Anti-CEA antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human CEA, pharmaceutical compositions comprising the antibodies, and uses of the anti-CEA antibodies or compositions to treat diseases such as cancer.

Description

[Technical field]

[0001] Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human CEA and methods of use for treating cancer. [Background technology]

[0002] Carcinoembryonic antigen (CEA, also known as CEACAM5 or CD66e) is a glycoprotein with a molecular weight of approximately 70-100 kDa depending on the amount of glycosylation present. The presence of CEA in human adenocarcinomas as a cancer-specific antigen was first reported by Gold et al., J. Exp. Med., 121, 439 (1965). CEA is normally expressed in various glandular epithelial tissues, such as the gastrointestinal, respiratory, and urogenital tracts, and is thought to be localized to the apical surface of cells (Hammarstrom, S. Semin. Cancer Biol. 9, 67-81 (1999)). For example, it is found in columnar epithelium and goblet cells of the colon (Fraengsmyr et al., Tumor Biol. 20:277-292 (1999)). In tumors generated from these tissue types, CEA expression increases from the apical membrane to the cell surface and enters the bloodstream when removed from the cell surface (Hammarstrom, S. Semin. Cancer Biol. 9, 67-81; (1999); see also Fraengsmyr et al., Tumor Biol. 20:277-292 (1999)). Overexpression of CEA has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatocellular, breast, and thyroid cancers. Thus, CEA is useful as a diagnostic tumor marker to determine elevated CEA levels in the blood of cancer patients in cancer prognosis and management (Chevinsky, AH (1991) Semin. Surg. Oncol. 7, 162-166; Shively, JE et al., (1985) Crit. Rev. Oncol. Hematol. 2, 355-399).

[0003] CEA is considered a tumor-associated antigen useful for targeted therapy (Kuroki M, et al., (2002) Anticancer Res 22:4255-64). One approach has been the generation of retroviral constructs displaying anti-CEA scFv and delivering the nitric oxide synthase (iNOS) gene to cancer cells expressing CEA (Kuroki M. et al., (2000) Anticancer Res. 20(6A):4067-71). Another approach has been to attach radioisotopes to anti-CEA antibodies and demonstrate that radiation is specifically directed to CEA-expressing tumors (Wilkinson et al., PNAS USA 98, 10256-60 (2001); Goldenberg et al., Am. J. Gastroenterol., 86: 1392-1403 (1991); Olafsen T. et al., Protein Engineering, Design & Selection, 17, 21-27, (2004); Meyer et al., Clin. Cancer Res. 15: 4484-4492 (2009); Sharkey et al., J. Nucl. Med. 46: 620-633 (2005)). The radioisotope approach has also been extended to anti-CEA antibody-drug conjugates (ADCs). For example, Shinmi et al. reported an anti-CEA antibody conjugated to monomethylauristatin E (MMAE) (Shinmi et al., Cancer Med. 6(4):798-808(2017)).

[0004] However, one of the problems of anti-CEA antibodies is cross-reactivity. CEA shows high homology with other CEACAM family members. For example, human CEA shows 84% ​​homology with CEACAM6, 77% homology with CECAM8, and 73% homology with CEACAM1. The present disclosure provides an anti-CEA antibody specific for CEA. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure is directed to anti-CEA antibodies and antigen-binding fragments thereof. The present disclosure includes the following embodiments:

[0006] An anti-CEA antibody or antigen-binding fragment thereof, comprising an antibody or binding fragment thereof that specifically binds to human CEA at amino acids 596 to 674 of SEQ ID NO:52.

[0007] The anti-CEA antibody or antigen-binding fragment thereof, which does not bind to other CEACAM family members.

[0008] (i) a heavy chain variable region comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 7, (b) an HCDR2 of SEQ ID NO: 8, and (c) an HCDR3 of SEQ ID NO: 9, and (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 10, (e) an LCDR2 of SEQ ID NO: 11, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO:24, (b) an HCDR2 of SEQ ID NO:25, and (c) an HCDR3 of SEQ ID NO:26, and (d) an LCDR1 of SEQ ID NO:27, (e) an LCDR2 of SEQ ID NO:28, and (f) an LCDR3 of SEQ ID NO:23; or (iii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 41, (b) an HCDR2 of SEQ ID NO: 42, and (c) an HCDR3 of SEQ ID NO: 43, and (d) an LCDR1 of SEQ ID NO: 44, (e) an LCDR2 of SEQ ID NO: 45, and (f) an LCDR3 of SEQ ID NO: 40. The anti-CEA antibody or antigen-binding fragment of claim 1, comprising:

[0009] (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 14, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 31, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 32; or (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 48, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 49. The anti-CEA antibody or the antigen-binding fragment thereof.

[0010] The anti-CEA antibody or antigen-binding fragment thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 14, 15, 31, 32, 48, or 49 have been inserted, deleted, or substituted.

[0011] (i) a heavy chain variable region (VH) comprising SEQ ID NO: 14, and a light chain variable region (VL) comprising SEQ ID NO: 15; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 31, and a light chain variable region (VL) comprising SEQ ID NO: 32; or (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 48, and a light chain variable region (VL) comprising SEQ ID NO: 49 The anti-CEA antibody or the antigen-binding fragment thereof.

[0012] The anti-CEA antibody or the antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0013] The anti-CEA antibody or the antigen-binding fragment thereof, which has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0014] The anti-CEA antibody or antigen-binding fragment thereof, which has reduced glycosylation, no glycosylation or is hypofucosylated.

[0015] The anti-CEA antibody or antigen-binding fragment thereof, which comprises increased bisecting GlcNac structures.

[0016] The anti-CEA antibody or antigen-binding fragment thereof, wherein the Fc domain is IgG1.

[0017] The anti-CEA antibody or antigen-binding fragment thereof, wherein the antibody is conjugated to a toxin.

[0018] A pharmaceutical composition comprising the anti-CEA antibody or antigen-binding fragment thereof, said pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0019] A method of treating cancer comprising administering to a patient in need thereof an effective amount of said antibody or said antigen-binding fragment.

[0020] The method, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0021] The above methods, wherein the antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

[0022] The method, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

[0023] The method, wherein the therapeutic agent is an anti-PD1 antibody or an anti-PDL1 antibody.

[0024] An isolated nucleic acid encoding the anti-CEA antibody or antigen-binding fragment.

[0025] A vector comprising the nucleic acid.

[0026] A host cell comprising said nucleic acid or said vector.

[0027] A method for producing said anti-CEA antibody or said antigen-binding fragment thereof comprising culturing said host cell and recovering the antibody or antigen-binding fragment from the culture.

[0028] In one embodiment, the anti-CEA antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:40.

[0029] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising one or more complementarity determining regions (HCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43; and / or (b) a light chain variable region comprising one or more complementarity determining regions (LCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:6, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:40.

[0030] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs): HCDR1 comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:24, or SEQ ID NO:41; HCDR2 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:25, or SEQ ID NO:42; and HCDR3 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:26, or SEQ ID NO:43; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs): LCDR1 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:27, or SEQ ID NO:44; LCDR2 comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:28, or SEQ ID NO:45; and LCDR3 comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:23, or SEQ ID NO:40.

[0031] In another embodiment, the anti-CEA antibody or antigen-binding fragment thereof comprises (a) three complementarity determining regions, HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and HCDR3 comprising the amino acid sequence of SEQ ID NO:9, or three complementarity determining regions, HCDR1 comprising the amino acid sequence of SEQ ID NO:24, HCDR2 comprising the amino acid sequence of SEQ ID NO:25, and HCDR3 comprising the amino acid sequence of SEQ ID NO:26, or three complementarity determining regions (HCDR1 comprising the amino acid sequence of SEQ ID NO:41, HCDR2 comprising the amino acid sequence of SEQ ID NO:42, and HCDR3 comprising the amino acid sequence of SEQ ID NO:43) (HCDR1 comprising the amino acid sequence of SEQ ID NO:41, HCDR2 comprising the amino acid sequence of SEQ ID NO:42, and HCDR3 comprising the amino acid sequence of SEQ ID NO:43). ), and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs): LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or three complementarity determining regions (LCDRs): LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, LCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 23, or three complementarity determining regions (LCDRs): LCDR1 comprising the amino acid sequence of SEQ ID NO: 44, LCDR2 comprising the amino acid sequence of SEQ ID NO: 45, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 40.

[0032] In another embodiment, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 7, (b) an HCDR2 of SEQ ID NO: 8, (c) an HCDR3 of SEQ ID NO: 9, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 10, (e) an LCDR2 of SEQ ID NO: 11, and (f) an LCDR3 of SEQ ID NO: 6.

[0033] In another embodiment, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 23.

[0034] In yet another embodiment, the anti-CEA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 41, (b) an HCDR2 of SEQ ID NO: 42, (c) an HCDR3 of SEQ ID NO: 43, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 44, (e) an LCDR2 of SEQ ID NO: 45, and (f) an LCDR3 of SEQ ID NO: 40.

[0035] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO:14, SEQ ID NO:31, or SEQ ID NO:48, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:14, SEQ ID NO:31, or SEQ ID NO:48, and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:32, or SEQ ID NO:49, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:15, SEQ ID NO:32, or SEQ ID NO:49.

[0036] In another embodiment, an anti-CEA antibody or antigen-binding fragment thereof of the present disclosure comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 31, or SEQ ID NO: 48, or an amino acid sequence that contains one, two, or three amino acid substitutions in the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 31, or SEQ ID NO: 48, and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 32, or SEQ ID NO: 49, or an amino acid sequence that contains one, two, three, four, or five amino acid substitutions in the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 32, or SEQ ID NO: 49. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0037] In one embodiment, the antibodies of the disclosure are of the IgG1, IgG2, IgG3, or IgG4 isotype. In more specific embodiments, the antibodies of the disclosure comprise a wild-type human IgG1 (also called human IgG1wt or huIgG1) or IgG2 Fc domain. In another embodiment, the antibodies of the disclosure comprise a human IgG4 Fc domain with S228P and / or R409K substitutions (according to the EU numbering system).

[0038] In one embodiment, the anti-CEA antibody of the present disclosure is administered at a concentration of 1×10 -6 M~1×10 -10 Binding affinity of M (K D In another embodiment, the antibody of the disclosure binds to CEA at about 1×10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, or approximately 1 x 10 -10 Binding affinity of M (K D ) binds to CEA.

[0039] In another embodiment, the anti-human CEA antibodies of the present disclosure exhibit cross-species binding activity to cynomolgus CEA.

[0040] In one embodiment, the antibodies of the present disclosure have potent Fc-mediated effector function: the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing CEA.

[0041] The present disclosure relates to an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of an anti-CEA antibody or antigen-binding fragment. In one embodiment, the isolated nucleic acid comprises a VH nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 50, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 50, and encodes a VH region of an antibody or antigen-binding fragment of the present disclosure. Alternatively or additionally, the isolated nucleic acid comprises a VL nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 34, or SEQ ID NO: 51, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17, SEQ ID NO: 34, or SEQ ID NO: 51, and encodes a VL region of an antibody or antigen-binding fragment of the present disclosure.

[0042] In another aspect, the disclosure relates to a pharmaceutical composition comprising an anti-CEA antibody or antigen-binding fragment thereof, and optionally a pharma- ceutically acceptable excipient.

[0043] In yet another aspect, the present disclosure relates to a method for treating a disease in a subject, comprising administering a therapeutically effective amount of an anti-CEA antibody or antigen-binding fragment thereof, or an anti-CEA antibody pharmaceutical composition to a subject in need thereof. In another embodiment, the disease treated by the antibody or antigen-binding fragment is cancer.

[0044] The present disclosure relates to the use of anti-CEA antibodies or antigen-binding fragments thereof, or anti-CEA antibody pharmaceutical compositions to treat diseases such as cancer. [Brief description of the drawings]

[0045] [Figure 1]Schematic diagram of shedding CEA (sCEA), chimeric CEA (CHIM), CEACAM6, and CEA mutant (CEA-v). In CEA, domains N, A1, Bl, A2, B2, A3, B3, and the GPI linker (GPI) are labeled, and in CEACAM6, domains N', A', and B' are labeled. [Figure 2A] Phylogenetic tree of VH region of anti-CEA domain B3 antibody. VH sequences of candidate anti-CEA antibodies were aligned using DNASTAR's Megaalign™ software. Sequence homology was displayed in the phylogenetic tree. [Figure 2B] Phylogenetic tree of VL region of anti-CEA domain B3 antibody. VL sequences of candidate anti-CEA antibodies were aligned using DNASTAR's Megaalign™ software. Sequence homology was displayed in the phylogenetic tree. [Diagram 3] A shows the affinity measurement of purified mouse anti-CEA antibody BGA13 on a chimeric construct (CHIM) by surface plasmon resonance (SPR) (B) shows the binding profile of BGA13 by antigen ELISA. [Figure 4A] Figure 1 shows the effect of soluble CEA (sCEA) on CEA antibody binding to MKN45 cells.Figure 2 shows the binding profile of domain B3 antibody in the presence or absence of soluble CEA (sCEA). [Figure 4B] Figure 1 shows the effect of soluble CEA (sCEA) on CEA antibody binding to MKN45 cells.The antibody binding profile in A is shown as a histogram. [Diagram 5] FIG. 5A-B show randomization sites for generating an antibody library for affinity maturation of the light chain CDR (LCDR) region (A) and heavy chain CDR (HCDR) region (B) of the humanized BGA13 antibody. [Figure 6] FIG. 1 shows the amino acid changes in the BGA13 light chain CDR regions after four rounds of selection. [Figure 7] FIG. 1 shows binding of affinity matured humanized BGA13 mutants to LOVO cells by flow cytometry. [Figure 8] FIG. 13 shows binding of optimized humanized BGA113 variants to MKN45 cells by flow cytometry. [Figure 9] A and B demonstrate the lack of off-target binding of antibody BGA113K to various CEACAM family members by flow cytometry (A) and antigen ELISA (B). [Figure 10] FIG. 1 shows the effect of soluble CEA on BGA113K binding to CEA-expressing MKN45 cells in the presence of various concentrations of soluble CEA. [Figure 11] FIG. 1 demonstrates that antibody BGA113 kills cells by ADCC in vitro. [Figure 12] FIG. 1 shows the reduction in tumor volume in a mouse cancer model when treated with BGA113 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0047] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include references to their corresponding plurals unless the context clearly dictates otherwise.

[0048] The term "or" is used to mean and is used synonymously with the term "and / or," unless context clearly dictates otherwise.

[0049] The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0050] The term "carcinoembryonic antigen" or "CEA" refers to a glycoprotein of approximately 70-100 kDa. CEA is also known as CEACAM5 or CD66e. The amino acid sequence of human CEA (SEQ ID NO:52) can also be found under Accession No. P06731 or NM_004363.2.

[0051] The terms "administration", "administering", "treating" and "treatment" as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell as well as contact of a reagent to a fluid, which in turn contacts the cell. The terms "administration" and "treatment" also refer to in vitro and ex vivo treatment, e.g., of a cell with a reagent, diagnostic, binding compound, or with another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human. In one aspect, treating any disease or disorder refers to alleviating the disease or disorder (i.e., delaying or preventing or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat", "treating", or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat", "treating", or "treatment" refers to modulating a disease or disorder, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. In yet another embodiment, "treat", "treating", or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0052] The term "subject" in the context of this disclosure is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein).

[0053] The term "affinity" as used herein refers to the strength of interaction between an antibody and an antigen. Within an antigen, the variable region of an antibody interacts with the antigen at many sites through non-covalent forces. In general, the more interactions, the stronger the affinity.

[0054] The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers that contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0055] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0056] In some embodiments, the anti-CEA antibody comprises at least one antigen binding site, at least the variable region. In some embodiments, the anti-CEA antibody comprises an antigen binding fragment from a CEA antibody described herein. In some embodiments, the anti-CEA antibody is isolated or recombinant.

[0057] The term "monoclonal antibody" or "mAb" or "Mab" as used herein refers to a population of substantially homogenous antibodies, i.e., the antibody molecules in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences in the variable domains, particularly in the complementarity determining regions (CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogenous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; US Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof, such as IgG1, IgG2, IgG3, IgG4, etc. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High titer monoclonal antibodies can be obtained by in vivo production by intraperitoneally injecting cells from individual hybridomas into mice (such as primed Balb / c mice) to produce ascites fluid containing high concentrations of the desired antibodies. Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluid or culture supernatants using column chromatography methods well known to those skilled in the art.

[0058] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100-110 or more amino acids in length that is primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is primarily responsible for effector function. Human light chains are typically classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and the antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids.

[0059] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally identical in primary sequence.

[0060] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity determining regions (CDRs)", located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions and allow binding to a specific epitope. Generally, from N-terminus to C-terminus, both light chain variable domains and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering scheme)).Definitions of antigen-binding sites are also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. In IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (Kabat numbering).In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0061] The term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. The hypervariable region comprises amino acid residues from the "CDRs" (e.g., LCDR1, LCDR2, and LCDR3 of the light chain variable domain and HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain). See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., which define antibody CDR regions by sequence. See also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, which define antibody CDR regions by structure. The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0062] Unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv); nanobodies and antibodies formed from antibody fragments.

[0063] As used herein, an antibody "specifically binds" to a target protein means that the antibody exhibits preferential binding to its target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody "specifically binds" or "selectively binds" is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, and refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, such as a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as compared to background levels, and does not specifically bind in significant amounts to other antigens present in the sample. In one aspect, under specified immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding, and does not specifically bind in significant amounts to other antigens present in the sample.

[0064] As used herein, the term "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0065] The term "humanized" or "humanized antibody" refers to forms of antibodies that contain sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). When necessary to distinguish the humanized antibody from a rodent parent antibody, the prefixes "hum," "hu," "Hu," or "h" are added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences as the rodent parent antibody, but can contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0066] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity determined with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. Corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that include variable regions. Sequence identity can be determined using methods described herein, such as aligning the two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0067] “Equilibrium dissociation constant (K D The term "dissociation rate constant (kd, time -1 ) as the binding rate constant (ka, time -1 , M -1 The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than M or 10 -8 Less than m, e.g., about 10-9 Less than M or 10 -10 M or less, and in some embodiments, about 10 -11 Less than M, 10 -12 Less than M or 10 -13 M

[0068] The term "cancer" or "tumor" as used herein has the broadest meaning as understood in the art and refers to a physiological condition in a mammal that is typically characterized by unregulated cell proliferation. In the context of this disclosure, cancer is not limited to a particular type or location.

[0069] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the replacement of an original amino acid with a new amino acid that does not substantially alter the chemical, physical and / or functional properties of the antibody or fragment, e.g., its binding affinity to CEA. In particular, common conservative changes of amino acids are well known in the art.

[0070] The term "knob-into-hole" technology as used herein refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide (at the interface where they interact). For example, knob-into-hole is used to engineer the Fc:Fc binding interface of an antibody, C L :C H I interface, or V H / V L(See, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, the knob-into-hole ensures the correct pairing of two different heavy chains together during antibody production. For example, antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain that pairs with a similar or different light chain variable domain. The knob-into-hole technology can also be used with the VH or VL regions to ensure correct pairing.

[0071] The term "knob," as used herein, in the context of "knob-into-hole" technology, refers to an amino acid change that introduces a knob into a polypeptide at the interface where it interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0072] The term "hole," as used herein, in the context of "knob-into-hole," refers to an amino acid change that introduces a socket or hole in a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0073] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as a value for initiating searches to find longer HSPs that contain them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from its maximum achieved value, or when the cumulative score becomes zero or below zero due to the accumulation of one or more negative scoring residue alignments, or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.For amino acid sequences, the BLAST program uses as defaults a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) of 50. Alignment (B), expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0074] BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence when the minimum total probability when comparing test nucleic acid with reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0075] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program of the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0076] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0077] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0078] In some embodiments, the present disclosure provides compositions, e.g., pharma- ceutically acceptable compositions, comprising an anti-CEA antibody described herein, formulated with at least one pharma- ceutically acceptable excipient. As used herein, the term "pharma-ceutically acceptable excipient" includes any and all solvents, dispersion media, isotonicity agents, and absorption delaying agents, etc., that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0079] The compositions disclosed herein can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and suppositories. The suitable form depends on the intended mode of administration and therapeutic application. Exemplary suitable compositions are in the form of injection or infusion solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0080] The term "therapeutically effective amount" as used herein refers to an amount of an antibody that, when administered to a subject for treating a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to achieve such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary with the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case may be apparent to one skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combination for effective treatment of a disease, disorder, or condition.

[0081] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in this disclosure. Such administration includes the simultaneous administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes the simultaneous administration in multiple containers or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration also includes the sequential use of various therapeutic agents, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in treating the condition or disorder described herein.

[0082] As used herein, the phrase "in combination with" means that the anti-CEA antibody is administered to the subject simultaneously with, immediately before, or immediately after the administration of the additional therapeutic agent. In certain embodiments, the anti-CEA antibody is administered as a co-formulation with the additional therapeutic agent.

[0083] The term "toxin" or "payload" or "cytotoxic agent" is used herein as a molecule that inhibits or reduces the expression of a molecule in a cell, the function of a cell, induces apoptosis of a cell, and / or causes the destruction of a cell. The term includes radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoids, pyrrolobenzodiazepines (PBDs), ricin, ricin A chain, comblastatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, cyclosporine ... Examples of toxins include, but are not limited to, sarcin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogenin, letostrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0084] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present disclosure provides anti-CEA antibodies and antigen-binding fragments thereof. Additionally, the present disclosure provides antibodies that have desirable pharmacokinetic properties and other desirable attributes and thus can be used to reduce the likelihood of cancer or to treat cancer. The present disclosure further provides pharmaceutical compositions comprising the antibodies, as well as methods of making and using such pharmaceutical compositions, for the prevention and treatment of cancer and related disorders.

[0085] Anti-CEA antibody The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CEA. The antibody or antigen-binding fragment of the present disclosure includes, but is not limited to, an antibody or antigen-binding fragment thereof generated as described below.

[0086] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having the amino acid sequence of SEQ ID NO: 14, 31 or 48 (Table 1). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibody or antigen-binding fragment comprises an HCDR having the amino acid sequence of any one of the HCDRs listed in Table 1. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibody comprises one, two, three or more HCDRs (or consists of one, two, three or more HCDRs) having the amino acid sequence of any of the HCDRs listed in Table 1.

[0087] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise a VL domain having the amino acid sequence of SEQ ID NO: 15, 32 or 49 (Table 1). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise an LCDR having the amino acid sequence of any one of the LCDRs listed in Table 1. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CEA, wherein the antibodies or antigen-binding fragments comprise (or consist of) one, two, three or more LCDRs having the amino acid sequence of any of the LCDRs listed in Table 1.

[0088] Other antibodies or antigen-binding fragments of the present disclosure contain amino acids in the CDR regions that are altered but have at least 60%, 70%, 80%, 90%, 95% or 99% identity with the CDR regions disclosed in Table 1. In some embodiments, this includes amino acid changes in which no more than 1, 2, 3, 4 or 5 amino acids are altered in the CDR regions when compared to the CDR regions shown in the sequences set forth in Table 1.

[0089] Other antibodies of the disclosure include those in which the amino acids, or the nucleic acids encoding the amino acids, have been altered but have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences set forth in Table 1. In some embodiments, this includes changes in the amino acid sequence in which no more than 1, 2, 3, 4 or 5 amino acids are altered in the variable regions while retaining substantially the same therapeutic activity when compared to the variable regions set forth in the sequences set forth in Table 1.

[0090] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds CEA, which can be optimized for expression in mammalian cells.

[0091] [Table 1-1]

[0092] [Table 1-2]

[0093] [Table 1-3]

[0094] [Table 1-4]

[0095] [Table 1-5]

[0096] [Table 1-6]

[0097] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CEA. In certain embodiments, the antibodies and antigen-binding fragments can bind to the same epitope of CEA.

[0098] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CEA antibodies described in Table 1. Thus, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in a binding assay. The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CEA demonstrates that the test antibody can compete with that antibody or its antigen-binding fragment for binding to CEA. Without being bound to any one theory, such antibodies can bind to the same or related (e.g., structurally similar or spatially proximal) epitope on CEA as the competing antibody or its antigen-binding fragment. In certain embodiments, an antibody that binds to the same epitope on CEA as an antibody or its antigen-binding fragment of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0099] Linker It is also understood that the domains and / or regions of the polypeptide chains of an antibody can be separated by linker regions of various lengths. In some embodiments, the antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by a linker region. For example, a linker region such as VL1-CL-(linker)VH2-CH1 can contain a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see US2009 / 0155275).

[0100] In different embodiments, a linker can be used to attach the compound between the toxin and the antibody, and in some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the toxin from the antibody in the intracellular environment. In still other embodiments, the linker unit is not cleavable, such that the toxin is released, for example, by antibody degradation. The linker can be, without limitation, a cleavable linker, a non-cleavable linker, a hydrophilic linker, a precharged linker, and a dicarboxylic acid-based linker.

[0101] Dimerization specific amino acids In one embodiment, the antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change results in "knobs into holes" interactions and increases the population of correct antibodies. The dimerization-specific amino acid can be in the CH1 domain or the CL domain or a combination thereof. The dimerization-specific amino acid is used to pair the CH1 domain with another CH1 domain (CH1-CH1) and the CL domain with another CL domain (CL-CL) and can be found at least in the disclosures of WO2014082179, WO2015181805 family and WO2017059551. The dimerization-specific amino acid can be in the Fc domain or combined with the dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides an antibody comprising at least one dimerization-specific amino acid pair.

[0102] Further modifications to the Fc region framework In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has a modified affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is modified can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0103] In another embodiment, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0104] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in Publication WO94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of the antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and the kappa isotype. The allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses, as well as the constant regions of the light chains of the kappa isotype as described by Jefferis et al., MAb.1:332-338 (2009).

[0105] In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for the Fcγ receptor by modifying one or more amino acids. This approach is described, for example, in publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and mutants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0106] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks or has reduced glycosylation). The glycosylation can be altered to, for example, increase the affinity of the antibody for an "antigen". Such carbohydrate modification can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0107] Additionally or alternatively, antibodies can be made with modified types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such modified glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with a modified glycosylation pathway. Cells with modified glycosylation pathways have been described in the art and can be used as host cells to express recombinant antibodies to produce antibodies with modified glycosylation. For example, EP 1,176,195 by Hang et al. describes cell lines with a functionally disrupted FUT8 gene encoding fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit an increase in bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0108] In another embodiment, when reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). However, native IgG4 has been found to be less stable under stress conditions such as in acidic buffer or at elevated temperature (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al., 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably binding the antibody to an IgG4 Fc engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form half antibodies, which generates bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al., 2007 Science, 317: 1554-157). Mutation of serine to proline at position 228 (EU numbering system) appeared to be inhibitory to IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al., 2002 Immunol, 105: 9-19).Some of the amino acid residues in the hinge and gamma Fc regions have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al, 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev immunol, 25:21-50). Furthermore, some IgG4 isoforms occurring rarely in the human population can also induce different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To generate antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce several modifications. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of US Patent No. 8,735,553 by Li et al.

[0109] Antibody production Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including but not limited to recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0110] The disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding a heavy or light chain variable region or segment comprising a complementarity determining region as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 33, or SEQ ID NO: 50. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 34, or SEQ ID NO: 51.

[0111] The polynucleotides of the present disclosure can encode the variable region sequences of the anti-CEA antibodies. The polynucleotides of the present disclosure can also encode both the variable and constant regions of the antibodies. Some of the polynucleotide sequences encode polypeptides that include both the heavy and light chain variable regions of the exemplified anti-CEA antibodies.

[0112] Also provided in the present disclosure are expression vectors and host cells for producing anti-CEA antibodies. The choice of expression vector depends on the intended host cell in which the vector is expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-CEA antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except when under the control of an inducing condition. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population in favor of coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of anti-CEA antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be increased by including enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0113] Host cells for carrying and expressing anti-CEA antibody chains may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be made, which typically contain expression control sequences compatible with the host cell (e.g., origin of replication). In addition, there will be any number of different known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express anti-CEA antibodies. Insect cells in combination with baculovirus vectors can also be used.

[0114] In other embodiments, mammalian host cells are used to express and produce the anti-CEA antibodies of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines with exogenous expression vectors. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters, and enhancers (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoter (such as human immediate early CMV promoter), constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0115] Detection and Diagnostic Methods The antibody or antigen-binding fragment of the present disclosure is useful in various applications, including, but not limited to, a method for detecting CEA. In one embodiment, the antibody or antigen-binding fragment is useful for detecting the presence of CEA in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues. In other embodiments, such tissues include normal tissues and / or cancerous tissues that express CEA at higher levels compared to other tissues.

[0116] In one embodiment, the present disclosure provides a method for detecting the presence of CEA in biological sample.In a particular embodiment, the method comprises contacting biological sample with anti-CEA antibody under conditions that allow antibody to bind to antigen, and detecting whether complex is formed between antibody and antigen.Biological sample can include, but is not limited to, urine, tissue, sputum, or blood sample.

[0117] Also included is a method for diagnosing a disorder associated with CEA expression. In a particular embodiment, the method comprises contacting a test cell with an anti-CEA antibody, determining the expression level (either quantitatively or qualitatively) of CEA expressed by the test cell by detecting the binding of the anti-CEA antibody to a CEA polypeptide, and comparing the expression level by the test cell with the CEA expression level in a control cell (e.g., a normal cell or a non-CEA-expressing cell of the same tissue origin as the test cell), wherein a higher level of CEA expression in the test cell compared to the control cell indicates the presence of a disorder associated with CEA expression.

[0118] Treatment method The antibodies or antigen-binding fragments of the present disclosure are useful in a variety of applications, including, but not limited to, methods for treating a CEA-associated disorder or disease. In one embodiment, the CEA-associated disorder or disease is cancer.

[0119] In one embodiment, the present disclosure provides a method for treating cancer.In a particular embodiment, the method comprises administering an effective amount of anti-CEA antibody or antigen-binding fragment to a patient in need thereof.Cancer includes, but is not limited to, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0120] The antibodies or antigen-binding fragments disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, including localized treatment, intralesional administration, if desired. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various times, bolus administration, and pulse infusion.

[0121] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or any dosages empirically / clinically determined to be appropriate, by any route.

[0122] The appropriate dosage of the antibody or antigen-binding fragment of the present disclosure for preventing or treating a disease will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 100 mg / kg of antibody may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several or more days depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives from about 2 to about 20, or for example about 6, doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0123] Combination therapy In one embodiment, the anti-CEA antibodies of the present disclosure can be used in combination with other therapeutic agents. Other therapeutic agents that can be used with the anti-CEA antibodies of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents (e.g., Abraxane®), docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20 targeting agents (e.g., rituximab, opioids, tumumab, RO5072759, LFB-R603), CD52 targeting agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomine, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19 targeting agents (e.g., MEDI-551, MOR208 ), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, Polo-like kinase 1 inhibitors (e.g., BI 672).

[0124] In another embodiment, anti-CEA antibodies can be used in combination with anti-PD1 antibodies. Anti-PD1 antibodies can include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab is disclosed in US8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed in US8,354,509 and US8,900,587 by Merck, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is under clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. 8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0125] Pharmaceutical Compositions and Formulations Also provided are compositions, including pharmaceutical preparations, that contain anti-CEA antibodies or their antigen-binding fragments, or polynucleotides that contain sequences that code for anti-CEA antibodies or antigen-binding fragments. In certain embodiments, the compositions contain one or more anti-CEA antibodies or antigen-binding fragments, or one or more polynucleotides that contain sequences that code for one or more anti-CEA antibodies or antigen-binding fragments. These compositions can further contain suitable carriers, such as pharma- ceutically acceptable excipients, including buffers, that are well known in the art.

[0126] Pharmaceutical formulations of the anti-CEA antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. Examples of suitable pharmacopoeitic carriers include, but are not limited to, hydrophilic polymers such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Pat. Nos. 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0127] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0128] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0129] Preparations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes. EXAMPLES

[0130] Example 1: Generation of anti-CEA monoclonal antibodies CEA recombinant protein for immunization and binding assays To discover novel antibodies against CEA that cross-react with both human and Macaca mulatta CEA in the perimembrane region containing domain B3 (amino acids 596-674 of SEQ ID NO:52; see Beauchemin et al., Mol. Cell Bio., 1987, 7(9):3321-3330) but do not exhibit off-target binding with other human CEACAM members, several recombinant proteins were designed and expressed for antibody screening (see Table 2).

[0131] The cDNA coding regions of full-length human CEA (SEQ ID NO: 52), Macaca CEA (SEQ ID NO: 53), and full-length human CEACAM6 (SEQ ID NO: 54) were sequenced based on the GenBank sequence. For human CEA (Accession No.: NM_004363.2), the gene is available from Sinobio (Cat. No. HG11077-UT). For Macaca CEA (Accession No.: NM_001047125), the gene is available from Genscript (Cat. No. OMb23865D). For human CEACAM6 (Accession No.: NM_002483.4), the gene is available from Sinobio (Cat. No. HG10823-UT). A schematic diagram of the CEA fusion proteins is shown in FIG. 1. It has been reported that splice variants of human CEA are co-expressed with full-length CEA on tumors (Peng et al., PloS one, 7, e36412-e36412 (2012)), and the variant (CEA-v) was prepared accordingly. To generate this construct, the coding region of the extracellular domain (ECD) consisting of amino acids (AA) 1-687 (SEQ ID NO: 55) of huCEA, the region of amino acids (AA) 1-690 (SEQ ID NO: 56) of monkey CEA, and the region of amino acids (AA) 1-320 (SEQ ID NO: 57) of CEACAM6 were PCR amplified. The regions of amino acids (AA) 1-78 (SEQ ID NO: 58) of CEA and amino acids 398-687 (SEQ ID NO: 59) of CEA were PCR amplified and then combined by overlap PCR to generate the CEA variant (CEA-v) (SEQ ID NO: 60). Alternatively, the region of CEACAM6 amino acids (AA) 1-273 (SEQ ID NO: 61) and the peri-membrane region including domain B3 of CEA amino acids (AA) 596-687 (SEQ ID NO: 62) were PCR amplified and then combined by overlap PCR to generate a chimeric construct (CHIM) (SEQ ID NO: 63). All constructs were then individually cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA) with the C-terminus fused to a 6xHis tag, resulting in five recombinant fusion protein expression plasmids for CEA, monkey CEA, CEACAM6, CEA-v, and CHIM.For production of recombinant fusion proteins, CEA, monkey CEA, CEACAM6, CEA-v, and CHIM plasmids were transiently transfected into HEK293-based mammalian cell expression system (generated in-house) and cultured for 5–7 days in a CO2 incubator equipped with a rotating shaker. The supernatant containing the recombinant proteins was collected and removed by centrifugation. The recombinant proteins were purified using Ni-NTA agarose (catalog no. R90115, Invitrogen). All recombinant proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots in a -80°C freezer.

[0132] Stable expression in cell lines To establish a stable cell line expressing full-length human CEA (accession number: NM_004363.2), a cDNA expressing CEA was cloned into the retroviral vector pFB-Neo (catalog number 217561, Agilent, USA). Dual-tropic retroviral vectors were generated according to a previous protocol (Zhang et al., Blood. 2005 106(5):1544-51). To generate human CEA-expressing cell lines, the viral vector containing human CEA was transduced into L929 (ATCC, Manassas, VA, USA) and CT26 cells (ATCC, Manassas, VA, USA). High-expressing cell lines were selected by culturing in complete RPMI1640 medium containing 10% FBS with G418, and then verified by FACS binding assay.

[0133] Immunization, hybridoma fusion and cloning Balb / c mice aged 8–12 weeks (HFK BIOSCIENCE CO., LTD, Beijing, China) were immunized with 500 μl of 1 × 10 7Mice were immunized intraperitoneally (ip) with 1000 L929 / huCEA cells. To stimulate antibody production, this procedure was repeated 2 weeks later. Two weeks after the third immunization, mouse sera were assessed for soluble CEA (sCEA) binding by ELISA and FACS. Splenocytes were isolated and fused to mouse myeloma cell line SP2 / 0 cells (ATCC, Manassas, VA, USA) using standard techniques (Colligan JE, et al., CURRENT PROTOCOLS IN IMMUNOLOGY, 1993).

[0134] Evaluation of antibody CEA binding activity by ELISA and FACS To screen for antibodies that bind to human CEA but not to CEACAM6 or sCEA, we screened and counter-screened for antibodies that bind to CHIM but not to sCEA, CEACAM6 and CEA-v, and antibodies that bind to CHIM, sCEA and CEA-v but not to CEACAM6. Supernatants of hybridoma clones were first screened by ELISA as described in (Methods in Molecular Biology (2007) 378:33-52) with some modifications. Briefly, sCEA, CHIM, CEACAM6 or CEA-v was individually coated on 96-well plates at a low concentration of 3 μg / ml. HRP-conjugated anti-mouse IgG antibody (Cat. No. 7076S, Cell Signaling Technology, USA) and substrate (Cat. No. 00-4201-56, eBioscience, USA) were used in the development, and the absorbance signal at a wavelength of 450 nm was measured using a plate reader (SpectraMax Paradigm™, Molecular Devices, USA). ELISA-positive clones were further verified by FACS using L929 / huCEA and / or MKN45 cells (ATCC). MKN45 cells are derived from human gastric carcinoma. CEA-expressing cells (10 5Cells / well) were incubated with ELISA-positive hybridoma supernatants and subsequently bound with Alexa Fluro-647-labeled goat anti-mouse IgG antibody (Cat. No. A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA).

[0135] Conditioned media from hybridomas that showed positive signals in FACS screening and did not bind to CEACAM6 and sCEA but did bind to CHIM were subjected to functional assays to evaluate the presence of sCEA in the binding of CEA antibodies to CEA-expressing cells (see Examples below). Antibodies with the desired binding specificity and functional activity were further subcloned and characterized.

[0136] Subcloning of hybridomas and adaptation to serum-free or low-serum medium After screening mainly by ELISA, FACS, and functional assays, positive hybridoma clones were subcloned by limiting dilution. Top antibody subclones verified through functional assays were adapted for growth in CDM4MAb medium (Cat. No. SH30801.02, Hyclone, USA) containing 3% FBS.

[0137] Expression and purification of monoclonal antibodies Hybridoma cells were cultured in CDM4MAb medium (catalog no. SH30801.02, Hyclone) and incubated at 37°C in a CO2 incubator for 5–7 days. Prior to purification, conditioned medium was collected by centrifugation and filtration through a 0.22 μm membrane. Supernatant containing mouse antibodies was applied and bound to a Protein A column (catalog no. 17127901, GE Life Sciences) according to the manufacturer's guide protocol. This procedure typically yielded antibodies with purity greater than 90%. Protein A affinity purified antibodies were dialyzed against PBS or further purified using a HiLoad 16 / 60 Superdex™ 200 column (catalog no. 17531801, GE Life Sciences) to remove aggregates. Protein concentration was determined by measuring absorbance at 280 nm. The final antibody preparation was stored in aliquots in a -80°C freezer.

[0138] [Table 2-1]

[0139] [Table 2-2]

[0140] [Table 2-3]

[0141] [Table 2-4]

[0142] [Table 2-5]

[0143] [Table 2-6]

[0144] [Table 2-7]

[0145] [Table 2-8]

[0146] [Table 2-9]

[0147] [Table 2-10]

[0148] [Table 2-11]

[0149] [Table 2-12]

[0150] Example 2: Cloning and sequence analysis of CEA antibodies Total RNA was prepared from mouse hybridoma cells using Ultrapure RNA kit (Cat. No. 74104, QIAGEN, Germany) according to the manufacturer's protocol. First-strand cDNA was synthesized using Invitrogen's cDNA synthesis kit (Cat. No. 18080-051), and PCR amplification of VH and VL genes of mouse monoclonal antibodies was performed using a PCR kit (Cat. No. CW0686, CWBio, Beijing, China). Oligo primers used for antibody cDNA cloning of heavy chain variable region (VH) and kappa light chain variable region (VL) were synthesized based on previously reported sequences (Brocks et al., Mol Med. 2001 7(7):461-9.). The PCR products were then subcloned into pEASY-Blunt cloning vector (Cat. No. CB101-02, TransGen, China) and sequenced. The amino acid sequences of the VH and VL regions were determined from the results of DNA sequencing.

[0151] The monoclonal antibodies were analyzed by comparing sequence homology and grouped based on sequence similarity (Figure 2). The complementarity determining regions (CDRs) were defined based on the IMGT (Lefranc et al., 1999 Nucleic Acids Research 27:209-212) system of sequence annotation. The amino acid sequence of a representative clone, BGA13, is shown in Table 3.

[0152] [Table 3]

[0153] Example 3: Determination of the binding profile of purified mouse anti-CEA antibodies CEA antibodies with specific binding to CEA as shown by ELISA and FACS, as well as CEA antibodies without sCEA interference, were characterized for their binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences) (Figure 3A). Briefly, anti-mouse IgG antibodies were immobilized on an activated CM5 biosensor chip (catalog no. BR100530, GE Life Sciences). Purified mouse antibodies were flowed over the chip surface and captured by the anti-mouse IgG antibodies. Then, serial dilutions (6.0 nM to 2150 nM) of purified CHIM, CEA-v, CEA, or monkey CEA recombinant proteins were flowed over the chip surface and the changes in surface plasmon resonance signal were analyzed to determine the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity profile of BGA13 is shown in Table 4 below.

[0154] The binding profile of BGA13 was checked by antigen ELISA to observe the binding of purified BGA13 to huCEA and monkey CEA. These indicated that BGA13 was a weak binder to soluble huCEA and monkey CEA or had a different conformation when soluble CEA was immobilized (Figure 3B). In this experiment, sCEA, CHIM, monkey CEA, CEA-v, and BSA were coated on 96-well plates at a high concentration of 10 μg / ml overnight at 4°C. BGA13 or control antibody ab4451 (catalog no. ab4451, abcam, USA) was incubated at a concentration of 2 μg / ml for 1 hour. HRP-conjugated anti-mouse IgG antibody (Cat. No. 7076S, Cell Signaling Technology, USA) and substrate (Cat. No. 00-4201-56, eBioscience, USA) were used for development, and the absorbance signal at a wavelength of 450 nm was measured using a plate reader (SpectraMax Paradigm, Molecular Devices, USA).

[0155] [Table 4]

[0156] Example 4: Effect of recombinant soluble CEA on the binding of BGA13 to CEA-expressing cells The presence of soluble CEA in the specific binding of various CEA antibodies to CEA-expressing cells was evaluated by flow cytometry. Briefly, human CEA-expressing cells (10 5Cells / well) were incubated with 2 μg / ml purified CEA mouse monoclonal antibody in the presence of 20 μg / ml super recombinant soluble CEA protein, and then bound with Alexa Fluro-647-labeled goat anti-mouse IgG antibody (Cat. No. A0473, Beyotime Biotechnology, China). The fluorescence of the cells was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). As shown in Figure 4A and Figure 4B, the binding of BGA13 to CEA-expressing cells was not affected by the presence of soluble CEA.

[0157] Example 5: Humanization of mouse anti-human CEA antibody Humanization and Engineering of mAbs For humanization of BGA13, human germline IgG genes were searched for sequences that shared high homology with the cDNA sequence of the BGA13 variable region by sequence comparison in the IMGT and NCBI human immunoglobulin gene databases. Human IGVH and IGVL genes, which are frequently present in the human antibody repertoire (Glanville et al., 2009 PNAS 106:20216-20221) and have high homology with BGA13, were selected as templates for humanization. Prior to humanization, the BGA13 heavy and light chain variable domains were fused to the wild-type human IgG1 constant region, designated as human IgG1wt (SEQ ID NO: 87), and the human kappa constant (CL) region (SEQ ID NO: 88), respectively.

[0158] [Table 5]

[0159] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press), and the BGA13 antibody was engineered in a human IgG1 format. In the first round of humanization, mutations from mouse to human amino acid residues in the framework regions were guided by the simulated 3D structure, and structurally important mouse framework residues to maintain the canonical structure of the CDRs were retained in the first version of the humanized antibody BGA13, BGA131 (heavy and light chain amino acid sequences are shown in SEQ ID NOs: 89 and 90).

[0160] [Table 6]

[0161] Specifically, the CDRs of BGA13 VL were grafted into the framework of the human germline variable gene IGVK1-27 (amino acid sequence of the light chain variable domain shown in SEQ ID NO: 92) while retaining two mouse framework residues (N66 and V68).The CDRs of BGA13 VH were grafted into the framework of the human germline variable gene IGVH1-46 (amino acid sequence of the heavy chain variable domain shown in SEQ ID NO: 91) while retaining five mouse framework residues (L39, I53, Y55, N66, S68).

[0162] [Table 7]

[0163] BGA13-1 was constructed as a human full-length antibody format using an in-house developed expression vector containing the constant region of wild-type human IgG1 with easily adaptable subcloning sites. Expression and preparation of BGA13-1 antibody was achieved by co-transfection of the above two constructs into 293G cells and purification using a Protein A column (catalog no. 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots in a -80°C freezer.

[0164] Additional single or multiple amino acid changes were made using BGA131 to convert human residues in the framework regions of VH and VL to the corresponding mouse germline residues, including V68A, R72A, and V79A in VH and V43S in VL, respectively, resulting in BGA132 (V68A, R72A in VH), BGA133 (V79A in VH), BGA134 (V68A, R72A, V79A in VH), BGA135 (V43S in VL), BGA136 (V68A, R72A in VH, and V43S in VL), BGA137 (V79A in VH, V43S in VL), and BGA138 (V68A, R72A, V79A in VH, V43S in VL). All antibodies containing modifications had similar binding activity to BGA131, and none of the changes disrupted binding.

[0165] Further engineering was performed by introducing mutations into the CDR and framework regions based on the BGA131 sequence to remove post-translational modification (PTM) sites. This included N52T, N54Q, N59S, N102G, N104Q, and S61A amino acid changes in the VH region. This resulted in BGA131A (N52T(VH)), BGA131B (N54Q(VH)), BGA131C (N59S(VH)), BGA131D (N102G(VH)), BGA131E (N104Q(VH)), and BGA131F (N54Q, N59S, S61A(VH)), all antibodies had similar binding specificity to BGA131, and none of the changes disrupted binding. While maintaining specificity, amino acid composition and expression level were also taken into consideration. All humanized mutations were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog no. FM111-02, TransGen, Beijing, China). The desired mutations were confirmed by sequence analysis. Compared with BGA13-1, BGA13-1F had no glycosylation site and significantly reduced binding affinity, but higher expression level (Table 8).

[0166] [Table 8]

[0167] Example 6: Generation of affinity maturation libraries The phagemid vector pCANTAB 5E (GE Healthcare) was used by standard molecular biology techniques to construct a phagemid designed to display the BGA13-1F Fab fragment on the surface of M13 bacteriophage as a fusion to the N-terminus of a fragment of the minor coat protein of gene 3. The gene 3 sequence was preceded by an amber stop codon to allow expression of the Fab fragment directly from the phagemid clone. The phagemid was used as a template to express 10 8 A phage display library containing unique members was constructed.

[0168] Two libraries (H-AM, L-AM) were constructed by randomizing the CDR positions of the heavy and light chains, respectively. All three CDRs were randomized in each library, but each CDR had a maximum of one mutation in each clone, except for HCDR3, which could have two simultaneous mutations. Each position was randomized with the NNK codon (IUPAC code) encoding any amino acid or an amber stop codon. The combined heavy and light chain library design included 5.0 × 10 6 There was a potential diversity of 100 unique full-length clones, with the expected distribution of clones with 0, 1, 2, and 3 mutations, respectively, being approximately 0.02%, 1.1%, 17%, and 82%. A minority portion of heavy chain clones was expected to have 4 mutations due to primer design in the HCDR3 region. As a first step, DNA fragments were amplified using pCANTAB 5E as a template and primers containing randomized CDR3 positions (see Figure 5, A and B). Then, the PCR products were gel purified and assembled using primers containing randomized CDR2 positions. This procedure was repeated using primers directed to randomized CDR1 positions. The resulting PCR products of heavy or light chains were then assembled with the corresponding CH or CL fragments by overlap PCR. The fragments were further assembled with the non-mutated light or heavy chains by overlap PCR. The resulting fragments were then gel purified and ligated with pCANTAB 5E after NcoI / NotI digestion. Purified ligations were transformed into TG1 bacteria by electroporation. Sequencing of 48 clones from each library confirmed randomization at each position (data not shown). However, due to limited sampling depth, not all amino acid mutations were observed at all positions. Approximately 52% and 55% of the light and heavy chain libraries had full-length randomized clones, representing 10% of the total clones generated even with moderate incorporation bias in oligonucleotide synthesis and library construction. 8independent clones were sufficient to cover all the potential diversity of the design.

[0169] Example 7: Generation of affinity matured humanized BGA13 variants Library Selection and Screening Generation of affinity matured humanized BGA13 Fab was performed by phage display using standard protocols (Silacci et al., (2005) Proteomics, 5, 2340-50; Zhao et al., (2014) PLoS One, 9, e111339). In the first and second rounds of selection, competitive selection was performed against immobilized CHIM in immunotubes (cat. no. 470319, ThermoFisher). Briefly, immunotubes were coated with 1 ml of CHIM (5 μg / ml in PBS) overnight at 4 °C. All affinity maturation libraries were incubated with the coated immunotubes for 1 h in the presence of various concentrations of BGA13-1F IgG (round 1, 1 μg / ml; round 2, 5 μg / ml). In the third and fourth rounds of selection, L929 / huCEA cells (round 3) or LOVO cells (ATCC CCL-229) (round 4) were used, and cell panning was performed using HEK293 cells as depletion cells. After four rounds of selection, individual clones were selected and phage-containing supernatants were prepared using standard protocols. ELISA-positive clones were sequenced and mutation sites were analyzed.

[0170] Analysis of CDR mutation frequency The frequency of mutations in each CDR after four rounds of selection was relatively high, ranging from 17% in HCDR3 to 95% in LCDR2. For the heavy chain, approximately half of the clones identified in the H-AM library were identical to the parental clone. The other clones contained one backmutation at Q54N in HCDR2.

[0171] When the light chains were analyzed, the mutations were more diverse. Two sites were mutated in almost all clones in LCDR1, respectively. Light chain residues 29 and 31 were mutated from Ile to Gln and Gly to Gln in 47.09% and 35.29% of clones, respectively. Position 29 not only had a high frequency of Gln mutations, but also had a subset of clones with a mutation to tyrosine. Position 31 not only had a high frequency of Gln mutations, but also had a ∼12.5% ​​chance of being mutated to Leu. Due to the constraints of the library design, mutations at positions 29 and 31 were not seen in combination with each other. However, mutations at each of these two sites were often combined with mutations in other CDRs. For LCDR2, only A51 was mutated in at least 64.71% of clones, but without any obvious pattern, and included large hydrophobic polar residues such as Tyr, Phe, Thr, and Asn. For LCDR3, two sites were mutated in at least 50% of the clones. Light chain residues 90 and 92 were mutated from His to Leu and Tyr to Leu in 11.76% and 47.06% of the clones, respectively. Figure 6 shows the sequence variance of the light chain CDR regions after four rounds of selection.

[0172] Expression of selected humanized BGA13 variants A combination of mutations was performed. The light chain variable regions from selected phage clones were subcloned into a mammalian expression vector expressing human kappa light chain. The light chain expression vector was co-transfected into 293G cells at a 1:1 ratio with a mammalian expression vector expressing the BGA13-1F heavy chain. A version of the CEA antibody was purified from the culture supernatant by protein A affinity chromatography (catalog no. 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5–5 mg / mL in PBS, aliquoted, and stored in a -80 °C freezer.

[0173] Characterization of affinity matured humanized BGA13 variants Affinity comparison of BGA13-1F and other affinity matured clones was performed by SPR assay (Table 9) using BIAcore™ T-200 (GE Life Sciences) and flow cytometry (Figure 7). In this experiment, anti-human IgG (Fc) antibody was immobilized on an activated CM5 biosensor chip (catalog no. BR100839, GE Life Sciences). Anti-CEA antibody was flowed over the chip surface and captured by anti-human Fab antibody. Then, serial dilutions of CHIM (1.37 nM to 333 nM) were flowed over the chip surface and the change in surface plasmon resonance signal was analyzed to determine the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off ) was calculated. In flow cytometry, CEA-expressing cells (10 5 Cells / well) were incubated with various concentrations of purified affinity matured antibodies and then bound with Alexa Fluro-647-labeled anti-hu IgG Fc antibody (Cat. No. 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). The equilibrium dissociation constant (K D ) as the ratio k off / k on BGA131F-ph-L (SEQ ID NO: 95) and BGA131F-ph-M (SEQ ID NO: 96) were shown to have improved affinity for the huCEA surface protein (Table 10).

[0174] [Table 9]

[0175] [Table 10]

[0176] Example 8: Further engineering of affinity matured humanized BGA13 variants Further engineering was performed by introducing mutations into the CDRs based on the BGA131F-ph-M template, including W33Y, Q54N, and S59N in VH, and T51Y in VL. This resulted in BGA1132A (W33Y(VH)), BGA1132B (Q54N(VH)), BGA1132C (S59N(VH)), and BGA1131A (T51Y(VL)), all of which showed improved binding activity to BGA-1131F. The most improved antibody finally resulted in the BGA113 antibody (Table 11) with (W33Y(VH), T51Y(VL)) changes, the sequence of which is shown in Table 12.

[0177] [Table 11]

[0178] [Table 12]

[0179] Example 9: Optimization of BGA113 To further improve the biochemical / biophysical properties, BGA113 was optimized by introducing substitutions in the CDR and framework regions (Table 13). Large hydrophobic residues were selected and changed to polar residues, with the exception of K13 and Q53, which were selected based on differences observed between human VH germlines. Amino acid composition, thermal stability (Tm), surface hydrophobicity, and isoelectronic point (pI) were considered while maintaining functional activity. Mutants were expressed in Fab format by cloning into vector pCANTAB-5E as described in Example 6. Fab-containing supernatants were then screened for CEA binding by ELISA and SPR analysis. Mutants without significant affinity loss were selected to identify residues that could tolerate substitutions. L92E in the light chain, K13E, Q54E, Y57D / E, and Y57K in the heavy chain were shown to have minimal impact on affinity. Therefore, BGA113 mutants in IgG format with single identified mutations or combinations were expressed and purified as described in Example 8. SPR studies and FACS analysis were performed and summarized in Table 14. It was confirmed that the introduced amino acid substitutions did not cause any change in specificity and epitope (data not shown). In summary, these results showed that these single or combined mutations (K13E, Q54E, Y57D and Y57K in the heavy chain, L92E in the light chain) had minimal effect on affinity, except for L92E, which slightly reduced the binding affinity to CEA. In summary, the Y57K change optimized the expression, CEA binding and affinity of the BGA113 antibody, resulting in BGA113K (Table 1).

[0180] [Table 13]

[0181] [Table 14]

[0182] Example 10: Binding profile of anti-CEA antibody BGA113K BGA113K and a previously disclosed CEA antibody designated antibody 2F1 in US2012 / 0251529 were produced in human IgG1 format and characterized for binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences).

[0183] To obtain this data, anti-human IgG (Fc) antibodies were immobilized on an activated CM5 biosensor chip (catalog no. BR100839, GE Life Sciences). BGA113K antibodies were flowed over the chip surface and captured by anti-human Fab antibodies. Serial dilutions (1.37 nM to 2150 nM) of soluble huCEA or cynoCEA (catalog no. CE5-C52H5, Acrobiosystem) were then flowed over the chip surface and the change in surface plasmon resonance signal was analyzed to estimate the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity of BGA113K and the 2F1 control antibody was calculated as follows: BGA113K and the 2F1 control antibody showed different binding affinities. As shown in Table 15 below, BGA113K has a very high affinity for human CEA and a comparable affinity for cynomolgus CEA.

[0184] In flow cytometry, CEA-expressing MKN45 cells (10 5 Cells / well) were incubated with various concentrations of purified affinity matured antibodies and then bound with Alexa Fluro-647 labeled anti-hu IgG Fc antibody (Cat. No. 409320, BioLegend, USA). Fluorescence of cells was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). As shown in Figure 8, BGA-113K was shown to specifically bind to native CEA on live cells in a dose-response manner with an EC50 of 2.92ug / ml.

[0185] [Table 15]

[0186] Example 11: Assessment of off-target specificity The off-target specificity of BGA113K was evaluated by ELISA and flow cytometry. In flow cytometry, CEACAM3 (SEQ ID NO: 65), CEACAM7 (SEQ ID NO: 66), or CEACAM8 (SEQ ID NO: 67) was transfected into HEK293 cells (10 5 Cells / well) were transiently transfected and then incubated with 2 μg / ml of purified BGA113K, followed by binding with Alexa Fluor-647-labeled anti-huIgG Fc antibody (Cat. No. 409320, BioLegend, USA). The fluorescence of the cells was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA). For antigen ELISA, CEACAM1 (SEQ ID NO: 64) (Cat. No. 10822-H08H, Sino Biological, China), CHIM (SEQ ID NO: 63), CEA (SEQ ID NO: 55), or CEACAM6 (SEQ ID NO: 57) were coated on 96-well plates at a concentration of 10 μg / ml overnight at 4°C. HRP-conjugated anti-human Fc (Fc-specific) IgG antibody (catalog number A0170, Sigma, USA) and substrate (catalog number 00-4201-56, eBioscience, USA) were used in the development, and the absorbance signal at a wavelength of 450 nm was measured using a plate reader (SpectraMax Paradigm, Molecular Devices, USA). As shown in Figure 9A-B, no cross-reactivity to other CEACAM family members was observed, and thus BGA113K showed specificity only for CEA (CEACAM5 in Figure 9A-B).

[0187] Example 12: Effect of soluble huCEA on the binding of BGA113K to CEA-expressing cells To determine whether soluble CEA (sCEA) has any effect on the specific binding of BGA113K, various concentrations (0, 0.5, 1, 2 μg / ml) of recombinant soluble CEA were premixed with BGA113K (0.01-100 μg / ml) and incubated for 5 min. These mixtures were then transferred to 2 × 10 MKN45 cells or other cells. 5 The cells were incubated with CEA-expressing cells for 30 min at 4°C. The cells were stained with secondary antibody anti-huFc-APC (cat. no. 409320, BioLegend, USA) and analyzed by flow cytometry. In the presence of 2 μg / ml recombinant sCEA, the binding of BGA113K to CEA-expressing cells was not affected. The results are shown for MKN45 cells (Figure 10), demonstrating the specificity of BGA113K for membrane-bound CEA.

[0188] Example 13: BGA113 is CEA + Induces strong ADCC effect against tumor cells To determine whether BGA113 in wild-type IgG1 format can induce antibody-dependent cellular cytotoxicity (ADCC), CD16(V158)-expressing NK92MI cells (NK92MI / CD16V) were used as effector cells and co-cultured with CEA-expressing mouse colon cancer cells (CT26-ATCC CRL-2638). Co-culture was performed for 5 h at an E:T ratio of 1:1 in the presence of BGA113 at the indicated concentrations (0.00005–5 μg / ml), and cytotoxicity was determined by lactate dehydrogenase (LDH) release. The amount of LDH in the supernatant was measured using a CytoTox™ 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI), and the amount of specific lysis was calculated according to the manufacturer's instructions. As shown in Figure 11, BGA113 had an EC of approximately 6.7 ng / ml. 50 were able to induce ADCC in vitro.

[0189] Example 14: In vivo antitumor effect of BGA113 CEA +To measure the in vivo effect of BGA113 on tumor cells, NK92MI / CD16V cells (5x10 6 ) to CT26 / CEA cells (10 6 ) and injected subcutaneously into NCG mice. BGA113 (0.12, 0.62 or 3.1 mg / kg) or vehicle control was given twice a week starting from the day of tumor injection (7 mice per group). Compared to vehicle, BGA113 at a dose of 3.1 mg / kg showed a low amount of tumor inhibition, although the difference from the vehicle control was not statistically significant (P>0.05) (Figure 12).

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Claims

1. 1. An anti-CEA antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof that specifically binds to human CEA at amino acids 596-674 of SEQ ID NO:52, The anti-CEA antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (VH) comprising (a) a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 7, (b) a HCDR2 of SEQ ID NO: 8, and (c) a HCDR3 of SEQ ID NO: 9, and (d) a light chain variable region (VL) comprising (a) a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, (b) a LCDR2 of SEQ ID NO: 11, and (c) a LCDR3 of SEQ ID NO: 6; (ii) a VH comprising (a) an HCDR1 of SEQ ID NO:24, (b) an HCDR2 of SEQ ID NO:25, and (c) an HCDR3 of SEQ ID NO:26, and (d) an LCDR1 of SEQ ID NO:27, (e) an LCDR2 of SEQ ID NO:28, and (f) an LCDR3 of SEQ ID NO:23; or (iii) a VH comprising (a) an HCDR1 of SEQ ID NO: 41, (b) an HCDR2 of SEQ ID NO: 42, and (c) an HCDR3 of SEQ ID NO: 43, and (d) an LCDR1 of SEQ ID NO: 44, (e) an LCDR2 of SEQ ID NO: 45, and (f) an LCDR3 of SEQ ID NO:

40. An anti-CEA antibody or antigen-binding fragment thereof comprising:

2. An anti-CEA antibody or antigen-binding fragment thereof, (i) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 14, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15; (ii) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 31, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 32; or (iii) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 48, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

49. The anti-CEA antibody or antigen-binding fragment thereof of claim 1 .

3. 3. The anti-CEA antibody or antigen-binding fragment thereof of claim 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 14, 15, 31, 32, 48, or 49 have been inserted, deleted, or substituted.

4. An anti-CEA antibody or antigen-binding fragment thereof, (i) a VH comprising SEQ ID NO: 14, and a VL comprising SEQ ID NO: 15; (ii) a VH comprising SEQ ID NO: 31, and a VL comprising SEQ ID NO: 32; or (iii) a VH comprising SEQ ID NO: 48, and a VL comprising SEQ ID NO: 49 The anti-CEA antibody or antigen-binding fragment thereof of claim 1 .

5. The anti-CEA antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab') 2 The anti-CEA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a fragment.

6. The anti-CEA antibody or antigen-binding fragment thereof of claim 1 , wherein the anti-CEA antibody or antigen-binding fragment thereof has at least one of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

7. The anti-CEA antibody or antigen-binding fragment thereof of claim 1, wherein the anti-CEA antibody or antigen-binding fragment thereof has reduced glycosylation, no glycosylation, or is hypofucosylated.

8. The anti-CEA antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof contains increased bisecting GlcNac structures.

9. The anti-CEA antibody or antigen-binding fragment thereof of claim 1 , wherein the Fc domain is IgG1.

10. The anti-CEA antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is conjugated to a toxin.

11. A pharmaceutical composition comprising the anti-CEA antibody or antigen-binding fragment thereof of claim 1 and a pharma- ceutically acceptable carrier.

12. A pharmaceutical composition for treating cancer, comprising the anti-CEA antibody or antigen-binding fragment thereof described in claim 1.

13. 13. The pharmaceutical composition of claim 12, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma or sarcoma.

14. The pharmaceutical composition of claim 12, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

15. 15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

16. The pharmaceutical composition of claim 14, wherein the therapeutic agent is an anti-PD1 antibody or an anti-PDL1 antibody.

17. 2. An isolated nucleic acid encoding the anti-CEA antibody or antigen-binding fragment of claim 1.

18. A vector comprising the nucleic acid of claim 17.

19. 19. A host cell comprising the nucleic acid of claim 17 or the vector of claim 18.

20. 20. A method for producing an anti-CEA antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 19 and recovering the anti-CEA antibody or antigen-binding fragment thereof from the culture.

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