LGR5 reporter gene has demonstrated that LGR5 intestinal stem cells are a pluripotent, self-renewing population of adult intestinal stem cells capable of producing uninterrupted bands of lacZ + daughter cells that originate from the crypt floor and extend to the tip of the villus (villustip).

Specific expression of LGR5 on Cancer Stem Cells (CSCs) provides the opportunity to selectively and efficiently target CSCs. LGR5 is highly overexpressed in CRC, pancreatic tumors, and most other solid tumors compared to normal tissue, providing a broad therapeutic window targeting CSCs in CRC, pancreatic, breast, ovarian, lung, gastric, and liver cancers.

A gatekeeper gene mutation (gate keep mutation) in CRC is a loss of adenomatous colon polyps (APCs), resulting in aberrant activation of Wnt signaling that normally regulates the balance between stem cell self-renewal and differentiation in the crypts of the colon. Deregulated Wnt signaling in intestinal stem cells leads to the formation of adenomatous polyps in the colon that are precursors to malignant CRC. The strategy of crossing (cross) of an inducible APC knockout mouse with its LGR5 stem cells with a specific and randomly labeled mouse via one of 4 (GFP/YFP/ECFP/RFP) fluorescent genetic markers confirmed that LGR5 stem cells are the source or root of intestinal tumors in these mice. The appearance of monochromatic tumors (i.e., all GFP or all RFP) 4 weeks after APC-induced depletion confirmed that these tumors originated from a single LGR5 stem cell. Furthermore, this model also allowed the fluorescent genetic markers in LGR5 stem cells to be converted to different colors, so that RFP + LGR5 cancer stem cells producing red tumors could be converted in the middle to ECFP + LGR5 cancer stem cells, which still seeded (seed) to the tumor, but now produce blue tumor cells that invaded the GFP + tumor mass that was previously all red. This shift experiment provided further evidence that not only did LGR5 CSCs originate from intestinal tumors and be able to initiate and inoculate growth of intestinal tumors, but also provided further evidence that they continuously maintained tumor formation, i.e., had the ability to regenerate for a long period of time (repopulating).

The functional role of LGR5 in cancer has been demonstrated by ribonucleic acid interference (RNAi) knockdown studies. Knock-down of LGR5 in one set of CRC tumor cell lines significantly inhibited the growth of soft agar colonies, and also inhibited the growth of HCT116 colon tumor xenografts in vivo. It was subsequently shown that LGR5RNAi knockdown could also reduce the in vitro growth of CSC colonies from patient-derived CRC tumor cells (data not shown). Finally, sorted LGR5+ patient-derived xenograft CRC tumor cells were found to be highly tumorigenic in vivo compared to control LGR 5-cells.

CSCs are thought to be responsible for the high incidence of tumor recurrence in many cancer patients treated with surgery and standard-of-care chemotherapy. For example, breast cancer patients were found to be enriched for CD44+ CSCs following chemotherapy, and high levels of CSCs were associated with poor clinical response to chemotherapy. Similarly, LGR5 expression was upregulated in metastatic CRC in injured liver following chemotherapy, suggesting that LGR5 CSCs increased in response to chemotherapy are able to initiate and/or exacerbate metastatic disease. Indeed, it was found that LGR5 expression was significantly higher in metastatic sites compared to primary CRC tumors.

anti-LGR5 antibodies

As used herein, the term "antibody" includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single chain Fvs (scFv), Fab fragments, F (ab') fragments, disulfide linked Fvs (sdFv) (including bispecific sdFv) and anti-idiotypic (anti-Id) antibodies, as well as epitope-binding fragments of any of the above. The antibodies in several embodiments provided herein can be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies may be specific for different epitopes of a polypeptide, or may be specific for a polypeptide as well as for a heterologous epitope (such as a heterologous polypeptide or a solid support material). See, for example, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; tutt et al, J.Immunol.147:60-69 (1991); us patent No. 4,474,893; us patent No. 4,714,681; U.S. Pat. No. 4,925,648; us patent No. 5,573,920; us patent No. 5,601,819; kostelny et al, J.Immunol.148:1547-1553 (1992); each of which is incorporated by reference herein in its entirety.

As used herein, LGR5 includes, but is not limited to, human LGR5 comprising a polypeptide of NCBI accession No. NP _003658.1 or a fragment thereof, encoded by the encoding nucleotide sequence in NM _003667.2 or a fragment thereof. The amino acid sequence and complete entry of NCBI accession No. NP _003658.1 and the nucleotide sequence and complete entry of NM _003667.2 are all incorporated herein by reference. Examples of LGR5 fragments contemplated herein include the extracellular, transmembrane or intracellular domain of LGR5 and portions thereof.

Several embodiments relate to hybridomas that produce light and/or heavy chains of anti-LGR5 antibodies, including anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below. In one aspect, the hybridoma produces a light chain and/or heavy chain of a humanized or fully human monoclonal antibody, such as 18G7H6A3 and 18G7H6a1, as prepared and described in the examples below.

Some embodiments relate to nucleic acid molecules encoding a light chain or a heavy chain of an anti-LGR5 antibody, which includes any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6a1, prepared and described in the examples below. In some aspects, the nucleic acid molecule encodes a light or heavy chain of a humanized or fully human monoclonal, such as antibodies 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

Various embodiments relate to vectors comprising one or more nucleic acid molecules encoding the light and/or heavy chains of an anti-LGR5 antibody, including any of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

In various embodiments, the glycosylation of an antibody can be modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of an antibody for a target antigen. Modification of such carbohydrates can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such deglycosylation can increase the affinity of the antibody for the antigen. Such methods are described in more detail in U.S. Pat. nos. 5,714,350 and 6,350,861; each of which is incorporated by reference herein in its entirety.

In several embodiments, the antibody specifically binds a polypeptide comprising or consisting of an LGR5 polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 97% identity, or at least 99% identity, or 100% identity to a human LGR5 polypeptide or fragment thereof of NCBI accession No. NP _003658.1(SEQ ID NO: 47). Such fragments may be, for example, at least about 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 contiguous or non-contiguous amino acids of the LGR5 polypeptide, or any number of contiguous or non-contiguous amino acids between any of the foregoing lengths.

In several embodiments, the antibody is antibody 18G7H6A3 and comprises the heavy chain amino acid sequence of SEQ ID NO. 13 and the DNA sequence of SEQ ID NO. 11. In some embodiments, the antibody is antibody 18G7H6a3 and has a heavy chain variable domain comprising SEQ ID No. 19. In several embodiments, the antibody is antibody 18G7H6A3 and comprises the light chain sequence of SEQ ID NO. 14. In other embodiments, the antibody is antibody 18G7H6A3 and comprises the light chain variable domain of SEQ ID NO: 21.

In some embodiments, the antibody comprises a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence of the above sequences. In some embodiments, the antibody comprises 100% identity to the sequence of the antibody over residues 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118 of the heavy, light, or variable domains of the sequences described above.

In some embodiments, the antibody comprises a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the antibody sequence. In some embodiments, the antibody comprises a sequence that is 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the antibody. In some embodiments, the antibody comprises a sequence with 100% identity to the sequence of the antibody over a range of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, or 111 residues.

In some embodiments, an anti-LGR5 antibody provided herein comprises a heavy chain CDRL comprising GYSFTAYW (SEQ ID NO:23), a heavy chain CDR2 comprising ILPGSDST (SEQ ID NO:2), and a heavy chain CDR3 comprising ARSGYYGSSQY (SEQ ID NO: 3). In some embodiments, an anti-LGR5 antibody provided herein comprises a light chain CDRL comprising ESVDSYGNSF (SEQ ID NO:4), a light chain CDR2 comprising LTS, and a light chain CDR3 comprising QQNAEDPRT (SEQ ID NO: 33).

In some embodiments, an anti-LGR5 antibody provided herein comprises: (a) heavy chain CDR1 comprising a variant of the above sequence having 1, 2, 3, or4 amino acid substitutions. The antibody may also have a heavy chain CDR2 with variants having 1, 2, 3, or4 amino acid substitutions. The antibody may also have a heavy chain CDR3 with variants having 1, 2, 3, or4 amino acid substitutions. In addition to these modifications of the heavy chain, the antibody may also have a light chain CDR1 with variants having 1, 2, 3, or4 amino acid substitutions. The antibodies may also have a light chain CDR2 with variants having 1, 2, 3, or4 amino acid substitutions. The antibody may also have a light chain CDR3 with 1, 2, 3, or4 amino acid substitutions. In some embodiments, the amino acid substitution is a conservative amino acid substitution.

In some embodiments, the anti-LGR5 antibodies provided herein comprise an antibody comprising a heavy chain variable region having at least 80% or 90% sequence identity to a sequence described in the sequence listing appended hereto. The antibody may also have a light chain variable region having at least 80% or 90% sequence identity to the antibody sequence described herein.

The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined, for example, by sequence alignment for optimal alignment purposes (e.g., gaps (gaps) can be introduced in the sequence of the first sequence). The amino acids or nucleotides at the corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity ═ identical position #/total position # × 100). The actual comparison of the two sequences can be accomplished by well known methods, such as by using mathematical algorithms. Specific non-limiting examples of such mathematical algorithms are described in Karlin et al, proc.natl.acad.sci.usa,90:5873-5877(1993), which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the BLASTN and BLASTX programs (version 2.2), which are incorporated herein by reference in their entirety, as described in Schafer et al, Nucleic Acids Res.,29:2994-3005 (2001). When BLAST and Gapped BLAST (Gapped BLAST) programs are used, the default parameters of the respective programs (e.g., BLASTN) can be used. See http:// www.ncbi.nlm.nih.gov, provided at 4/10/2002. In one embodiment, the database retrieved is a non-redundant (NR) database and the parameters for sequence comparison may be set to: no filtration is carried out; the expected value is 10; the word size is 3; the matrix is BLOSUM 62; and a gap existence penalty of 11 and a gap extension penalty of 1.

Several embodiments also include variants of the above antibodies, including any of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below, comprising a variable region light chain (V) and a light chain of variable regions_L) Domain and/or variable region heavy chain (V)_H) One or more amino acid residue substitutions in the domain. Several embodiments also include variants of the above antibodies, the variants at one or more V_LCDR and/or one or more V_HThe CDRs have one or more additional amino acid residue substitutions therein. V can be tested in vitro and in vivo by the antibodies described above_HDomain, V_HCDR、V_L(ii) Domain and/or V_LAntibodies generated by introducing substitutions into the CDRs, for example, are tested for their ability to bind LGR5 (by, for example, immunoassays including, but not limited to, ELISA and BIAcore).

Various embodiments include an antibody that specifically binds LGR5, the antibody comprising V of an anti-LGR5 antibody that specifically binds LGR5_HDomain, V_HCDR、V_LDomain or V_LDerivatives of CDRs, any of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below. Mutations (e.g., additions, deletions, and/or substitutions) can be introduced into the nucleotide sequence encoding the antibody using standard techniques known to those skilled in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions. In one embodiment, relative to the original V_HAnd/or V_LCDR, said V_HAnd/or V_LCDR derivatives comprise less than 25 amino acidsSubstitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions. In another embodiment, said V_HAnd/or V_LThe CDR derivatives have conservative amino acid substitutions (e.g., as above) made at one or more predicted nonessential amino acid residues (i.e., amino acid residues not critical for antibody specific binding to LGR 5). Alternatively, all or part of V may be followed_HAnd/or V_LMutations are introduced randomly into the CDR coding sequences, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

Several embodiments also include an antibody that specifically binds LGR5 or a fragment thereof, the antibody comprising an amino acid sequence of a variable heavy and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy and/or light chain of any of the antibodies described herein, including any of the anti-LGR5 antibodies, including those anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the following examples.

Another embodiment includes introducing conservative amino acid substitutions in any portion of the anti-LGR5 antibody, such as any of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below. It is well known in the art that "conservative amino acid substitutions" refer to amino acid substitutions that substitute functionally equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids with similar polarity are used as functional equivalents and cause silent changes in the amino acid sequence of the peptide. Substitutions that are neutral in charge and where a residue is replaced with a smaller residue may also be considered "conservative substitutions," even if the residue is in a different grouping (e.g., replacement of phenylalanine with a smaller isoleucine). Families of amino acid residues with similar side chains have been defined in the art. Some families of conservative amino acid substitutions are shown in table 1.

TABLE 1

Family of	Amino acids
		Non-polar	Trp、Phe、Met、Leu、Ile、Val、Ala、Pro
Polarity without electric charge	Gly、Ser、Thr、Asn、Gln、Tyr、Cys
		Acidic/negatively charged	Asp、Glu
Basic/positively charged	Arg、Lys、His
		with β -branches	Thr、Val、Ile
Residues influencing chain orientation	Gly、Pro
		Of aromatic type	Trp、Tyr、Phe、His

Blocking cancer stem cell growth with anti-LGR5 antibodies

Several embodiments relate to the use of anti-LGR5 antibodies to block cancer stem cell growth in vitro and in vivo. In some embodiments, a method of blocking growth of a cancer stem cell comprises administering to a cancer stem cell an effective amount of an anti-LGR5 antibody, wherein the effective amount of the anti-LGR5 antibody is sufficient to reduce growth of the cancer stem cell.

In some embodiments, the method of blocking growth of a cancer stem cell comprises administering to the cancer stem cell an effective amount of an anti-LGR5 antibody, wherein the effective amount of the anti-LGR5 antibody is sufficient to reduce or block proliferation of the cancer stem cell, or reduce or block growth of the cancer stem cell.

In some aspects, an effective amount of an anti-LGR5 antibody is administered to cancer stem cells in vitro. In other aspects, an effective amount of an anti-LGR5 antibody is administered in vivo to cancer stem cells in a patient in need of such treatment.

As used herein, the term "cancer stem cell" refers to a cell that is capable of extensive or unlimited proliferation and produces a large proportion of cancer cells in a cancer. In some aspects, the large proportion of cancer cells represents the majority of cancer cells in a given cancer. For purposes of example and not limitation, cancer stem cells can be the starting cells of a tumor or the progenitor cells of cancer cells that comprise a majority of the cancer mass. In some aspects, a cancer stem cell refers to a cell that is capable of dividing to form one or more tumors when implanted into an immunocompromised individual without any other mutation or introduction of exogenous cell proliferation-inducing or carcinogenic agents. In some aspects, cancer stem cell division results in additional cancer stem cells and ultimately differentiation into cancer cells or cancer tissue.

In some embodiments, the growth, proliferation, or viability of the cancer stem cells is hindered without interfering with LGR5-RSpo binding or signaling. In some embodiments, the growth, proliferation, or viability of the cancer stem cell is hindered without interfering with LGR5-RSpo binding or signaling by blocking or inhibiting LGR5 signaling through Wnt.

As used with respect to blocking cancer stem cell growth, the term "effective amount" refers to an amount of anti-LGR5 antibody sufficient to reduce the growth of cancer stem cells to any degree. Any assay known in the art can be used to measure the growth of cancer stem cells. For example, cancer stem cell growth can be measured by colony number, total cell number, or volume/size of cell population or colony. In several embodiments, cancer stem cell growth can be measured by the tumor sphere growth assay described in example 1 below.

In certain embodiments, an effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% reduction in growth of a cancer stem cell population or tumor sphere, or any percentage between any of the foregoing numbers. In some aspects, the anti-LGR5 antibody is any one of or a combination of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

For example, in some embodiments, an effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by at least about a 5% -99%, 5% -80%, 5% -40%, 10% -99%, 10% -80%, 10% -60%, 10% -40%, 20% -99%, 20% -80%, 20% -60%, 20% -40%, 50% -98%, 50% -80%, or 60% -99% reduction in cancer stem cell population or tumor sphere growth. In some aspects, the anti-LGR5 antibody is any one of or a combination of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

In other embodiments, an effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by a reduction in cancer stem cell population or tumor sphere growth of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 25, 50, 75, 100, 200, or 1000-fold, or any multiple between any of the foregoing numbers. In some aspects, the anti-LGR5 antibody is any one of or a combination of the anti-LGR5 antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

In some embodiments, an effective amount of anti-LGR5 antibody sufficient to block growth of a cancer stem cell to any of the degrees described above is at a concentration of about 1nM, 50nM, 75nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 500nM, 550nM, 600nM, 700nM, 800nM, 900nM, 1 μ M, 50 μ M, 75 μ M, 100 μ M, 150 μ M, 200 μ M, 250 μ M, 300 μ M, 350 μ M, 400 μ M, 500 μ M, 550 μ M, 600 μ M, 700 μ M, 800 μ M, 900 μ M, 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 75mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM, 1M, 5M, 10M, 15mM, 25M, 35mM, 40mM, 45mM, 50mM, 75mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM, 1M, 5M, or any number between any two of the above concentrations. In some aspects, an anti-LGR5 antibody composition may comprise two antibodies designated 18G7H6A3 and 18G7H6a1, prepared and described in the examples below.

In some embodiments, an anti-LGR5 antibody provided herein binds human LGR5 with a KD of less than about 200nM, less than about 100nM, less than about 80nM, less than about 50nM, less than about 20nM, less than about 10nM, less than about 1nM, and ranges between any of the foregoing. In some embodiments, an anti-LGR5 antibody provided herein binds LGR5 with an affinity of less than about 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, and any range of values described above. In some embodiments, an anti-LGR5 antibody provided herein binds LGR5 with an affinity of greater than about 0.0001nM, 0.001nM, 0.01nM, and any range of values described above.

In some embodiments, the anti-LGR5 antibodies provided herein bind an epitope comprising or consisting of, or within: 47, amino acids T175, E176, Q180, R183, S186, A187, Q189, D247, E248, T251, R254, S257, N258, K260. In some embodiments, the anti-LGR5 antibodies provided herein bind to an epitope that comprises, consists of, or is within leucine-rich repeats 6-9 (see, e.g., Chen et al genesdv.27 (12):1345-50, which is incorporated herein by reference in its entirety). In some embodiments, the anti-LGR5 antibodies provided herein bind to an epitope that comprises or consists of the convex surface of the extracellular domain of LGR5 or is within the convex surface of the extracellular domain of LGR5 (see, e.g., Chen et al genes dev.27(12):1345-50, which is incorporated herein by reference in its entirety).

Some embodiments include methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGR5 antibody provided herein. In some embodiments, the cancer is selected from: pancreatic cancer, colorectal cancer, lung cancer, pancreatic cancer, and breast cancer such as triple negative breast cancer. In some embodiments, the colorectal cancer comprises an inactivating mutation of an Adenomatous Polyposis Coli (APC) gene, does not comprise an inactivating mutation of an APC gene, or comprises a wild-type APC gene. In some embodiments, the cancer is. In some embodiments, the cancer comprises elevated levels of LGR5 protein. In some embodiments, the cancer is colon cancer that expresses elevated levels of LGR 5. In some embodiments, the cancer is pancreatic cancer expressing elevated levels of LGR5, in some embodiments, the cancer is breast cancer expressing elevated levels of LGR 5.

some embodiments include methods of treating a disease in a subject, wherein the disease is associated with β -catenin activation and/or aberrant β -catenin signaling some embodiments include administering to a subject in need thereof a therapeutically effective amount of an anti-LGR5 antibody provided herein.

Some embodiments include methods of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGR5 antibody provided herein in combination with at least one other therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent. In some embodiments, the additional therapeutic agent comprises a biological agent. Some embodiments include administering an anti-LGR5 antibody provided herein in combination with a chemotherapeutic agent and a biological agent. In some embodiments, administration of an anti-LGR5 antibody provided herein in combination with a chemotherapeutic agent may increase the expression level of LGR5 in a cancer, such as a tumor. Some embodiments of the methods provided herein comprise determining the expression level of LGR5 protein in a tumor or cancer.

Some embodiments of the methods provided herein comprise identifying a subject for treatment with an anti-LGR5 antibody provided herein. Some embodiments include determining whether the subject has a tumor with an elevated level of LGR5 expression compared to the expression of the same LGR5 protein in normal tissue. Some embodiments include: selecting the subject for treatment if the tumor has an elevated expression level of LGR 5. Some embodiments further comprise determining whether the subject has a tumor comprising an inactivating mutation of an APC gene. Some embodiments further comprise: if the tumor contains an inactivating mutation of the APC gene, the subject is selected for treatment.

Methods, compositions, and related disclosures related to the foregoing are provided in, for example, PCT publication No. WO2013/067055, published on 5/10 of 2013 (the contents of which are incorporated herein by reference in their entirety), and PCT publication No. WO 2013/067054, published on 5/10 of 2013 (the contents of which are incorporated herein by reference in their entirety), and PCT publication No. WO 2013/067057, published on 5/10 of 2013 (the contents of which are incorporated herein by reference in their entirety), and PCT publication No. WO 2013/067060, published on 5/10 of 2013 (the contents of which are incorporated herein by reference in their entirety).

Pharmaceutical composition

The humanized monoclonal antibodies or antigen-binding fragments thereof provided herein that specifically bind LGR5 may be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise the humanized monoclonal antibody, or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion vehicles, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration. Suitable carriers are described in the latest edition of the art standard reference Remington's pharmaceutical Sciences, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and water insoluble carriers such as fixed oils may also be used. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional vehicle or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention are prepared to suit their intended route of administration. Examples of routes of administration include parenteral routes, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may comprise the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted using an acid or base (e.g., hydrochloric acid or sodium hydroxide). The parenteral formulation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil (TM) (Cremophor el.tm.) (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

It is particularly advantageous to prepare the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms described herein depends on and directly depends on the unique characteristics of the active compounds and the particular therapeutic effect to be achieved, as well as limitations inherent in the art of combining such active compounds for the treatment of individuals.

The pharmaceutical composition may be packaged in a container, package, or dispenser with instructions for administration.

Reagent kit

Some embodiments provided herein include kits. In some embodiments, a kit can comprise a humanized antibody provided herein. In some embodiments, the antibody is lyophilized. In some embodiments, the antibody is in an aqueous solution. In some embodiments, the kit comprises a pharmaceutical carrier for administration of the antibody. In some embodiments, the kit further comprises a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from: folinic acid, fluorouracil, irinotecan, gemcitabine and nanoparticulate albumin-bound paclitaxel (ABRAXANE).

Some embodiments include a container containing a pharmaceutical composition comprising an administered dose of a humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds LGR5, and a suitable pharmaceutical carrier, wherein the dose is suitable for treating a subject having cancer. The humanized monoclonal antibody or antigen binding fragment thereof can be administered at a dose greater than, less than, or equal to about 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, or a range between any two of the foregoing doses. In some embodiments, the dose of the humanized monoclonal antibody or antigen-binding fragment thereof is from about 2.5mg/kg to about 20mg/kg, or from about 2.5mg/kg to about 15 mg/kg. In some embodiments, the pharmaceutical composition is suitable for intravenous administration. In some embodiments, the pharmaceutical composition is suitable for intraperitoneal injection.

Method of treatment

Some embodiments of the methods, compositions, and kits include methods of treating a subject having cancer. Some such methods comprise administering to a subject in need thereof an effective amount of a humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds LGR 5. The subject may be a mammal, such as a human.

In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer may be colon cancer, colorectal cancer, pancreatic cancer, breast cancer, or lung cancer. In some embodiments, the cancer may be colon cancer comprising a mutation in APC, colon cancer comprising a mutation in KRAS, metastatic colorectal cancer, metastatic pancreatic cancer, triple negative breast cancer, or small cell lung cancer.

The humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds LGR5 can include heavy chain CDRs, such as heavy chain CDR1 comprising SEQ ID NO:23, heavy chain CDR2 comprising SEQ ID NO:25, and/or heavy chain CDR3 comprising SEQ ID NO: 27. In some embodiments, the monoclonal antibody or antigen binding fragment thereof can include a heavy chain variable domain comprising SEQ ID NO 19. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a heavy chain comprising SEQ ID NO 13. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include light chain CDRs such as light chain CDR1 comprising SEQ ID No. 29, light chain CDR2 comprising SEQ ID No. 31, and/or light chain CDR3 comprising SEQ ID No. 33. In some embodiments, the monoclonal antibody or antigen binding fragment thereof can include a light chain variable domain comprising SEQ ID NO 21. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a light chain comprising SEQ ID NO. 14. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a heavy chain comprising SEQ ID NO. 13 and a light chain comprising SEQ ID NO. 14. In some embodiments, the humanized monoclonal antibody or antigen binding fragment thereof is 18G7H6a 3.

The dose of the humanized monoclonal antibody or antigen-binding fragment thereof for treating a subject with cancer can be greater than, less than, or equal to about 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, or a range between any two of the foregoing doses. In some embodiments, the dose of the humanized monoclonal antibody or antigen-binding fragment thereof is from about 2.5mg/kg to about 20mg/kg, or from about 2.5mg/kg to about 15 mg/kg.

The humanized monoclonal antibody or antigen binding fragment thereof can be administered daily, weekly, or monthly. In some embodiments, administration may be once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, or once every 6 days. In some embodiments, administration may be once weekly, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In some embodiments, the administration may be monthly.

The route of administration of the humanized monoclonal antibody or antigen-binding fragment thereof can be adapted for biological administration. For example, administration may be by intraperitoneal injection, or by intravenous route.

Administration of the humanized monoclonal antibody or antigen-binding fragment thereof can be administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include folinic acid (leucovorin), fluorouracil (5-FU), irinotecan, gemcitabine, and nanoparticulate albumin-bound paclitaxel (ABRAXANE). In some embodiments, the FOLFIRI combination: folinic acid, fluorouracil and irinotecan, may be administered in combination with a humanized monoclonal antibody or antigen-binding fragment thereof. In some embodiments, an initial dose of a chemotherapeutic agent in combination with a humanized monoclonal antibody or antigen-binding fragment thereof in a method of treating cancer may be administered prior to administration of the initial dose of the humanized monoclonal antibody or antigen-binding fragment thereof. In some embodiments, an initial dose of a chemotherapeutic agent in combination with a humanized monoclonal antibody or antigen binding fragment thereof in a method of treating cancer may be administered after an initial dose of the humanized monoclonal antibody or antigen binding fragment thereof is administered.

In some embodiments, the initial dose of irinotecan may be about 180mg/m²About 90 minutes of administration; the initial dose of folinic acid is about 400mg/m²Administered for about 120 minutes and concurrently with an initial dose of irinotecan; and/or initial dose of fluorouracil of about 400mg/m²Administered after administration of the initial dose of folinic acid. In some embodiments, folinic acid, fluorouracil, and irinotecan are administered every 14 days.

In some embodiments, the humanized monoclonal antibody or antigen-binding fragment thereof can be administered in combination with other therapeutic agents. Examples of other therapeutic agents include bevacizumab, aflibercept, cetuximab, and panitumumab.

Examples

Example 1 humanization of LGR5 antibody

Human germline sequences were used as acceptor frames for humanizing the murine antibody 18G 7.1. To find the closest germline sequences, the most similarly expressing light chains and the most similar heavy chains were identified in the NCI IgBLAST germline sequence database (ncbi. In this search, the CDR sequence of 18G7.1 was masked. The selection of the most suitable expressed sequence includes checking for sequence identity of classical residues and interfacial residues, and checking for similarity in CDR loop length.

To determine possible structural conflicts in critical structural box residues between the candidate humanized sequence and the parent murine monoclonal antibody 18G7.1, a three-dimensional model was generated. Synthesis of antibody structures was used to make homology models with candidate humanized sequences for transplantation, followed by molecular energy minimization. Structural analysis using computer software Pymol will be used to determine residues that could potentially negatively affect correct folding.

From this analysis, 6 candidate VH chains were constructed, comprising: 1) a functional human framework containing selected substitutions within the candidate humanized framework regions based on analysis of the possible effects on folding, and ii) the parental 18G7.1 murine antibody CDRs (SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3). In frame fusions of the constant region of human IgG1 were chemically synthesized.

Similarly, two candidate VL chains were constructed, comprising: 1) a functional human framework containing selected substitutions within candidate humanized framework regions based on analysis of possible effects on folding, and ii) parental 18G7.1 murine antibody CDRs (SEQ ID NO:4, SEQ ID NO5 and SEQ ID NO 6). The candidate VL chains and candidate VH chains were chemically synthesized with in-frame fusions to the constant region of human IgG 1.

The selected candidate variant humanized heavy and light chain combinations were tested for function by co-transfection into mammalian cells. Each of the 6 candidate humanized 18G7.1 heavy chains described above were co-transfected into HEK293 cells along with each of the candidate 18G7.1 light chains and the conditioned cultures were assayed for LGR5 antigen binding activity by flow cytometry. In addition, the 3 candidate humanized 18G7.1 heavy chains described above were co-transfected into HEK293 cells along with a second candidate 18G7.1 light chain and the conditioned cultures were assayed for LGR5 antigen binding activity by flow cytometry. The combination of 18G7.1 candidate heavy/light chains, designated 18G7H6 and exhibiting the most robust binding (humanized variant) was chosen for affinity maturation.

Example 2 affinity maturation of humanized LGR5 antibody

To increase the affinity of the selected humanized variant 18G7H6, a combination of alanine scanning mutagenesis and saturation mutagenesis was used. Residues in the heavy chain CDR1 and light chain CDRs 1 and 3 were mutated to alanine, transfected into HEK293 cells, and the resulting conditioned cultures were assayed for LGR5 antigen binding activity by flow cytometry. Saturation mutagenesis was performed on the heavy chain CDR3, in which each residue of CDR3 was mutated to each of the 19 naturally occurring amino acids, unless the original amino acid at that position was identical. The various mutants were transfected into HEK293 cells and the resulting conditioned cultures were assayed for LGR5 antigen binding activity by flow cytometry.

These mutations were combined in increasing numbers into 3 constructs. These 3 constructs were then transfected into HEK293 cells and the resulting conditioned cultures were assayed for LGR5 antigen binding activity by flow cytometry. 2 constructs 18G7H6a1 and 18G7H6A3 were selected for further characterization. Table 1A lists certain sequences of antibodies.

TABLE 1A

Example 3-preparation of humanized LGR5 antibody

A GS single gene vector (designated 18G7Ch) for 18G7H6a1, 18G7H6A3 and chimera 18G7.1 (murine Fab from 18G7.1 fused to human IgG1Fc) was constructed, amplified, and transiently co-transfected into chinese hamster ovary cells (CHOK1SV GS-KO) in 200ml volumes using transient transfection for expression evaluation. Large scale transient transfection was then initiated, with a final volume of 5 liters for CHOK1SV GS-KO cells and 2.5 liters for 18G7H6A1 and 18G7H6A 3. The clarified culture supernatant was purified using one-step protein a chromatography. Product quality analysis in the form of SE-HPLC, SDS-PAGE and endotoxin measurements was performed using a 1mg/ml concentration of purified material containing internal human antibodies as a control sample. The results show high purity of the recovered product (> 95.7%).

Example 4 construction of a cell line for humanized LGR5 antibody

A pool of stable GS-CHO transfectants expressing 18G7H6A3 antibody was generated by transfecting CHOK1SV GS-KO host cells with the expression vector p18G7H6A 3/DGV. DGV containing the gene encoding the antibody was constructed, transfected and the resulting cells were subsequently generated by single cell sorting of the transfectant pool using FACS methodCloning of the cell line. The 96-well plates generated during cloning were screened once a week for the presence of single colonies. After about 2 weeks, useSystematic method, supernatants from up to 1000 colonies were screened for antibody production. Of the 1000 colonies screened, 991 produced detectable levels of antibody. The Octet data were ranked and the highest productivity colonies were picked for further processing.

The highest grade colonies were suspension cultured in 96 deep well plates in CD CHO medium and subsequently adapted to subculture medium. The productivity assay of the selected cell line was performed using a feeding scheme that mimics the bioreactor process as much as possible. The culture was harvested on day 12 and usedThe system method determines the antibody concentration. The concentration range of the harvested antibody is<20mg/L to 3000 mg/L. Based on the ranking position in the productivity screen, the parental pool from which the cell line was derived, and the evidence that each cell line was derived from a single colony, 20 cell lines were selected for further evaluation. Cultures of the selected 20 cell lines were expanded from 96 deep well plates to shake flasks by serial subculture. Rank position based on productivity screening in "simplified" fed-batch (fed-batch) suspension culture, and acceptable growth characteristics in shake flask cultures during conventional subculture (conventional subculture, always ≧ 1 × 10 per mL)⁶Individual living cells), in two 10L laboratory scale stirred-tank bioreactors, a lead (lead) cell line was selected for evaluation. The leader cell line shows consistently high growth and viability during conventional subculture and has at harvest time>A titer of 2000 mg/L. This Cell line was used to prepare a primary Cell Bank (Master Cell Bank, MCB) and evaluated in a 10L laboratory scale bioreactor.

Example 5-humanized LGR5 antibody binding to human LGR5

FACS-based assays were used to measure binding of purified 18G7H6a1 and 18G7H6A3 to recombinant human LGR5 overexpressed on the surface of CHO cells. CHO and CHO-LGR5 cells were stained at 4 ℃ with serial dilutions of 18G7H6a1 or 18G7H6A3, surface stained with PE-conjugated anti-human IgG secondary antibody detection, and analyzed on FACScalibur. 18G7H6a1 and 18G7H6A3 bound less than 10nM of EC50 to human LGR 5. Antibody control (MOPC) and wild-type CHO without LGR5 were used as negative controls in this experiment. 18G7H6a3 showed no binding to wild-type CHO, and the isotype control did not show any measurable binding to human LGR 5.

To determine potential animal model species for studying the efficacy and safety of 18G7H6A3, cross-reactivity of 18G7H6A3 with LGR5 expressed by homologous species was determined in a series of in vitro binding studies. Referring to fig. 1, as shown, antibody 18G7H6a3(BNC101) was found to strongly bind to human LGR5 and cynomolgus monkey LGR5, but not to rat or mouse LGR 5.

Example 6-binding of humanized LGR5 antibody to recombinant human LGR5 extracellular domain bound to plate

Binding of 18G7H6a1 and 18G7H6A3 to human LGR5 was assessed in vitro using an ELISA-based plate binding assay. This assay measures the binding of antibodies to purified recombinant LGR5 ectodomain-IgG-Fc fusions bound to ELISA plates, and antibodies that bind LGR5 were detected by using a horseradish peroxidase-conjugated anti-human IgG-CH1 secondary antibody. The EC50 of 18G7H6A3 for human LGR5-Fc was found to be 300 pM.

Example 7-binding characteristics of humanized LGR5 antibody to tumor cells

The binding characteristics of 18G7H6A3 to human cancer cell lines expressing different levels of LGR5 were analyzed by flow cytometry to determine the potential targeting characteristics of 18G7H6A3 to heterogeneous tumor populations. The expression levels of LGR5 in multiple tumor cell lines were quantified by flow cytometry.

Human tumor cell lines analyzed in these studies include colon cancer cell lines (CT1(Bionomics), CT3(Bionomics), DLD1(ATCC), Ls174T (ATCC), LoVo (ATCC), SW48(ATCC), SW480(ATCC), SW620(ATCC) and HCT116 (ATCC)); triple negative breast cancer cell lines (Hs578T (ATCC) and MDA-MB-231 (ATCC)); pancreatic cancer cell lines (AspC-1(ATCC), BxPC3(ATCC), Capan2(ATCC), HPAFII (ATCC), SW1990(ATCC), CFPAC (ATCC), PanC10.05(ATCC) and PANC-1 (ATCC)); cisplatin-sensitive ovarian cancer cell lines (OVCAR3(ATCC) and SK-OV-3 (ATCC)); cisplatin-resistant ovarian cancer cell lines (SK-OV-3/CP, OVCAR8/CP, Igrov1/CP and A2780/CP (TGEN)), and the lung adenocarcinoma cell line HOP62 (ATCC).

Cells grown to near confluence were digested (lift) with TrypLE cell dissociation buffer (Life Technologies), counted and grown at1 × 10⁵Individual cells/well were seeded into 96-well V-shaped bottom plates. The assay starting concentration of 18G7H6A3 was 100nM and serial dilutions were made in staining buffer (PBS/0.8% bovine serum albumin). The samples were incubated on ice for 30 minutes, then centrifuged at 1800rpm for 2 minutes at 4 ℃ and washed 3 times with staining buffer. Mu.l of a secondary goat anti-human IgG-PE conjugate (Southern Biotech) diluted 1:250 was added to each corresponding well of staining buffer. The samples were incubated on ice for an additional 15 minutes, then washed as above, and resuspended in 100. mu.l of staining buffer (Life Technologies) containing Propidium Iodide (PI) to exclude dead cells. Samples were analyzed on a FACScalibur flow cytometer using CellQuest (Becton Dickinson) and FlowJo (TreeStar, Inc) software.

Cell surface expression levels of LGR5 in various tumor cell lines were quantified by flow cytometry. CT1 colorectal and pancreatic cancer cell lines Panc-1, Capan2 and CFPAC belong to the highest LGR5 expression. Moderate expression levels were observed in pancreatic cancer cell lines (AspC-1, SW1990, HPAFII), cis-platinum-resistant ovarian cancer cell lines (OVCAR8/CP, A2780/CP and Igrov1/CP), and colon, breast and ovarian cancer cell lines (SW48, Hs578T and OVCAR 3). Low but detectable levels of cell surface expression of LGR5 were observed in colon cancer cell lines (SW480, LoVo) and breast cancer cell lines (MDA-MB-231). Table 2 summarizes the data for 18G7H6A3FACS binding to tumor cell lines.

TABLE 2

Example 8-humanized anti-LGR5 antibody inhibits malignant colorectal tumor growth in vivo

The primary CRC xenograft model of CT1 was derived from stage IV metastatic colon cancer patients. DNA sequencing of this tumor identified common colon cancer mutations for a variety of genes, including K-Ras, PI3K, PTEN, p53, and APC. On day 0, low passage CT1 tumor spheres stored in serum-free medium were injected subcutaneously in artificial basement membrane (Matrigel) into SCID/Bg mice and tumor size and body weight were monitored twice weekly. On day 25, when the tumor reached 120mm³At this time, CT1 subcutaneous tumors were randomly assigned to groups of 10 mice. Mice were treated with PBS, antibody control MOPC, 18G7H6a1, 18G7H6A3, or human/mouse chimera 18G7 Ch. Mice were dosed at 15mg/kg twice weekly (BIW) for 2.5 weeks (5 doses total).

Antibody 18G7H6a3 showed significant anti-tumor activity in vivo during the course of 4 doses (15mg/kg twice weekly) compared to PBS and MOPC antibody controls. Although antibody 18G7H6a1 showed anti-tumor activity, monoclonal 18G7H6A3 showed superior activity compared to 18G7H6a1 and the parent murine chimera 18G7Ch antibody. Table 3 shows the percentage of tumor volume reduction (group vs MOPC) in CT1 after 1-4 doses of Lgr5+ Ab.

TABLE 3

Dosage #:	1	2	3	4
					18G7Ch	9.2％	30.6％	19.5％	29.0％
18G7H6A1	17.5％	19.1％	14.2％	19.0％
					18G7H6A3	38.8％	42.0％	28.9％	35.4％

example 9-humanized anti-LGR5 antibody inhibits colorectal tumor growth in vivo

The primary CRC xenograft model of CT3 was derived from stage III mCRC patients and has mutations in K-Ras, H-Ras, APC, PI3K, PTEN, STK11, RB1, TP53, FGFR2, VANGL2, and ISCO. Low passage cryopreserved CT3 primary xenograft tumor fragments were implanted into 5 SCID/Bg mice. On day 41 post-implantation, SCID mice carrying CT3 primary xenografts were removed on average from 5 and pooled to-1150 mm³Dissociated and reimplantated subcutaneously in artificial basement membrane into cb.17scid mice and monitored twice weekly for tumor size and body weight. When the tumor reached an average of 130mm³Mice were randomly assigned (34 days post-implantation). Mice were treated with PBS, antibody control MOPC, 18G7H6A3, 18G7H6a1, or human/mouse chimera 18G7 Ch. Mice were dosed at 15mg/kg twice weekly (BIW) starting on day 34 for 2.5 weeks (5 doses). All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance, until completion.

After 4 doses (15mg/kg twice weekly), monoclonal 18G7H6A3 showed significant anti-tumor activity, although antibody 18G7H6a1 showed anti-tumor activity compared to PBS and MOPC antibody controls. Compared with the parent murine chimera 18G7Ch antibody, 18G7H6A3 showed superior activity, and showed equivalent activity to 18G7H6a 1. Table 4 shows the percentage of tumor volume reduction in CT3 (group vs MOPC) after n doses of test Ab.

TABLE 4

Ab dose #:	1	2	3	4
					18G7Ch	22.6％	8.9％	17.0％	13.8％
18G7H6A1	18.3％	12.6％	28.8％	28.7％
					18G7H6A3	34.2％	38.1％	23.4％	28.2％

example 10-combination of humanized anti-LGR5 antibody with FOLFIRI inhibits colorectal tumor growth in vivo

CT3 cells grown under CSC conditions were implanted into cb.17scid mice. At day 40 post-implantation, when tumors reached-160 mm³When mice were randomized into treatment groups comprising i) PBS, ii) FolFiri (5FU 30mg/kg, leucovorin 90mg/kg and irinotecan 24mg/kg), administered every 5 days for 15 days (total of 3 doses); and iii) FolFiri (as in ii) in combination with 18G7H6A3(15mg/kg twice weekly). Tumor volume analysis showed that the combination of 18G7H6a3 and FolFiri attenuated CT3 tumor growth compared to the FolFiri protocol. Combination treatment reduced tumor volume by about 58%, 53%, 45%, 33%, and 3 on days 61, 65, 68, 71, and 75, respectively7% (fig. 2).

Example 11-humanized anti-LGR5 antibody inhibits the growth of pancreatic cancer tumors in vivo

To assess the efficacy of 18G7H6a3 as a single agent or in combination with standard of care, a pancreatic cancer xenograft model was tested. AspC-1 cells (in a 1:1 ratio of artificial basement membrane + RPMI) were implanted into CB17.SCID mice. On day 20 post-implantation, tumors were randomized into 5 groups: i) PBS, ii) MOPC (15mg/kg, twice weekly, ip), iii)18G7H6A3(15mg/kg, twice weekly, ip), iv) gemcitabine (90mg/kg, twice weekly, ip), and v) gemcitabine and 18G7H6A3 were combined in parallel at the doses described above.

Single agent 18G7H6a3 was found to inhibit tumor growth by up to approximately 40% on day 41 post-implantation compared to saline and/or control IgG. In addition, the combination of 18G7H6a3 with gemcitabine significantly inhibited tumor growth in the AsPC-1 model compared to gemcitabine alone (up to 36% on day 61 post-implantation). Compared to PBS and control IgG, single agent 18G7H6a3 provided some inhibition of tumor growth until day 65.

Example 12-humanized anti-LGR5 antibody inhibits in vivo triple negative breast cancer tumor growth

This in vivo study was performed using low passage triple negative breast cancer cells (ER-, PR-, without HER2 overexpression). MDA-MB-231-LM3 cells were cultured in adherent cultures of DMEM/10% FBS/anti-anti medium. On day 0, the ratio in RPMI: MDA-MB-231-LM3 cells in artificial basement membrane (1:1) were injected into the 4 th mammary fat pad of CB.17SCID mice and tumor size and body weight were monitored twice weekly. On day 27, tumors reached-155 mm³At this time, MDA-MB-231-LM3 tumors were randomized into groups of 4 groups of 10 mice. Mice were treated with PBS, antibody control MOPC, or 18G7H6a 3. Mice were dosed at 15mg/kg twice weekly (BIW) for 3.5 weeks (7 doses). Found to be comparable to PBS (60.7% tumor growth inhibition) or MOPC antibody (49.3% tumor growth inhibition) control, antibody 18G7H6a3, showed significant anti-tumor activity (fig. 3).

Example 13 inhibition of LGR5 expression in colorectal cancer cells treated with SN38 or PI3K/mTOR inhibitors

A series of CRC cell lines (including DLD1, HCT116, LS174t, LoVo, SW48, SW480 and SW620) were treated with either a PI3K/mTOR dual inhibitor (NVP) or 2 different cytotoxic agents, including SN38 (active metabolite of irinotecan) or 5FU (5 fluorouracil). Cells were treated with 1um of the above reagent and harvested after 72 hours. Cells were then stained with anti-LGR 5Mab conjugated to AlexaFluor647 and data analyzed by flow cytometry using FACScalibur.

Flow cytometry analysis of CRC cell lines showed higher expression of LGR5 in LoVo, HCT116, LS174t, SW48, SW480 and SW620 cells when treated with PI3K/mTOR inhibitor. In addition, treatment with SN38 increased expression of LGR5 in HCT116, LS174t, SW48, SW480, and particularly SW620 cells. However, 5FU treatment did not induce LGR5 expression in any of these cell lines, suggesting that there is a unique underlying mechanism for modulating LGR5 expression in these cell lines. These data indicate that LGR5+ cells are more resistant to treatment with the above agents, as treatment mostly targets LGR 5-negative non-cancer stem cell populations. To understand whether these reagent treatments up-regulated LGR5 expression in these cells, we analyzed LGR5 cell surface expression in all cell lines by flow cytometry. LGR5 expression was significantly upregulated in LoVo following treatment with PI3K/mTOR inhibitor. These data indicate that treatment with small molecule inhibitors or cytotoxic agents targets LGR5 negative cells and results in increased expression of LGR5 in these cells.

Example 14-LGR 5 expression is promoted in pancreatic cancer cell lines treated with Small molecule inhibitors or cytotoxic Agents

in addition to CRC cell lines, to further broaden the above findings, the expression of LGR5 in a range of pancreatic cell lines treated with relevant standard of care (including nanoparticulate albumin-bound paclitaxel (nab-paclitaxel), gemcitabine and taxol) and small molecule inhibitors targeting most relevant pathways of pancreatic cancer (such as inhibitors of PI3K, MEK and GSK3 β) pancreatic cell lines examined include AsPc1, HPAFII, PANC1, BxPC3, CFPAC, PANC10.05, Capan2 and SW1990 as assessed by flow cytometry, treatment with nanoparticulate albumin-bound paclitaxel resulted in the upregulation of LGR5 in PANC1, BxPC3 and PANC10.05, treatment with gemcitabine upregulated LGR5 in PANC1, whereas treatment with AFLGphenol resulted in the increased expression of LGR5 in HPPI 3/mTOR 25 resulted in the upregulation of CFLGR 5, and treatment with MEK 5 in MEK inhibitor 857.

Example 15 LGR5 in colorectal cancer tumors treated with the FOLFIRI regimen (5FU, folinic acid and irinotecan) Is adjusted upwards

To investigate whether chemotherapy altered LGR5 expression in colorectal tumors, mice were treated every 5 days with 5FU (30mg/kg, i.p), folinic acid (90mg/kg) and 2 different doses of irinotecan (24mg/kg or 8 mg/kg). The results of these studies indicate that while the CT3 tumor was sensitive to the chemotherapy regimen, the CT1 tumor did not completely regress and showed some tolerance to the regimen (fig. 4). To examine the effect of FOLFIRI treatment on LGR5 expression, total mRNA was extracted from tumors derived from CT1 and CT3 patients, and LGR5 expression was determined by qRT-PCR and analyzed by subtracting the CT value (cycle threshold) of LGR5 in each sample from the corresponding GAPDH transcript to yield a DCT (Δ CT) value. The data is represented as 2 to the power of DCT. Analysis of LGR5 abundance showed that LGR5 transcripts were increased in CT1 (about 2 fold) and CT3 tumors (about 3.5 fold) compared to the corresponding saline-treated tumors.

Example 16 treatment with gemcitabine alone and gemcitabine in combination with nanoparticle albumin-bound paclitaxel LGR5 is upregulated in pancreatic cancer tumors

To investigate whether standard-of-care chemotherapy treatment of pancreatic cancer altered LGR5 expression in pancreatic tumors, mice were treated twice weekly with a combination of gemcitabine and nanoparticle albumin-bound paclitaxel (in JH109 primary xenografts). At the time of endpoint analysis, qRT-PCR data using tumor cDNA showed a significant increase in LGR5 expression in chemotherapy-treated tumors compared to the corresponding saline-treated tumors, indicating that treatment with standard of care resulted in upregulation of LGR5 in tumor cells.

LGR5 expression in JH109 model, a xenograft model of patient-derived pancreatic tumors. The mice were implanted with tumor masses that continued to passage within the recipient but were never exposed to in vitro culture conditions. Treatment of tumor-bearing mice with a chemotherapeutic regimen (a combination of gemcitabine and nanoparticulate albumin-bound paclitaxel) resulted in significant inhibition of tumor growth. Consistent with the colon cancer model, chemotherapy resulted in upregulation (greater than 4-fold) of LGR5 in JH109 tumors, further suggesting that the cancer stem cell population was enriched upon chemotherapy treatment. See, for example, fig. 5.

Example 17-humanized anti-LGR5 antibody inhibits pancreatic tumor growth in vivo

The efficacy of 18G7H6a3 was also studied in a pancreatic cancer xenograft model. PANC1 cells were implanted into cb.17scid mice (1E 6/mouse implanted subcutaneously in artificial basement membrane + RPMI at a 1:1 ratio) and randomized into treatment groups on day 41 post-implantation: i) PBS, ii) IgG control (15mg/kg, twice weekly, ip), iii)18G7H6A3(15mg/kg, twice weekly, ip), iv) gemcitabine (90mg/kg, twice weekly, ip), and v) a concurrent combination of gemcitabine and 18G7H6A3(15mg/kg, twice weekly, ip). Gemcitabine was administered in the partition group for 3 weeks to inhibit tumor growth. All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance.

Analysis of tumor volumes showed that while a single agent of 18G7H6A3 had a trend favorable to inhibit tumor growth (up to 30% at day 70 post-implantation), the combination of 18G7H6A3 and gemcitabine significantly inhibited the growth of PANC1 tumors (up to 52% at day 80 post-implantation) compared to the gemcitabine-only group. See fig. 6.

In this example, the significant activity of 18G7H6a3 observed when administered in combination with chemotherapy (gemcitabine) may be attributed to the increased expression of the target antigen LGR5 in response to gemcitabine treatment.

Example 18-humanized anti-LGR5 antibody inhibits growth of pancreatic tumors pretreated in vivo

In addition to cell lines, we also investigated the efficacy of 18G7H6a3 as a single agent or in combination with standard of care in a JH109 primary patient derived pancreatic cancer xenograft model. The JH109 xenograft model is from patients who have received 4 treatment regimens including 5-FU, gemcitabine, Erbitux and radiation. The original patient tumors have been serially passaged in immunodeficient mice without exposure to in vitro culture. To examine the efficacy of 18G7H6a3 in the JH109 model, tumor-bearing mice were treated with the following protocol (n-7): control IgG (15mg/kg, i.p., twice weekly), 18G7H6A3(15mg/kg, i.p., twice weekly) single agent, standard of care chemotherapy (a combination of gemcitabine (50mg/kg, i.p., once weekly) and nanoparticulate albumin-bound paclitaxel (30mg/kg, i.v., once weekly)), a combination of chemotherapy and control IgG, and a combination of chemotherapy and 18G7H6 A3. While the single 18G7H6A3mAb did not affect tumor growth, the combination of 18G7H6A3 with nanoparticle albumin-bound paclitaxel and gemcitabine chemotherapy resulted in a significantly greater degree of tumor inhibition compared to chemotherapy alone. The combination of 18G7H6a3 and chemotherapy resulted in 77% greater tumor growth inhibition compared to chemotherapy alone. The tumors of 3 mice treated with the combination of 18G7H7a3 chemotherapy were completely eliminated (no detectable tumor). The 18G7H6a3 chemotherapy combination group continued to inhibit tumor growth even after treatment discontinuation, and 1 mouse had no measurable tumor three months after discontinuation of chemotherapy. In this example, the significant activity of 18G7H6a3 observed when administered in combination with chemotherapy (gemcitabine plus nanoparticle albumin-bound paclitaxel) may be attributed to increased expression of the target antigen LGR5 in response to gemcitabine nanoparticle albumin-bound paclitaxel treatment and demonstrated prevention of regrowth or recurrence of the primary tumor in vivo after chemotherapy treatment to eliminate the primary tumor mass.

Example 19-treatment of humanized LGR5 antibody to deplete the population of cancer stem cells

For flow cytometry analysis, cells from 5 individual tumors were stained with multiple antibodies against the stem cell specific markers CD44 and CD 166. The tumors were dissociated, mouse cells were depleted, and viable cells were then counted. Dissociated cells were used to analyze the expression of cell surface stem cell markers by flow cytometry.

The presence of cancer stem cell populations was reduced as determined by the CD166+/CD44+, LGR5+/CD166+, or LGR5+/CD166+/CD44+ subpopulations (fig. 7).

Example 20-treatment of humanized LGR5 antibody reduces recurrence of colon cancer tumors and cancer stem cells in vivo Frequency of

The effect of 18G7H6A3 in combination with FolFiri was tested in a model of colon cancer CT3 (example 10). The results of this primary tumor efficacy study showed that the combination of 18G7H6a3 with 3 rounds of FolFiri regimen was more effective in reducing tumor growth than FolFiri alone. To determine whether the 18G7H6A3FOLFIRI combination regimen was also effective in reducing Cancer Stem Cell (CSC) frequency, tumors from day 78 were harvested, dissociated, pooled and reimplanted in a limiting dilution assay with a new population of tumor-initiating cb17.scid mice at 10, 30, 100 cells/flank. The mice were then monitored twice weekly for tumor growth and allowed to grow without further treatment.

Cells isolated from mice treated with the anti-LGR5 antibody 18G7H6A3 in combination with FOLFIRI had greatly reduced tumorigenicity compared to cells isolated from mice treated with FOLFIRI alone (fig. 8). In addition, reimplanted cells from the 18G7H6A3FOLFIRI combination had significantly slower tumor growth characteristics and delayed progression time compared to FOLFIRI alone (fig. 9). Finally, treatment with 18G7H6A3 reduced the frequency of cancer stem cells at day 40 by linear regression analysis of factor 6 (1/856.3 for 18G7H6A3/FOLFIRI versus 1/138.6 for FOLFIRI). These data indicate that 18G7H6a3 in combination with FOLFIRI effectively targets tumor initiating or cancer stem cell populations. For the 30 cells/animal data, day 68 was the last day. The data is significant, p is 0.0039.

Example 21-treatment of humanized LGR5 antibody reduces recurrence of pancreatic cancer tumors and the frequency of cancer stem cells in vivo Rate of change

The effect of 18G7H6A3 in combination with gemcitabine was tested in the PANC1 model for pancreatic cancer. This study showed that 18G7H6a3 in combination with gemcitabine significantly inhibited tumor growth in the PANC1 model compared to gemcitabine alone. Tumor cells from these treatment groups were harvested, dissociated, pooled and replanted in a limiting dilution assay (500, 1500, 4500 or 13500 cells/animal) with a new population of cb.17scid mice and allowed to grow without treatment.

In re-implantation with the limiting dilution assay, cells isolated from mice treated with the anti-LGR5 antibody 18G7H6A3 in gemcitabine combination had greatly reduced tumorigenicity compared to cells isolated from mice treated with gemcitabine alone. Reimplantation of PANC1 tumors treated with gemcitabine in combination with 18G7H6a3 showed a reduction in frequency of transplantation in 4500 cell implanted mice (20% of gemcitabine 40% versus combination) and in 13500 cell implanted mice (70% of gemcitabine 100% versus combination). Using linear regression, the frequency of gemcitabine engrafting into tumors was about 1.5-fold higher in gemcitabine compared to gemcitabine in the combination group (1 in 14883 versus 1 in 21336). These data indicate that 18G7H6a3 in combination with gemcitabine is effective in targeting tumor initiating or cancer stem cell populations.

In addition to PANC1 tumors, we also analyzed the percentage of engraftment and cancer stem cell frequency in AsPC-1 tumor-bearing mice treated with gemcitabine as a single agent or in combination with 18G7H6A3 in limiting dilution experiments (using 500, 1500, 4500, and 13500 cells). Tumor volume measurements at day 40 post-treatment showed a reduction in the percentage of tumor bearing mice in the combination group compared to gemcitabine in mice implanted with 4500 or 13500 cells (40% and 80% compared to 30% and 50%, respectively). The frequency of cancer stem cells was also greater, greater than 1.5-fold, in gemcitabine compared to the combined group, further suggesting that the combination of 18G7H6a3 with gemcitabine targets cancer stem cell populations of pancreatic cancer.

Example 22-treatment of humanized LGR5 antibody reduces recurrence of triple negative breast cancer tumors and cancer stem cells in vivo Frequency of cells

The effect of 18G7H6A3 in combination with paclitaxel was tested in the triple negative breast cancer MDA-MB-231-LM3 model (example 12). This study showed that 18G7H6a3 in combination with paclitaxel had the lowest additional inhibition of tumor growth compared to paclitaxel alone. These tumors were harvested, dissociated, pooled and reimplanted in a limiting dilution assay with a new population of cb.17scid mice at 10, 30, 100 cells/flank and allowed to grow without treatment.

Cells isolated from mice treated with the anti-LGR5 antibody 18G7H6A3 in combination with paclitaxel had greatly reduced tumorigenicity compared to cells isolated from mice treated with paclitaxel alone. In addition, reimplanted cells from 18G7H6a3 plus paclitaxel tumors had significantly slower tumor growth characteristics and delayed progression time compared to paclitaxel alone. Finally, 18G7H6a3 plus paclitaxel treatment reduced the frequency of cancer stem cells by linear regression analysis. These data indicate that 18G7H6a3 in combination with paclitaxel effectively targets tumor initiating or cancer stem cell populations.

Example 23-prophylactic treatment with humanized anti-LGR5 antibody and chemotherapy inhibits metastatic colorectal metastasis in vivo Cancer growth

In vivo studies were performed using low passage colorectal cancer cells (BMCRC086) derived from liver metastases (met) of colorectal cancer patients. On day 0, BMCRC086 cells were thawed, suspended in RPMI: artificial basement membrane (1:1) and injected subcutaneously into the posterior side of cb.17scid mice. Animals were monitored twice weekly for tumor size and body weight. On day 7, mice were treated with PBS, 18G7H6A3, FOLFIRI, or FOLFIRI in combination with 18G7H6 A3. Mice were dosed with 15mg/kg of PBS and 18G7H6A3 twice weekly (BIW) for 7.5 weeks (16 doses). Mice were dosed with FOLFIRI (30mg/kg fluorouracil and 90mg/kg folinic acid on days 7, 12, 17, 22, 27 and 32; 24mg/kg irinotecan on days 8, 13, 18, 23, 28 and 33) for 4 weeks (6 doses). The combination of 18G7H6a3 with FOLFIRI showed significant anti-tumor activity compared to FOLFIRI alone (figure 10).

Example 24-treatment of humanized LGR5 antibodies inhibits the Wnt signaling pathway

in an in vivo tumor efficacy study, 18G7H6A3 treated tumors from colon cancer CT1 (example 8) and CT3 (example 9) were characterized by western blot analysis after sacrifice tumor samples were excised from each treated mouse (n ═ 5-10 mice per group), immediately frozen in a liquid nitrogen cooled mortar, pestle crushed (cryo-crushed), snap frozen in liquid nitrogen and stored at-80 ℃ until use ℃. cryo-crushed tumors were lysed using ice-cold lysis buffer (reduced RIPA buffer containing phosphatase and protease inhibitors) for 30 minutes, occasionally vortexed several times. supernatants containing tumor lysis proteins were run on SDS-PAGE gels followed by western blots against various signaling proteins (and their phosphorylated forms). in CT3 tumors, a large number of significant differences between the treated groups were observed.in fig. 11, phosphorylation-Thr 41/Ser 41/45- β -catenin (Wnt signaling protein) was demonstrated to be a marker for Wnt signaling and was able to be degraded by tumor cells in vivo, with Wnt signaling proteins inhibiting activity of 3618 r 8678.

Example 25-humanized LGR5 antibody treatment did not inhibit the Wnt-signaling pathway in vitro

Parental HEK-293T cells and HEK-293T cells stably expressing LGR5 were transduced with lentiviruses (GFP cognal, QIAGEN) containing TCF-LEF reporter vectors and stably expressing reporters were selected. Parental lines and a stable reporter line expressing LGR5 were inoculated at 25,000/well in 96-well plates, attached overnight, serum starved, and treated with antibody or vehicle for 6 hours, followed by recombinant human Wnt3a (3nM) and recombinant human R-spondin (R-spondin) for 18 hours. Two concentrations of each R-vertebrate 1-3 and one concentration of R-spo4(100pM, 300pM, 1nM, 3nM or 10nM) were tested based on assays of the activity of different R-vertebrates in activated TCF/LEF reporter cell lines. Reporter-driven GFP signal was measured on a microplate reader. For the R-spondyloproteins tested, all experiments shown are summary data (data mean + SD) from three independent experiments (each experiment was performed in duplicate).

As shown in figure 12, increasing concentrations of soluble 18G7H6a3 did not affect induction of TCF/LEF promoter-driven GFP expression by combination with Wnt3a plus RSPO1, RSPO2, or RSPO 3. Also shown is positive control antibody 76C12, which shows that it inhibits the induction of signaling activity by LGR4 and LGR5 in the presence of RSPO and Wnt. This data demonstrates that the anti-LGR5 antibody 18G7H6A3 does not block RSPO driven activation of the TCF/LEF promoter.

Example 26-humanized LGR5 antibody targeting tumor cells by ADCC (antibody dependent cellular cytotoxicity) mechanism

CHO-LGR5 cells were cultured to confluence and spun down, resuspended in PBS and counted. An aliquot of cells (approximately 10 ten thousand) was added to another tube containing 100 μ M preheated (37 ℃) CFSE (carboxyfluorescein succinimidyl ester) and the mixture was incubated for 15 minutes in a cell incubator. The final CFSE concentration was about 1. mu.M. Next, the cells were washed and resuspended in pre-warmed medium and placed in an incubator for another 30 minutes, then washed with PBS. The stained cells were then stained with 18G7H6A3 (100. mu.M). To ensure that the antibody binds to the CHO-LGR5 cells, in some studies, an aliquot of the cells were also stained with a second goat anti-human PE conjugated antibody and analyzed on a laboratory calibur instrument. U937 cells were stained with DDAO-SE (DDAO succinimidyl ester; 2. mu.M dye for 10 ten thousand cells) for 15 minutes at room temperature in a laboratory protected location. The cells were then incubated with 1ml of FBS (fetal bovine serum) followed by 5 minutes in a dark position. Next, cells were washed with PBS supplemented with FBS (10%) and resuspended in RPMI supplemented with FBS (2.5%). CHO-LGR5-18G7H6A3 and U937-DDAO-SE labeled cells were co-incubated in a cell culture incubator for 5 hours and analyzed on a laboratory calibur instrument. As a negative control, an aliquot of CHO-LGR5-CFSE cells (no 18G7H6A3 staining) was also co-incubated with U937 and analyzed on a calibur instrument.

Analysis of flow cytometry data indicated that most CHO-LGR5 cells stained with CFSE and 18G7H6A3 were viable and could be detected on calibur instruments. In addition, U937(U937 (human monocyte cell line; effector cells) and CHO-LGR5 cells were both detectable when stained and obtained separately-finally, co-incubation of U937-DDAO-SE with CHO-LGR5-CFSE-18G7H6A3 identified a double positive population, however, co-incubation of U937 with CHO-LGR5-CFSE lacking 18G7H6A3 did not yield a double positive population the presence of which indicates cross-binding of U937 (which expresses FcR) to CHO-LGR5-18G7H6A3 (which expresses the Fc portion) and further suggests that ADCC is one of the mechanisms of the anti-tumor activity of 18G7H6A 3.

Example 27-humanised LGR5 antibody internalizes LGR5

Internalization of 18G7H6A3 was detected on CHO cells overexpressing LGR 5. Cells were stained with 100nM antibody at 4 ℃ for 30 minutes to 2 hours, excess antibody was washed away, and stained cells were incubated at 4 ℃ or 37 ℃. Internalization of cell surface bound antibody was monitored by staining cells with a secondary antibody conjugated to AlexaFluor488 at various time points. The internalization rate has a measured t1/2 value of 6.703 ± 1.282 min surface localization upon incubation at 37 ℃. Internalization was largely blocked upon incubation at 4 ℃, although some reduction in surface bound antibody was observed.

Example 28-humanized LGR5 antibody does not competitively block the binding of soluble RSPO to LGR5

The interaction of biotin-18G 7H6A3 with hLGR5-Fc in the presence of human R-spondin 1/2/3/4 protein was tested using a competitive ELISA format. LGR5-Fc was coated at 2. mu.g/mL onto a 96-well high binding ELISA plate and incubated overnight at 4 ℃. Plates were blocked with PBS + 1% BSA. Biotin-18G 7H6A3 was diluted to 1. mu.g/mL in binding buffer. Concentrations were selected from a direct binding ELISA between the previous LGR5-Fc and biotin-18G 7H6A3 to produce a strong signal above the EC50 concentration. Competitor proteins were added to the ELISA plates at different concentrations simultaneously with biotin-18G 7H6a 3. A1: 1,000 dilution of streptavidin-HRP (R & D Systems, cat #890803) was used for detection. The plates were developed with TMB (Thermo) and data collected at 450nm on a SpectraMax Plus 384 microplate reader. Data analysis was performed using GraphPad Prism 6 program. ELISA was repeated 3 times with some modifications to biotin-mAb and competitor concentrations.

As a positive control, LGR5-Fc competed on the plate for binding to biotin-18G 7H6A3 and hLGR 5-Fc. The ability of R-spondyloin 1/2/3/4 to block the binding of biotin-18G 7H6A3 to LGR5-Fc coated on a plate was examined. The proteins were purchased from R & D Systems and were full-length constructs expressed in mammalian cells. At the highest concentration of R-spondyloproteins, no complete blocking of antibody binding to LGR5 was observed (fig. 13).

Example 29-humanized LGR5 antibody does not competitively block the binding of soluble RSPO to LGR5

Binding of ligand (RSPO or Norrin) alone to LGR5 was insufficient to induce LGR5 signaling. In contrast, LGR5 forms ternary complexes with multiple co-receptors to drive signaling. To examine the effect of 18G7H6A3 on LGR5 ternary complex formation, the binding of LGR5 to RNF43, ZNRF3 and LRP6 in the presence of R-spondyloprotein 1/2/3/4 and Norrin was examined using an ELISA format. RNF43-Fc, ZNRF3-Fc and LRP6-Fc were coated at 4. mu.g/mL (in 1 × PBS) on 96-well high binding plates. Plates were incubated overnight at 4 ℃ and blocked with PBS + 1% BSA. LGR5-Fc was diluted to 1. mu.g/mL in a first buffer, all in the presence or absence of 1. mu.g/mL of R-spondyloprotein 1/2/3/4 or 0.5. mu.g/mL of Norrin. R-spondyloprotein 1/2/3/4 or Norrin was pre-incubated with hLGR5-Fc prior to addition to the ELISA plates. Three replicates of wells were used for each condition and three replicates were tested. Bound hLGR5-Fc was detected using a 1:2,000 anti-FLAG mAb (Cell Signaling). Anti-mouse IgG HRP (JIR) diluted 1:10,000 was used for detection. The plates were developed with TMB (Thermo) and data collected at 450nm on a SpectraMaxPlus 384 microplate reader. Data analysis was performed using GraphPad Prism 6 program. Ternary complexes with LGR5, ligand RSPO or Norrin, and co-receptors (RNF43-Fc, ZNRF3-Fc and LRP6-Fc) were observed.

Next, 18G7H6A3 was additionally added to the ELISA plates in the presence of LGR5-Fc and RSPO or Norrin. 18G7H6a3 significantly reduced LGR5 ternary complexes formed with both RSPO and Norrin ligands, as well as all three co-receptors (RNF43, ZNRF3, and LRP 6). See fig. 14. Since 18G7H6a3 does not compete directly or competitively for ligand binding, this data is evidence of an allosteric model of inhibition.

Example 3Epitope mapping of 0-anti-LGR 5 antibody 18G7H6A3

To further characterize the specific region of LGR5 to which antibody 18G7H6a3 binds, epitope mapping experiments were performed using deuterium and hydrogen exchange mass spectrometry. Test digests prepared with non-deuterated buffers in various concentrations of guanidine hydrochloride (GdnHCl) were used to optimize the proteolytic conditions for optimal peptide coverage of LGR5 alone prior to performing hydrogen-deuterium exchange experiments. For pepsin digestion of DXMS, samples were thawed at 5 ℃ and immediately digested on a protease column packed with porcine pepsin (Sigma) using a flow rate of 100. mu.l/min and 0.05% trifluoroacetic acid. Digested fragments were collected on a C18 collection column and separated on a C18 reverse phase column (Vydac) using a linear acetonitrile gradient of 6% to 38%. The eluate from the column was directly electrospray into an LCQClasic mass spectrometer (Thermo Finnigan, Inc.) or a Q-TOF mass spectrometer (Micromass). The determination of pepsin-produced peptides from the MS/MS data set was aided by the use of SEQUEST (Thermo Finnigan, Inc.). The peptide pool was then further validated by dxms explorer (Sierra Analytics inc., Modesto, CA). Peptide coverage maps of different concentrations of GdnHCl were compared and the conditions of optimal coverage maps for each individual protein or protein complex were used for subsequent deuterium exchange experiments. All steps were performed at 0 ℃ as described previously.

The exchange experiment was started by mixing LGR5-Fc in protein buffer or LGR5-Fc pre-incubated with 18G7H6A3 with D2O buffer to 50% D2O final concentration. The mixture was incubated at 0 ℃ for 10, 30, 100, 300, 1,000, 3,000 or 10,000 seconds and then the exchange reaction was quenched by adding ice cold quench solution (0.96% formic acid, 0-0.8M guanidine hydrochloride) so that the sample had a final concentration of 0.58% formic acid and 0-0.5M guanidine hydrochloride, pH 2.5. The samples were then immediately frozen on dry ice and stored at-80 ℃. As previously described, data processing for DXMS experiments utilized specialized software (DXMSExplorer, Sierra Analytics Inc.).

The hydrogen/deuterium (H/D) -exchange data provides details about the changes in solvent exposure due to the binding of 18G7H6a3 and the buried surface-exposed residues when the antibody binds to the antigen. Analysis of the HD exchange data shows that 18G7H6A3 binds to amino acids T175, E176, Q180, R183, S186, A187, Q189, D247, E248, T251, R254, S257, N258, K260 of SEQ ID NO:47 within the leucine rich repeat 6-9 convex on the surface opposite the R-spondin binding site as determined by X-ray crystallization studies (see, e.g., Chen et al, human Gene Dev.27(12):1345-50, which is incorporated herein by reference in its entirety). These data indicate that the residues involved in LGR5 binding to R-spondyloproteins are not involved in binding to 18G7H6 A3. These preliminary results do not exclude the fact that: other structural elements in LGR5 may be involved in the binding site of 18G7H6 A3.

Example 31 administration of 18G7H6A3 to human patients with Colon cancer

A population of human patients with colon cancer is treated with chemotherapy and tumor volume is monitored. It was observed that at the start of chemotherapy, the mean tumor volume stopped increasing and actually decreased. After a longer duration, the tumor volume stabilizes and eventually begins to increase.

A second population of human patients suffering from colon cancer is treated with chemotherapy and 18G7H6a3 co-administration. The mean tumor volume was again monitored. It was observed that tumor volume stopped increasing at the start of chemotherapy and actually decreased. The tumor volume was observed to decrease to a minimum volume much smaller than the volume of the first population. It was also found that tumor volume remained low for a considerable period of time relative to the first population.

Example 32 administration of 18G7H6A3 to human patients with Colon cancer

Chemotherapy is administered only to a first population of human patients with colon cancer. Administering a combination of chemotherapy and 18G7H6a3 to a second population of human patients with colon cancer.

The first population showed a temporary reduction in tumor size and growth, after which tumor growth continued and symptoms recovered. Tumor growth after chemotherapy treatment is resistant to subsequent chemotherapy treatment.

The second population showed a reduction in tumor size to baseline levels and tumor growth stopped. Tumor growth did not resume during and after completion of the treatment regimen. After completion of the treatment regimen, growth in the second population is not restored and the symptoms of cancer no longer exist.

Example 33-administration of 18G7H6A3 to human patients with Colon cancer increases survival

Chemotherapy is administered only to a first population of human patients with colon cancer. Administering a combination of chemotherapy and 18G7H6a3 to a second population of human patients with colon cancer.

Patient survival was monitored for a fixed duration (1 year) after treatment. It was observed that patient survival in the second population was much higher than in the first population. That is, a significantly higher proportion of the second population survived the first year after treatment than the survival rate of the first population.

Similar observations were made at later intervals and it was observed that members of the second group were significantly more likely to survive to the second interval (2 years after treatment) in survivors for the first interval than first group members that were alive 1 year after treatment.

Example 34 administration of 18G7H6A3 to human patients with Breast cancer

A population of human patients with breast cancer is treated with chemotherapy and tumor volume is monitored. It was observed that the mean tumor volume stopped increasing at the start of chemotherapy and actually decreased. After a prolonged period of time, the tumor volume stabilizes and eventually begins to increase.

A second population of human patients suffering from breast cancer is treated with chemotherapy administered in conjunction with 18G7H6a 3. The mean tumor volume was again monitored. It was observed that when chemotherapy was initiated, tumor volume stopped increasing and actually decreased. A reduction in tumor volume to a minimal volume that is substantially reduced compared to the first population was observed. It was also found that the tumor size remained low for a considerable period of time relative to the first population.

Example 35 administration of 18G7H6A3 to human patients with Breast cancer

Chemotherapy is administered only to a first population of human patients with breast cancer. Administering a combination of chemotherapy and 18G7H6a3 to a second population of human patients having breast cancer.

The first population showed a temporary reduction in tumor size and growth, after which tumor growth resumed and symptoms recovered. Tumor growth after chemotherapy treatment is resistant to subsequent chemotherapy treatment.

The second population showed a reduction in tumor size to baseline levels and tumor growth stopped. Tumor growth did not resume during and upon completion of the treatment regimen. After completion of the regimen, growth did not recover in the second population and symptoms of cancer no longer existed.

Example 36 administration of 18G7H6A3 to human patients with Breast cancer increases survival

Chemotherapy is administered only to a first population of human patients with breast cancer. Administering a combination of chemotherapy and 18G7H6a3 to a second population of human patients having breast cancer.

Patient survival was monitored for a fixed duration (1 year) after treatment. It was observed that the survival of patients in the second population was substantially higher than the survival of patients in the first population. That is, a significantly higher proportion of the second population survives the first year after treatment than the survival of the first population.

Similar observations were made at later intervals and it was observed that members of the second group were significantly more likely to survive to the second interval (2 years after treatment) in survivors for the first interval than first group members that were alive 1 year after treatment.

Example 37-administration of 18G7H6A3 to human patients with Colon cancer reduces side effects

An anti-LGR5 antibody that chemotherapy and blocks LGR5-RSPO binding and signaling is administered to a first population of human patients with colon cancer. A second population of human patients with colon cancer is administered chemotherapy and 18G7H6a 3.

The first population showed non-therapeutic side effects associated with interference with RSPO1 signaling through LGR 5. These side effects are detrimental to the patient's health.

The second population administered 18G7H6a3 in combination with chemotherapy showed no non-therapeutic side effects associated with interfering with RSPO1 signaling through LGR 5.

Example 38 expression of LGR5 in advanced CRC tumors.

The expression of LGR5 transcripts was studied using the RNAscope technology using LGR 5-specific probes. LGR5 transcript was detectable in tissues including colon, intestine, cerebellum and pancreas. LGR5 transcripts were also detectable in patient-derived xenograft (PDX) tissues including CT1CRC and JH109 pancreatic tumors. The expression of LGR5 was studied in CRC patient samples isolated at different stages of tumorigenesis, including early (grade I) and late (metastatic) lesions. LGR5 transcripts were expressed in CRC grade I, II and II lesions, and were highly expressed in CRC metastatic lesions.

Example 39 expression of LGR5 in xenografts derived from metastatic pancreatic patients

LGR5 expression was studied in xenografts derived from metastatic pancreatic patients using Quantitative Polymerase Chain Reaction (QPCR). Tumor tissue samples were either snap frozen or added to cryovials containing RNAlater (Qiagen, CA) and transferred to-70 ℃ after incubation at 4 ℃ for several hours. Total RNA was extracted using Qiagen RNeasy extraction kit (Qiagen, CA) and cDNA was synthesized using SuperScriptIII kit (Life Technologies, CA) and the manufacturer's protocol. Human specific LGR5 and GAPDH primers were used in a StepOne thermal cycler (Life Technologies, CA) and the following thermal conditions were used to measure human LGR5 transcript abundance: 50 ℃ (2 min); 90 deg.C (2 min), and 90 deg.C (15 sec) and 60 deg.C (1 min) for 40 cycles, and melting curve evaluation (65 deg.C-95 deg.C). LGR5 abundance was quantified using the 2^ delta Ct equation.

LGR5 was highly expressed in xenografts derived from patients with metastatic pancreas. Chemotherapy treatment resulted in increased LGR5 expression in pancreatic tumors. Using human specific primers, LGR5 transcripts from a range of pancreatic patient-derived xenografts can be measured using QPCR. Although LGR5 was detectable in most tumors, there was an increased trend of LGR5 expression in metastatic tumors, further suggesting a role for LGR5 in late-stage tumorigenesis.

LGR5 expression was studied in a series of pancreatic tumors including JH109, ASPC1, and PANC 1. Treatment with standard of care therapy (SOC) (Gemzar and Abraxane in JH109, and Gemzar alone in PANC1 and ASPC1) resulted in the induction of LGR5 expression in each of the above tumors (fig. 15). Clearly, LGR5 expression was reduced to levels comparable to controls (saline or MOPC) in tumors treated with the combination of 18G7H6a3 and SOC. These data further indicate that expression of LGR5 can be used as a biomarker in PANC tumors in response to combined treatment (18G7H6A3+ SOC).

Example 40-CTNNB1 is one of the target genes for CRC and 18G7H6A3 in pancreatic tumors

Potential targets for 18G7H6a3 in the Wnt pathway were investigated. Wnt QPCR plates (Qiagen, CA) were prepared with primers for approximately 80 Wnt pathway genes in 96-well PCR plates. Cdnas from 18G7H6a3 or MOPC (control) treated tumors were pooled and QPCR was performed in Wnt plates. The data for each plate was normalized to the corresponding GAPDH and the abundance of each gene was measured using the 2^ delta Ct equation. To measure fold difference, data in each 18G7H6a 3-treated tumor was divided by the corresponding value from the MOPC-treated group. Values greater than 1 or less than 1 indicate up-or down-regulation of the 18G7H6a3 treatment group, respectively. Preliminary assessment of the number of up-or down-regulated genes showed that in both tumor models (CT1 and CT3), the down-regulated genes were more than the up-regulated genes, indicating that 18G7H6A3 had an inhibitory effect on gene expression. Detailed analysis identified several differentially expressed genes, including FZDB, FZD7, WNT7B, FBXW11, FZD1, DVL1, CSNK2a1, and CTNNB 1.

in cervical cancer, there may be a close association between LGR5 expression and CTNNB1 in other studies, LGR5 overexpression (using LGR5 recombinant vector) or downregulation (using shRNA) resulted in upregulation or downregulation, respectively, of CTNNB1 (Chen Q, Cao HZ, Zheng ps.2014.oncotarget 5: 9092-105). additionally, analysis of immunohistochemical patches from cervical cancer patients showed a significant correlation between LGR5 and CTNNB1 expression in this study, expression of CTNNB1 was further studied using QPCR (to measure transcript levels) and western blot (to assess protein expression) using human specific primers, investigating ctb 1 expression in pancreatic and CRC tumors, nnr 5 expression similarly as explained in example 45, increased CTNNB1 expression with SOC treatment, and combined expression of 18G7H6a 2 and CRC tumors with csa 638 as a combined inhibition of CTNNB 638 and Wnt 19 β expression of protein expression in Wnt pathway 18-19, showed that inhibition of CTNNB expression in these tumors was reduced by QPCR 2-18 β -19, 8, and 18 β -c 3- β -expression as a combined inhibition of protein kinase inhibition by QPCR 2-19, 2- β -expression in this study, whereby inhibition of protein-19 inhibition of the expression of the Wnt pathway.

in a series of CRC, pancreatic and breast tumors, the quantification of other components of the Wnt pathway, including p- β -catenin, GSK-3 β (total as well as phosphorylated) and lrp 6. western blot data showed that Wnt pathway signaling was significantly inhibited in ASPC1 and PANC1 tumors, but also revealed some trends in other models favoring Wnt pathway down-regulation.bmcrc 086 tumors that were not responsive to 18G7H6A3 treatment were also negative for LGR5 expression and components of the Wnt signaling pathway, further supporting the conclusion that the mechanism of action of 18G7H6A3 is specifically targeting LGR5 and inhibiting Wnt signaling.

Expression of Wnt pathway genes in pancreatic tumors including ASPC1, PANC1, and JH109 were studied. Based on in vivo data, there was a difference in tumor volume between the 18G7H6 A3-treated tumor and the PBS-treated tumor in PANC1 and ASPC 1. In contrast, JH109 tumors did not respond to standard treatment regimens with 18G7H6a3 single agent or SOC chemotherapy combination. Differences in Wnt gene expression in responsive cells (PANC1 and ASPC1) and non-responsive cells (JH109) were studied. In the combination treatment group, Wnt6, FZD8, FOSL1, Wnt11, NFATC and FZD5 were all down-regulated in ASPC1 and PANC1 combination treated tumors and up-regulated in JH109 tumors. In pancreatic and CRC data, genes including WNT11, WNT6, FRZB and PRICKEL were all down-regulated in PANC1, ASPC1, CT1 and CT3 cells, but not in JH109 cells.

Gene tree analysis identified potential genes that were co-regulated in pancreatic tumors treated with 18G7H6a3, including Wnt11, FRAT1, LEF1, GSK3B, FZD8, and LRP 6. Analysis of differentially expressed transcripts in each treatment also identified genes that were up/down regulated by more than 2-fold in pancreatic tumors (fig. 17). 18G7H6a3 treatment some genes such as Wnt7A were common in all tumors relative to control-treated tumors.

Examples 41-18G7H6A3 inhibition of transcription in CT1 tumors

The expression of the gene targeted by 18G7H6a3 was studied in both early and late tumorigenesis. Mice were implanted with CT1 and tumors were harvested from the control, 18G7H6A3, FOLFIRI, or combined group on day 3, day 10, and day 17. Total RNA was harvested and prepared from each tumor on day 3 for gene array hybridization using Illumina human chips. A global analysis of differentially expressed genes (greater than 1.5 or 2 fold, p <0.05) showed that there were more genes downregulated than those upregulated in tumors treated with 18G7H6a3 (either as single agent or in combination with FOLFIRI). This suggests that 18G7H6a3 treatment may have a stronger inhibitory effect on the whole cell transcription machinery. PCA (principal component analysis) also showed that 18G7H6a3 was close to overall gene expression in control-treated tumors. However, when 18G7H6a3 was added to FOLFIRI (i.e., the combined group), there was a clear difference in the combined group relative to FOLFIRI, indicating that targeting LGR5 may significantly alter gene expression in FOLFIRI-treated tumors.

Analysis of the differentially expressed genes in 18G7H6A3 versus vehicle identified several tumor promoters that were down-regulated in 18G7H6A3 treated tumors, such as ANGPT2, AKAP12 and ADM, and several tumor suppressors that were up-regulated in 18G7H6A3 treated tumors, such as DAB1, MIR655, NKX1-2 (fig. 18). In contrast, FOLFIRI treatment appeared to up-regulate tumor promoters (FBN2, HKDC1, ABCB1, FGF2), as well as some tumor suppressors such as TRIB3, ATF3, and TIMP3 (fig. 19). The combination of FOLFIRI and 18G7H6a3 resulted in down-regulation of more tumor promoters such as ALDOC, CDH5, ITGA2, and also in up-regulation of more tumor suppressors such as ZBTB11, ITPKA, PSMC3IP and BAK1 (fig. 20).

Example 42-treatment of 18G7H6A3 in an in situ model of pancreatic patient-derived xenografts was significantly reduced Human CTC in peripheral blood

To investigate the role of 18G7H6a3 in inhibiting primary tumor growth and metastasis, LGR5 expression was examined in a series of pancreatic patient-derived xenograft samples and PANC1424 cells as well as PANC1427 cells.

Tumor samples were implanted subcutaneously into NOD/SCID (non-obese diabetic severe combined immunodeficiency) mice and subsequently into mice destined for in vivo useIn the pancreas of the studied receptors. Tumor volume was measured ultrasonically weekly and will have a value of-100 mm³Tumor mice were included in efficacy studies and treated with the following reagents: 1-MOPC isoform (15mg/kg, twice weekly; ip); 2-18G7H6A3(15mg/kg twice weekly; ip); 3-SOC (Gemzar 50 mg/kg; ip, twice weekly; and Abraxane30mg/kg, iv, twice weekly); 4-18G 7H6a3 and SOC combination at the doses described above. At the end of the study, peripheral blood was collected from each tumor-bearing mouse for CTC (using flow cytometry) and circulating DNA assessment. For flow cytometry, blood samples were treated using RBC lysis buffer (ACK buffer, Life Tech, CA) using the manufacturer's protocol and stained with human HLA-FITC (eBiosciences, CA) and human LGR5-AF647(BD Pharmingen, CA) for 30 minutes at 4 ℃. Cells were washed twice with staining buffer (PBS-FBS 3%) and 7AAD (7-amino actinomycin), then harvested in a laboratory FACScalibur instrument and data analyzed using FCS Express software (De Novo, CA).

LGR5 was expressed in xenograft samples from different pancreatic patient sources. Human CTCs were detected in peripheral blood. Although the percentage of HLA + cells was not significantly altered in MOPC relative to 18G7H6A3, the percentage of circulating HLA + LGR5+ cells was significantly reduced in 18G7H6A3 treated mice (fig. 21).

The percentage of HLA + cells in chemotherapy-treated mice did not change significantly relative to combination-treated mice, however, the combination of 18G7H6a3 with SOC almost completely ablated HLA + LGR5+ cells in a parallel (concurrent) and subtractive (debulk) setting (fig. 22A and 22B). Treatment of 18G7H6a3 (either as a single agent or in combination with SOC) significantly reduced human CTCs in peripheral blood in a pancreatic patient-derived xenograft orthotopic model.

Example 43 LGR5 expression in other models

LGR5 expression was studied in skin samples from Cynomolgus macaques (Cynos) using flow cytometry and RNAscope. Skin samples from cynomolgus macaques were treated with vehicle or different doses of 18G7H6a3(G2:10 mg/kg; G3:50 mg/kg; and G4:150mg/kg) on days 0, 7, 14 and 21. At the end of the study, skin samples were placed in DMEM supplemented with antibiotics (penicillin and streptomycin) and antifungal solutions (anti-100X, Life Technologies, CA). Skin samples were digested with a mixture of collagenase and thermolysin (Liberase, roche inc, CA). After incubation overnight with Liberase and mechanical disruption, skin progenitor cells were isolated (progenitor, SP). SP was stained with rat anti-human LGR5(AF647, BD Pharmingen, CA) and analyzed in a laboratory calibur instrument. Data analysis using FCS Express (Denovo Software, CA) showed that LGR5 was detectable in cynomolgus monkey SP, however, there was no significant difference in the frequency of LGR5 between 18G7H6A3 (at different doses) and the vehicle treated group. Using RNAscope, LGR5 was detectable in skin areas, particularly in hair follicles, and to a much lesser extent LGR5 in skin endothelial cells. There was no significant difference in LGR5 positive regions in the vehicle-treated samples relative to the 18G7H6a 3-treated samples.

Gene expression of peripheral blood mononuclear cells isolated from cynomolgus macaques was studied. Total RNA was extracted using Qiagen RNeasy kit and cDNA was synthesized using Superscript cDNA synthesis kit (Life Technologies, CA). The cDNAs from each treatment were pooled and added to RT²Sybergreen qPCR master mix (SAbiosciences, MA). The final mix was added to each well of a 96-well plate containing cynomolgus QPCR primers for chemokines or inflammatory cytokines. PCR thermal profiles include: 95 ℃, 10 minutes, and 40 cycles of 95 ℃, 15 seconds, and 60 ℃, 1 minute before entering the melting curve phase. The data (Ct values) in each plate were normalized by subtracting from the corresponding GAPDH, and the abundance of each transcript was calculated using the 2^ DCT equation. Analysis of the number of transcripts differentially expressed (greater than 2-fold) between the 18G7H6a3 group and the vehicle-treated group showed that, consistent with the gene array data, there were more downregulated genes than upregulated genes. As the dose increases, there are fewer up-regulated genes and more down-regulated genes. It is composed ofAfter the final dose of 18G7H6a3, the G4 recovery (G4R) group of cynomolgus monkeys that did not receive any treatment for 4 weeks showed nearly similar numbers of up-and down-regulated genes. Detailed analysis determined differentially expressed genes (CCL11, IL3, SPP1, CCL13, CXCL6 and TNFRSF11b) whose expression is negatively correlated with the dose of 18G7H6a3, i.e., highest in 10mg/kg and lowest in 150 mg/kg.

Genes that are typically down-regulated between treatments include CCL1, IFN γ, CCR8, IL2, IL3, and IL4, some of which are enriched in M1 or M2 macrophages.

Example 44-humanized anti-LGR5 antibody inhibits Small cell Lung cancer tumor growth in vivo

Patient derived small cell lung cancer xenograft models. Dissociated tumor cells from BLG293 tumors were implanted subcutaneously in cb.17scid mice in artificial basement membrane and tumor size and body weight were monitored twice weekly. When the tumor reaches 130mm³At mean, mice were randomly assigned. Mice were treated with PBS, antibody control MOPC, or 18G7H6a 3. Mice were dosed twice weekly (BIW) at 15 mg/kg. All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance, until completion.

18G7H6a3 showed significant anti-tumor activity compared to PBS (24.9% tumor growth inhibition) and MOPC antibody (24.7% tumor growth inhibition) controls.

Examples 45-18G7H6A3 increase survival of mice that have subtracted pancreatic tumor recurrence following chemotherapy

Panc1427(UCSD1427) tumors were completely depleted (regressed) by treatment with chemotherapy (gemcitabine/Abraxane) and 18G7H6a 3. When the tumor was regressed, the chemotherapy was removed and the mice were treated with 18G7H6a3 or untreated. Animals treated with 18G7H6a3 were significantly healthier compared to control animals, of which several mice had to be euthanized due to severe health observations such as lameness or weight loss. On day 150, mice treated with 18G7H6A3 and chemotherapy survived relative to 4/8 mice treated with chemotherapy alone. Fig. 23 summarizes these results.

Example 46 administration of 18G7H6A3 to patients

Phase I dose escalation studies of BNC101 (a humanized monoclonal antibody against LGR5) were performed in metastatic colorectal cancer patients as described below. Name of the finished product: BNC101Solution for infusion (BNC101Solution for infusion). Name of active ingredient: BNC101, 18G7H6a3, ET101, LGR5 antibodies.

Purpose of study

To determine the Maximum Tolerated Dose (MTD), recommended phase II dose (RP2D), safety, tolerability, and Pharmacokinetic (PK) profile of BNC101 for patients with metastatic colorectal cancer administered intravenously. The main objective was to determine the MTD of BNC101 in metastatic colorectal cancer patients as a single agent and in combination with chemotherapy. A second objective is to determine RP2D in patients with metastatic colorectal cancer for BNC101 as a single agent and in combination with chemotherapy as follows. BNC101 is evaluated for safety and tolerability [ Adverse Events (AE), dose omission (omision), or delay ]. The immunogenicity of BNCs 101 (antibody production against BNCs 101) was assessed. The Pharmacokinetics (PK) (half-life, delivery volume and clearance) of BNC101 as single agent and in combination with chemotherapy were determined. Preliminary assessment of Overall Response Rate (ORR), Progression Free Survival (PFS) and Overall Survival (OS) of metastatic colorectal cancer patients treated with BNC 101. An exemplary objective is to assess changes in disease-associated biomarkers (CEA). Biomarkers for activity [ pharmacodynamics, e.g. Circulating Tumor Cells (CTC), LGR5+ cells, circulating tumor DNA ] were assessed.

Safety endpoint

Treatment emergencies (clinical and experimental data) were evaluated. Dose discontinuation and discontinuation was evaluated.

Selection criteria for key patients

1. Sign the written informed consent. 2. Age (age)>Age 18. 3. The eastern cooperative tumor group (ECOG) physical strength status score is 0-1. 4. Histologically or cytologically validated colorectal cancer patients who have failed at least 2 lines of chemotherapy (single treatment cohort) or at least 1 line of chemotherapy (combination treatment cohort) for metastatic disease and from the point of view of physician and patient, it is not unreasonable to attempt experimental treatment. The adjuvants FOLFOX (folinic acid; fluorouracil (5-FU); and oxaliplatin) within 6 months were considered a treatment. After treatment 1, the maintenance strategy was not considered as an additional treatment. 5. The patient must have an accessible, easy biopsy without placing the patient or his treatment in a dangerous tumor lesion. The monotherapy escalation cohort 3 and subsequent patients, monotherapy expansion cohort and all combination therapy patients agree and are willing to provide 2 series of tumor lesion biopsies (possibly, preferably at least 2 fresh nuclei/open cells (punches)). The biopsy may be from liver metastases, replacing the primary tumor. The presence of tumor tissue in the fresh biopsy is verified by a trained pathologist using appropriate provisional histological or cytological procedures. 6. Measurable disease according to the solid tumor efficacy evaluation criteria (RECIST), version 1.1. 7. There is no known brain metastasis. 8. The life expectancy is at least more than or equal to 12 weeks. 9. Normal organ and bone marrow function: a. absolute neutrophil count without growth factor support within 14 days prior to inclusion>1,500/mL. b. Platelets in transfusion-free conditions within 14 days prior to inclusion>100,000/mL. c. Hemoglobin > 9.0 g/dL-patients can be transfused or receive erythropoiesis therapy to meet this criteria. d. Total bilirubin<1.5x convention (institutional) Upper Limit of Normal (ULN) (for subjects with Gilbert syndrome,<2x ULN). e. The serum albumin is more than or equal to 3 g/dL. 10. Aspartate Aminotransferase (AST) (serum glutamic-oxaloacetic transaminase, SGOT) and alanine Aminotransferase (ALT) (serum glutamic-pyruvic transaminase, SGPT)<2.5x convention ULN (for subjects with liver involvement)<5x convention ULN, but cannot be associated with increased bilirubin). 11. Prothrombin Time (PT) and activation site for patients receiving biopsyThe fractional thrombin time (APTT)/International Standard ratio (INR) falls within the normal limits (+ -15%). 12. Creatinine<1.5x customary ULN or creatinine clearance for subjects with creatinine levels above the customary normal value>60mL/min/1.73m². 13. Women and men with fertility potential have adequate contraceptive measures.

Administration and scheduling

The incrementing occurs in two separate sets of queues in an interleaved fashion: single agent BNC101 dose escalation occurred in dose escalation in the cohort of combination chemotherapy. In the latter, BNC101 is combined with FOLFIRI to increase the dosage. Until a monotherapy cohort of RP2D has been determined, the combination chemotherapy cohort does not begin treatment. The increase in the combination chemotherapy cohort began at a level lower than the single agent RP2D of the single chemotherapy cohort. Although BNC101 in combination with chemotherapy is not expected to exhibit additional toxicity, two additional decreasing levels below the initial dose may be achieved in case they are needed.

BNC101 monotherapy

Standard phase 3+3I study design was used. The starting dose is 1/30 of the highest no significant damaging effect level (NOAEL) dose in animals (2.5 mg/kg in humans), which is calculated to account for species differences in receptor binding. Subsequent BNC101 dosage levels were 5, 10, and 15 mg/kg.

The dosing schedule was weekly (q1 w).

Cycle duration was 4 weeks (28 days) (4 weeks infusion) with no rest weeks in between cycles.

Dose escalation was performed to determine MTD. No increments or decrements of the dose are allowed within the dosing queue. In all cases, BNC101 should be administered in the morning to be able to prepare and transport blood samples in time for transformation studies conducted on the same day.

Dose escalation was started with a cohort of 3 patients at a dose of 2.5mg/kg (1/30 of the highest NOAEL dose in the animals). If at the end of 28 days (4 BNC101 administrations) no CTCAE grade ≧ 2AE attributed to the study drug was observed, the cohort of the second 3 patients was treated at the next dose level (5 mg/kg). Further dose escalation (starting at 10mg/kg followed by 15mg/kg) was continued in the cohort of 3 patients.

If 1/3 of the subjects in the 3 subject cohort presented dose-limiting buoyancy (DLT), the dose was increased to 6 subjects. If 2 or more subjects develop a DLT, no further dose at that level will be administered to the other subjects, and 3 additional subjects will be added to the previous dose cohort, unless 6 subjects have been treated at that dose level.

From the time of the first dose until 28 days, the subject was evaluated for DLT. Dose escalation for newly enrolled subjects, when appropriate, occurs after all subjects in the cohort have completed their 28-day DLT assessment. Subjects with stable disease or response at day 56 (2 cycles) and after day 56 would be allowed to continue to receive weekly BNC101 dosing until disease progression.

If no DLT is reported at the highest BNC101 dose combination tested, the patient cohort showing PK characteristics with BNC101 exposure comparable to the maximal exposure demonstrated in laboratory animals is RP2D for combination chemotherapy.

At least 9 additional subjects will be included further after <2 of 6 subjects had grade 3 (excluding grade 3 infusion response resolution within 24 hours) or grade 4 AE (DLT) at the highest dose level, or had achieved adequate PK exposure in the absence of such toxicity.

BNC101 in combination with FOLFIRI

After completing BNC101 monotherapy dose escalation, achieving the stated safety and RP2D, treatment of metastatic colorectal cancer patients can be initiated on their combined chemotherapy cohort with BNC101 at1 dose level below RP2D established in monotherapy. These queues each contain 3 objects. Increments are made according to the same rules as used for the monotherapy cohort. If the initial BNC101 dose was shown to exhibit DLT in 2 of the maximum of 6 patients, 2 additional descending cohorts would be provided according to the 3+3 rule to identify RP2D for combination chemotherapy.

If no DLT is reported at the highest BNC101 dose combination tested, the patient cohort showing PK characteristics with BNC101 exposure comparable to the maximum exposure demonstrated in laboratory animals is RP2D combined with chemotherapy.

At least 9 additional subjects will be included further after <2 of 6 subjects had grade 3 (excluding grade 3 infusion response resolution within 24 hours) or grade 4 AE (DLT) at the highest dose level, or had achieved adequate PK exposure in the absence of such toxicity.

FOLFIRI component: irinotecan (IRI) -initial dose 180mg/m²(90 minutes on day 1); folinic acid (LV) -initial dose 400mg/m²(120 minutes of administration, on day 1, with IRI); 5-FU bolus-initial dose 400mg/m²(post-LV dosing on day 1); 5-FU infusion-initial dose 2400mg/m²(48 hours of administration, starting on day 1). The FOLFIRI cycle is repeated every 14 days.

DLT definition

DLT is defined as any of the following occurring at cycle 1 (day 0-day 28) of any given incremental queue: grade 3 or4 non-hematologic toxicity (including allergic reactions) using NCI CTCAE v 4.0; grade 3 nausea or grade 4 vomiting or grade 3 diarrhea for a duration of greater than 48 hours, which occurs despite appropriate treatment; grade 4 thrombocytopenia of any duration; and grade 4 non-concurrent neutropenia (i.e., no cold or infection), for any duration in the monotherapy cohort, or for >7 days in the combination chemotherapy cohort. Grade 4 febrile neutropenia requiring hospitalization, and any grade 3 hematologic toxicity requiring treatment delay for more than 3 weeks and prolongation of QTc interval to ≧ 500msec or an increase of 60msec from baseline mean QTc interval. Any other drug-related grade 3 non-hematologic adverse events (including allergic reactions), except hyperlipidemia in subjects not receiving maximum medical management or electrolyte abnormalities that can be controlled with supplements. All AEs were considered potentially relevant to treatment unless there was a clear correlation between observed toxicity and disease Progression (PD). Adverse events meeting these criteria would not be considered for the purpose of reporting DLT or determining MTD.

Duration of treatment

Patients were treated until PD, intolerable toxicity, withdrawal of informed consent, or sponsor termination of the study, whichever occurred first.

Sample size

Up to about 54 patients were tested in this study to determine the toxicity profile of BNC101, DLT and MTD, and/or RP2D in monotherapy and in combination chemotherapy. It is expected that for each dose level cohort, 1-6 evaluable patients will provide sufficient data to evaluate the toxicity and PK profile of BNC 101. Once RP2D was identified, expansion of these cohorts occurred in both monotherapy and combination chemotherapy groups (to a minimum of 9 additional patients). If no DLT is reported at the highest BNC101 dose tested, the cohort of patients showing PK profiles with BNC101 exposure comparable to the maximum exposure demonstrated in laboratory animals is RP 2D.

Frequency of follow-up visits

Every 7(± 3) days for each week of a 4 week cycle. After termination of treatment, survival follow-up information and subsequent anti-cancer treatment was collected every 3 months until death, loss of follow-up, withdrawal of patient informed episodes, or sponsor termination of study.

Security assessment

From day 0 to day 28 (cycle 1), DLT of subjects was evaluated. Adverse events were reported up to 30 days after the last dose. Adverse events were reported continuously. Adverse events occurring between day 28 and day 30 after the last dose were reported, but were not integrated into the determination of MTD. Physical examination was sorted weekly for performance status and performed at baseline, at the beginning of each cycle (e.g., every 4 weeks), at study End (EOS) follow-up, and at resolution of any AEs.

Electrocardiogram (ECG) detection

ECG for all 12 leads were obtained in triplicate and separated by at least 5 minutes. Baseline (within 14 days prior to the first dose) ECGs were closely obtained on days 1 and 15 of cycle 1, and pre-dose ECGs were obtained on days 8 and 22. On day 1 of cycle 2 and subsequent cycles, a pre-dose ECG was obtained. EOS ECGs must be collected. The ECG will be sent to the local laboratory for evaluation.

Response assessment

Computed Tomography (CT) was performed at baseline and every 8 weeks. The first response was evaluated on day 56 (2 cycles). The radiologic study of the anti-tumor response was repeated at EOS follow-up if not performed within the previous 28 days. A telephone follow-up or record check is performed on a monthly basis for a period of 6 months and every 3 months thereafter for a period of 18 months (24 months total).

Pharmacokinetic assessment

Pharmacokinetic samples were taken densely on day 1 and day 15 and sparsely on day 8 and day 22 in cycle 1. Samples were taken sparsely on day 1 of cycle 2 and subsequent cycles. Blood samples were taken before BNC101 administration (before infusion started), during BNC101 infusion, and after BNC101 infusion (after infusion ended).

Cellular and molecular biomarker assessment

At each week of cycle 1, day 1 of each subsequent cycle, and at the end of treatment, baseline blood was collected from the patient to measure CTC and levels of biomarkers (including but not limited to LGR5) that are indicators of pharmacodynamic effects. The patient also had 2 matched skin biopsies (2 fresh nuclei/punch per biopsy) at baseline and cycle 1, day 22. Additional hair samples of the patient may be collected (including collecting hair follicles).

Tumor focus biopsy

For monotherapy increment cohorts 1 and 2, no biopsy is required. For the monotherapy increment cohort 3 and subsequent cohorts, and the monotherapy expansion cohort, matching biopsies (baseline and day 22) were mandatory. For all combination treated patients, matched biopsies were mandatory. Where possible, a minimum of 2 fresh nuclei per biopsy per opening is preferred. The biopsy may be from liver metastases, replacing the primary tumor. The presence of tumor tissue in the fresh biopsy is verified by a trained pathologist using appropriate provisional histological or cytological procedures. All samples were de-identified. The identity of the individual is not determined by the researcher or sponsor. The password protected data stored in the central reference laboratory is not revealed to the patient or to the physician treating the patient. Patient information is strictly used to anonymously report study data. Such study data would not be placed in a patient chart or accessed by a physician and would not nor be available for diagnostic or therapeutic purposes.

Immunogenicity assessment

The presence of anti-BNC 101 antibody was detected at baseline, before each dose, at the end of treatment, and every 4 weeks after termination of treatment for a period of 12 weeks. Due to the different characteristics and expected evolution of monotherapy versus patients in the combined treatment cohort, different numbers of samples can be obtained in each cohort.

As used herein, the term "comprising" is synonymous with "including", "containing", or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The above specification discloses several methods and materials of the present invention. The present invention allows for modifications in the methods and materials, as well as changes in the manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from consideration of the present disclosure or practice of the invention disclosed herein. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed herein, but that the invention will include all modifications and alterations falling within the true scope and spirit of the invention.

All references cited herein, including but not limited to published and unpublished applications, patents, and literature citations, are hereby incorporated by reference in their entirety and are hereby incorporated as part of the present application. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Sequence listing

<110> ecology Co., Ltd (Bionomics, Inc.)

Kristesfer L Riyes (Christopher L. Reyes)

Peter Zhu (Peter Chu)

Elizabeth Doolin (Elizabeth Doolin)

Jose Iglesias (Jose Iglesias)

<120> administration of anti-LGR5 monoclonal antibody

<130>BIONO.014WO

<150>62/311631

<151>2016-03-22

<160>49

<170>FastSEQ for Windows Version 4.0

<210>1

<211>8

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 heavy chain CDR1 amino acids

<400>1

Gly Tyr Thr Phe Ser Gly Tyr Trp

1 5

<210>2

<211>8

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 heavy chain CDR2 amino acids

<400>2

Ile Leu Pro Gly Ser Asp Ser Thr

1 5

<210>3

<211>11

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 heavy chain CDR3 amino acids

<400>3

Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr

1 5 10

<210>4

<211>10

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 light chain CDR1 amino acids

<400>4

Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe

1 5 10

<210>5

<211>3

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 light chain CDR2 amino acids

<400>5

Leu Thr Ser

1

<210>6

<211>10

<212>PRT

<213> Mouse (Mouse)

<220>

<221>

<222>

<223>18G7.1 light chain CDR3 amino acids

<400>6

Met Gln Gln Asn Asn Glu Asp Pro Arg Thr

1 5 10

<210>7

<211>354

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain DNA

<400>7

gaggtgcagc tggtgcagag cggagccgag gtgaagaagc ccggcgagag cctgaggatc 60

agctgcaagg gcagcggcta cagcttcacc gcgtactgga tcgagtgggt gaggcaggct 120

cccggcaagg gcctggagtg gatcggcgag atcctgcccg gcagcgacag caccaactac 180

aacgagaagt tcaagggcca cgtgaccatc agcgccgaca agagcatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggccagcgac accgccgtgt actactgcgc ccgcagcggc 300

tactacggca gcagccagta ctggggccag ggcaccctgg tgaccgtgag cagc 354

<210>8

<211>333

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain DNA

<400>8

gacatcgtgc tgacccagag ccccgccagc ctggccgtga gccccggcca gagggccacc 60

atcacctgcc gcgccagcga gagcgtggac agctacggca acagcttcat gcactggtat 120

cagcagaagc ccggccagcc ccccaagctg ctgatctacc tgaccagcaa cctggagtcc 180

ggcgtgcccg acaggttcag cggcagcggc agcggcaccg acttcaccct gaccatcaac 240

cccgtggagg ccaacgacgc cgccacctac tactgccagc agaacgccga ggaccccagg 300

accttcggcg gcggcaccaa gctggagatc aag 333

<210>9

<211>466

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain amino acids

<400>9

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

35 40 45

Thr Ala Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

85 90 95

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly

465

<210>10

<211>238

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain amino acids

<400>10

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Pro Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Asn Ala Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>11

<211>1423

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain DNA

<400>11

aagcttgccg ccaccatgga atggtcctgg gtgttcctgt tcttcctgtc cgtgaccacc 60

ggcgtgcact ccgaagtgca gctggtgcag tctggcgccg aagtgaagaa gcctggcgag 120

tccctgcgga tctcctgcaa gggctccggc tactccttca ccgcctactg gattgagtgg 180

gtgcgacagg cccctggcaa gggcctggaa tggatcggag agatcctgcc cggctccgac 240

tccaccaact acaacgagaa gttcaagggc cacgtgacca tctccgccga caagtccatc 300

tctaccgcct acctgcagtg gtcctccctg aaggcctctg acaccgccgt gtactactgc 360

gccagatccg gcctgtacgg ctcctctcag tattggggcc agggcaccct cgtgaccgtg 420

tcctctgctt ctaccaaggg cccaagcgtg ttccccctgg cccccagcag caagagcacc 480

agcggcggca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 540

gtgtcctgga acagcggagc cctgacctcc ggcgtgcaca ccttccccgc cgtgctgcag 600

agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc 660

cagacctaca tctgtaacgt gaaccacaag cccagcaaca ccaaggtgga caagaaggtg 720

gagcccaaga gctgtgacaa gacccacacc tgccccccct gcccagcccc cgagctgctg 780

ggcggaccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 840

acccccgagg tgacctgtgt ggtggtggac gtgtcccacg aggacccaga ggtgaagttc 900

aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 960

tacaacagca cctacagggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 1020

ggcaaggagt acaagtgtaa ggtgtccaac aaggccctgc cagccccaat cgaaaagacc 1080

atcagcaagg ccaagggcca gccaagagag ccccaggtgt acaccctgcc acccagcagg 1140

gacgagctga ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctacccaagc 1200

gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1260

ccagtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 1320

agatggcagc agggcaacgt gttcagctgc tccgtgatgc acgaggccct gcacaaccac 1380

tacacccaga agagcctgag cctgtcccca ggctgatgaa ttc 1423

<210>12

<211>739

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain DNA

<400>12

aagcttgccg ccaccatgtc cgtgcctacc caggtgctgg gactgctgct gctgtggctg 60

accgacgcca gatgcgacat cgtgctgacc cagagccctg cctctctggc tgtgtctcct 120

ggccagaggg ccaccatcac ctgtagagcc tccgagtccg tggactccta cggcaactcc 180

ttcatgcact ggtatcagca gaagcccggc cagcccccca agctgctgat ctacctgacc 240

tccaacctgg aatccggcgt gcccgacaga ttctccggct ctggctctgg caccgacttc 300

accctgacca tcaaccccgt ggaagccaac gacgccgcca cctactactg ccagcagaac 360

gccgaggacc ccagaacctt tggcggaggc accaagctgg aaatcaagcg tacggtggcc 420

gctcccagcg tgttcatctt ccccccaagc gacgagcagc tgaagagcgg caccgccagc 480

gtggtgtgtc tgctgaacaa cttctacccc agggaggcca aggtgcagtg gaaggtggac 540

aacgccctgc agagcggcaa cagccaggag agcgtcaccg agcaggacag caaggactcc 600

acctacagcc tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg 660

tacgcctgtg aggtgaccca ccagggcctg tccagccccg tgaccaagag cttcaacagg 720

ggcgagtgct gatgaattc 739

<210>13

<211>466

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain amino acids

<400>13

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

35 40 45

Thr Ala Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

85 90 95

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Leu Tyr Gly Ser Ser Gln Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly

465

<210>14

<211>238

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain amino acids

<400>14

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Pro Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Asn Ala Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>15

<211>354

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7Ch heavy chain DNA

<400>15

caggttcagc tgcagcagtc tggagctgag ctggtgaagc ctggggcctc agtgaagata 60

tcctgcaagg ctactggcta cacattcagt ggctactgga tagagtgggt aaagcagagg 120

cctggacatg gccttgagtg gattggagag attttgcctg gaagtgatag tactaactac 180

aatgagaagt tcaagggcaa ggccacattc actgcagatacatcctccaa cacagtctac 240

atgcaattca gcagcctgac atctgaggac tctgccgtct attactgtgc aagatcgggt 300

tactacggta gtagtcagta ctggggccaa ggcaccactc tcacagtctc ctca 354

<210>16

<211>334

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7Ch light chain DNA

<400>16

aacattgtgc tgacccaatc tcctgcttct ttggctgtgt ctctagggca gagggccacc 60

atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120

cagcagaaac caggacagcc acccaaactc ctcatctatc ttacatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattgat 240

cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatcctcgg 300

acgttcggtg gaggcaccaa gctggaaatc aaac 334

<210>17

<211>466

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7Ch heavy chain amino acids

<400>17

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe

35 40 45

Ser Gly Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn

85 90 95

Thr Val Tyr Met Gln Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly

465

<210>18

<211>238

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7ch light chain amino acids

<400>18

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 1015

Asp Ala Arg Cys Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Asn Asn Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>19

<211>137

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain variable domain amino acids

<400>19

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

35 40 45

Thr Ala Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

85 90 95

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210>20

<211>411

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain variable domain DNA

<400>20

atggaatggt cctgggtgtt cctgttcttc ctgtccgtga ccaccggcgt gcactccgaa 60

gtgcagctgg tgcagtctgg cgccgaagtg aagaagcctg gcgagtccct gcggatctcc 120

tgcaagggct ccggctactc cttcaccgcc tactggattg agtgggtgcg acaggcccct 180

ggcaagggcc tggaatggat cggagagatc ctgcccggct ccgactccac caactacaac 240

gagaagttca agggccacgt gaccatctcc gccgacaagt ccatctctac cgcctacctg 300

cagtggtcct ccctgaaggc ctctgacacc gccgtgtact actgcgccag atccggcctg 360

tacggctcct ctcagtattg gggccagggc accctcgtga ccgtgtcctc t 411

<210>21

<211>131

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain variable domain

<400>21

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Pro Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Asn Ala Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210>22

<211>393

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain variable domain DNA

<400>22

atgtccgtgc ctacccaggt gctgggactg ctgctgctgt ggctgaccga cgccagatgc 60

gacatcgtgc tgacccagag ccctgcctct ctggctgtgt ctcctggcca gagggccacc 120

atcacctgta gagcctccga gtccgtggac tcctacggca actccttcat gcactggtat 180

cagcagaagc ccggccagcc ccccaagctg ctgatctacc tgacctccaa cctggaatcc 240

ggcgtgcccg acagattctc cggctctggc tctggcaccg acttcaccct gaccatcaac 300

cccgtggaag ccaacgacgc cgccacctac tactgccagc agaacgccga ggaccccaga 360

acctttggcg gaggcaccaa gctggaaatc aag 393

<210>23

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR1 amino acids

<400>23

Gly Tyr Ser Phe Thr Ala Tyr Trp

1 5

<210>24

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR1 DNA

<400>24

ggctactcct tcaccgccta ctgg 24

<210>25

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR2 amino acids

<400>25

Ile Leu Pro Gly Ser Asp Ser Thr

1 5

<210>26

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR2 DNA

<400>26

atcctgcccg gctccgactc cacc 24

<210>27

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR3 amino acids

<400>27

Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr

1 5 10

<210>28

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 heavy chain CDR3 DNA

<400>28

gccagatccg gcctgtacgg ctcctctcag tat 33

<210>29

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR1 amino acids

<400>29

Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe

1 5 10

<210>30

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR1 DNA

<400>30

gagtccgtgg actcctacgg caactccttc 30

<210>31

<211>3

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR2 amino acids

<400>31

Leu Thr Ser

1

<210>32

<211>9

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR2 DNA

<400>32

ctgacctcc 9

<210>33

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR3 amino acids

<400>33

Gln Gln Asn Ala Glu Asp Pro Arg Thr

1 5

<210>34

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A3 light chain CDR3 DNA

<400>34

cagcagaacg ccgaggaccc cagaacc 27

<210>35

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR1 amino acids

<400>35

Gly Tyr Ser Phe Thr Ala Tyr Trp

1 5

<210>36

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR1 DNA

<400>36

ggctactcct tcaccgccta ctgg 24

<210>37

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR2 amino acids

<400>37

Ile Leu Pro Gly Ser Asp Ser Thr

1 5

<210>38

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR2 DNA

<400>38

atcctgcccg gcagcgacag cacc 24

<210>39

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR3 amino acids

<400>39

Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr

1 5 10

<210>40

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain CDR3 DNA

<400>40

gcccgcagcg gctactacgg cagcagccag tac 33

<210>41

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR1 amino acids

<400>41

Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe

1 5 10

<210>42

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR1 DNA

<400>42

gagagcgtgg acagctacgg caacagcttc 30

<210>43

<211>3

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR2 amino acids

<400>43

Leu Thr Ser

1

<210>44

<211>9

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR2 DNA

<400>44

ctgaccagc 9

<210>45

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR3 amino acids

<400>45

Gln Gln Asn Ala Glu Asp Pro Arg Thr

1 5

<210>46

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain CDR3 DNA

<400>46

cagcagaacg ccgaggaccc caggacc 27

<210>47

<211>561

<212>PRT

<213> human (Homo Sapiens)

<400>47

Met Asp Thr Ser Arg Leu Gly Val Leu Leu Ser Leu Pro Val Leu Leu

1 5 10 15

Gln Leu Ala Thr Gly Gly Ser Ser Pro Arg Ser Gly Val Leu Leu Arg

20 25 30

Gly Cys Pro Thr His Cys His Cys Glu Pro Asp Gly Arg Met Leu Leu

35 40 45

Arg Val Asp Cys Ser Asp Leu Gly Leu Ser Glu Leu Pro Ser Asn Leu

50 55 60

Ser Val Phe Thr Ser Tyr Leu Asp Leu Ser Met Asn Asn Ile Ser Gln

65 70 75 80

Leu Leu Pro Asn Pro Leu Pro Ser Leu Arg Phe Leu Glu Glu Leu Arg

85 90 95

Leu Ala Gly Asn Ala Leu Thr Tyr Ile Pro Lys Gly Ala Phe Thr Gly

100 105 110

Leu Tyr Ser Leu Lys Val Leu Met Leu Gln Asn Asn Gln Leu Arg His

115 120 125

Val Pro Thr Glu Ala Leu Gln Asn Leu Arg Ser Leu Gln Ser Leu Arg

130 135 140

Leu Asp Ala Asn His Ile Ser Tyr Val Pro Pro Ser Cys Phe Ser Gly

145 150155 160

Leu His Ser Leu Arg His Leu Trp Leu Asp Asp Asn Ala Leu Thr Glu

165 170 175

Ile Pro Val Gln Ala Phe Arg Ser Leu Ser Ala Leu Gln Ala Met Thr

180 185 190

Leu Ala Leu Asn Lys Ile His His Ile Pro Asp Tyr Ala Phe Gly Asn

195 200 205

Leu Ser Ser Leu Val Val Leu His Leu His Asn Asn Arg Ile His Ser

210 215 220

Leu Gly Lys Lys Cys Phe Asp Gly Leu His Ser Leu Glu Thr Leu Asp

225 230 235 240

Leu Asn Tyr Asn Asn Leu Asp Glu Phe Pro Thr Ala Ile Arg Thr Leu

245 250 255

Ser Asn Leu Lys Glu Leu Gly Phe His Ser Asn Asn Ile Arg Ser Ile

260 265 270

Pro Glu Lys Ala Phe Val Gly Asn Pro Ser Leu Ile Thr Ile His Phe

275 280 285

Tyr Asp Asn Pro Ile Gln Phe Val Gly Arg Ser Ala Phe Gln His Leu

290 295 300

Pro Glu Leu Arg Thr Leu Thr Leu Asn Gly Ala Ser Gln Ile Thr Glu

305 310 315 320

Phe Pro Asp Leu Thr Gly Thr Ala Asn Leu Glu Ser Leu Thr Leu Thr

325 330 335

Gly Ala Gln Ile Ser Ser Leu Pro Gln Thr Val Cys Asn Gln Leu Pro

340 345 350

Asn Leu Gln Val Leu Asp Leu Ser Tyr Asn Leu Leu Glu Asp Leu Pro

355 360 365

Ser Phe Ser Val Cys Gln Lys Leu Gln Lys Ile Asp Leu Arg His Asn

370 375 380

Glu Ile Tyr Glu Ile Lys Val Asp Thr Phe Gln Gln Leu Leu Ser Leu

385 390 395 400

Arg Ser Leu Asn Leu Ala Trp Asn Lys Ile Ala Ile Ile His Pro Asn

405 410 415

Ala Phe Ser Thr Leu Pro Ser Leu Ile Lys Leu Asp Leu Ser Ser Asn

420 425 430

Leu Leu Ser Ser Phe ProIle Thr Gly Leu His Gly Leu Thr His Leu

435 440 445

Lys Leu Thr Gly Asn His Ala Leu Gln Ser Leu Ile Ser Ser Glu Asn

450 455 460

Phe Pro Glu Leu Lys Val Ile Glu Met Pro Tyr Ala Tyr Gln Cys Cys

465 470 475 480

Ala Phe Gly Val Cys Glu Asn Ala Tyr Lys Ile Ser Asn Gln Trp Asn

485 490 495

Lys Gly Asp Asn Ser Ser Met Asp Asp Leu His Lys Lys Asp Ala Gly

500 505 510

Met Phe Gln Ala Gln Asp Glu Arg Asp Leu Glu Asp Phe Leu Leu Asp

515 520 525

Phe Glu Glu Asp Leu Lys Ala Leu His Ser Val Gln Cys Ser Pro Ser

530 535 540

Pro Gly Pro Phe Lys Pro Cys Glu His Leu Leu Asp Gly Trp Leu Ile

545 550 555 560

Arg

<210>48

<211>137

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 heavy chain variable amino acids

<400>48

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

35 40 45

Thr Ala Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

85 90 95

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Ser Ser Gln Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210>49

<211>131

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223>18G7H6A1 light chain variable amino acids

<400>49

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Pro Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Asn Ala Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

Claims (28)

1. A method of treating a human subject having metastatic colorectal cancer, comprising administering to a subject in need thereof an effective amount of a humanized monoclonal antibody that specifically binds to leucine repeat rich G protein-coupled receptor 5(LGR5), wherein:

the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14;

the monoclonal antibody is administered weekly for at least 4 weeks;

the monoclonal antibody is administered intravenously; and is

The dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg.

2. The method of claim 1, wherein the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan.

3. The method of claim 2, wherein an initial dose of monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.

4. The method of claim 3, wherein: the initial dose of irinotecan is about 180mg/m²About 90 minutes of administration; the initial dose of folinic acid is about 400mg/m²(ii) is administered for about 120 minutes and concurrently with the initial dose of irinotecan; the initial dose of fluorouracil is about 400mg/m²(ii) administered after administration of the initial dose of folinic acid; and folinic acid, fluorouracil and irinotecan are administered every 14 days.

5. A method of treating a subject having cancer, comprising administering to a subject in need thereof an effective amount of a humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds leucine rich repeat G-protein coupled receptor 5(LGR5), wherein:

the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14;

the monoclonal antibody is administered weekly for at least 4 weeks;

the monoclonal antibody is administered intravenously; and is

The dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg.

6. The method of claim 5, wherein the monoclonal antibody is administered in combination with a chemotherapeutic agent.

7. The method of claim 6, wherein the chemotherapeutic agent is selected from the group consisting of folinic acid, fluorouracil, irinotecan, gemcitabine, and nanoparticulate albumin-bound paclitaxel (ABRAXANE).

8. The method of claim 7, wherein an initial dose of monoclonal antibody is administered prior to administration of the chemotherapeutic agent.

9. The method of claim 7, wherein the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan.

10. The method of claim 7, wherein an initial dose of monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.

11. The method of claim 7, wherein the initial dose of irinotecan is about 180mg/m²Administration was for about 90 minutes.

12. The method of claim 11, wherein the initial dose of folinic acid is about 400mg/m²Administered for about 120 minutes and concurrently with the initial dose of irinotecan.

13. The method of claim 12, wherein the initial dose of fluorouracil is about 400mg/m²Administered after administration of the initial dose of folinic acid.

14. The method of claim 13, wherein the folinic acid, fluorouracil, and irinotecan are administered every 14 days.

15. The method of any one of claims 1-14, wherein the monoclonal antibody is administered in combination with an additional therapeutic agent selected from bevacizumab, aflibercept, cetuximab, and panitumumab.

16. The method of any one of claims 5-14, wherein the cancer comprises a solid tumor.

17. The method of any one of claims 5-14, wherein the cancer is selected from colon cancer, colorectal cancer, pancreatic cancer, breast cancer, and lung cancer.

18. The method of any one of claims 5-14, wherein the cancer is selected from colon cancer with APC mutations, colon cancer with KRAS mutations, metastatic colorectal cancer, metastatic pancreatic cancer, triple negative breast cancer, and small cell lung cancer.

19. The method of any one of claims 5-14, wherein the cancer is metastatic colorectal cancer.

20. The method of any one of claims 1-14, wherein the subject has a characteristic selected from the group consisting of: prior to administration of the monoclonal antibody, prior chemotherapy for metastatic disease failed at least line 1 (line); no known brain metastases; has an expected life of 12 weeks or more; (ii) has an absolute neutrophil count of greater than about 1500 cells/mL without growth factor support within 14 days prior to administration of the monoclonal antibody; (ii) has a platelet count of greater than 100,000 platelets/mL under transfusion-free conditions within 14 days prior to administration of the monoclonal antibody; has a hemoglobin content of greater than or equal to 9.0 g/dL; and serum albumin having greater than or equal to 3 g/dL.

21. The method of any one of claims 1-14, wherein the subject is a mammal.

22. The method of any one of claims 1-14, wherein the subject is a human.

23. A container comprising a pharmaceutical composition comprising an administered dose of a humanized monoclonal antibody that specifically binds to leucine rich repeat G-protein coupled receptor 5(LGR5) and a suitable pharmaceutical carrier, wherein the administered dose of the monoclonal antibody is from about 2.5mg/kg to about 15 mg/kg.

24. The container of claim 23, wherein the pharmaceutical composition is suitable for intravenous administration.

25. A humanized monoclonal antibody that specifically binds to leucine rich repeat G protein-coupled receptor 5(LGR5), for use in the treatment of metastatic colorectal cancer, wherein:

the monoclonal antibody comprises a heavy chain comprising SEQ ID NO 13 and a light chain comprising SEQ ID NO 14;

the monoclonal antibody is administered weekly for at least 4 weeks;

the monoclonal antibody is administered intravenously; and is

The dosage of the monoclonal antibody is about 2.5mg/kg to about 15 mg/kg.

26. The humanized monoclonal antibody for treating metastatic colorectal cancer of claim 25, wherein the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan.

27. The humanized monoclonal antibody for treating metastatic colorectal cancer of claim 26, wherein an initial dose of the monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.

28. The humanized monoclonal antibody for treating metastatic colorectal cancer of claim 25, wherein: the initial dose of irinotecan is about 180mg/m²About 90 minutes of administration; the initial dose of folinic acid is about 400mg/m²(ii) is administered for about 120 minutes and concurrently with the initial dose of irinotecan; the initial dose of fluorouracil is about 400mg/m²(ii) administered after administration of the initial dose of folinic acid; and folinic acid, fluorouracil and irinotecan are administered every 14 days.