Antibody against ceacam5, and use thereof

Abstract

Disclosed are an antibody against CEACAM5, and a use thereof, particularly an antibody binding to CEACAM5 or an antigen-binding fragment thereof, a nucleic acid comprising a nucleotide sequence encoding the antibody, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. Also disclosed are a pharmaceutical composition, a conjugate and a chimeric antigen receptor each comprising the antibody, and a treatment method using the antibody.

Description

Antibodies against ceacam5 and uses thereof

[0001] This international patent application claims priority to Chinese patent application No. 202410635805.7, filed May 21, 2024, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention relates to antibodies binding to CEACAM5 and uses thereof, in particular in the treatment of cancer. BACKGROUND

[0003] CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5), also known as Carcinoembryonic antigen (CEA), is a membrane protein widely used for tumor marker monitoring. CEACAM5 belongs to the immunoglobulin superfamily and is mainly expressed on gastrointestinal cells and on cancer cells of various cancers, such as colorectal cancer, breast cancer and lung cancer. Structurally, CEACAM5 is composed of a large N-terminal ectoplasmic region, one or more immunoglobulin-like domains, a transmembrane region and a short intracellular region. The extracellular region is composed of multiple repeat immunoglobulin-like (Ig-like) domains, with abundant glycosylation sites. According to structural similarity, the extracellular region immunoglobulin-like domain can be divided into N, A, B domains, and CEACAM5 has seven domains, N, A1-A3, B1-B3. This unique structure enables CEACAM5 to participate in intercellular adhesion processes and regulate various biological functions such as cell proliferation, differentiation and apoptosis.

[0004] Due to the high expression of CEACAM5 on most tumor cells and its low expression in normal tissues, it has become a popular candidate for tumor therapy targets. Several CEACAM5 antibody-based ADCs have entered clinical trials, some of which have shown considerable efficacy and manageable safety. Labetuzumab (also known as hMN-14) developed by Immunomedics has carried out development attempts for colorectal cancer indications by coupling SN-38. However, due to poor efficacy (1.1% PR, 48.8% SD, mPFS 3.6 months, mOS 6.9 months), Immunomedics stopped its development in colorectal cancer indications (Cancer. JCO 35, 3338-3346 (2017).). Nevertheless, the exploration of colorectal cancer indications using the CEACAM5 target continues.

[0005] The immunoglobulin superfamily to which CEACAM5 belongs has multiple homologous membrane proteins, among which CEACAM1 (uniprot ID: P13688), CEACAM6 (uniprot ID: P40199), CEACAM7 (uniprot ID: Q14002) and CEACAM8 (uniprot ID: P31997) have high homology with CEACAM5 (uniprot ID: P06731), with sequence homology as high as 62%, 79%, 60% and 71% respectively. Researchers Laura A Strickland et al. found that CEACAM6 is expressed in human neutrophils and granulocyte precursor cells, and that ADC targeting CEACAM6 causes dose-dependent neutropenia in non-human primates (Laura A Strickland et al. J Pathol 2009; 218: 380-390). Therefore, antibodies and ADCs against CEACAM5 should avoid cross-reacting with homologous proteins such as CEACAM1 / 6 / 7 / 8 to avoid normal tissue toxicity, which undoubtedly poses a challenge to the screening of antibodies. SUMMARY

[0006] The present application aims to provide novel antibodies targeting CEACAM5 membrane proteins. The antibodies of the present application can specifically bind to human CEACAM5 and have high affinity, good intracellular endocytosis activity and other properties.

[0007] Accordingly, in one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0008] (1) the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises a LCDR1, a LCDR2 and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively; or

[0009] (2) the VH comprises a HCDR1, a HCDR2 and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises a LCDR1, a LCDR2 and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively; or

[0010] (3) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively; or

[0011] (4) the VH comprises HCDR1, HCDR2, HCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively.

[0012] In some embodiments, (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 38, 48, 50, 52, 54, 56, 58, 61, 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 43, 65, 67, 69, 71, 73, 75; or

[0013] (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; or

[0014] (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; or

[0015] (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0016] In some embodiments, (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; or

[0017] (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or

[0018] (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73; or

[0019] (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; or

[0020] (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; or

[0021] (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0022] In some embodiments, (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 43; or

[0023] (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 69; or

[0024] (3) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73; or

[0025] (4) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13; or

[0026] (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0027] (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33.

[0028] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0029] In some embodiments, the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0030] In some embodiments, the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0031] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; or (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0032] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 79, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 77; or (ii) the light chain comprises the amino acid sequence of SEQ ID NO: 83, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 81.

[0033] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

[0034] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0035] In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

[0036] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof disclosed herein.

[0037] In some embodiments, the nucleic acid comprises:

[0038] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 39, 49, 51, 53, 55, 57, 59, 62, 64, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 44, 66, 68, 70, 72, 74, 76; or

[0039] (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; or

[0040] (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; or

[0041] (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0042] In some embodiments, the nucleic acid comprises

[0043] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44; or

[0044] (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70; or

[0045] (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; or

[0046] (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; or

[0047] (5) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; or

[0048] (6) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0049] In some embodiments, the nucleic acid comprises:

[0050] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78; or

[0051] (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0052] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 80 and the nucleotide sequence of SEQ ID NO: 78; or comprises the nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 82.

[0053] In yet another aspect, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

[0054] In yet another aspect, the present disclosure provides a host cell comprising the nucleic acid or vector disclosed herein.

[0055] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0056] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0057] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0058] In yet another aspect, the present disclosure provides a conjugate comprising the antibody or antigen-binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

[0059] In some embodiments, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0060] In yet another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof disclosed herein.

[0061] In yet another aspect, the present disclosure provides a method for treating a disease in a subject comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein.

[0062] In some embodiments, the disease is a cancer, e.g., a cancer associated with CEACAM5 expression.

[0063] In some embodiments, the cancer is selected from pancreatic cancer, lung cancer, small intestine cancer, colorectal cancer, gastric cancer, cervical cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0064] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0065] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1: Affinity of anti-CEACAM5 humanized antibodies.

[0067] Figure 2: Endocytosis activity of anti-CEACAM5 humanized antibodies DT3C.

[0068] Figure 3: Binding of anti-CEACAM5 humanized antibodies to CEACAM5-expressing tumor cells.

[0069] Figure 4: Immunogenicity of anti-CEACAM5 humanized antibodies to human PBMC cells.

[0070] Figure 5: PK parameters of anti-CEACAM5 humanized antibodies in FcRn humanized mouse model. DETAILED DESCRIPTION

[0071] The above features and advantages of the present application, and additional features and advantages thereof, will be more clearly understood from consideration of the following detailed description when read in conjunction with the accompanying drawings.

[0072] The embodiments described herein with reference to the drawings are explanatory, illustrative, and used to generally understand the present application. The embodiments should not be interpreted as limiting the scope of the present application. Identical or similar elements and elements having identical or similar functions are denoted by the same reference numerals throughout the specification.

[0073] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and phrases related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunohistochemistry, and the like, as used herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the relevant art, unless otherwise indicated. Furthermore, for better understanding of the present application, the definitions and explanations of the related terms are provided below.

[0074] Definitions

[0075] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "the antibody" includes multiple antibodies, and so forth.

[0076] The term "comprising" and variations thereof as used herein are intended to mean including, but not limited to, the elements or steps listed after the term. The term "comprising" is used to include both the presence of the stated elements or steps and the absence of one or more of the stated elements or steps.

[0077] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Such molecules are typically comprised of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding sites for the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0078] The heavy chain of an immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (fragment crystallizable). The Fd region comprises the VH and CH1 domains and binds to the light chain to form the Fab (fragment antigen binding) portion of an antibody. The Fc fragment is responsible for the effector functions of an immunoglobulin, including, for example, complement binding and binding to Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. Hinge domains are structurally diverse, varying in sequence and length between immunoglobulin classes and subclasses.

[0079] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the antibody fragment. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexible conformational changes allowed by the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0080] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is composed of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an epitope of an antigen. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains comprise, from amino-terminus to carboxy-terminus, the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0081] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure can use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined according to the Kabat definition.

[0082] Based on the amino acid sequences of the constant regions of the heavy chains of the antibodies, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Based on the amino acid sequences of the light chains, the light chains of the antibodies can be assigned to lambda (l) chains and kappa (K) chains.

[0083] As used herein, the term "antibody" shall be construed in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bi-specific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0084] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, nor is it intended to be limited to antibodies produced by any specific method.

[0085] The term "bispecific antibody" is understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be understood in its broadest sense, including full-length bispecific antibodies and antigen binding fragments thereof. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification of the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0086] The term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody elicits a reduced likelihood of adverse immune reactions in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0087] The term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., murine antibody) are replaced with corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids can also be permissible, as long as they still retain the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody comprising sequences derived from non-human immunoglobulin. In some cases, CDR region residues in the human immunoglobulin (recipient antibody) are replaced by CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that have the desired properties, affinities, and / or capabilities. In some cases, FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody can contain amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0088] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0089] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); (ix) a Nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, this term also includes "linear antibodies", comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions, as well as modified forms of any of the foregoing fragments that retain antigen binding

[0090] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for whole antibodies.

[0091] As used herein, the term "binds" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0092] Affinity is a measure of the strength of binding between an antibody and the associated antigen. Affinity involves both the avidity between the antigenic determinant and the antigen binding site of the antibody and the number of associated binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or has a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D M indicates non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and different variations thereof known in the art.

[0093] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein or proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually include at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are therefore the specific targets of an antibody or antigen binding fragment thereof.

[0094] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions when aligned. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0095] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions that replace an amino acid residue with another amino acid residue having similar chemical structure that have little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0096] Such conservative substitutions are preferably substitutions of one amino acid by another amino acid residue in one of the following groups (a) to (e): (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, lie, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0097] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin or to Glu; Met to Leu, to Tyr or to lie; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie or to Leu.

[0098] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0099] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0100] The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or that such carrier or excipient is approved or approvable by a regulatory agency of the Federal or a state government of the United States for inclusion in a pharmaceutical composition intended for parenteral administration to humans.

[0101] As used herein, the terms "treatment," "therapy," "treat," and the like, refer to the application of an agent or performance of a procedure for the purpose of obtaining an effect. These effects can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual that can be predisposed to the disease (e.g., including a disease that can be associated with or caused by a primary disease) but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameters, such as a decrease in symptoms; alleviation of symptoms; elimination of symptoms or a disease condition; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the end point less detrimental. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease or a side effect of a disease in a subject.

[0102] As used herein, the term "effective amount" refers to an amount of an agent administered to a subject to treat a disease sufficient to achieve treatment of the disease.

[0103] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0104] Anti-CEACAM5 antibodies

[0105] The present disclosure provides an antibody or antigen-binding fragment thereof that binds CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0106] In some embodiments, the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively.

[0107] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively.

[0108] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively.

[0109] In some embodiments, the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively.

[0110] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 38, 48, 50, 52, 54, 56, 58, 61, 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 43, 65, 67, 69, 71, 73, 75.

[0111] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.

[0112] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0113] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0114] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0115] In some embodiments, the VH comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5. In some embodiments, the VL comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5.

[0116] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0117] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0118] In some embodiments, the insertion, deletion, and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3, and / or FR4.

[0119] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within a group (a) to (e) below: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0120] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0121] In preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0122] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 69.

[0123] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73.

[0124] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0125] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0126] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33.

[0127] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody. In preferred embodiments, the antibody is a humanized antibody.

[0128] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to lambda (l) chains and kappa (K) chains. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0129] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl antibody.

[0130] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; or (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0131] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 79 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 77; or (ii) the light chain comprises the amino acid sequence of SEQ ID NO: 83 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 81.

[0132] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to IL-13. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen binding activity.

[0133] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0134] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.

[0135] A number of tumor-associated antigens associated with particular cancers have been identified in the art. In some embodiments, a tumor-associated antigen is an antigen that can elicit a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. Numerous other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0136] Examples of tumor-associated antigens include, but are not limited to, 5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0137] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen binding region binds to any of the gamma, delta, epsilon, zeta, and eta chains of CD3.

[0138] An antibody disclosed herein can comprise an Fc region. The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgGl Fc.

[0139] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, FcyRI, FcyRII, or FcyRIIIA binding. For example, the Fc region can be of the IgGl isotype, or of a non-IgGl type, e.g., IgG2, IgG3, or IgG4, which has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall’Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, the Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred embodiment the Fc region comprises L234A and L235A (LA mutations).

[0140] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation receptor site or is otherwise manipulated to alter the glycosylation properties. For example, in an IgGl Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Thus, in a particular embodiment, the Fc region comprises an IgGl sequence with the N297Q mutation.

[0141] In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, e.g., by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, e.g., as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the method described in Shields et al. (1999) Nature Biotech 17:176 can be used to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, e.g., as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0142] ​In other embodiments, the Fc region has an extended serum half-life. For examples of altering (e.g., decreasing or increasing) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745, all of which are incorporated herein by reference in their entireties. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to decrease the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to increase the in vivo half-life of the antibody. In a particular embodiment, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and / or the third constant (CH3) domain (residues 341-447 of human IgGl) (numbered according to the EU numbering system). In a particular embodiment, the constant region of an antibody IgGl described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254; and a substitution of threonine (T) to glutamic acid (E) at position 256 (numbered according to the EU numbering system). See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. Such mutant IgGs, termed “YTE mutants,” have been demonstrated to exhibit a four-fold increase in half-life compared to the wild-type version of the same antibody (see Dall Acqua WF et al. (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the Fc region comprises M252Y, S254T, and T256E (YTE mutations).

[0143] Nucleic acid

[0144] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0145] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoramidate.

[0146] In some embodiments, the present application provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0147] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 39, 49, 51, 53, 55, 57, 59, 62, 64, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 44, 66, 68, 70, 72, 74, 76.

[0148] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

[0149] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; or

[0150] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; or

[0151] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0152] In some embodiments, the nucleic acid comprises: (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0153] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 80 and the nucleotide sequence of SEQ ID NO: 78; or comprises the nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 82.

[0154] In some embodiments, the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In some embodiments, the present application provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid (DNA) comprising a deoxyribonucleotide sequence encoding an antibody disclosed herein.

[0155] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into a human cell in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is comprised in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is integrated into the genome of a cell.

[0156] In some embodiments, the ribonucleic acid (RNA) can be introduced into a human cell in vivo. In some embodiments, the ribonucleic acid (RNA) of the present application is comprised in a vector or delivery agent.

[0157] Vectors

[0158] The present disclosure provides vectors comprising the nucleic acids disclosed herein.

[0159] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an antibody. For example, the present application provides an expression vector comprising any of the nucleic acid molecules described above.

[0160] Any vector can be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0161] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as lambda GT10, lambda GT11, lambda Zap II (Stratagene), lambda EMBL4, and lambda NM1149, can also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0162] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from COLEL, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0163] For example, the vector can be an adenoviral vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered into a subject, then in vivo into a cell of the subject, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

[0164] Host cells

[0165] The present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0166] Any cell can be used as a host cell for the nucleic acids or vectors of the disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0167] The host cells of the application are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the application can be administered to a subject in vivo, and the host cells express the antibodies disclosed herein in vivo.

[0168] The present application provides host cells into which any of the above-described vectors have been introduced. The present application also provides methods of making the antibodies of the application, comprising a) culturing the host cells disclosed herein under conditions suitable for production of the antibodies; and b) obtaining the antibodies from the culture.

[0169] Pharmaceutical compositions

[0170] The present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0171] The antibodies or antigen-binding fragments thereof of the present application (also referred to herein as“active compounds”) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise the antibody or antigen-binding fragment thereof, together with a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such vehicles and agents for use in

[0172] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0173] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0174] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0175] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0176] Oral composition generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash in which the compound is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0177] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide.

[0178] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into

[0179] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository

[0180] In one embodiment, the active compounds are prepared with an agent that protects the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0181] The present application provides therapeutic compositions comprising the antibodies or antigen binding fragments thereof of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions with

[0182] Conjugates

[0183] The present disclosure provides conjugates comprising the antibodies or antigen binding fragments thereof disclosed herein, and a chemical moiety conjugated thereto.

[0184] In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC."

[0185] The term "conjugate" or "linkage" can refer to joining two polypeptides into one contiguous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, in which a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0186] The chemical moiety can be linked to the antibody of the present application using any number of means known to those of skill in the art. Both covalent and non-covalent means of attachment can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with a suitable functional group on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of a number of known linker molecules. The linker can be any molecule that serves to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond to both the antibody and the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0187] In certain cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a linkage that is cleavable in the vicinity of the target site.

[0188] The conditions experienced by the enzymatic activity or immunoconjugate within the target cell or in the vicinity of the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0189] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0190] Antibodies disclosed herein can be derivatized or linked to another molecule, such as another peptide or protein. Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detector agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule, such as a streptavidin core region or a polyhistidine tag.

[0191] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type). Suitable cross-linking agents include heterobifunctional or homobifunctional cross-linking agents having two distinctly reactive moieties separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or a sulfosuccinimidyl propionate. Such linkers are commercially available.

[0192] In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0193] In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulatory agent, a radioactive compound, an enzyme (e.g., a perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin. In some embodiments, the therapeutic agent can be, for example, a maytansinoid, a geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin) or a minor groove binder such as a calicheamicin. In some embodiments, the therapeutic agent can be, for example, MMAE, Dxd, or a derivative thereof.

[0194] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0195] Antibodies can be conjugated to detectable labels; for example, detectable labels that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0196] Antibodies or antigen-binding fragments can also be conjugated to enzymes that are useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When antibodies or antigen-binding fragments are conjugated to detectable enzymes, detection can be achieved by the addition of additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when horseradish peroxidase reagents are present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0197] Antibodies can be fused to self-labeling protein tags, such as HaloTag. For example, the protein tag can be cloned to the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0198] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0199] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter group (such as a leucine zipper pair sequence, a binding site for biotin, a metal binding domain, an epitope tag) that are recognized by a second reporter group. In some embodiments, the tag is attached by a spacer arm of various lengths to reduce potential steric hindrance.

[0200] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of a target antigen by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0201] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-gamma, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulatory protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0202] Chimeric antigen receptor

[0203] The present disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or antigen binding fragment thereof disclosed herein. The present disclosure also provides a genetically modified cell comprising a chimeric antigen receptor disclosed herein.

[0204] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule engineered to contain an antigen binding domain that targets a specific antigen and, when bound to that antigen, activates an immune cell (e.g., a T cell or NK cell, such as a naive T cell, central memory T cell, effector memory T cell, or a combination thereof) to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells.

[0205] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen binding domain to an intracellular signaling domain. The antigen binding domain is typically an antigen binding fragment derived from a monoclonal antibody (mAb) (e.g., scFv), but it can be based on other formats containing antibody-like antigen binding sites or antigen binding domains derived from natural ligands of the antigen. A hinge domain is often required to separate the antigen binding domain from the membrane and allow for its proper orientation. A common hinge domain used is the Fc of IgGl. Depending on the antigen, more compact spacers can suffice, such as the stalk from CD8a, and even just the IgGl hinge alone. The transmembrane domain anchors the protein in the cell membrane and links the hinge domain to the intracellular domain (endodomain).

[0206] According to at least one non-limiting view, there have been at least three “generations” of CAR molecules. In the first generation CARs, they were designed to have an intracellular domain derived from the intracellular portion of the gamma chain of Fc epsilon Rl or CD3 zeta. Thus, these first generation CARs transmit immune signal 1, which is sufficient to trigger T cell killing of cognate target cells, but is not able to fully activate T cell proliferation and survival. To overcome this limitation, second generation CARs have been constructed, which have a composite intracellular domain generated by fusing the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3 zeta, so that both an activation signal and a costimulatory signal can be transmitted upon antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, immune signal 2, which triggers T cell proliferation. Some CARs have also been described that include TNF receptor family intracellular domains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third generation CARs have now been described, which have intracellular domains capable of transmitting activation, proliferation, and survival signals.

[0207] Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more costimulatory domains.

[0208] An “antigen binding domain” refers to the portion of a chimeric antigen receptor that recognizes an antigen. In classic CARs, the antigen binding domain comprises a single chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been created with domain antibodies (dAbs), VHH antigen binding domains, or antigen binding domains derived from natural ligands of the antigen. In the present application, the antigen binding domain can be an antibody or antigen binding fragment thereof as described herein.

[0209] A “hinge domain” positions the antigen binding domain away from the surface of the effector cell to enable proper cell / cell contact, antigen binding, and activation to function. CARs optionally include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise an amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular region of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4-1BB, and CD7, which can be from the wild-type hinge region of these molecules or can be altered.

[0210] As used herein, a "transmembrane domain" (TM domain) refers to a portion of a CAR that, optionally via a hinge domain, fuses an extracellular binding moiety with intracellular moieties (e.g., a costimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of an immune effector cell. A transmembrane domain is typically a hydrophobic region of a CAR that spans the plasma membrane. The TM domain can be a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein) or a fragment thereof, an artificial hydrophobic sequence, or a combination thereof.

[0211] An intracellular domain (endodomain) is the signaling portion of a chimeric antigen receptor. It contains a signaling domain and one or more costimulatory domains. Upon antigen recognition, the receptor clusters naturally CD45 and CD148 away from the synapse and signals to the cell, thereby activating one or more immune cell effector functions (e.g., a native immune cell effector function). The most commonly used endodomain component is the endodomain component of CD3 zeta, which contains 3 ITAMs. Upon antigen binding, it transmits an activation signal to the T cell. CD3 zeta can not provide a fully sufficient activation signal and can require additional costimulatory signaling. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0212] An "intracellular signaling domain" refers to a portion of a CAR polypeptide that participates in transducing the information that the CAR is bound to a target antigen, inside the immune effector cell, to elicit effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to CAR-bound target cells, or other cellular responses elicited upon antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signaling domains containing immunoreceptor tyrosine-based activation motifs (ITAMs) include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 delta, CD3 eta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0213] A "costimulatory domain" refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding antigen. The costimulatory domain can be a costimulatory domain of, e.g., 4-1BB, CD27, CD28, or OX40.

[0214] Therapeutic methods and uses

[0215] The present disclosure provides methods for treating a disease in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein.

[0216] The present disclosure also provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0217] The present disclosure also provides an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein for use in treating a disease in a subject.

[0218] In some embodiments, the disease is a cancer, for example a cancer associated with CEACAM5 expression or a CEACAM5-positive cancer. In preferred embodiments, wherein the cancer is selected from pancreatic cancer, lung cancer, small intestine cancer, colorectal cancer, gastric cancer, cervical cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

[0219] In some embodiments, the dose administered to a subject can vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dose should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of the antibody, composition, conjugate, or chimeric antigen receptor to be administered to a human or other subject to treat a medical condition. The precise amount required can depend on many factors such as the activity of the antibody and the route of administration.

[0220] The antibody, composition, conjugate, or chimeric antigen receptor described herein can be administered to a mammal in a single dose or in a series of sub-doses over a suitable period of time, for example, as needed, once a day, once every half week, once a week, once every two weeks, once every half month, once every two months, once every half year, or once a year. A dosage unit comprising an effective amount of the antibody, composition, conjugate, or chimeric antigen receptor can be administered in a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.

[0221] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, conjugate, or chimeric antigen receptor is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, for example, orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0222] In some embodiments, the methods further comprise administering a second therapeutic agent to the subject. In some embodiments, the antibodies or antigen-binding fragments thereof, pharmaceutical compositions, conjugates, or chimeric antigen receptors disclosed herein are used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, the antibodies, compositions, conjugates, or chimeric antigen receptors disclosed herein are administered prior to, substantially simultaneously with, or following administration of the second therapeutic agent.

[0223] Kits / Drug Delivery Devices

[0224] The present disclosure provides kits or drug delivery devices comprising the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acid molecules disclosed herein, the vectors disclosed herein, the host cells disclosed herein, the conjugates disclosed herein, the pharmaceutical compositions disclosed herein, or the chimeric antigen receptors disclosed herein.

[0225] In some embodiments, the kits or drug delivery devices include one or more containers with one or more of the components of a pharmaceutical composition as described herein, such as the antibodies or antigen-binding fragments disclosed herein.

[0226] In particular embodiments, the kits include a first container comprising an antibody disclosed herein. In particular embodiments, the kits include a first container that is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

[0227] In particular embodiments, injection devices containing an antibody are provided herein. In particular embodiments, the injection devices comprise an antibody in a sterile solution. In particular embodiments, the injection devices are syringes.

[0228] In one embodiment, the kits include instructional materials that disclose the manner in which the antibodies of the disclosure are used. The instructional materials can be written, electronic (e.g., computer diskette or CD-ROM), or visual (e.g., video file). The kits can further include additional components to facilitate the use of the kits for which they are designed. Thus, for example, the kits can additionally contain tools for detecting a label (e.g., enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as secondary antibodies, etc.). The kits can also include buffers and other reagents commonly used to practice particular methods. Such kits and suitable contents are well known to those of skill in the art.

[0229] Examples

[0230] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art. Those skilled in the art will readily understand that the application can be practiced with materials and reagents other than those of the examples. The present application is directed to any and all such variations and equivalents. Further, the entire disclosure of any patent or patent application cited herein is incorporated by reference.

[0231] The control targeting CEACAM5 antibody is Tusamitamab, prepared according to the patent WO2014079886A1, hereinafter ref1.

[0232] Example 1, Preparation of antigens and stable cell lines

[0233] 1.1 Expression of recombinant CEACAM5 antigen protein and CEACAM family antigen proteins (CEACAM1, 6, 7, 8)

[0234] The nucleic acid sequence encoding the antigen protein with his, Fc tag was integrated into a mammalian cell expression vector, and after bacterial infection, lysis, plasmid extraction, and washing, the antigen protein expression plasmid was obtained. P11 was subcultured in HEK293F cells to the appropriate number, and the complex formed by PEI and the plasmid was added dropwise to the HEK293F cells subcultured at a cell density of 1.5 x 10 6 The HEK293F cells were placed in a cell incubator shaker at 37°C, 120 rpm, and 5% CO2 for culture. The first day of transfection was day 0. On the first and third days of transfection, 5% OPM-293Profeed (Opmi, P82019) was added to the volume of cells on the day of transfection. On the fourth day after transfection, the cell viability after transfection was detected using a full-automatic cell analyzer (Beckman, Vi-cell). When the cell viability decreased to about 70%, the fermentation broth was collected and filtered to obtain the antigen protein.

[0235] The relevant antigen protein amino acid sequences are as follows:

[0236] Human CEACAM5 (SEQ ID NO. 1)

[0237] Cynomolgus monkey CEACAM5 (SEQ ID NO. 2)

[0238] Rat CEACAM5 (SEQ ID NO. 3)

[0239] Human CEACAM1 (SEQ ID NO. 4)

[0240] Human CEACAM6 (SEQ ID NO. 5)

[0241] Human CEACAM7 (SEQ ID NO. 6)

[0242] Human CEACAM8 (SEQ ID NO. 7)

[0243] 1.2 Construction of HEK293 stable cell lines expressing CEACAM5 antigen protein on membrane

[0244] Integrate the nucleic acid sequence encoding the membrane protein into the lentivirus expression vector, after bacterial infection, lysis, plasmid extraction, and washing, obtain the plasmid expressing the antigen protein. After the HEK293 cells (ATCC, Cat No. CRL-3216) are recovered for 1-2 days, when the confluence reaches 70-80%, the cells are passaged, the complete medium, trypsin solution, and PBS are preheated at 37°C, the cells are gently washed with PBS, an appropriate amount of trypsin solution is added (to ensure complete coverage of the cells by gently shaking the culture bottle, about 2-3 mL for a 10 cm culture dish, and about 3-4 mL for a 15 cm culture dish), and incubated at 37°C or room temperature for 2-4 minutes. Under the microscope, the cells become rounded, and the cells are detached from the culture surface by gently tapping the side of the culture dish. Add 2-3 times the volume of trypsin to the complete medium to terminate the digestion and resuspend the cells, transfer to a centrifuge tube, centrifuge at 300g for 3-5 minutes, resuspend the cells with complete medium, count the cells, and inoculate into new culture bottles at a 1:3-1:10 passage ratio. Remove the cells, discard the supernatant, slowly add 9 mL of Opti-MEM medium (Gibco, Cat No. 31985070), try not to blow up the cells, and drop the prepared PEI / DNA complex evenly into the cells. Gently shake the culture bottle left and right and front and back, and place it in a 37°C, 5% CO2 incubator for culture. After 16-24 hours of transfection, discard the supernatant, add 30 mL of complete medium, and continue to culture in a 37°C, 5% CO2 incubator; transfection detection: after 24-72 hours of transfection, detect the expression of the relevant receptor by flow cytometry. After pressurization, sorting, and retesting, obtain the monoclonal HEK293 cells expressing human / monkey CEACAM5 membrane protein (hereinafter referred to as HEK293-hCEACAM5 / HEK293-cyCEACAM5).

[0245] Example 2, Production of Anti-CEACAM5 Chimeric Antibody

[0246] 2.1 Mouse immunization and hybridoma cell preparation

[0247] Take 6-8 weeks old female Balb / c mice (Vital River, strain code: 211), after weighing and anesthesia, immunize with plasmid encoding CEACAM5 protein or CEACAM5 antigen protein, or alternately immunize. When immunizing with DNA, take 20 μg plasmid mixed with 1 μg CpG, then directly inject into the abdomen of the mouse with a gene gun (Biorad) at 40 psi, immunize once a week. Three days before fusion, use CEACAM5 antigen protein for tail vein injection for impact immunization. When immunizing with protein, take 79 μl antigen protein mixed with 401 μL PBS, 3 mL syringe to take 480 μL of the prepared antigen mixture, another syringe to take 480 μL CFA, mix with a T-shaped tee until a white emulsion of water-in-oil type is formed. The prepared antigen is injected subcutaneously at a dose of 50 μg per mouse at the root of the thigh. Three days before fusion, immunize with 10 μg CEACAM5 antigen protein per mouse by footpad injection.

[0248] After immunization, the mice are sacrificed by cervical dislocation, and the mice are soaked in 75% alcohol solution for 5 min before collecting the spleen, popliteal lymph node, inguinal lymph node and iliac lymph node, and grinding in DMEM medium to obtain a lymphocyte-rich suspension. Take Sp2 / 0 cells and mix with an equal proportion of lymphocytes, centrifuge and resuspend, then use an electrofusion instrument to fuse the cells to obtain hybridoma cells.

[0249] 2.2 Hybridoma clone screening

[0250] 384-well plate ELISA screening

[0251] Dilute hCEACAM5 antigen protein to a concentration of 1 μg / mL with coating solution, 30 μL / well into the enzyme-labeled plate, 4°C overnight. Take the enzyme-labeled plate out of the refrigerator and wash three times with 0.05% PBST. Add 80 μL / well of blocking solution 2% milk to the plate, incubate at room temperature for 1 h. Wash once with 0.05% PBST. Add samples and controls to the enzyme-labeled plate at 30 μL / well, incubate at room temperature for 1 h. Wash five times with 0.05% PBST. Dilute the secondary antibody goat anti-mouse IgG-Fc HRP (Jackson, Cat: 115-035-071) 4000 times with 0.5% milk, 30 μL / well into the enzyme-labeled plate, incubate at room temperature for 1 h. Wash five times with 0.05% PBST. Add color developing solution, 30 μL / well, and incubate in a 37°C constant temperature incubator, develop for 10 min. Add 2M concentrated sulfuric acid stop solution, 15 μL / well, avoid bubbles, and read on the enzyme-labeled instrument within 15 min (wavelength: 450 nm).

[0252] As shown in Table 1, mAb1, mAb2, mAb3, and mAb4 clones exhibit strong binding activity to human CEACAM5 protein. Furthermore, mAb1, mAb3, and mAb4 clones show stronger binding activity to cynomolgus monkey CEACAM5 protein than the ref1 antibody. The binding of mAb1, mAb2, mAb3, and mAb4 clones to a mixture of CEACAM1, 6, 7, and 8 proteins from the same family (CEACAM1, 6, 7, and 8) is weaker than that of the ref1 antibody, demonstrating that the hybridoma clones possess better specificity for binding to CEACAM5 protein.

[0253] Table 1. ELISA binding activity of different hybridoma clone supernatants for human or cynomolgus monkey CEACAM5 protein and mixtures of related proteins.

[0254] FACS screening

[0255] HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells were labeled with CFSE (Thermo Fisher, catalog number: C34554) and the final density was 1×10⁻⁶. 7 cells / mL, per 1×10 7 Incubate HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells at room temperature in the dark for 10 min using 10 μL CFSE per cell. After incubation, wash three times with 0.5% BSA, centrifuge at 200g for 3 min, discard the supernatant, and gently break up the cell clumps. Add 3% BSA to HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells respectively, block at 4℃ for 30 min, and incubate at 5×10⁻⁶ cells / mL. 6 500 μL 3% BSA per cell; after blocking, HEK293-hCEACAM5 and HEK293-cyCEACAM5 cells were mixed together, and 0.5% BSA was added to the plate according to the number of cells to be plated, 100 μL / well; after centrifugation at 200g for 3 min, the aggregated cell clumps were gently broken up; the hybridoma supernatant culture medium and control antibody were added at 50 μL / well and incubated for 60 min; after incubation, 0.2 ml 0.5% BSA was added to wash twice, and after centrifugation at 200g for 3 min, the supernatant was discarded and the cell clumps were broken up; anti-human IgG or anti-mouse IgG was added at 30 μL / well for incubation. After incubation with secondary antibody for 30 min, 0.2 ml 0.5% BSA was added to wash three times, and after centrifugation at 200g for 3 min, the supernatant was discarded and the cells were broken up; finally, 50 μL / well PBS was added to resuspend the cells and flow cytometry was performed.

[0256] As shown in Table 2, the clones and expressions of mAb1, mAb2, mAb3, and mAb4 all exhibited strong binding activity to HEK293 cells from humans or cynomolgus monkeys.

[0257] Table 2. FACS binding activity of supernatant culture of different hybridoma clones to human or cynomolgus CEACAM5 target on HEK293 cell surface

[0258] 2.3 Hybridoma murine anti-sequence determination and chimeric antibody construction

[0259] cDNA synthesis

[0260] The hybridoma clones that bind to CEACAM5 in the selection screening were amplified, and the RNA was extracted, and the RNA (mRNA) was reverse transcribed into cDNA under the action of reverse transcriptase. The first strand of cDNA was used as a template for PCR amplification. The gene-specific upstream and downstream primers for PCR amplification were designed according to the target gene. The gene-specific upstream primer was annealed with the first strand of cDNA, and the second strand of cDNA was synthesized under the action of Taq DNA polymerase. Then, the first and second strands of cDNA were used as templates for PCR amplification with gene-specific upstream and downstream primers to obtain a large amount of cDNA.

[0261] Reverse transcription and amplification of cDNA, 65°C for 5 min, and incubation on ice for 2 min. The reaction system is as follows:

[0262] Prepare the cDNA synthesis reaction solution (5 μl reaction system), and add the following according to the table:

[0263] Take 5 μl of cDNA synthesis reaction solution into 200 μl PCR tube, mix thoroughly, spin, and react at 50°C for 50 min. After 85°C termination reaction for 5 min, immediately insert into ice. After spin, add 1 μl of RNase H to the PCR tube, and react at 37°C for 20 min. Dilute the synthesized cDNA 5 times, and store at -20°C.

[0264] Chimeric antibody plasmid construction

[0265] The reaction system for PCR amplification of V region fragments is as follows:

[0266] The amplified fragments and the vector plasmid were placed at the optimum enzyme digestion reaction temperature, and the restriction endonuclease was added for enzyme digestion reaction for 1 hour. After electrophoretic separation of the target fragments, the desired fragments were purified and recovered according to the NucleoSPIN Gel and PCR Clean-up recovery kit instructions. The recovered linearized gel recovery product of the vector and the target fragment recovery product were added with 2x HiFi Assembly ligase, and placed in a 50°C water bath for connection reaction for 15 min to obtain the chimeric expression plasmid. After competent cell transfection, amplification, lysis, filtration collection, and washing purification, the monoclonal chimeric plasmid was obtained. The sequences of the variable regions of the heavy chain and light chain of the chimeric antibody are shown in Table 3.

[0267] Table 3 Anti-CEACAM5 chimeric antibody light / heavy chain variable region sequences

[0268] The variable region sequences of the heavy chain / light chain of the anti-CEACAM5 antibody are as follows (wherein the CDR sequences are defined according to the Kabat definition rule):

[0269] mAb1

[0270] The amino acid sequence of the heavy chain VH of mAb1 is shown as SEQ ID NO. 38, the encoding nucleic acid is shown as SEQ ID NO. 39, and the CDR1, CDR2, and CDR3 are shown as SEQ ID NO. 40, 41, and 42, respectively.

[0271] Nucleotide sequence

[0272] HCDR1: SYWMHW (SEQ ID NO. 40)

[0273] HCDR2: YINPNTGYTEYSQKFKD (SEQ ID NO. 41)

[0274] HCDR3: ERGNYNYPLDY (SEQ ID NO. 42)

[0275] The amino acid sequence of the light chain VL of mAb1 is shown as SEQ ID NO. 43, the encoding nucleic acid is shown as SEQ ID NO. 44, and the CDR1, CDR2, and CDR3 are shown as SEQ ID NO. 45, 46, and 47, respectively.

[0276] Nucleotide sequence

[0277] LCDR1: RASENIYSYLA (SEQ ID NO. 45)

[0278] LCDR2: NAKTLAE (SEQ ID NO. 46)

[0279] LCDR3: QHHYGISWT (SEQ ID NO. 47)

[0280] mAb2

[0281] The amino acid sequence of the heavy chain VH of mAb2 is set forth in SEQ ID NO. 8, the encoding nucleic acid is set forth in SEQ ID NO. 9, and the CDR1, CDR2, and CDR3 thereof are set forth in SEQ ID NO. 10, 11, 12, respectively.

[0282] Nucleotide sequence

[0283] HCDR1: SDYAWN (SEQ ID NO. 10)

[0284] HCDR2: YINYIGSTSYNPSLKS (SEQ ID NO. 11)

[0285] HCDR3: ADYDEDY (SEQ ID NO. 12)

[0286] The amino acid sequence of the light chain VL of mAb2 is set forth in SEQ ID NO. 13, the encoding nucleic acid is set forth in SEQ ID NO. 14, and the CDR1, CDR2, and CDR3 thereof are set forth in SEQ ID NO. 15, 16, 17, respectively.

[0287] Nucleotide sequence

[0288] LCDR1: SASSIVHYMH (SEQ ID NO. 15)

[0289] LCDR2: DTSKLAS (SEQ ID NO. 16)

[0290] LCDR3: HQRSSYPFT (SEQ ID NO. 17)

[0291] mAb3

[0292] The amino acid sequence of the heavy chain VH of mAb3 is set forth in SEQ ID NO. 18, the encoding nucleic acid is set forth in SEQ ID NO. 19, and the CDR1, CDR2, and CDR3 thereof are set forth in SEQ ID NO. 20, 21, 22, respectively.

[0293] Nucleotide sequence

[0294] HCDR1 : NYWMNW (SEQ ID NO. 20)

[0295] HCDR2: EIRLKSDNYATHYAESVKG (SEQ ID NO. 21 )

[0296] HCDR3: DNSGWFAY (SEQ ID NO. 22)

[0297] The amino acid sequence of the VL of the light chain of mAb3 is shown in SEQ ID NO. 23, the encoding nucleic acid is shown in SEQ ID NO. 24, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 25, 26, 27 respectively.

[0298] Nucleotide sequence

[0299] LCDR1 : RASESVEYYGISLMQ (SEQ ID NO. 25)

[0300] LCDR2: AASKVES (SEQ ID NO. 26)

[0301] LCDR3: QQSRKVPSRT (SEQ ID NO. 27)

[0302] mAb4

[0303] The amino acid sequence of the VH of the heavy chain of mAb4 is shown in SEQ ID NO. 28, the encoding nucleic acid is shown in SEQ ID NO. 29, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 30, 31, 32 respectively.

[0304] Nucleotide sequence

[0305] HCDR1 : DYNVYW (SEQ ID NO. 30)

[0306] HCDR2: YIDPYNDGSNYNQKFKG (SEQ ID NO. 31 )

[0307] HCDR3: GKVDYRYAEPAWFAY (SEQ ID NO. 32)

[0308] The amino acid sequence of the VL of the light chain of mAb4 is shown as SEQ ID NO. 33, the encoding nucleic acid is shown as SEQ ID NO. 34, and the CDR1, CDR2 and CDR3 thereof are shown as SEQ ID NO. 35, 36 and 37, respectively.

[0309] Nucleotide sequence

[0310] LCDR1: RASESVEYYGTSLME (SEQ ID NO. 35)

[0311] LCDR2: AASYVES (SEQ ID NO. 36)

[0312] LCDR3: QQSRKFPFT (SEQ ID NO. 37)

[0313] Example 3, Characterization of Anti-CEACAM5 Chimeric Antibody

[0314] 3.1 Binding test of chimeric antibody to tumor cells and stable cells

[0315] The binding of chimeric antibody to stable cell lines was detected by FACS. The CEACAM5 stable cell lines HEK293-hCEACAM5 and HEK293-cyCEACAM5 in logarithmic growth phase and the CEACAM5 positive tumor cells MKN45 (National Experimental Cell Resource Sharing Platform, item number: 1101HUM-PUMC000229), BxPc-3 (ATCC, item number: CRL-1687), Caco-2 (ATCC, item number: HTB-37) and KatoIII (ATCC, item number: HTB-103) were adjusted to 5x10 5 cells / ml with PBS containing 1% BSA, 100 μl / well of cell suspension was added to a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, the supernatant was discarded, 100 μl of gradient-diluted chimeric antibody (initial concentration 200 nM, 4-fold dilution) was added to each well, and incubated at 4°C for 60 minutes. Secondary antibody was added with 50 μl / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), and incubated on ice for 20 minutes. The binding activity of CEACAM5 chimeric antibody to cells was detected by flow cytometry.

[0316] The cell binding results are shown in Table 4. The mAb1 clone has strong binding activity to tumor cells expressing CEACAM5 and HEK293 cells expressing human or cynomolgus CEACAM5 antigen protein.

[0317] Table 4. EC50 of anti-CEACAM5 chimeric antibodies to tumor cells and CEACAM5 stable cell binding

[0318] 3.2 Endocytosis activity of anti-CEACAM5 chimeric antibodies

[0319] DT3C is a fusion protein composed of diphtheria toxin and protein G 3C fragment that binds IgG, which can bind to antibody Fc, enter cells when the antibody is endocytosed, and release diphtheria toxin DT under the action of furin protease, leading to cell death. Developed by researchers at Sapporo Medical University Miki Yamaguchi in 2014, it has been widely used as an evaluation method for ADC screening (Yamaguchi, Miki, et al. Biochem Biophys Res Commun 454.4 (2014): 600-603.).

[0320] MKN45 cells expressing CEACAM5 were used as target cells, and the cell density was adjusted to 1 x 10 4 cells / well, and plated one day in advance. The next day, the DT3C protein (homemade) was gradient diluted (133 nM as the starting concentration, 5-fold gradient, a total of 9 dilutions), and incubated with 10 ug / mL experimental antibody at 37°C for 30 min to form antibody-DT3C complexes, and then the antibody-DT3C complexes were added to the cell culture medium and incubated with the cells for 3 days. The number of viable cells in each experimental group was detected by CCK-8 kit, the cell survival rate was calculated, and the antibody concentration-survival rate curve was fitted by four-parameter model. The results of cell endocytosis activity are shown in Table 5, and mAb1 clone has good cell endocytosis activity.

[0321] Table 5. IC50 of anti-CEACAM5 chimeric antibody DT3C endocytosis activity

[0322] Example 4, Humanization of anti-CEACAM5 chimeric antibodies

[0323] The germline gene with high homology to mAb1 was selected as the template, the humanized light chain template was selected as IGKV1-39*01 and IGKV3-15*01, and the J region was selected as IGKJ2*01; the humanized heavy chain template was selected as IGHV1-69*01 and IGHV3-30*01, and the J region was selected as IGHJ4*01. The CDR region of the murine anti-CEACAM5 antibody was transplanted into the corresponding humanized gene template, and part of the amino acids were mutated according to the in silico simulation results to improve the degree of humanization.

[0324] The heavy chain variable region and light chain variable region sequences of the anti-CEACAM5 humanized antibody are shown in Table 6 and Table 7, respectively.

[0325] Table 6, Heavy chain variable region sequence of the anti-CEACAM5 humanized antibody

[0326] Table 7, Light chain variable region sequence of the anti-CEACAM5 humanized antibody

[0327] The heavy chain / light chain variable region sequences of the anti-CEACAM5 humanized antibody are as follows:

[0328] The amino acid sequence of mAb1 H1 is shown as SEQ ID NO. 48, the encoding nucleic acid is shown as SEQ ID NO. 49, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 41, 42, respectively.

[0329] Nucleotide sequence

[0330] The amino acid sequence of mAb1 H2 is shown as SEQ ID NO. 50, the encoding nucleic acid is shown as SEQ ID NO. 51, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 41, 42, respectively.

[0331] Nucleotide sequence

[0332] The amino acid sequence of mAb1 H3 is shown as SEQ ID NO. 52, the encoding nucleic acid is shown as SEQ ID NO. 53, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 41, 42, respectively.

[0333] Nucleotide sequence

[0334] The amino acid sequence of mAb1 H4 is shown as SEQ ID NO. 54, the encoding nucleic acid is shown as SEQ ID NO. 55, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 41, 42, respectively.

[0335] Nucleotide sequence

[0336] The amino acid sequence of mAb1 H5 is shown as SEQ ID NO. 56, the encoding nucleic acid is shown as SEQ ID NO. 57, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 41, 42, respectively.

[0337] Nucleotide sequence

[0338] The amino acid sequence of mAb1 H6 is shown as SEQ ID NO. 58, the encoding nucleic acid is shown as SEQ ID NO. 59, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, SEQ ID NO. 60 (YINPNTGYTEYSQKFKG), SEQ ID NO. 42, respectively.

[0339] Nucleotide sequence

[0340] The amino acid sequence of mAb1 H7 is shown as SEQ ID NO. 61, the encoding nucleic acid is shown as SEQ ID NO. 62, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 60, 42, respectively.

[0341] Nucleotide sequence

[0342] The amino acid sequence of mAb1 H8 is shown as SEQ ID NO. 63, the encoding nucleic acid is shown as SEQ ID NO. 64, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 40, 60, 42, respectively.

[0343] Nucleotide sequence

[0344] The amino acid sequence of mAb1 K1 is shown as SEQ ID NO. 65, the encoding nucleic acid is shown as SEQ ID NO. 66, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47, respectively.

[0345] Nucleotide sequence

[0346] The amino acid sequence of mAb1 K2 is shown as SEQ ID NO. 67, the encoding nucleic acid is shown as SEQ ID NO. 68, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0347] Nucleotide sequence

[0348] The amino acid sequence of mAb1 K3 is shown as SEQ ID NO. 69, the encoding nucleic acid is shown as SEQ ID NO. 70, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0349] Nucleotide sequence

[0350] The amino acid sequence of mAb1 K4 is shown as SEQ ID NO. 71, the encoding nucleic acid is shown as SEQ ID NO. 72, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0351] Nucleotide sequence

[0352] The amino acid sequence of mAb1 K5 is shown as SEQ ID NO. 73, the encoding nucleic acid is shown as SEQ ID NO. 74, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0353] Nucleotide sequence

[0354] The amino acid sequence of mAb1 K6 is shown as SEQ ID NO. 75, the encoding nucleic acid is shown as SEQ ID NO. 76, and the CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 45, 46, 47 respectively.

[0355] Nucleotide sequence

[0356] The heavy chain amino acid sequence of mAb1 H6K3 is shown as SEQ ID NO. 77, the encoding nucleic acid is shown as SEQ ID NO. 78; and the light chain amino acid sequence is shown as SEQ ID NO. 79, the encoding nucleic acid is shown as SEQ ID NO. 80.

[0357] Heavy chain amino acid sequence

[0358] Heavy chain nucleic acid sequence

[0359] Light chain amino acid sequence

[0360] Light chain nucleic acid sequence

[0361] The heavy chain amino acid sequence of mAb1 H6K5 is set forth in SEQ ID NO. 81, and the encoding nucleic acid is set forth in SEQ ID NO. 82; the light chain amino acid sequence is set forth in SEQ ID NO. 83, and the encoding nucleic acid is set forth in SEQ ID NO. 84.

[0362] Heavy chain amino acid sequence

[0363] Heavy chain nucleic acid sequence

[0364] Light chain amino acid sequence

[0365] Light chain nucleic acid sequence

[0366] Example 5, Affinity characterization of anti-CEACAM5 humanized

[0367] 5.1 Affinity of anti-CEACAM5 humanized antibodies to hCEACAM5

[0368] After the full sequence synthesis of the light chain variable region and the heavy chain variable region, they were cloned into eukaryotic expression vectors containing the antibody kappa chain constant region Ckappa or human IgG1 constant region CH1-CH3. After the combination and pairing of the light chain and heavy chain plasmids, the cells were transfected to express the antibody. The Gator (BLI, Bio-Layer Interferometry) was used to detect the affinity between the antibody and the antigen. The commercial Protein A or HFC (anti-hIgG Fc) probe was used to fix the antibody, and then the antigen was combined to record the intermolecular binding and dissociation process in real time to measure the affinity between the antigen and the antibody. One column of Protein A probes was placed on a MAX plate containing 260 μl of buffer, soaked for 10 minutes, and one column of regeneration solution and buffer was added to the MAX plate as a neutralizing solution. The antibody was diluted with buffer to 5 μg / ml, and 200 μl / well was transferred to the 96-well sample. The antibody solidification height was 1.5 nm. The monovalent antigen was diluted with buffer to a maximum concentration of 10×KD, and then diluted with a 3-fold concentration gradient, a total of 3 concentration gradients and one blank buffer as a negative control. Two columns of buffer were set in the 96-well plate for balancing the probe. The sample plate and the MAX plate were placed in the corresponding positions of the instrument, and the sample plate was set to tilt mode. The program baseline1 was set to 30 seconds, loading was set to 100 seconds, baseline2 was set to 30 seconds, association was set to 150 seconds, dissociation was set to 150 seconds, and regeneration / neutralization was set to 5 seconds each and repeated 3 times.

[0369] The affinity characterization of the humanized VH and VL pair is shown in Table 8. The H5, H6 heavy chain and K3, K4, K5, K6 light chain pair has a higher affinity.

[0370] Table 8. Affinity of humanized light and heavy chain paired antibodies

[0371] 5.2 Binding of anti-CEACAM5 humanized antibodies to tumor cells

[0372] The binding of the candidate antibodies to CEACAM5-positive tumor cells was detected by FACS. The MKN45 cells in the logarithmic growth phase were adjusted to 5×10 5 cells / ml with PBS containing 1% BSA, 100 μl / well of cell suspension was added to the 96-well U-shaped plate, centrifuged at 300g for 5 minutes, the supernatant was discarded, and 100 μl of gradient-diluted chimera was added to each well. Incubate at 4°C for 60 minutes. The secondary antibody was added with 50 μl / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), and incubated on ice for 20 minutes. The binding activity of the CEACAM5 candidate antibody to the cells was detected by flow cytometry.

[0373] The results are shown in Table 9. The humanized antibodies have strong binding activity to CEACAM5-positive tumor cells.

[0374] Table 9. EC50 of humanized light-heavy chain paired antibody binding to tumor cells

[0375] 5.3 Binding of anti-CEACAM5 humanized antibodies to various species of CECAM5 antigens and CEACAM family proteins

[0376] The monoclonal antibody was digested into Fab using papain, and the affinity to various species of CECAM5 antigens and CEACAM family proteins was detected. The antibody and activated papain were mixed at a mass ratio of 10:1, incubated at 37°C for 3 hours, then an appropriate amount of Protein A filler (40 μg of protein was added to 1 μL of Protein A filler) was added to the digestion product and incubated for 20 min, and the undigested IgG and digested Fc were removed, and finally the Fab fragment was obtained.

[0377] Using surface plasmon resonance technology (Biacore T200 / SPR), Anti-His monoclonal antibody was coupled to the chip surface by amino coupling on the CM5 chip surface, and various species of antigen proteins were captured to the chip surface as ligands by specific binding of the 6*His tag of Anti-His monoclonal antibody, then the gradient diluted Fab was flowed through the chip surface as the analyte, the monovalent affinity of Fab to the antigen was detected, and finally the data was analyzed by Biacore analysis software 3.0 analysis software, and the 1:1 affinity between the antigen and antibody was obtained by langmiur 1:1 kinetics fitting processing.

[0378] The affinity detection results are shown in Figure 1. The affinity of mAb1 H6K3 and mAb1 H6K5 to human and cynomolgus CEACAM5 proteins is stronger than that of ref1, and they do not bind to mouse CEACAM5 and CEACAM5 family proteins. The affinity difference of CEACAM5 mAb1 H6K3 to human and cynomolgus CEACAM5 proteins is within 10 times.

[0379] Example 6, endocytosis activity of anti-CEACAM5 humanized antibodies in high-CEACAM5-expressing tumor cells

[0380] The density of CEACAM5-expressing cells MKN45 was adjusted to 1×10 4Cells were plated at 1,000 cells / well the day before. The next day, DT3C protein was gradient diluted (133 nM as the starting concentration, 5-fold gradient, a total of 9 dilutions), and incubated with the experimental group antibodies (10 ug / mL) at 37°C for 30 min to form antibody-DT3C complexes, and then the antibody-DT3C complexes were added to the cell culture medium and incubated with the cells for 3 days. The CCK-8 kit was used to detect the number of viable cells in each experimental group, the cell survival rate was calculated, and the antibody concentration-survival rate curve was obtained by fitting with a four-parameter model.

[0381] The results are shown in Figure 2. The mAb1 H6K3 and mAb1 H6K5 antibodies have good endocytosis activity, and the endocytosis activity is better than that of ref1.

[0382] Example 7, FACS binding of anti-CEACAM5 humanized antibodies to tumor cells highly expressing CEACAM5

[0383] FACS was used to detect the binding of candidate antibodies to tumor cells highly expressing CEACAM5. MKN45 (National Experimental Cell Resource Sharing Platform, Catalog No: 1101HUM-PUMC000229), BxPc-3 (ATCC, Catalog No: CRL-1687), Caco-2 (ATCC, Catalog No: HTB-37), KatoIII (ATCC, Catalog No: HTB-103), SNU16 (ATCC, Catalog No: CRL-5974), HPAC (ATCC, Catalog No: CRL-2119), and NCI-H2110 (ATCC, Catalog No: CRL-5924) cells in the logarithmic growth phase were adjusted to 5x10 5 Cells were plated at 1,000 cells / well the day before. The next day, DT3C protein was gradient diluted (133 nM as the starting concentration, 5-fold gradient, a total of 9 dilutions), and incubated with the experimental group antibodies (10 ug / mL) at 37°C for 30 min to form antibody-DT3C complexes, and then the antibody-DT3C complexes were added to the cell culture medium and incubated with the cells for 3 days. The CCK-8 kit was used to detect the number of viable cells in each experimental group, the cell survival rate was calculated, and the antibody concentration-survival rate curve was obtained by fitting with a four-parameter model.

[0384] The results are shown in Figure 3. The mAb1 H6K3 antibody has good binding ability to tumor cells expressing CEACAM5.

[0385] Example 8, immunogenicity stimulation of anti-CEACAM5 humanized antibodies to PBMC

[0386] Peripheral venous blood was collected from 24 healthy volunteers, and PBMC was collected by Ficoll centrifugation. The cells were washed once with PBS, counted, and the cell density was adjusted to 2.5x106 The cells were diluted to 1 x 105cells / ml, 100 μl was added to a 96-well U-shaped plate. The diluted antibody sample 100 μl was added to the cells, and incubated at 37°C, 5% CO2for 48 hours. Donanemab (homemade) was used as a positive control, mcKLH (Thermo Fisher, item number 77600) was used as a positive control 2, and the wells without sample were used as a negative control.

[0387] The supernatant was discarded, and the 96-well plate was washed once with 200 μl / well of flow buffer, 50 μl of blocking solution was added, and incubated on ice for 30 minutes. 1 μl / well of FITC anti-human CD4, APC anti-human CD137 (OX40), and PE anti-human CD134 (4-1BB) fluorescent antibodies were added, and incubated on ice for 45 minutes. 50 μl of 5 μg / ml PI was added for staining for 5 minutes, and the flow buffer was washed twice. The cells were resuspended in 100 μl of PBS, and analyzed by flow cytometry.

[0388] The results are shown in Figure 4. The CEACAM5 humanized candidate antibody mAb1 H6K3 had no obvious immunogenicity reaction.

[0389] Example 9, PK parameters of anti-CEACAM5 humanized antibodies in FcRn humanized mice

[0390] 6-8-week-old female B-hFcRn mice (Bioss, item number: 110001) were raised in a sterile animal room at 25°C with 12-hour alternating day and night, and were allowed to eat and drink freely. The experimental group mice (4 mice per group) were injected with 10 mg / kg of antibody via the tail vein, and the antibody was detected for endotoxin and diluted in about 100 μL of sterile saline. The orbital venous plexus blood 100-200 μL was collected before injection (0h) and 5 min, 7h, D1, D3, D5, D7, D14, D21, D28, D35 after injection, and the serum was separated by centrifugation and stored at -80°C for detection.

[0391] hCEACAM5 (cHis) was diluted to 0.5 pg / ml with coating solution, 100 pL / well was added to the enzyme-labeled plate, and coated overnight at 2-8°C. Then the plate was washed 3 times with 0.05% PBST (plate 1), 200 pL / well of 2% milk was added to the plate, and blocked at room temperature for 60 min, and the plate was washed once with 0.05% PBST. The standard was diluted by 2-fold gradient from 10 pg / mL with blank C57 mouse serum, then diluted with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. The standard was diluted to high concentration quality control (3750 ng / mL), medium concentration quality control (800 ng / mL), and low concentration quality control (58.5 ng / mL) with blank mouse serum, then diluted with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. The sample was diluted with 0.5% milk, then diluted with C57 mouse serum to the corresponding fold, and then the plate was washed 5 times with 0.05% PBST. Anti-hlgGl HRP (biolegend, cat: 410603) was diluted 6000-fold with 0.5% milk, 100 uL / well was added to the enzyme-labeled plate, and incubated at room temperature for 1 h. The plate was washed 5 times, 100 pL / well of color developing solution was added, bubbles were avoided, the sealing film was pasted, and color development was carried out in a 37°C constant temperature incubator for 10 min in the dark. 2M sulfuric acid stop solution, 50 pL / well, was added to avoid bubbles, mixed well, and the enzyme-labeled instrument was read within 5 min (wavelength: 450 nm).

[0392] The results are shown in Figure 5, the half-life of CEACAM5 humanized antibody mAb1 H6K3 hlgG1 in FcRn humanized mice was 165.7 h, and the half-life of ref1 antibody was 116.5 h. The blood clearance rate of mAb1 H6K3 hlgG1 in humanized mice was lower than that of ref1 antibody.

[0393] While the preferred embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that many changes, modifications, and substitutions can be made thereto without departing from the application. It is to be understood that various alternatives to the embodiments described herein are possible. The following claims are intended to define the scope of the application and encompass methods and structures within the scope of the claims and equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that binds CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 41 or 60, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, 46, 47, respectively; or (2) the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10, 11, 12, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 15, 16, 17, respectively; or (3) the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20, 21, 22, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, 26, 27, respectively; or (4) the VH comprises a HCDR1, a HCDR2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 30, 31, 32, respectively, and the VL comprises a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 35, 36, 37, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 38, 48, 50, 52, 54, 56, 58, 61, 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 43, 65, 67, 69, 71, 73, 75; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; or (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

33.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein: the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 38, 56, 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 43, 69, 73.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein: the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69 or 73.

5. The antibody or antigen-binding fragment thereof of claim 2, wherein: (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 43; or (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 69; or (3) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73; or (4) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13; or (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

33. (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23; or (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

33.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein: (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; or (ii) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

81.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

13. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-12.

14. The nucleic acid according to claim 13, comprising: (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 39, 49, 51, 53, 55, 57, 59, 62, 64, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 44, 66, 68, 70, 72, 74, 76; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; or (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; or (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

34.

15. The nucleic acid according to claim 14, comprising (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70; or (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 59, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 74; or (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14; or (5) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24; or (6) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

34.

16. The nucleic acid according to claim 15, comprising (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 80, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 78; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 84, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

82.

17. A vector comprising the nucleic acid according to any one of claims 13-16.

18. A host cell comprising the nucleic acid according to any one of claims 13-16 or the vector according to claim 17.

19. A pharmaceutical composition comprising (i) the antibody or antigen binding fragment thereof according to any one of claims 1-12; and (ii) a pharmaceutically acceptable carrier or excipient.

20. The pharmaceutical composition according to claim 19, further comprising a second therapeutic agent.

21. The pharmaceutical composition according to claim 20, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

22. A conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-12, and a chemical moiety conjugated thereto.

23. The conjugate according to claim 22, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

24. A chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-12.

25. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-12, a pharmaceutical composition according to any one of claims 19-21, a conjugate according to claim 22 or 23, or a CAR according to claim 24.

26. The method of claim 25, wherein, The disease is a cancer, e.g., a cancer associated with CEACAM5 expression.

27. The method of claim 26, wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, small intestine cancer, colorectal cancer, gastric cancer, cervical cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, and skin cancer.

28. The method according to any one of claims 25-27, further comprising administering to the subject a second therapeutic agent.

29. The method according to claim 28, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

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