Anti-CEACAM5 / 6 antigen-binding molecule and method of treatment thereof

Abstract

The present invention relates generally to the field of antibody technology. Specifically, the present invention is directed to anti-CEACAM5 / 6 antigen-binding molecules and methods of treatment thereof.

Description

[Technical field]

[0001] The present invention relates generally to the field of antibody technology. Specifically, the present invention is directed to anti-CEACAM5 / 6 antigen-binding molecules and methods of treatment thereof. [Background technology]

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) belong to the carcinoembryonic antigen (CEA) family. CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)-anchored cell surface glycoproteins that are known to be highly expressed in a wide variety of cancers, including gastric cancer, breast cancer, pancreatic cancer, colon cancer, and non-small cell lung cancer (NSCL).

[0003] Post-translational glycosylation of CEACAM is a cell type- and species-dependent process that can alter the dimerization properties of CEACAM. In oral squamous cell carcinoma, N-glycosylated CEACAM6 is a tumor marker associated with recurrence and is required for enhanced cell migration and invasion. N-terminally glycosylated CEACAM5 is upregulated in colorectal cancer (CRC). Furthermore, protein expression of N-glycosylated CEACAM species is found predominantly on the apical membrane of CRC cells, whereas it is predominantly present in the interior of normal colonocytes.

[0004] Although CEACAM5 and CEACAM6 are promising cancer targets, there are only a limited number of anti-CEACAM5 and anti-CEACAM6 monoclonal antibodies currently in development for human cancer treatment, so there is a need to develop novel antibodies against these targets that are effective as cancer therapies.

[0005] Antibody humanization is a crucial step in bringing antibodies to clinical therapeutic use in humans. Antibody humanization of mouse antibodies involves replacing the mouse IgG backbone of the constant regions and the framework regions of the variable regions with human versions while retaining the original complementarity determining regions (CDRs) responsible for antigen binding. Antibody humanization reduces the risk of rejection and human anti-mouse antibody (HAMA) responses that are known to occur with therapeutic antibodies of mouse origin. However, this process often compromises the original function and / or specificity of the antibody.

[0006] It would therefore be generally desirable to overcome or ameliorate one or more of the above-mentioned challenges. Summary of the Invention

[0007] Disclosed herein is an antigen-binding molecule comprising: (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 1, a VHCDR2 amino acid sequence of SEQ ID NO: 2, and a VHCDR3 amino acid sequence of SEQ ID NO: 3; and (ii) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 4, a VLCDR2 amino acid sequence of SEQ ID NO: 5, and a VLCDR3 amino acid sequence of SEQ ID NO: 6; wherein the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10 or 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17 or 13.

[0008] Disclosed herein is an isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule as defined herein.

[0009] Disclosed herein are constructs comprising a polynucleotide defined herein in operative linkage with one or more regulatory sequences.

[0010] Disclosed herein are host cells containing the constructs defined herein.

[0011] Disclosed herein is a composition comprising an antigen-binding molecule as defined herein and a pharma- ceutically acceptable carrier.

[0012] Disclosed herein is an antigen-binding molecule or composition as defined herein for use as a medicament.

[0013] Disclosed herein is a method of treating or preventing cancer or an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of an antigen binding molecule or composition defined herein.

[0014] Disclosed herein is an antigen-binding molecule or composition as defined herein for use in the treatment or prevention of cancer or an inflammatory disease in a subject.

[0015] Disclosed herein is the use of an antigen-binding molecule or composition as defined herein in the manufacture of a medicament for treating or preventing cancer or an inflammatory disease in a subject.

[0016] Disclosed herein is a method for detecting cancer or an inflammatory disease in a subject, comprising: contacting a sample obtained from the subject with an antigen-binding molecule defined herein, wherein an increase in the binding level of the antigen-binding molecule in the sample compared to a reference is indicative of cancer or an inflammatory disease.

[0017] Disclosed herein is a method for identifying a subject predisposed to cancer or an inflammatory disease, comprising contacting a sample obtained from the subject with an antigen-binding molecule defined herein, wherein an increase in the level of binding in the sample compared to a reference indicates that the subject is predisposed to cancer or an inflammatory disease.

[0018] Disclosed herein are kits comprising an antigen-binding molecule as defined herein together with instructions for use, when used in the methods defined herein.

[0019] Embodiments of the invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows potential variable heavy (VH) framework translated sequences with the CDRs underlined. [Diagram 2] FIG. 2 shows a potential variable light (VL) framework translation sequence with the CDRs underlined. [Diagram 3] FIG. 3 shows A) the screening workflow and B) the results of the screening workflow. [Figure 4] Figure 4 shows: A) Summary of screening outcomes, and B)-C) Key changes in variable heavy (VH) and variable light (VL) framework sequences that resulted in reduced specificity, highlighted in yellow. Variable heavy (VH) and variable light (VL) chains that retained specificity are in italics. [Diagram 5] Figure 5 shows the heavy and light chain sequences of the AB1 antibody. The AB1 antibody contains the LPH1 VH and LPL2 VL sequences as shown in Figures 1 and 2. The AB2 antibody (not shown) contains the AH1 VH and AL1 VL sequences. The variable heavy (VH) and variable light (VL) chains are in bold and the CDRs are underlined. [Figure 6] FIG. 6 shows a translated sequence alignment of human CEACAM5 (amino acids 1-360 of SEQ ID NO:86) and human CEACAM6 (amino acids 1-344 of SEQ ID NO:87) with the conserved N256 glycosylation site. [Figure 7]Figure 7 shows the characterization of AB1 antigen. AB1 detects two bands in A549 cell lysates, a 75 kDa band and a 180 kDa band. A549 cells were subjected to single and double siRNA knockdown of CEACAM5 and CEACAM6. (A) Western blot analysis of siRNA-treated cell lysates shows that AB1 does not bind to the 180 kDa band in CEACAM5 single or double knockdown samples. It is further shown that AB1 does not bind to the 75 kDa band in CEACAM6 single or double knockdown samples. AB1 detects 75 kDa and 180 kDa bands in scrambled gene siRNA knockdown cell lysates and untreated cells (mock samples). CEACAM5 was knocked down less efficiently than CEACAM6 in single and double knockdown samples. Detection of the housekeeping gene GAPDH is used as a loading control. (B). The efficiency of siRNA knockdown was monitored by gene expression analysis. CEACAM5 was knocked down less efficiently than CEACAM6 in both single and double knockdown samples. [Figure 8] Figure 8 shows the N-glycosylation dependency of AB1 antigen recognition. A549 cell lysates were reduced, denatured, and treated with PNGase F to remove N-linked glycosylation, and probed with AB1 and commercial CEACAM5 and CEACAM6 antibodies. Western blot analysis shows that AB1 binding to the 75 kDa band (CEACAM6) and 180 kDa band (CEACAM5) is lost after reduction and PNGase F treatment. This also appears to be N-glycan dependent, as the commercial CEACAM6 antibody detects the protein reduced to 75 kDa and CEACAM6 deglycosylated to a smaller size (37 kDa), while the commercial CEACAM5 antibody does not detect any deglycosylated CEACAM5. Detection of the housekeeping gene GAPDH is used as a loading control. [Figure 9]Figure 9 shows the N256 glycosylation dependency of AB1 recognition. The lung cancer cell line NCI-H1299, which does not express CEACAM5 and CEACAM6, was transfected with CEACAM5 or CEACAM6 sequences and mutant sequences of CEACAM5 or CEACAM6 with mutation at N-glycosylation position 256 (N256A). This mutation abolishes N-glycosylation at position N256. Mean fluorescence intensity (MFI) is normalized (nMFI) to human IgG1 isotype control (see table in Figure 9A and B). Flow cytometry binding of AB1 is compared with commercially available CEACAM5 and CEACAM6 antibodies and competitor antibodies. Competitor antibodies include CEACAM6-specific tinurilimab (Bayer), CEACAM5-specific tusamitamab (Sanofi), N-glycosylated CEACAM5 and N-glycosylated CEACAM6-specific NEO-201 (Precision Biologics) and biosimilars to CEACAM5 and CEACAM6-specific EBC-123. EBC-123 is a biosimilar to L-DOS47 (Helix Biopharma Corp), which is an original monomeric camelid single domain VHH 2A3 grafted onto a human Fc. AB1 has been shown to bind to NCI-H1299 cells expressing CEACAM5 or CEACAM6 (A), but not to cells expressing N256A mutant CEACAM5 or N256A mutant CEACAM6 (B). This indicates that N256 glycosylation of CEACAM5 and CEACAM6 is important for AB1 binding to CEACAM5 or CEACAM6. None of the CEACAM6-specific competitor antibodies (tinurilimab, EBC-123, and NEO-201) showed this dependency on N256 glycosylation of CEACAM6 (Figure 9A). [Figure 10]Figure 10 shows flow cytometry binding of AB1, AB2 and AB3 to different populations of human primary peripheral blood cells (top) or human primary bone marrow leukocytes (bottom) in comparison with commercial and competitor antibodies. Primary cells along with lysed red blood cells are incubated with various primary antibodies targeting CEACAM5 and CEACAM6. Cells are labeled with fluorescently labeled lineage markers for granulocytes (CD15+), T cells (CD3+) or B cells (CD19+). Mean fluorescence intensity (MFI, top) or normalized MFI (bottom) (right) are shown for 3 donors / 2 replicates for primary peripheral blood cells and 1 donor / 3 replicates for primary human bone marrow leukocytes. No binding (i.e. negligible binding) by AB1, AB2 or AB3 to CD3+ T cells is seen. AB1, AB2 and AB3 are found to bind poorly to peripheral blood granulocytes (CD15+ cell population) or B cells (CD19+ population) in comparison to CEACAM6-specific antibodies such as tinurilimab (Bayer), NEO-201 (Precision Biologics) and EBC-123 (Helix Biopharma Corp). The CEACAM5-specific tusamitamab (Sanofi) shows no binding to human leukocytes. [Figure 11]FIG. 11 shows the affinity measurements by Biolayer Interferometry for (A) CEACAM5 and N256A mutant CEACAM5 and (B) CEACAM6 and N256A mutant CEACAM6. In-house Avi-tagged CEACAM5, CEACAM6, and N256A mutant CEACAM5 and N256A mutant CEACAM6 are immobilized on Dip and Read SA biosensor. Antibody AB1 and competitor antibodies EBC-123 (Helix Biopharma Corp), NEO-201 (Precision Biologics) and Tusamitamab (Sanofi) are used as analytes. The results show that AB1 binds to both CEACAM5 and CECAM6 with comparable affinity constants (KD) in the double-digit nanomolar range. The KD of AB1 is 10-100 times smaller for N256A mutant CEACAM5 and N256A mutant CEACAM6 compared to wild-type CEACAM5 and CEACAM6 proteins. Competitor antibodies bind to CEACAM5, CEACAM6, N256A mutant CEACAM5 and N256A mutant CEACAM6 with similar affinity. [Figure 12]Figure 12 shows the internalization of antibody AB1 and competitor antibodies into antigen-positive and antigen-negative cell lines. A) shows the internalization of AB1 in NCI-H1299 and NCI-H1299 cells overexpressing CEACAM5 or CEACAM6 or N256A mutant CEACAM5 or N256A mutant CEACAM6. B) shows the internalization of AB1 in NCI-H1299 cells and NCI-H1299 cells overexpressing CEACAM5 or CEACAM6, compared to an IgG1 isotype control antibody and a competitor antibody specific for CEACAM5 and / or CEACAM6. AB1 is internalized by NCI-H1299 expressing CEACAM5 or CEACAM6, but not by NCI-H1299 or N256A mutant CEACAM5 or N256A mutant CEACAM6 (A). CEACAM6-specific tinurilimab (Bayer) is nonspecifically internalized into NCI-H1299 cells. CEACAM5-specific tusamitamab (Sanofi) is internalized only into NCI-H1299 overexpressing CEACAM5, whereas CEACAM5- and CEACAM6-specific NEO-201 (Precision Biologics) and EBC-123 (Helix Biopharma Corp) are internalized at a rate comparable to AB1 into NCI-H1299 overexpressing CEACAM5 or CEACAM6 (B). [Figure 13] Figure 13 shows conjugated antibody AB3, which consists of AB1 conjugated to monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocarproylvalinecitrulline (vc-PAB) linker. [Figure 14]FIG. 14 shows in vitro functionality by CellTiterGlo™ viability assay and dose-response curves estimating IC50 after 72 hours of cell incubation. A) Left panel shows dose-response curves of AB3 with NCI-H1299 compared to NCI-H1299 overexpressing CEACAM6 (clonal cell line 12) or mutant N256A CEACAM6 (clonal cell line 19). A) Right panel shows dose-response curves of AB3 with NCI-H1299 compared to NCI-H1299 overexpressing CEACAM5 (clonal cell line 2F3) or mutant N256A CEACAM5 (clonal cell line 6). B) shows the IC50 obtained with AB3, IgG1 isotype control-MMAE and free MMAE using NCI-H1299 cells and NCI-H1299 cells overexpressing CEACAM5, CEACAM6, N256A mutant CEACAM5 and N256A mutant CEACAM6. Double-digit nanomolar IC50s are obtained for NCI-H1299 cells expressing membrane-bound CEACAM5 or CEACAM6, whereas higher IC50s are obtained for NCI-H1299 cells expressing neither CEACAM5 nor CEACAM6 and N256A mutant CEACAM5 or N256A mutant CEACAM6. Comparably high IC50s are obtained with the IgG1-MMAE isotype control for all cell lines, demonstrating the specificity of AB3. Free MMAE has an IC50 in the picomolar range. C) IC50 for AB3 compared to CEACAM5-specific competitor antibody Tusamitamab (Sanofi) conjugated to MMAE. IC50 for both AB3 and Tusamitamab-MMAE are compared for cancer cell lines Capan-1, CFPAC-1, HCC4006, SNU-16 and HT-29. AB3 targets a broader range of cancer indications due to its dual specificity for N256 glycosylated CEACAM5 and N256 glycosylated CEACAM6. [Figure 15]Figure 15 shows AB3 in a gastric xenograft model. SNU-16 cells express both AB1 antigens CEACAM5 and CEACAM6. Female Balb / c nude mice (n=10 / group) are implanted with SNU-16 cells and treated IV once (or once a week x 3 for docetaxel). AB3 is given at 1, 3 or 5 mg / kg on day 0 with only a single dose. AB3 gives a TGI of 154%, 147% and 114% on day 35, respectively. The treatment is well tolerated and no weight loss is observed in this case. Treatment with IgG1-MMAE at 5 or 1 mg / kg has a statistically significant (p=0.05) lower TGI (123% and 95%, respectively). Furthermore, only AB3-treated mice showed complete responses in a dose-dependent manner, with 6 / 10, 3 / 10 and 1 / 10 mice being tumor-free after a single dose of 5, 3 and 1 mg / kg AB3, respectively, whereas no tumor-free animals were observed after treatment with IgG1-MMAE alone. Anova: One-way ANOVA test followed by Bonferroni multiple comparison test, significance levels shown compared to G1: p<0.0001=****, p<0.001=**, p<0.05=*, ns=not significant; NA=not applicable; BW, body weight (set as 100% for each animal at day 0 / inoculation, group mean values ​​shown as %); MTV, mean tumor volume of all / remaining animals; n=number of animals; PR, partial response; CR, complete response; TGI, tumor growth inhibition rate=mean tumor volume (control day 35-treatment day 35) / (control day 21-control day 0); TRD, treatment-related death; NTRD, non-treatment-related death (e.g. ulcerated tumor, medication error). [Figure 16]Figure 16 shows AB3 in the pancreatic xenograft model Capan-1. Capan-1 cells express both AB1 antigens CEACAM5 and CEACAM6. Female NSG mice (n=10 / group) are implanted with Capan-1 cells and treated once IV. AB3 is given at 5 mg / kg on day 0 with only a single dose. AB3 gives a TGI of 110% on day 21. The treatment is well tolerated and no weight loss is observed. Treatment with IgG1-MMAE at 5, 3 or 1 mg / kg has a statistically significant (p=0.05) lower TGI (28%, 185 and -8%, respectively). Anova: One-way ANOVA test followed by Bonferroni multiple comparison test, significance levels shown compared to G1: p<0.0001=****, p<0.001=**, p<0.05=*, ns=not significant; BW, body weight (set as 100% for each animal at day 0 / inoculation, group mean values ​​shown as %); MTV, mean tumor volume of all / remaining animals; n=number of animals; PR, partial response; CR, complete response; TGI, tumor growth inhibition rate=mean tumor volume (control day 21-treatment day 21) / (control day 21-control day 0); TRD, treatment-related death; NTRD, non-treatment-related death (e.g. ulcerated tumor, medication error). [Figure 17]Figure 17 shows AB3 in pancreatic xenograft model BxPC-3. BxPC-3 cells express both AB1 antigens CEACAM5 and CEACAM6. Female NOD-SCID mice (n=8 / group) are implanted with BxPC-3 cells and treated once IV. AB3 is given at 1 or 5 mg / kg on day 0 with only a single dose. AB3 gives a TGI of 107% and 21%, respectively, on day 21. Treatment is well tolerated and no weight loss is observed. Treatment with IgG1-MMAE at 5 mg / kg has a statistically significant (p=0.05) lower TGI of 6%. Anova: One-way ANOVA test followed by Bonferroni multiple comparison test, significance levels shown compared to G1: p<0.0001=****, p<0.001=**, p<0.05=*, ns=not significant; BW, body weight (set as 100% for each animal at day 0 / inoculation, group mean values ​​shown as %); MTV, mean tumor volume of all / remaining animals; n=number of animals; PR, partial response; CR, complete response; TGI, tumor growth inhibition rate=mean tumor volume (control day 21-treatment day 21) / (control day 21-control day 0); TRD, treatment-related death; NTRD, non-treatment-related death (e.g. ulcerated tumor, medication error). [Figure 18] Figure 18 shows the tumor growth profile of antigen-negative NCI-H1299 and NCI-H1299 overexpressing N256A mutant CEACAM5 or N256A mutant CEACAM6 versus antigen-positive NCI-H1299 overexpressing CEACAM5 or CEACAM6. Female NOD-SCID mice (n=6 / group) are implanted with NCI-H1299 cells and tumor volumes are measured three times weekly up to 75 days. No differences are observed in tumor onset or doubling time for NCI-H1299, NCI-H1299 overexpressing CEACAM6, or NCI-H1299 overexpressing N256A mutant CEACAM6. Early tumor onset is observed for NCI-H1299 overexpressing CEACAM5, whereas tumor onset is delayed by 35 days for NCI-H1299 overexpressing N256A mutant CEACAM5. After tumor onset, tumor doubling times are comparable for all cell lines. [Figure 19]Figure 19 shows AB3 in xenograft model using antigen-negative NCI-H1299 cells and NCI-H1299 cells overexpressing N256A mutant CEACAM5 vs. antigen-positive NCI-H1299 cells overexpressing CEACAM5 or CEACAM6. Female NOD-SCID mice (n=6 / group) are implanted with NCI-H1299 cells and treated once IV. AB3 is given at 5mg / kg on day 0 with only a single dose. AB3 gives 32% and 28% TGI on day 21 for antigen-negative cells NCI-H1299 and NCI-H1299 overexpressing N256A mutant CEACAM5, respectively. AB3 gives 109% and 110% TGI on antigen-positive cells NCI-H1299 overexpressing CEACAM5 or CEACAM6, respectively. The treatment is well tolerated and no weight loss is observed in this case. Anova: One-way ANOVA test followed by Bonferroni multiple comparison test, significance levels shown compared to G1: p<0.0001=****, p<0.001=**, p<0.05=*, ns=not significant; BW, body weight (set at 100% for each animal at day 0 / inoculation, group mean values ​​in % are shown); MTV, mean tumor volume of all / remaining animals; n=number of animals; PR, partial response; CR, complete response; TGI, tumor growth inhibition rate=mean tumor volume (control day 21-treatment day 21) / (control day 21-control day 0); TRD, treatment-related death; NTRD, non-treatment-related death (e.g. ulcerated tumor, medication error). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present specification teaches a humanized antigen-binding molecule that binds to CEACAM5 and / or CEACAM6. The present specification teaches a humanized antigen-binding molecule comprising: (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of GNTFTSYVMH (SEQ ID NO: 1), a VHCDR2 amino acid sequence of YINPYNDGTKYNEKFKG (SEQ ID NO: 2), and a VHCDR3 amino acid sequence of STARATPYFYAMDY (SEQ ID NO: 3); and (ii) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of KSSQSLLWSVNQNSYLS (SEQ ID NO: 4), a VLCDR2 amino acid sequence of GASIRES (SEQ ID NO: 5), and a VLCDR3 amino acid sequence of QHNHGSFLPYT (SEQ ID NO: 6).

[0022] Disclosed herein is an antigen-binding molecule comprising: (1) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 1, a VHCDR2 amino acid sequence of SEQ ID NO: 2, and a VHCDR3 amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 4, a VLCDR2 amino acid sequence of SEQ ID NO: 5, and a VLCDR3 amino acid sequence of SEQ ID NO: 6; wherein the VH defined in (1) has at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity with at least one region other than the CDR of the VH amino acid sequence set forth in SEQ ID NO: 10 or 7, and the VL defined in (1) has at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity with at least one region other than the CDR of the VL amino acid sequence set forth in SEQ ID NO: 17 or 13.

[0023] Without wishing to be bound by theory, the inventors found that during the process of converting mouse antibodies to humanized antibodies, many variants exhibited reduced specificity and / or function compared to the parental mouse and chimeric forms. Several key criteria were identified to rank the humanized variants resulting from in silico humanization optimization. Importantly, it was the aggregation and site specificity readouts that allowed the elimination of problematic motifs and allowed for a rapid shortlisting of the final three molecules for further preclinical evaluation.

[0024] In one embodiment, the antigen binding molecule binds to CEACAM5 and / or CEACAM6. In one embodiment, the antigen binding molecule binds to CEACAM5. In one embodiment, the antigen binding molecule binds to CEACAM6. In one embodiment, the antigen binding molecule binds to CEACAM5 and CEACAM6.

[0025] In one embodiment, the antigen binding molecule binds to CEACAM5 and / or CEACAM6 when they are glycosylated at the N256 position. In one embodiment, the antigen binding molecule binds to CEACAM5 when CEACAM5 is glycosylated at the N256 position. In one embodiment, the antigen binding molecule binds to CEACAM6 when CEACAM6 is glycosylated at the N256 position. In one embodiment, the antigen binding molecule binds to CEACAM5 and CEACAM6 when both CEACAM5 and CEACAM6 are glycosylated at the N256 position.

[0026] The antigen-binding molecules of the present invention may be in isolated, purified, synthetic, or recombinant form. Suitable antigen-binding molecules may be selected from antibodies and their antigen-binding fragments, including monoclonal antibodies (MAbs), chimeric antibodies, humanized antibodies, human antibodies, and antigen-binding fragments of such antibodies. Antigen-binding molecules may be multivalent (e.g., bivalent) or monovalent. In some embodiments, the antigen-binding molecule comprises an Fc domain. In other embodiments, the antigen-binding molecule lacks an Fc domain. In some embodiments, the antigen-binding molecule is a monovalent antigen-binding molecule (e.g., Fab, scFab, Fab', scFv, one-armed antibody, etc.).

[0027] "Antigen-binding molecule" refers to a molecule that has binding affinity for a target antigen. It will be understood that the term covers immunoglobulins, immunoglobulin fragments, and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. Exemplary antigen-binding molecules that are useful in the practice of the present invention include antibodies and antigen-binding fragments thereof. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0028] The term "antibody", as used herein, is understood to mean any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a target antigen. The term "antibody" includes full-length immunoglobulin molecules that contain two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (HCVR, sometimes abbreviated as VH) and a heavy chain constant region. The heavy chain constant region usually contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (LCVR, sometimes abbreviated as VL) and a light chain constant region. The light chain constant region will usually contain one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with highly conserved regions also called framework regions (FRs). Each VH and VL typically contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antigen-binding molecules described herein may be identical to the FRs of the germline sequence of the target species (i.e., the species to which the antigen-binding molecules or antigen-binding fragments described herein will be administered). In some embodiments, the FRs may be naturally or artificially modified. In general, it is desirable for each of the FR sequences to be identical to the FR sequences derived from the immunoglobulin molecules of the target species, including minimizing the immune response against the binding molecules upon administration to a subject of the target species, while in some embodiments, the antigen-binding molecules or antigen-binding fragments thereof may contain one or more amino acid residues throughout one or more of their FR sequences that are foreign to the corresponding positions in one or more FRs derived from the target species.

[0029] Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and do not require that the antibody be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the various immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various immunoglobulin classes are well known to those skilled in the art. Light chains can be classified into kappa light chains and lambda light chains.

[0030] In one embodiment, the antigen-binding molecule of the present invention has an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region may be a wild-type human Fc region or a human Fc region containing one or more amino acid substitutions. The antibody may have a mutation that stabilizes the disulfide bond between the two heavy chains of the immunoglobulin, such as a mutation in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al., 1993. Mol. Immunol., 30:105-08). See also, e.g., US2005 / 0037000. The heavy chain constant region may also have a substitution that modifies the properties of the antigen-binding molecule (e.g., reduces one or more of Fc receptor binding, glycosylation of the antigen-binding molecule, deamidation, binding to complement, or methionine oxidation). In some examples, the antigen-binding molecule may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the antigen-binding molecule is modified to reduce or eliminate effector function. The heavy chain constant region may be chimeric, for example, the Fc region may include the CH1 and CH2 domains of an IgG antibody of IgG4 isotype, and the CH3 domain from an IgG antibody of IgG1 isotype (see, for example, U.S. Patent Application Publication No. 2012 / 0100140 A1).

[0031] As used herein, the term "complementarity determining region" (CDR; i.e., CDR1, CDR2 and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is required for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may be, for example, selected from amino acid residues from a "complementarity determining region" defined by Kabat [i.e., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] and / or amino acid residues from a "hypervariable loop" [i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)]. In some instances, the complementarity determining regions can include amino acids from both the CDR regions and the hypervariable loops as defined by Kabat.

[0032] "Antigen-binding site" (or paratope) refers to a site, i.e., one or more amino acid residues, of an antigen-binding molecule that allows interaction with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen-binding sites, and a Fab molecule typically has a single antigen-binding site. The antigen-binding site of the antigen-binding molecules described herein typically specifically binds to an antigen, more specifically to an epitope of the antigen.

[0033] The terms "antigen-binding fragment," "antigen-binding portion," "antigen-binding domain," and "antigen-binding site" are used interchangeably herein to refer to a portion of an antigen-binding molecule that participates in antigen binding. These terms include any naturally occurring, enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0034] Antigen-binding fragments of antibodies can be derived from full-length antibody molecules, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and, where appropriate, constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and engineered chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, form cysteine ​​residues, modify, add or delete amino acids, etc.

[0035] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, one-arm antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), camelid VHHs, and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0036] Antigen-binding fragments of antibodies will usually contain at least one variable domain. The variable domain may be of any size and amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be placed in any suitable configuration relative to each other. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, antigen-binding fragments of antibodies may contain monomeric VH or VL domains.

[0037] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2, (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present disclosure may comprise a homodimer or heterodimer (or other multimer) of any of the configurations of the variable and constant domains listed above that are non-covalently associated (e.g., by disulfide bonds) with each other and / or with one or more monomeric VH or VL domains. A multispecific antigen-binding molecule will usually comprise at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, can be constructed for use in the context of the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0038] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antigen-binding molecule to an antigen. The variable domains of heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, where each domain contains four conserved framework regions (FR) and three hypervariable regions (or CDRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0039] The term "constant domain" or "constant region" as used herein refers to the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding, but exhibits various immune effector functions.

[0040] In one embodiment, the antigen-binding molecule or antigen-binding fragment thereof is humanized. "Humanized" means that the antigen-binding molecule comprises an amino acid sequence that is compatible with humans, such that the amino acid sequence is unlikely to be regarded as foreign by the immune system of a human subject. In one embodiment, the humanized antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more human immunoglobulin molecules. In some embodiments, all of the framework regions of the humanized antigen-binding molecule will be derived from one or more human immunoglobulin molecules. A humanized antibody may comprise at least a portion of an antibody constant region derived from a human immunoglobulin molecule.

[0041] The phrases "specifically bind" or "specific binding" refer to a binding reaction between two molecules that is at least twice background under physiological conditions, and more typically 10-100 times greater than background molecular association. When one or more detectable binding agents that are proteins are used, specific binding determines the presence of that protein in a heterogeneous population of multiple proteins and other biologics. Thus, under specified immunoassay conditions, a particular antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity for that determinant. Such selection can be performed by removing antigen-binding molecules that cross-react with other molecules. A variety of immunoassay formats can be used to select antigen-binding molecules (e.g., immunoglobulins) that are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods for determining binding affinity and specificity are also well known in the art (see, e.g., Harlow and Lane, supra; Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (WH Freeman and Co. 1976)).

[0042] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair, e.g., an antigen-binding molecule. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the sum of the dissociation rate constant and the association rate constant (k, respectively). off and k on ) ratio. Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR). Affinity can also be measured using biolayer interferometry (BLI). In another embodiment, the method for measuring affinity is determined using a cell-based affinity measurement technique.

[0043] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to refer to a linear series of amino acid residues linked one to the other by peptide bonds between the alpha amino group and the carboxy group of adjacent residues. The amino acid residues are normally in the naturally occurring "L" isomeric form. However, any L-amino acid residue can be substituted with a residue in the "D" isomeric form, so long as the desired functional property is retained by the polypeptide.

[0044] As used herein, the term "modified antibodies" includes synthetic forms of antibodies that have been modified to be non-naturally occurring, such as antibodies that contain at least two heavy chain portions but not two complete heavy chains (e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been modified to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, etc. scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. In addition, the term "modified antibodies" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen).

[0045] In one embodiment, a) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VL amino acid sequence set forth in SEQ ID NO: 17; b) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VL amino acid sequence set forth in SEQ ID NO: 13. c) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VL amino acid sequence set forth in SEQ ID NO: 17; or d) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDR of the VL amino acid sequence set forth in SEQ ID NO: 13.

[0046] In one embodiment, a) the VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 10 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 17 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; b) the VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 10 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 13 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13. c) the VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 7 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 17 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; or d) the VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 7 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 13 by the deletion, substitution or addition of one or more amino acids (e.g., 1, 2, 3, 4 or 5) in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13.

[0047] In one embodiment, the antigen binding molecule comprises: a) a VHFR1 that is distinct from the VHFR1 amino acid sequence set forth in QVQLVQSGVEVKKPGASVKVSCKAS (SEQ ID NO: 19) or QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 20) by one or more amino acid deletions, substitutions, or additions; b) a VHFR2 that is distinct from the VHFR2 amino acid sequence set forth in WVRQAPGQGLEWMA (SEQ ID NO: 21) or WVRQAPGQGLEWMG (SEQ ID NO: 22) by one or more amino acid deletions, substitutions, or additions; c) a VHFR3 that is distinct from the VHFR3 amino acid sequence set forth in RVTLTTDSSTTTAYMELKSLQFDDTAVYYCAR (SEQ ID NO: 23) or RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 24) by one or more amino acid deletions, substitutions, or additions; d) a VHFR4 that differs from the VHFR4 amino acid sequence set forth in YWGQGTLVTVSS (SEQ ID NO: 25) by one or more amino acid deletions, substitutions, or additions; e) a VLFR1 that is distinct from the VLFR1 amino acid sequence set forth in DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 26) or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 27) by the deletion, substitution, or addition of one or more amino acids; f) a VLFR2 that is distinct from the VLFR2 amino acid sequence set forth in WYQQKPGKAPKLLIY (SEQ ID NO:28) or WYQLKPGQPPKLLLY (SEQ ID NO:29) by one or more amino acid deletions, substitutions, or additions; g) a VLFR3 that is distinct from the VLFR3 amino acid sequence set forth in GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 30) or GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 31) by the deletion, substitution, or addition of one or more amino acids; and / or h) A VLFR4 that differs from the VLFR4 amino acid sequence set forth in FGQGTKVEIK (SEQ ID NO: 32) or FGGGTKLEIK (SEQ ID NO: 33) by the deletion, substitution or addition of one or more amino acids.

[0048] In one embodiment, the antigen binding molecule comprises: a) the VHFR1 amino acid sequence of QVQLVQSGX1EVKKPGASVKVSCKAS, where X1 is V or A (SEQ ID NO: 34); b) a VHFR2 amino acid sequence of WVRQAPGQGLEWMX2, where X2 is A or G (SEQ ID NO: 35); c) a VHFR3 amino acid sequence of RVTLTTDSSTTTAYMELKSLQFDDTAVYYCAR (SEQ ID NO: 23) or RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 24); d) the VHFR4 amino acid sequence of YWGQGTLVTVSS (SEQ ID NO: 25); e) the VLFR1 amino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 26) or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 27); f) a VLFR2 amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO:28) or WYQLKPGQPPKLLLY (SEQ ID NO:29); g) the VLFR3 amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 30) or GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 31); and / or h) A VLFR4 amino acid sequence of FGQGTKVEIK (SEQ ID NO: 32) or FGGGTKLEIK (SEQ ID NO: 33).

[0049] In one embodiment, the antigen binding molecule comprises the VH amino acid sequence of SEQ ID NO: 10 or 7 and the VL amino acid sequence of SEQ ID NO: 17 or 13.

[0050] In one embodiment, a) the antigen binding molecule comprises a VH amino acid sequence of SEQ ID NO: 10 and a VL amino acid sequence of SEQ ID NO: 17; b) the antigen binding molecule comprises a VH amino acid sequence of SEQ ID NO: 10 and a VL amino acid sequence of SEQ ID NO: 13; c) the antigen binding molecule comprises a VH amino acid sequence of SEQ ID NO: 7 and a VL amino acid sequence of SEQ ID NO: 17; or d) the antigen binding molecule comprises a VH amino acid sequence of SEQ ID NO: 7 and a VL amino acid sequence of SEQ ID NO: 13.

[0051] In one embodiment, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody, a substantially intact antibody, a Fab fragment, a scFab, a Fab', a single-chain variable fragment (scFv), or a one-arm antibody.

[0052] In one embodiment, the antigen binding molecule is a full-length antibody. In one embodiment, the full-length antibody is an IgG (e.g., IgG1) antibody.

[0053] In one embodiment, the antigen-binding molecule comprises a light chain sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 84, and a heavy chain sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to SEQ ID NO: 85.

[0054] The term "sequence identity" as used herein refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over a comparison window.Therefore, "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G and I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0055] An antigen-binding molecule as defined herein may contain one or more conservative amino acid substitutions.

[0056] A "conservative amino acid substitution" should be understood to mean a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and can be generally subclassified as shown in the table below, "Amino Acid Classifications": Amino Acid Subclassification

[0057] [Table 1]

[0058] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine and tryptophan; the group of amino acids with basic side chains is lysine, arginine and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that the replacement of leucine with isoleucine or valine, the replacement of aspartate with glutamate, the replacement of threonine with serine, or similar replacement of amino acids with structurally related amino acids will not significantly affect the properties of the resulting mutant polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by assaying its activity.

[0059] Conservative substitutions are also shown in the table below (exemplary and preferred amino acid substitutions). Amino acid substitutions that fall within the scope of the invention are made by selecting substitutions that are not significantly different overall in terms of (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining side chain bulk. After substitutions are introduced, mutants can be screened for their ability to specifically bind to antigens using methods known to those of skill in the art, including those described elsewhere herein. Exemplary and Preferred Amino Acid Substitutions

[0060] [Table 2]

[0061] In one embodiment, the antigen binding molecule is conjugated to a radioisotope or a cytotoxin.

[0062] In one embodiment, the antigen-binding molecule is a full-length IgG (e.g., IgG1) antibody conjugated to a cytotoxin. The antigen-binding molecule can be conjugated to the cytotoxin via a protease-cleavable maleimidocarproylvalinecitrulline (vc-PAB) linker.

[0063] In one embodiment, the antigen binding molecule consists of AB1 conjugated to monomethyl auristatin E (MMAE) via a maleimidocarproylvalinecitrulline (vc-PAB) linker (i.e., AB3 shown in FIG. 13). In one embodiment, the linker-toxin combination has the formula C 58 H 94 N 10 O 12 and has the chemical name L-valinamide, N-methyl-N-[[[4-[[L-valyl-N5-(aminocarbonyl)-L-ornityl]amino]phenyl]methoxy]carbonyl]-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl. Conjugation can be accomplished by a maleimide-cysteine ​​based method by first reducing the mAh interchain disulfide bond with TCEP at 37° C. and then linking the maleimide moiety of the drug to the reduced cysteine. The drug-antibody ratio (DAR) can be analyzed by hydrophobic interaction chromatography (HIC). For example, the DAR ratio can be between 3 and 4.

[0064] In another embodiment, an antigen-binding molecule or antigen-drug conjugate (ADC) of the following formula (I): Ab-(LD)n (I) or a pharma- ceutically acceptable salt thereof is provided. (In the formula, Ab is an antibody or antibody fragment thereof as defined herein: L is a linker; D is a cytotoxin).

[0065] In one embodiment, the invention relates to an ADC where L is of formula (II):

[0066] [ka] is the linker for [In the formula, L2 is cycloalkylene-carbonyl, (C2-C6)alkyl or (C2-C6)alkyl-carbonyl; W is an amino acid unit; w is an integer of 0 to 5; Y is PAB

[0067] [ka] is PAB-carbonyl; Even if x is H

[0068] [ka] It may be; y is 0 or 1; The asterisk indicates the attachment point to D; The wavy line indicates the point of attachment to Ab].

[0069] One embodiment of the present invention relates to an ADC, wherein L2 has the formula:

[0070] [ka] It is of [In the formula, Asterisk is (W) windicates the point of attachment to; The wavy line represents the formula

[0071] [ka] indicates the point of attachment to the nitrogen atom of the maleimide moiety].

[0072] In one embodiment of the present invention, w=0, or w=2, then (W)w is:

[0073] [ka] Selected from [In the formula, Asterisk is (Y) y indicates the point of attachment to; The wavy line indicates the attachment point to L2].

[0074] In one embodiment, the invention relates to an ADC where L is:

[0075] [ka] Selected from (where the asterisk indicates the point of attachment to D and the wavy line indicates the point of attachment to Ab).

[0076] In another embodiment, the invention relates to an ADC where L is of formula (III):

[0077] [ka] is the linker for [In the formula, L'2 is cycloalkylene-carbonyl, (C2-C6)alkylene, or (C2-C6)alkylene-carbonyl; W' is an amino acid unit; w' is an integer from 0 to 5; Y' is PAB:

[0078] [ka] is PAB-carbonyl; Even if x is H

[0079] [ka] It may be; y' is 0 or 1; R' is C1-C3 alkenyl or H.

[0080] In one embodiment, the compound of formula (III) has the formula (III'):

[0081] [ka] It is a compound of the formula:

[0082] In one embodiment, the compound of formula (III') is characterized in that L2' is a C2 alkylenecarbonyl and w' is 2.

[0083] In one embodiment, the linker compound of formula (III') is:

[0084] [ka] It is.

[0085] In another embodiment, the linker compound of formula (III') is:

[0086] [ka] It is.

[0087] In another embodiment, the linker compound of formula (III') is:

[0088] [ka] It is.

[0089] The linkers of the present invention can be synthesized using amide bond coupling. There are many methods for amide synthesis. Some methods are described, but are not limited to, in Montalbetti, Christian AG N [Tetrahedron 61(46), 2005, 10827-10852]. Alternatively, the linkers can be synthesized using standard stepwise addition of one or more residues, for example, using a peptide or protein synthesizer. Alternatively, other methods that can be used for amide formation include, but are not limited to, the Beckmann rearrangement, the Schmidt reaction, nitrile hydrolysis, the Wilgerott-Kindler reaction, the Passerini reaction, the Ughi reaction, the Boudreux reaction, the Chapman rearrangement, the Leuckart amide synthesis, the Ritter reaction, the ester aminolysis, the Schotten-Baumann reaction, ruthenium-based catalysis of alcohols and amines, or the photolytic addition of formamide to olefins.

[0090] In one embodiment, the cytotoxin is selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), mertansine (DM-1), saporin, gemcitabine, irinotecan, etoposide, vinblastine, pemetrexed, docetaxel, paclitaxel, platinum agents (e.g., cisplatin, oxaliplatin, and carboplatin), vinorelbine, capecitabine, mitoxantrone, ixabepilone, eribulin, 5-fluorouracil, trifluridine, and tipiracil.

[0091] The antigen-binding molecule can also be conjugated to a detectable moiety.

[0092] Detectable moieties contemplated by the present invention include any type known in the art that is suitable for diagnostic detection, including, for example, in vitro detection and in vivo imaging. Detectable moieties can be, for example, fluorophores, radionuclide reporters, metal-containing nanoparticles or microparticles, ultrasound contrast agents (e.g., nanobubbles or microbubbles), or optical imaging dyes. Detectable moieties also include contrast particles that are visible in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Fluorophores can be detected and / or imaged, for example, by fluorescence polarization, fluorescence activated cell sorting, and fluorescence microscopy, with or without electrospray ionization mass spectrometry (ESI-MS) detection, as well as fluorescence emission computed tomography (FLECT) imaging. Radionuclide reporters can be detected and imaged, for example, by radionuclide (nuclear) detection, such as single photon emission computed tomography (SPECT), positron emission tomography (PET) or scintigraphy imaging. Metal-containing nanoparticles or microparticles can be detected using optical imaging, including MRI, which is commonly used with paramagnetic nanoparticles or microparticles, and MPI, which is commonly used with superparamagnetic particles. Ultrasound contrast agents can be detected using ultrasound imaging, including contrast-enhanced ultrasound (CEU).

[0093] The detectable label can also be an enzyme substrate label. Enzymes can generally catalyze a chemical change in a chromogenic substrate that can be measured using various techniques. For example, enzymes can catalyze a chemical change in a chromogenic substrate that can be measured using various techniques. For example, an example can catalyze a color change in the substrate that can be measured spectrophotometrically. Alternatively, enzymes can change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. Chemiluminescent substrates can then be electronically excited by a chemical reaction and emit light that can be measured (e.g., using a chemiluminometer), or donate energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0094] Examples of enzyme-substrate combinations include, for example: 1) Horseradish peroxidase (HRP) utilizes hydrogen peroxide to oxidize dye precursors [e.g., orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)]; 2) alkaline phosphatase (AP) with paranitrophenyl phosphate as a chromogenic substrate; and 3) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactoside) or a fluorogenic substrate (4-methylumbelliferyl-β-D-galactoside) Examples include:

[0095] In another embodiment of the present invention, the antigen-binding molecule does not need to be labeled, and its presence can be detected using a labeled antibody that binds to the antigen-binding molecule. The antigen-binding molecule of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, immunohistochemistry assays, and immunoprecipitation assays.

[0096] In one embodiment, the antigen binding molecule selectively binds to gefitinib-resistant lung cancer cells, osimertinib-resistant lung cancer cells, non-small cell lung cancer cells, breast cancer cells, pancreatic cancer cells, stomach (or gastric) cancer cells, small intestine cancer cells, esophageal cancer cells or colorectal cancer cells.

[0097] Disclosed herein is an isolated polynucleotide comprising a nucleic acid sequence encoding an antigen-binding molecule as defined herein.

[0098] The terms "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides, which may be mRNA, RNA, cRNA, cDNA or DNA. The terms refer to a polymeric form of nucleotides, usually ribonucleotides or deoxynucleotides, or any type of modified nucleotide, at least 10 bases in length. The terms include single-stranded and double-stranded forms of DNA.

[0099] Also disclosed herein are vectors comprising a nucleic acid encoding the antigen-binding molecules described herein.

[0100] By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived from, for example, a plasmid, bacteriophage or virus, into which a nucleic acid sequence can be inserted or cloned. The vector preferably contains one or more unique restriction enzyme sites and may be capable of autonomous replication in a given host cell, including a target cell or tissue, or a precursor cell or tissue thereof, or may be capable of integrating into the genome of a given host, so that the cloned sequence can be reproduced. Thus, the vector may be a self-replicating vector, i.e. a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, for example, as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which the vector is integrated. The vector system may include a single vector or plasmid, two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or may include a transposon. The choice of vector will usually depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also contain a selection marker, such as an antibiotic resistance gene, that can be used to select suitable transformants. Examples of resistance genes are well known to those skilled in the art.

[0101] Disclosed herein are constructs comprising a polynucleotide defined herein in operative linkage with one or more regulatory sequences.

[0102] The term "construct" refers to a recombinant genetic molecule that contains one or more nucleic acid sequences isolated from various sources. Thus, a construct is a chimeric molecule that assembles two or more nucleic acid sequences of different origins into a single nucleic acid molecule, including any construct that contains: (1) a nucleic acid sequence that contains regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) a sequence that encodes a portion of a functional RNA molecule or protein that is not naturally associated with it, or (3) a portion of a promoter that is not naturally associated with it. Exemplary constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of integrating into a genome or replicating autonomously, and containing one or more nucleic acid molecules operably linked thereto. A construct of the invention will generally include the necessary elements for directing the expression of a nucleic acid sequence of interest, such as a target nucleic acid sequence or a modulator nucleic acid sequence, also contained in the construct. Such elements may include a control element or regulatory sequence, such as a promoter (to direct transcription of the nucleic acid sequence) operably linked to the nucleic acid sequence of interest, and often also a polyadenylation sequence. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as a prokaryotic and eukaryotic origin, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of the host cell. Two or more constructs may be included in a single nucleic acid molecule, such as a single vector, or in two or more separate nucleic acid molecules, such as two or more separate vectors.An "expression construct" generally comprises at least one control sequence operably linked to a nucleotide sequence of interest. Thus, for example, a promoter operably linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or part thereof, including a host cell. Conventional compositions and methods for preparing and using constructs and host cells for carrying out the present invention are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0103] "Control element", "control sequence", "regulatory sequence", and the like, as used herein, refer to nucleic acid sequences (e.g., DNA) necessary for the expression of an operably linked coding sequence in a particular host cell. Control sequences suitable for prokaryotic cells include, for example, promoters and may include cis-acting sequences such as operator sequences and ribosome binding sites. Control sequences suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, and targeting sequences that direct the product encoded by the transcribed polynucleotide to an intracellular compartment within a target cell or to the extracellular environment.

[0104] Disclosed herein are host cells containing the constructs defined herein.

[0105] The terms "host", "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", including the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as originally screened or selected in the transformed cell are included herein. A host cell is any type of cell line that can be used to produce the antigen-binding molecules of the present invention. Host cells include cultured cells, mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name a few, but also cells contained within transgenic animals, transgenic plants or cultured plants or cultured animal tissues.

[0106] Disclosed herein is a pharmaceutical composition comprising an antigen-binding molecule as defined herein and a pharma- ceutically acceptable carrier.

[0107] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle composed of materials that are not biologically or otherwise undesirable, i.e., the materials can be administered to a subject together with a selected active agent without causing any or substantial adverse side effects. Carriers can include excipients and other additives, such as diluents, surfactants, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0108] Representative pharma- ceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials and combinations thereof, as would be known to one skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0109] The pharmaceutical composition can be in various forms. This includes, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred dosage form depends on the intended mode of administration and therapeutic use. Suitable pharmaceutical compositions can be administered intravenously, subcutaneously or intramuscularly. In some embodiments, the composition is in the form of an injectable and infusible solution. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In certain embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In other embodiments, the pharmaceutical composition is administered by intramuscular or subcutaneous injection.

[0110] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0111] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In the present invention, pharma- ceutically acceptable carriers include, but are not limited to, 0.01-0.1M, preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, dextrose-Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on dextrose-Ringer's solution, and the like. Preservatives and other additives may be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0112] More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy needleability exists. The composition must be stable under the conditions of manufacture and storage, and preferably will be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin and / or by maintaining the required particle size. In certain embodiments, the agent of the present disclosure may be conjugated to a vehicle for cell delivery. In such embodiments, the agent can be encapsulated in a suitable vehicle to aid in the delivery of the agent to target cells, or to improve the stability of the agent, or to minimize the possible toxicity of the agent. As will be appreciated by those skilled in the art, various vehicles are suitable for delivering the agent of the present disclosure.Non-limiting examples of suitable structured fluid delivery systems can include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems.Methods for incorporating the agent of the present disclosure into delivery vehicles are known in the art.Various embodiments are shown below, but it will be understood that other methods known in the art for incorporating the antigen-binding molecules described herein into delivery vehicles are also contemplated.

[0113] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. The antigen-binding molecule of the present disclosure may be administered multiple times. The interval between single doses may be daily, weekly, monthly, or yearly. The interval may be irregular, as indicated by measuring the blood level of the modified polypeptide or antigen in the patient. Alternatively, the antigen-binding molecule may be administered as a sustained release formulation, which requires less frequent administration. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0114] For ease of administration and uniformity of dosage, it can be advantageous to formulate the composition in unit dosage form.Unit dosage form as used herein refers to a physically separate unit that is adapted as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect, in association with required pharmacologic acceptable carrier.The specification of the unit dosage form of the present invention is governed by and directly depends on (a) the unique characteristics of active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the art when preparing such active compound for the treatment of individual susceptibility.

[0115] The dosage and treatment regimen of the antigen-binding molecule can be determined by one skilled in the art. In certain embodiments, the antigen-binding molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 0.01-50 mg / kg, e.g., 0.01-0.1 mg / kg, e.g., about 0.1-1 mg / kg, about 1-5 mg / kg, about 5-25 mg / kg, about 10-50 mg / kg. The administration schedule can vary, e.g., from once a week to once every 2, 3 or 4 weeks.

[0116] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated.Furthermore, it should be understood that the specific dosage regimen for any particular subject should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0117] Disclosed herein is an antigen-binding molecule or composition as defined herein for use as a medicament.

[0118] Disclosed herein is a method of treating or preventing cancer or an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of an antigen binding molecule or composition defined herein.

[0119] The terms "treat," "treatment," and the like are used interchangeably herein to mean to alleviate, reduce, relieve, ameliorate, or otherwise inhibit a condition, including one or more symptoms of the condition. The terms "prevent," "preventing," "prevention," "prophylactic," "tending to prevent," and the like are used interchangeably herein to mean to prevent or delay the onset of a condition, or the risk of developing a condition.

[0120] The terms "treat", "treatment", and the like also include alleviating, reducing, ameliorating, or otherwise suppressing the effects of a condition for at least a period of time. It should also be understood that the terms "treat", "treatment", and the like do not imply that a condition or its symptoms will be permanently alleviated, reduced, ameliorated, or otherwise suppressed, and thus encompass temporary alleviation, reduction, relief, amelioration, or otherwise suppression of a condition or its symptoms.

[0121] The terms "patient," "subject," "host," or "individual," as used interchangeably herein, refer to any subject, particularly a vertebrate subject, and more particularly a mammalian subject, for whom treatment or prevention is desired. Suitable vertebrates within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys and apes, as well as monkey species from the genus Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and chimpanzees (Pan pantheons). troglodytes)], rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cows), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, companion birds such as ducks, geese, canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. In one embodiment, the subject is a human subject.

[0122] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is usually characterized in part by uncontrolled cell proliferation. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early and late stage cancers. "Non-metastatic" means a cancer that remains at the primary site and has not invaded the lymphatic or vascular system or tissues other than the primary site. The term "metastatic cancer" refers to a cancer that has metastasized or may metastasize from one part of the body to another. In general, a non-metastatic cancer is any cancer that is stage 0, I, II, and occasionally stage III. On the other hand, a metastatic cancer is usually a stage IV cancer.

[0123] The term "cancer" includes, but is not limited to, breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vulvar cancer, squamous cell carcinoma, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, , thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue tumors, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, CNS tumors, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumors, brain stem glioma, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cavity cancer, pharyngeal cancer, or a combination thereof.

[0124] In one embodiment, the cancer cells are solid cancer cells or hematological cancer cells.

[0125] The term "blood cancer" can refer to one or more of leukemia, lymphoma, chronic myeloproliferative disease, Langerhans cell histiocytosis, multiple myeloma / plasma cell neoplasms, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, or combinations thereof. In some embodiments, the leukemia is any one or more of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), or combinations thereof. In some embodiments, the lymphoma is any one or more of AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, T-cell lymphoma, cutaneous lymphoma, Waldenstrom's macroglobulinemia, B-cell lymphoma, or combinations thereof.

[0126] The term "solid cancer" can refer to one or more of the following: breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vulvar cancer, squamous cell carcinoma, vaginal cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue tumors, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, CNS tumors, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumors, brain stem glioma, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cavity cancer, pharyngeal cancer, sarcoma, or a combination thereof.

[0127] In one embodiment, the cancer is a metastatic cancer. The cancer can be a refractory cancer or a recurrent cancer.

[0128] In one embodiment, the cancer is selected from the group consisting of gefitinib-resistant lung cancer, osimertinib-resistant lung cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, gastric (or stomach) cancer, small intestine cancer, esophageal cancer and colorectal cancer.

[0129] In one embodiment, the cancer is a cancer associated with overexpression of CEACAM5 and / or CEACAM6. In one embodiment, the cancer is a cancer associated with overexpression of glycosylated CEACAM5 and / or CEACAM6. In one embodiment, the cancer is a cancer associated with overexpression of glycosylated CEACAM5. In one embodiment, the cancer is a cancer associated with overexpression of glycosylated CEACAM6. In one embodiment, the cancer is a cancer associated with overexpression of glycosylated CEACAM5 and CEACAM6. CEACAM5 and / or CEACAM6 may be glycosylated at position N256.

[0130] In one embodiment, the inflammatory disease is Crohn's disease or asthma. Asthma can be caused by neutrophilic inflammation.

[0131] The methods disclosed herein may include administration of a "therapeutically effective amount" of an agent (e.g., an antigen-binding molecule, a polynucleotide, a construct, a vector, a host cell, or a pharmaceutical composition) to a subject. As used herein, the term "therapeutically effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to produce the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, and the mode of administration. Thus, it is not possible to specify an exact "effective amount". However, in any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0132] Disclosed herein is an antigen-binding molecule or composition as defined herein for use in the treatment or prevention of cancer or an inflammatory disease in a subject.

[0133] Disclosed herein is the use of an antigen-binding molecule or composition as defined herein in the manufacture of a medicament for treating or preventing cancer or an inflammatory disease in a subject.

[0134] The medicament may be administered with one or more further active pharmaceutical ingredients. In one embodiment, the medicament is to be administered with chemotherapy.

[0135] Disclosed herein is a method for detecting cancer or an inflammatory disease in a subject, comprising: contacting a sample obtained from the subject with an antigen-binding molecule defined herein, wherein an increase in the binding level of the antigen-binding molecule in the sample compared to a reference is indicative of cancer or an inflammatory disease.

[0136] Disclosed herein is a method for identifying a subject predisposed to cancer or an inflammatory disease, comprising contacting a sample obtained from the subject with an antigen-binding molecule defined herein, wherein an increase in the level of binding in the sample compared to a reference indicates that the subject is predisposed to cancer or an inflammatory disease.

[0137] In one embodiment, the sample is a cell, tissue, or blood sample.

[0138] In one embodiment, the antigen-binding molecule comprises a detectable label. The detectable label can be selected from the group consisting of a fluorescent label, a chemiluminescent label, an enzyme label and a radionuclide label. The detectable label can be selected from the group consisting of biotin, alkaline phosphatase, horseradish peroxidase, FITC, PE and Cy Dye. The detectable label can be detected in an assay selected from flow cytometry, tissue sections, immunofluorescence, immunocytochemistry or immunohistochemistry.

[0139] Disclosed herein are kits comprising an antigen-binding molecule as defined herein together with instructions for use, when used in the methods defined herein.

[0140] Throughout this specification and subsequent statements, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are understood to imply the inclusion of a specified integer or step or group of integers or steps but not to the exclusion of other integers or steps or groups of integers or steps.

[0141] Reference herein to any prior publication (or information derived therefrom) or any known matter is not intended to be, and should not be construed as, an admission or acknowledgement, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge within the scope of the endeavor to which this specification pertains.

[0142] Those skilled in the art will understand that the invention described herein is susceptible to changes and modifications other than those specifically described. It is to be understood that the invention encompasses all such changes and modifications within its spirit and scope. The invention also encompasses all of the steps, features, compositions and compounds mentioned or shown herein, individually or collectively, and encompasses any and all combinations of any two or more of said steps or features.

[0143] Certain embodiments of the present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the generality of the above. EXAMPLES

[0144] Example 1 Generation of humanized variants GR6A04 is a previously described murine IgG1κ monoclonal antibody developed against NSCLC, with demonstrated specificity for colorectal, gastric and breast cancer lines by flow cytometry and cancer tissue samples. GR6A04 exhibits cytotoxic activity in vitro and in vivo as an antibody-drug conjugate conjugated to monomethyl auristatin E (MMAE) and as a naked antibody.

[0145] During the process of converting mouse antibodies into humanized antibodies suitable for clinical therapeutic use, it was discovered that, unlike typical antibody humanization processes, many variants exhibited reduced specificity and / or function compared to the parental mouse and chimeric forms. Several key criteria were identified to rank the humanized variants resulting from in silico humanization optimization. Importantly, it was the aggregation and site-specificity readouts that allowed the elimination of problematic motifs and allowed for a rapid shortlisting of the final three molecules for further preclinical evaluation.

[0146] Six framework heavy chain sequences and six framework light chain sequences resulted from in silico humanization, thus resulting in 17 unique heavy / light chain combinations of humanized variants containing the CDRs of the murine antibody, GR6A04, an anti-N-glycosylated CEACAM5 / 6 therapeutic antibody (see Figures 1-3), which specifically binds to CEACAM5 / 6 when glycosylated at N256.

[0147] To screen these variants for retention of original functionality and downstream developability, four key criteria were identified and evaluated in the following order (see Figure 3A): (1) flow cytometry binding of three cancer lines (two binding and one non-binding); (2) aggregation tendency by SEC; (3) cell-based binding affinity with one cancer line; and (4) epitope site specificity through the use of a CEACAM6 mutant with an amino acid switch from N (asparagine) to A (alanine) at position 256 to mutate the N-glycan binding site.

[0148] Importantly, unlike most standard humanization protocols, where CDR grafting into human frameworks is relatively straightforward and screening is primarily based on binding and / or affinity, our workflow is unique in that it complements this with early developability screening by SEC agglutination measurement and also epitope site specificity. The epitope site specificity assay was noteworthy because it eliminated several promising candidates that performed well in flow cytometry binding and affinity testing. These two criteria provided crucial specificity and manufacturability information that would otherwise be overlooked in simple binding and / or affinity screening.

[0149] Through the elimination of clones at each stage of the screening process, three final molecules were identified that passed all four key criteria and thereby retained the most similar functionality to the original murine antibody: AH1 / AL1, LPH1 / LPL2, and AH1 / LPL2. Further evaluation with stably expressed humanized antibodies and in vitro efficacy of directly conjugated antibody-drug conjugates will allow for the creation of a shortlist of final lead molecules to progress towards clinical trials.

[0150] Example 2 Characterization of humanized variants Alignment Test Alignment of CEACAM5 and CEACAM6 reveals a conserved N256 glycosylation site (Figure 6).

[0151] Western Blot Test Western blot analysis using A549 lysates detected two bands at 75 and 180 kDa with AB1 (having heavy and light chain sequences shown in FIG. 5).

[0152] To determine whether the two bands seen in the Western blot are CEACAM5 and CEACAM6, A549 cells were subjected to single and double siRNA knockdown of CEACAM5 and CEACAM6. Western blot analysis of siRNA-treated cell lysates shows that AB1 does not bind to the 180 kDa band in CEACAM5 single or double knockdown samples. It is further shown that AB1 does not bind to the 75 kDa band in CEACAM6 single or double knockdown samples (Figure 7A). This indicates that the antigen of AB1 is CEACAM5 and CEACAM6. The efficiency of siRNA knockdown was monitored by gene expression analysis (Figure 7B). CEACAM5 was knocked down less efficiently than CEACAM6 in single knockdown samples as well as double knockdown samples. This explains why the commercially available anti-CEACAM5 antibody still picked up CEACAM5 in single and double knockdown samples, albeit with lower intensity (Figure 7a). A commercially available anti-CEACAM6 antibody detected CEACAM6, but with significantly lower intensity in single and double knockdowns (FIG. 7a).

[0153] It was further shown that N-glycosylation is important for the binding of AB1 to CEACAM5 and CEACAM6. A549 cell lysates were reduced, denatured, and treated with PNGase F to remove N-linked glycosylation (Figure 8). Cell lysates were probed with AB1 antibody and commercial anti-CEACAM5 and anti-CEACAM6 antibodies. Western blot analysis showed that AB1 binding to the 75 kDa band (CEACAM6) and 180 kDa band (CEACAM5) disappeared after reduction and PNGase F treatment. The commercial anti-CEACAM6 antibody detects the protein reduced to 75 kDa and detects CEACAM6 deglycosylated to a smaller size (37 kDa). The commercial anti-CEACAM5 antibody binds reduced CEACAM5 with lower intensity, but this also appears to be N-glycan dependent, as deglycosylated CEACAM5 was not detected at all. This indicates that N-glycosylation is important for the binding of AB1 to CEACAM5 or CEACAM6.

[0154] FACS binding analysis The N256 glycosylation dependency of AB1 recognition was demonstrated by flow cytometry analysis. Flow binding analysis (Figure 9) was performed on NCI-H1299 or NCI-H1299 overexpressing N256A mutant CEACAM5 or NCI-H1299 overexpressing N256A mutant CEACAM6 or NCI-H1299 overexpressing CEACAM5 or NCI-H1299 overexpressing CEACAM6.

[0155] The lung cancer cell line NCI-H1299, which does not express CEACAM5 or CEACAM6, was transfected with CEACAM5 or CEACAM6 sequences and mutant sequences of CEACAM5 or CEACAM6 in which the N-glycosylation position N256 was mutated (N256A, asparagine to alanine). This mutation abolishes N-glycosylation. The mean fluorescence intensity (MFI) was normalized (nMFI) to the human IgG1 isotype control (see table in Figure 9A and B). The flow cytometry binding of AB1 was compared with commercially available anti-CEACAM5 and anti-CEACAM6 antibodies and competitor antibodies. Competitor antibodies included CEACAM6-specific tinurilimab (Bayer), CEACAM5-specific tusamitamab (Sanofi), N-glycosylated CEACAM5 and N-glycosylated CEACAM6-specific NEO-201 (Precision Biologics) and biosimilars to CEACAM5 and CEACAM6-specific EBC-123. EBC-123 is a biosimilar to L-DOS47 (Helix Biopharma Corp), which is an original monomeric camelid single domain VHH 2A3 grafted onto a human Fc. AB1 was shown to bind to NCI-H1299 cells expressing CEACAM5 or CEACAM6 (Figure 9A), but not to cells expressing N256A mutant CEACAM5 or N256A mutant CEACAM6 (Figure 9B). This indicates that N256 glycosylation of CEACAM6 is important for AB1 binding to its antigen recognition motif. None of the CEACAM6-specific competitor antibodies (tinurilimab, EBC-123, and NEO-201) showed this dependency on N256 glycosylation of CEACAM6 (Figure 9A).

[0156] granulocyte binding The humanized leads are differentiated from the tested competitor compounds by their lower binding to granulocytes (CD15+ cell population) or B cells (CD19+ population) in peripheral blood or bone marrow cells (Figure 10).

[0157] Flow cytometric binding of AB1, AB2 and AB3 to different populations of primary peripheral blood cells (Figure 10 top) or primary bone marrow leukocytes (Figure 10 bottom) in comparison to competitor antibodies. Primary cells along with lysed red blood cells were incubated with different primary antibodies targeting CEACAM5 and CEACAM6. Cells were labeled with fluorescently labeled lineage markers for granulocytes (CD15+), T cells (CD3+) or B cells (CD19+). Mean fluorescence intensity (MFI, top) or normalized MFI (nMFI) (bottom) are shown for 3 donors / 2 replicates for primary peripheral blood cells and 1 donor / 3 replicates for primary human bone marrow leukocytes. CD3 binding by AB1, AB2 or AB3 + No binding was observed to T cells (i.e., negligible binding was observed). AB1, AB2, and AB3 were found to bind very little to peripheral blood granulocytes (CD15+ cell population) or B cells (CD 19+ population) compared to CEACAM6-specific antibodies such as tinurilimab (Bayer), NEO-201 (Precision Biologics), and EBC-123 (Helix Biopharma Corp). The CEACAM5-specific tusamitamab (Sanofi) shows no binding to human leukocytes at all.

[0158] Affinity testing Affinity measurements of AB1 were performed by Biolayer Interferometry using Octet 384-Red (Figure 11). In-house Avi-tagged CEACAM5, CEACAM6 and N256A mutant CEACAM5 and N256A mutant CEACAM6 were immobilized on Dip and Read SA biosensors. Antibody AB1 and competitor antibodies EBC-123 (Helix Biopharma Corp), NEO-201 (Precision Biologics) and Tusamitamab (Sanofi) are used as analytes.

[0159] Results show that AB1 binds to both CEACAM5 and CECAM6 with comparable affinity constants (KD) in the double-digit nanomolar range. The KD of AB1 was 10-100-fold lower for N256A mutant CEACAM5 and N256A mutant CEACAM6 compared to the CEACAM5 and CEACAM6 proteins. Competitor antibodies bound to both wild-type and mutant CEACAM5 and CEACAM6 with similar affinities.

[0160] Internal migration test The internalization of AB1 and competitor antibodies into antigen-positive and antigen-negative cell lines was evaluated (Figure 12). Figure 12A shows the internalization of AB1 in NCI-H1299 overexpressing CEACAM5 or CEACAM6 or N256A mutant CEACAM5 or N256A mutant CEACAM6. Figure 12B) shows the internalization of AB1 into NCI-H1299 and NCI-H1299 overexpressing CEACAM5 or CEACAM6, compared to IgG1 isotype control antibody and competitor antibodies specific for CEACAM5 and / or CEACAM6. Cells were incubated with antibody and anti-human FabFluor pH Red antibody. The antibody-FabFluor pH Red complex exhibits red fluorescence at low pH. Internalization was monitored for up to 24 hours and the integrated intensity was plotted. Figure 12A shows that AB1 is internalized in NCI-H1299 expressing CEACAM5 or CEACAM6, but not in NCI-H1299 or N256A mutant CEACAM5 or N256A mutant CEACAM6 expressing NCI-H1299. Figure 12B shows that CEACAM6-specific tinurilimab (Bayer) is nonspecifically internalized in NCI-H1299 cells. The CEACAM5-specific tusamitamab (Sanofi) is internalized only into NCI-H1299 overexpressing CEACAM5, whereas the CEACAM5- and CEACAM6-specific NEO-201 (Precision Biologics) and EBC-123 (Helix Biopharma Corp) are internalized at rates comparable to AB1 into NCI-H1299 overexpressing CEACAM5 or CEACAM6.

[0161] In vitro cell killing data (CellTiterGlo™ Viability Assay) In vitro cell functionality of AB3 was evaluated using the CellTiterGlo™ viability assay and IC after 72 hours of cell incubation. 50This was demonstrated by dose-response curves estimating the IC10 values. The left panel of FIG. 14A shows dose-response curves of AB3 with NCI-H1299 compared to NCI-H1299 overexpressing CEACAM6 (clonal cell line 12) or mutant N256A CEACAM6 (clonal cell line 19). The right panel of FIG. 14A shows dose-response curves of AB3 with NCI-H1299 compared to NCI-H1299 overexpressing CEACAM5 (clonal cell line 2F3) or mutant N256A CEACAM5 (clonal cell line 6). FIG. 14B shows the IC10 values ​​obtained with AB3, IgG1 isotype control-MMAE and free MMAE using NCI-H1299 cells and NCI-H1299 cells overexpressing CEACAM5, CEACAM6, N256A mutant CEACAM5 and N256A mutant CEACAM6. 50 Double-digit nanomolar IC 50 was obtained for NCI-H1299 cells expressing membrane-bound CEACAM5 or CEACAM6, whereas higher IC was obtained for NCI-H1299 cells expressing neither CEACAM5 nor CEACAM6 and for NCI-H1299 cells expressing N256A mutant CEACAM5 or N256A mutant CEACAM6. 50 The same high IC 50 was obtained with the IgG1-MMAE isotype control for all cell lines, demonstrating the specificity of AB3. Free MMAE had IC in the picomolar range. 50 , demonstrating the specificity of AB3. Figure 14C shows the IC for AB3 compared to the CEACAM5-specific competitor antibody tusamitamab (Sanofi) conjugated to MMAE. 50 AB3 targets a broader range of cancer indications due to its dual specificity for N256 glycosylated CEACAM5 and N256 glycosylated CEACAM6.

[0162] In vivo data AB3 is demonstrated in vivo in one gastric cancer xenograft model with SNU-16 (Figure 15) and two pancreatic models (Capan-1, Figure 16 and BxPC-3, Figure 17). Efficacy study endpoints were tumor growth inhibition (TGI) observed in the treatment group, tolerability (monitored by body weight measurement and monitoring of clinical signs), while in some studies AB1 antigen levels on tumor cells were also monitored throughout the study (data not shown).

[0163] SNU-16, Capan-1, and BxPC-3 cells express both AB1 antigens CEACAM5 and CEACAM6. Female Balb / c nude mice (n=10 / group) were implanted with SNU-16 cells, female NSG mice (n=10 / group) with Capan-1 cells, and female NOD-SCID mice (n=8 / group) with BxPC-3 cells. Mice were treated IV once (or once weekly x 3 for docetaxel). AB3 was given at 1, 3, or 5 mg / kg for SNU-16 and Capan-1 with only a single dose on day 0. The 3 mg / kg dose was omitted for the BxPC-3 model. AB3 produced TGI of 154%, 147%, and 114%, respectively, at day 35 in the SNU-16 model. Treatment with HuIgG1-MMAE (isotype control) at 5 or 1 mg / kg had a statistically significant (p=0.05) lower TGI (123% and 95%, respectively). Furthermore, only AB3-treated mice showed a complete response in a dose-dependent manner, with 6 / 10, 3 / 10 and 1 / 10 mice being tumor-free after a single dose of 5, 3 and 1 mg / kg AB3, respectively, whereas no tumor-free animals were observed after treatment with HuIgG1-MMAE alone (see FIG. 15).

[0164] In the Capan-1 model, AB3 resulted in a TGI of 110% on day 21. Treatment with HuIgG1-MMAE at 5, 3 or 1 mg / kg had a statistically significant (p=0.05) lower TGI (28%, 185 and -8%, respectively) (see Figure 16).

[0165] In the BxPC-3 model, AB3 resulted in a TGI of 107% and 21%, respectively, on day 21. Treatment with HuIgG1-MMAE at 5 mg / kg had a statistically significant (p=0.05) lower TGI of 6%. Treatment was well tolerated, with no weight loss observed (see FIG. 17).

[0166] Expression of CEACAM5, CEACAM6, N256A mutant CEACAM5 and N256A mutant CEACAM6 was demonstrated with respect to tumor growth (see FIG. 18). Female NOD-SCID mice were implanted with cells and tumor volumes were measured three times a week after randomization in two dimensions using calipers. Volumes were calculated in mm using the formula: V=(L×W×W) / 2. 3 where V is the tumor volume, L is the tumor length (the longest dimension of the tumor), and W is the tumor width (the longest tumor dimension perpendicular to L). Tumor volumes were measured up to 75 days and are presented in FIG. 18. No differences were observed in tumor onset or doubling time for NCI-H1299, NCI-H1299 overexpressing CEACAM6, or NCI-H1299 overexpressing N256A mutant CEACAM6. Early tumor onset was observed for NCI-H1299 overexpressing CEACAM5, while tumor onset was delayed by 35 days for NCI-H1299 overexpressing N256A mutant CEACAM5. Tumor doubling time after tumor onset was comparable for all cell lines.

[0167] In vivo efficacy was demonstrated in a lung cancer xenograft model using NCI-H1299 cell line and NCI-H1299 overexpressing CEACAM5 or CEACAM6 or N256A mutant CEACAM5 (see FIG. 19). Female NOD-SCID mice (n=6 / group) were implanted with NCI-H1299 cells or NCI-H1299 cells overexpressing N256A mutant CEACAM5 or CEACAM5 or CEACAM6 and treated once by IV. AB3 was given at 5mg / kg on day 0 with only a single dose. AB3 resulted in 32% and 28% TGI for antigen-negative cells NCI-H1299 and NCI-H1299 overexpressing N256A mutant CEACAM5, respectively, on day 21. AB3 resulted in a TGI of 109% and 110% for antigen-positive cells NCI-H1299 overexpressing CEACAM5 or CEACAM6, respectively (see FIG. 19). Treatment was well tolerated and no weight loss was observed in this case.

Claims

**Claim 1** An antigen-binding molecule comprising: (1) a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO: 1, the VH CDR2 amino acid sequence of SEQ ID NO: 2, and the VH CDR3 amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising the VL CDR1 amino acid sequence of SEQ ID NO: 4, the VL CDR2 amino acid sequence of SEQ ID NO: 5, and the VL CDR3 amino acid sequence of SEQ ID NO: 6; wherein the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10 or 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17 or 13, An antigen-binding molecule. **Claim 2** a) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; b) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13; c) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; or d) the VH defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and the VL defined in (1) comprises at least 90% sequence identity with at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13, The antigen-binding molecule according to claim 1. **Claim 3** a) VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 10 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 17 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; b) VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 10 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 10, and VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 17 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13; c) VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 7 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 17 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 17; or d) VH is distinguished from the VH amino acid sequence set forth in SEQ ID NO: 7 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VH amino acid sequence set forth in SEQ ID NO: 7, and VL is distinguished from the VL amino acid sequence set forth in SEQ ID NO: 13 by deletion, substitution or addition of one or more amino acids in at least one region other than the CDRs of the VL amino acid sequence set forth in SEQ ID NO: 13, The antigen-binding molecule according to claim 1.

4. a) VHFR1 that is distinguished from the VHFR1 amino acid sequence set forth in QVQLVQSGVEVKRPASVKVSCKAS (SEQ ID NO: 19) or QVQLVQSGAEVKRPASVKVSCKAS (SEQ ID NO: 20) by deletion, substitution or addition of one or more amino acids; b) VHFR2 that is distinguished from the VHFR2 amino acid sequence set forth in WVRQAPGQGLEWMA (SEQ ID NO: 21) or WVRQAPGQGLEWMG (SEQ ID NO: 22) by deletion, substitution or addition of one or more amino acids; c) VHFR3 that is distinguished from the VHFR3 amino acid sequence set forth in RVTLTTDSSTTTAYMELKSLQFDDAVYYCAR (SEQ ID NO: 23) or RVTMTTDTSTSTVYMELSSLRS EDTAVYYCAR (SEQ ID NO: 24) by deletion, substitution, or addition of one or more amino acids; d) VHFR4 that is distinguished from the VHFR4 amino acid sequence set forth in YWGQGTVLVTVSS (SEQ ID NO: 25) by deletion, substitution, or addition of one or more amino acids; e) VLFR1 that is distinguished from the VLFR1 amino acid sequence set forth in DIQMTQSPSSLSASVGD RVTITC (SEQ ID NO: 26) or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 27) by deletion, substitution, or addition of one or more amino acids; f) VLFR2 that is distinguished from the VLFR2 amino acid sequence set forth in WYQQKPGKAPKLLIY (SEQ ID NO: 28) or WYQLKPGQPPKLLLY (SEQ ID NO: 29) by deletion, substitution, or addition of one or more amino acids; g) VLFR3 that is distinguished from the VLFR3 amino acid sequence set forth in GVPSRFSGSGSGTDFTLTISSLQ PEDFATYYC (SEQ ID NO: 30) or GVPD RFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 31) by deletion, substitution, or addition of one or more amino acids; and / or h) VLFR4 that is distinguished from the VLFR4 amino acid sequence set forth in FGQG TKVEIK (SEQ ID NO: 32) or FGGG TKLEIK (SEQ ID NO: 33) by deletion, substitution, or addition of one or more amino acids, comprising the antigen-binding molecule according to claim 1.

5. a) QVQLVQSGX １ EVKKPGASVKVSCKAS (in the sequence, X １ is V or A) (SEQ ID NO: 34) VHFR1 amino acid sequence; b) WVRQAPGQGLEWMX ２ (In the sequence, X ２ is A or G) (SEQ ID NO: 35) the VHFR2 amino acid sequence; c) The VHFR3 amino acid sequence of RVTLTTDSSTTTAYMELKSLQFDDAVYYCAR (SEQ ID NO: 23) or RVTMTTDTSTSTVYMELSSLRS EDTAVYYCAR (SEQ ID NO: 24); d) The VHFR4 amino acid sequence of YWGQGTVLVTVSS (SEQ ID NO: 25); e) The VLFR1 amino acid sequence of DIQMTQSPSSLSASVGD RVTITC (SEQ ID NO: 26) or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 27); f) The VLFR2 amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 28) or WYQLKPGQPPKLLLY (SEQ ID NO: 29); g) The VLFR3 amino acid sequence of GVPSRFSSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 30) or GVPDRFSSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 31); and / or h) The VLFR4 amino acid sequence of FGQGTVEIK (SEQ ID NO: 32) or FGGGTKLEIK (SEQ ID NO: 33) comprising The antigen-binding molecule according to claim 1.

6. The antigen-binding molecule according to claim 1, comprising the VH amino acid sequence of SEQ ID NO: 10 or 7 and the VL amino acid sequence of SEQ ID NO: 17 or 13.

7. a) comprising the VH amino acid sequence of SEQ ID NO: 10 and the VL amino acid sequence of SEQ ID NO: 17; b) comprising the VH amino acid sequence of SEQ ID NO: 10 and the VL amino acid sequence of SEQ ID NO: 13; c) comprising the VH amino acid sequence of SEQ ID NO: 7 and the VL amino acid sequence of SEQ ID NO: 17; or d) comprising the VH amino acid sequence of SEQ ID NO: 7 and the VL amino acid sequence of SEQ ID NO: 13, The antigen-binding molecule according to claim 1.

8. The antigen-binding molecule according to claim 1, which is an antibody or an antigen-binding fragment thereof.

9. The antibody or antigen-binding fragment thereof according to claim 8, which is a full-length antibody, a substantially intact antibody, a Fab fragment, scFab, Fab', a single-chain variable fragment (scFv) or a one-armed antibody.

10. The antigen-binding molecule according to claim 1, comprising a light chain sequence having at least 70% sequence identity with SEQ ID NO: 84 and a heavy chain sequence having at least 70% sequence identity with SEQ ID NO:

85.

11. The antigen-binding molecule according to claim 1, which binds to CEACAM5 and / or CEACAM6.

12. The antigen-binding molecule according to claim 1, which is conjugated to a radioisotope or a cytotoxin.

13. The cytotoxin is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM-1), saporin, gemcitabine, irinotecan, etoposide, vinblastine, pemetrexed, docetaxel, paclitaxel, platinum agents (e.g., cisplatin, oxaliplatin and carboplatin), vinorelbine, capecitabine, mitoxantrone, ixabepilone, eribulin, 5-fluorouracil, trifluridine and tipiracil, The antigen-binding molecule according to claim 12.

14. The antigen-binding molecule according to claim 1, which selectively binds to gefitinib-resistant lung cancer cells, osimertinib-resistant lung cancer cells, non-small cell lung cancer cells, breast cancer cells, pancreatic cancer cells, gastric (or stomach) cancer cells, small intestine cancer cells, esophageal cancer cells or colorectal cancer cells.

15. An isolated polynucleotide comprising a nucleic acid sequence encoding the antigen-binding molecule according to any one of claims 1 to 14.

16. A construct comprising the polynucleotide according to claim 15 operably linked to one or more control sequences.

17. A host cell containing the construct according to claim 16.

18. A composition comprising the antigen-binding molecule according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. The composition according to claim 18 for use as a medicament.

20. A composition comprising the antigen-binding molecule according to any one of claims 1 to 14 for treating or preventing cancer or an inflammatory disease in a subject.

21. The composition according to claim 20, wherein the cancer is selected from the group consisting of gefitinib-resistant lung cancer, osimertinib-resistant lung cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, gastric (or stomach) cancer, small intestine cancer, esophageal cancer and colorectal cancer.

22. The composition according to claim 20, wherein the inflammatory disease is Crohn's disease or asthma.

23. Use of the antigen-binding molecule according to any one of claims 1 to 14 in the manufacture of a medicament for treating or preventing cancer.

24. A method for detecting cancer in a subject, comprising contacting a sample obtained from the subject with the antigen-binding molecule according to any one of claims 1 to 14, wherein an increase in the binding level of the antigen-binding molecule in the sample as compared to a reference indicates cancer.

25. A method for identifying a subject susceptible to cancer, comprising contacting a sample obtained from the subject with the antigen-binding molecule according to any one of claims 1 to 14, wherein an increase in the binding level in the sample as compared to a reference indicates that the subject is susceptible to cancer.

26. The method according to claim 24, wherein the antigen-binding molecule comprises a detectable label.

27. The method according to claim 25, wherein the antigen-binding molecule comprises a detectable label.

28. A kit for use in the method according to claim 24, comprising the antigen-binding molecule according to any one of claims 1 to 14 together with instructions for use.

29. A kit for use in the method according to claim 25, comprising the antigen-binding molecule according to any one of claims 1 to 14 together with instructions for use.

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