CN119431584A - MUC1 binding molecules and their applications - Google Patents

Abstract

The present invention provides MUC1 binding molecules, including MUC1 single domain antibodies, that specifically bind to the extracellular domain of MUC 1.

Description

MUC1 binding molecules and uses thereof

Technical Field

The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a MUC1 binding molecule and application thereof. The MUC1 binding molecule of the invention can be combined with MUC1 extracellular region with high specificity, has higher affinity and biological activity, low immunogenicity, stable structure and good patentability.

Background

MUC1 is a mucin, expressed in a variety of epithelial cells, and highly expressed in a variety of solid and hematological tumor cells, MUC1 being one of the most potentially valuable targets in the field of tumor therapy. Due to the differences in the oxidative glycosylation modification of MUC1 and the occurrence of cleavage, MUC1 has a large difference in the structure of tumor cell surface from that of normal cell surface, and these differences determine that MUC1 will be a specific target for solid tumors.

The nano antibody has natural advantages of easy expression, high stability, high affinity and the like, and has very wide application prospect in developing the MUC1 nano antibody according to the self advantages of the nano antibody and the MUC1 biological mechanism.

Disclosure of Invention

The present invention aims to provide MUC1 binding molecules having affinity and uses thereof.

In a first aspect the invention provides a MUC1 binding molecule comprising a MUC1 single domain antibody having one or more amino acid mutations compared to a wild-type single domain antibody having a wt-CDR1 as shown in SEQ ID NO. 1, a wt-CDR2 as shown in SEQ ID NO. 10 and a wt-CDR3 as shown in SEQ ID NO. 17.

In one or more embodiments, the amino acid mutation is located in a CDR region of a wild-type single domain antibody;

In one or more embodiments, the MUC1 single domain antibody comprises CDR1, CDR2, and CDR3, the MUC1 single domain antibody having one or more of the following features (1) - (3):

(1) The CDR1 contains one or more mutations compared to SEQ ID NO 1 of T3Q, R D, R5H, R5K, R5E, R5Y, R6E, R6H, R6K, R6D, R Y, preferably one or more mutations selected from the group consisting of T3Q, R5D, R6E, R H and R6H, R K and R6K, R5D and R6D, R5E and R6E, R Y and R6Y,

(2) The CDR2 contains one or more mutations compared to SEQ ID NO 10 of I1G, T3Q, T3W, F4D, D6A, D6G, D7S, T Q, preferably one or more mutations selected from the group consisting of I1G, T3W, D6G, F D and D6A, T3Q and T8Q, D G and D7S,

(3) The CDR3 contains one or more mutations compared to SEQ ID NO 17 of T1Q, I3G, Y6H, Q8E, Q8Y, L9G, L9Y, L9S, S10N, D Q, preferably one or more mutations selected from the group consisting of T1Q, Q8E, Q8Y, L9G, I G and L9G, L9Y, L9S, 6H and S10N and D12Q.

In one or more embodiments, the MUC1 single domain antibody comprises CDR1, CDR2, and CDR3, the CDR1, CDR2, CDR3 comprising the mutations shown in any one of the following groups A1 to a 17:

In one or more embodiments, the MUC1 single domain antibody has one or more of the following characteristics:

(1) The CDR1 is shown in any one of SEQ ID NO 1-9,

(2) The CDR2 is shown in any one of SEQ ID NO 10-16,

(3) The CDR3 is shown in any one of SEQ ID NO 17-25;

and the CDR1, the CDR2 and the CDR3 are not SEQ ID NO. 1, SEQ ID NO. 10 and SEQ ID NO. 17 respectively.

In one or more embodiments, the MUC1 single domain antibody contains CDR1, CDR2, and CDR3 as set forth in any one of SEQ ID NOs of groups a1 to a17 below:

CDR1	CDR2	CDR3	Group of
				2	11	18	a1
3	12	19	a2
				6	10	17	a3
7	10	17	a4
				8	10	17	a5
4	10	17	a6
				9	10	17	a7
1	10	20	a8
				1	10	21	a9
5	10	22	a10
				1	10	24	a11
1	10	25	a12
				1	13	23	a13
2	10	17	a14
				1	14	17	a15
1	15	17	a16
				1	16	17	a17

In one or more embodiments, the wild-type single domain antibody is set forth in SEQ ID NO. 32.

In one or more embodiments, the anti-MUC 1 single domain antibody has reduced affinity for MUC1 as compared to a wild-type single domain antibody.

In one or more embodiments, the anti-MUC 1 single domain antibody binds MUC1 at K _D of about 1 x 10 ^-8～5×10^-7 M, preferably 1.5 x 10 ^-8～2×10^-7 M. The affinity is determined by Surface Plasmon Resonance (SPR).

In one or more embodiments, the anti-MUC 1 single domain antibody comprises FR1, FR2, FR3 and FR4, wherein FR1 is shown as SEQ ID NO 26 or 27, FR2 is shown as SEQ ID NO 28, FR3 is shown as SEQ ID NO 29 or 30, and FR4 is shown as SEQ ID NO 31.

In one or more embodiments, the MUC1 single domain antibody has an amino acid sequence as set forth in any one of SEQ ID NOS.33-49, preferably as set forth in SEQ ID NOS.33, 34, 40 or 45.

In one or more embodiments, the MUC1 binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment thereof comprising one, two, or more anti-MUC 1 single domain antibodies described herein.

In one or more embodiments, the multivalent or multispecific single domain antibody is linked to a plurality of single domain antibodies by a linker. The linker consists of 1-15 amino acids selected from G and S.

In one or more embodiments, the antigen binding fragment of the heavy chain antibody is a single chain heavy chain antibody.

In one or more embodiments, the heavy chain antibody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

In one or more embodiments, the heavy chain antibody comprises a MUC1 single domain antibody and a heavy chain constant region.

In one or more embodiments, the heavy chain constant region is a constant region of a camelid heavy chain antibody comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of human IgG Fc, e.g., CH2 and CH3 of IgG1 or IgG 4. Preferably, the heavy chain constant regions are CH2 and CH3 of IgG4, and the amino acid sequence is shown as SEQ ID NO. 50.

In one or more embodiments, the MUC1 binding molecules are MUC1 single domain antibodies and human IgG4 Fc.

In one or more embodiments, the heavy chain constant region is a constant region of a cartilaginous fish heavy chain antibody comprising CH1, CH2, CH3, CH4, and CH5.

In one or more embodiments, the antibody is an antibody comprising the anti-MUC 1 single domain antibody as a heavy chain variable domain.

In one or more embodiments, the antibody further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.

In one or more embodiments, the antigen binding fragment of the antibody is selected from the group consisting of Fab, F (ab') 2, fv, scFv.

In one or more embodiments, the binding molecules of any of the embodiments of the invention are chimeric antibodies or fully human antibodies, preferably fully human antibodies.

The invention also provides a polynucleotide selected from the group consisting of:

(1) The coding sequence of a single domain antibody described in any of the embodiments herein or an antibody or antigen binding fragment thereof described herein;

(2) The complement of (1);

(3) A fragment of 5-50bp of any one of the sequences (1) or (2).

In one or more embodiments, the fragments are primers.

The invention also provides a nucleic acid construct comprising a polynucleotide as described herein.

In one or more embodiments, the nucleic acid construct is a recombinant vector or an expression vector.

The invention also provides a bacteriophage comprising a MUC1 binding molecule according to any of the embodiments herein.

In one or more embodiments, the MUC1 binding molecule is displayed on the phage surface.

The invention also provides a host cell selected from the group consisting of:

(1) Expressing a MUC1 binding molecule as described in any of the embodiments herein;

(2) Comprising a polynucleotide as described herein, and/or

(3) Comprising the nucleic acid construct described herein.

The invention also provides a method of producing a MUC1 binding molecule comprising culturing a host cell as described herein under conditions suitable for the production of a MUC1 binding molecule (e.g., a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, an antibody, or an antigen binding fragment thereof), and optionally purifying the MUC1 binding molecule from the culture.

The invention also provides a pharmaceutical composition comprising a MUC1 binding molecule, polynucleotide, nucleic acid construct, phage or host cell as described in any of the embodiments herein, and a pharmaceutically acceptable adjuvant.

In one or more embodiments, the pharmaceutical composition is for use in treating cancer.

In one or more embodiments, the cancer is a MUC 1-associated cancer. Preferably, the cancer is selected from breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gall bladder cancer, oesophageal cancer, prostate cancer or glioblastoma.

The invention also provides the use of a MUC1 binding molecule as described in any of the embodiments herein in the manufacture of a medicament for the prevention or treatment of cancer.

In one or more embodiments, the cancer is a MUC 1-associated cancer. Preferably, the cancer is selected from breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gall bladder cancer, oesophageal cancer, prostate cancer or glioblastoma.

The invention also provides a method of treating or preventing cancer comprising administering to a patient in need thereof a therapeutically effective amount of a MUC1 binding molecule according to any of the embodiments of the invention, or a pharmaceutical composition comprising a MUC1 binding molecule according to any of the embodiments of the invention.

In one or more embodiments, the cancer is a MUC 1-associated cancer. Preferably, the cancer is selected from breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gall bladder cancer, oesophageal cancer, prostate cancer or glioblastoma.

The invention also provides a kit for detecting MUC1 for assessing the effect of a drug treatment or diagnosing cancer, said kit comprising a MUC1 binding molecule, polynucleotide, nucleic acid construct, phage, host cell as described in any of the embodiments herein.

In one or more embodiments, the kit further comprises reagents for detecting the binding of MUC1 to a single domain antibody, antibody or antigen binding fragment thereof. The bound reagent is detected, for example, by an enzyme-linked immunosorbent assay.

In one or more embodiments, the detection binding reagent is a detectable label, such as biotin, that is capable of linking to the MUC1 binding molecule. The detectable label is linked to the MUC1 binding molecule or is separately present in the kit.

The invention also provides a non-diagnostic method of detecting the presence of MUC1 in a sample, the method comprising incubating the sample with a MUC1 binding molecule as described in any of the embodiments herein, and detecting the binding of MUC1 to a single domain antibody, antibody or antigen binding fragment thereof, thereby determining the presence of MUC1 in the sample. The detection is an enzyme-linked immunosorbent assay.

The invention also provides the use of a MUC1 binding molecule as described in any of the embodiments herein in the manufacture of a kit for detecting MUC1 in a sample, assessing the effect of a drug treatment or diagnosing cancer.

Drawings

FIGS. 1-2 show ELISA detection results of candidate antibodies and MUC1 protein.

FIG. 3 shows the results of detection of binding of candidate antibodies to MB468 tumor cell lines.

FIG. 4 shows the results of binding assays of candidate antibodies to H226 tumor cell lines.

FIG. 5 shows the results of detection of binding of candidate antibodies to the RPMI8226 tumor cell line.

FIG. 6 shows the results of detection of binding of candidate antibodies to SKOV3 tumor cell lines.

Detailed Description

The present inventors have conducted extensive and intensive studies to screen wild-type single-domain antibodies that bind MUC1 with high specificity through a large number of screens. The present inventors have mutated wild-type single domain antibodies, screened for MUC1 single domain antibody mutants having affinity and high biological activity, and provided MUC1 binding molecules comprising anti-MUC 1 single domain antibody mutants. The MUC1 binding molecule of the invention can specifically bind MUC1, has better affinity and high biological activity, low immunogenicity, stable structure and good patentability.

Antibodies to

As used herein, a "MUC1 binding molecule" is a protein that specifically binds MUC1, including but not limited to antibodies, antigen binding fragments of antibodies, heavy chain antibodies, nanobodies (nanobodies), minibodies (minibodies), affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen binding fragments, e.g., fab, F (ab') 2, and Fv. The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetramer units and a further polypeptide called the J chain, containing 10 antigen binding sites, while IgA antibodies contain 2-5 basic 4 chain units that can polymerize in combination with the J chain to form multivalent assemblies. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds being dependent on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH 1, CH2 and CH3 for each alpha and gamma chain) and four (CH 1, CH2, CH3 and CH 4) constant domains (CH) for the mu and epsilon isoforms and a Hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL and VH are aligned together, while CL and the first constant domain of the heavy chain (CH 1) are aligned together. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic AND CLINICAL Immunology, eighth edition, daniel p.sties, abba i.terr and Tristram g.Parsolw editions, appleton & Lange, norwalk, CT,1994, pages 71 and chapter 6. Light chains from any vertebrate species can be classified, based on their constant domain amino acid sequences, into one of two distinct types called kappa and lambda. Immunoglobulins may be assigned to different classes or isotypes depending on their heavy chain constant domain (CH) amino acid sequence. There are five classes of immunoglobulins IgA, igD, igE, igG and IgM, with heavy chains called α, δ, ε, γ and μ, respectively. The gamma and alpha classes can be further divided into subclasses based on relatively small differences in CH sequence and function, e.g., human expressed subclasses IgG1, igG2A, igG2B, igG3, igG4, igA1, and IgA2.

The "heavy chain antibody" as referred to herein is an antibody derived from a camelidae or cartilaginous fish organism. In contrast to the 4-chain antibodies described above, the heavy chain antibody lacks the light and heavy chain constant region 1 (CH 1), comprising only 2 heavy chains consisting of a variable region (VHH) linked to the constant region by a hinge-like structure and other constant regions. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.

As used herein, the terms "single domain antibody", "anti-MUC 1 single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", "nanobody" are used interchangeably and refer to a single domain antibody that specifically recognizes and binds to MUC 1. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Single domain antibodies are the smallest functional antigen binding fragments. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

The binding molecules comprising two or more single domain antibodies are multivalent single domain antibodies and the binding molecules comprising two or more different specific single domain antibodies are multispecific single domain antibodies. Multivalent or multispecific single domain antibodies connect multiple single domain antibodies via linkers. The linker generally consists of 1-15 amino acids selected from G and S.

Herein, heavy chain antibodies and antibodies are intended to distinguish between different combinations of antibodies. Because of the similarity in structure, the following structural descriptions for antibodies are applicable to heavy chain antibodies as well as to light chains.

"Variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

The term "variable" refers to the case where certain segments in the variable domain differ widely in antibody sequence. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domains. Instead, it focuses on three segments called hypervariable regions (HVRs), both in the light and heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 for the heavy chain variable region (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 for the light chain variable region, respectively. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR 4) that mostly take on a β -sheet conformation, linked by three HVRs that form a loop linkage and in some cases form part of the β -sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen binding site of the antibody. Typically, the light chain variable region is of the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4 and the heavy chain variable region is of the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cell-mediated cytotoxicity.

"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1 q) of the classical complement system. In the IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody, and the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as the stretch of sequence from the amino acid residue at heavy chain position C226 or P230 to the carboxy-terminus, wherein the numbering is according to the EU index as in Kabat. As used herein, the Fc region may be a native sequence Fc or a variant Fc.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. The antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, scFv-Fc fragments, multispecific antibodies formed from antibody fragments, and any fragments that should be capable of increasing half-life by chemical modification or by incorporation into liposomes. Digestion of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, which has the ability to crystallize readily. The Fab fragment consists of the complete light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH 1). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of antibodies produced a larger F (ab') 2 fragment, roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F (ab ') 2 antibody fragments were initially generated as pairs of Fab ' fragments with hinge cysteines between the Fab ' fragments. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also the region recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for heavy and light chains) are highlighted from the fold of these two domains, contributing to the antigen-binding amino acid residues and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less avidity than the complete binding site. "Single chain Fv" may also be abbreviated "sFv" or "scFv" and is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen-binding structure.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention may be generated by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for producing human or human-like antibodies from animals having a portion or the entire human immunoglobulin locus or gene encoding a human immunoglobulin sequence, single cell sequencing methods.

Monoclonal antibodies herein also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"Humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged for sequences found in a human antibody. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are grafted into the β -sheet framework of the human antibody variable region to produce antibodies, the specificity of which is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies, and the like include humanized variants of each of the antibodies.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing a human antibody. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

In some embodiments, the invention also provides a single domain antibody, heavy chain antibody, antibody or antigen binding fragment thereof that binds to the same epitope of MUC1 as any of the anti-MUC 1 single domain antibodies of the invention, i.e., a single domain antibody, heavy chain antibody, antibody or antigen binding fragment thereof that is capable of cross-competing with any single domain antibody of the invention for binding to MUC 1.

The present invention provides a MUC1 binding molecule comprising a MUC1 single domain antibody having one or more amino acid mutations compared to a wild-type single domain antibody having a wt-CDR1 as shown in SEQ ID NO. 1, a wt-CDR2 as shown in SEQ ID NO. 10 and a wt-CDR3 as shown in SEQ ID NO. 17.

In one or more embodiments, the amino acid mutation is located in a CDR region of a wild-type single domain antibody;

In one or more embodiments, the MUC1 single domain antibody comprises CDR1, CDR2, and CDR3, the MUC1 single domain antibody having one or more of the following features (1) - (3):

(1) The CDR1 contains one or more mutations compared to SEQ ID NO 1 of T3Q, R D, R5H, R5K, R5E, R5Y, R6E, R6H, R6K, R6D, R Y, preferably one or more mutations selected from the group consisting of T3Q, R5D, R6E, R H and R6H, R K and R6K, R5D and R6D, R5E and R6E, or R5Y and R6Y,

(2) The CDR2 contains one or more mutations compared to SEQ ID NO 10 of I1G, T3Q, T3W, F4D, D6A, D6G, D7S, T Q, preferably one or more mutations selected from the group consisting of I1G, T3W, D6G, F D and D6A, T3Q and T8Q, or D6G and D7S,

(3) The CDR3 contains one or more mutations compared to SEQ ID NO 17 of T1Q, I3G, Y6H, Q8E, Q8Y, L9G, L9Y, L9S, S10N, D Q, preferably one or more mutations selected from the group consisting of T1Q, Q8E, Q8Y, L9G, I G and L9G, L9Y, L9S, or 6H and S10N and D12Q.

Wherein T3Q represents that threonine at position 3 is replaced by glutamine, R5H\R6H represents that the mutation of R5H and the mutation of R6H are simultaneously carried out, and other mutations are similar.

In one or more embodiments, the MUC1 single domain antibody comprises CDR1, CDR2, and CDR3, the CDR1, CDR2, CDR3 comprising the mutations shown in any one of the following groups A1 to a 17:

Wherein, the A1 group means that the CDR1, CDR2 and CDR3 of the MUC1 single domain antibody contain mutations at the same time, and the mutations contained in the CDR1, CDR2 and CDR3 are CDR1:R5D, CDR2:F4D and D6A, and CDR3:Y6H and S10N and D12Q respectively.

In one or more embodiments, the MUC1 single domain antibody has one or more of the following features (1) - (3):

(1) The CDR1 is shown in any one of SEQ ID NO 1-9,

(2) The CDR2 is shown in any one of SEQ ID NO 10-16,

(3) The CDR3 is shown in any one of SEQ ID NO 17-25;

and the CDR1, the CDR2 and the CDR3 are not SEQ ID NO. 1, SEQ ID NO. 10 and SEQ ID NO. 17 respectively.

In one or more embodiments, the MUC1 single domain antibody contains CDR1, CDR2, and CDR3 as set forth in any one of SEQ ID NOs of groups a1 to a17 below:

CDR1	CDR2	CDR3	Group of
				2	11	18	a1
3	12	19	a2
				6	10	17	a3
7	10	17	a4
				8	10	17	a5
4	10	17	a6
				9	10	17	a7
1	10	20	a8
				1	10	21	a9
5	10	22	a10
				1	10	24	a11
1	10	25	a12
				1	13	23	a13
2	10	17	a14
				1	14	17	a15
1	15	17	a16
				1	16	17	a17

。

In one or more embodiments, the wild-type single domain antibody is set forth in SEQ ID NO. 32.

In one or more embodiments, the anti-MUC 1 single domain antibody has reduced affinity for MUC1 as compared to a wild-type single domain antibody, and the anti-MUC 1 single domain antibody binds MUC1 with a K _D of about 1X 10 ^-8～5×10^-7 M, preferably 1.5X10 ^-8～2×10^-7 M. The affinity is determined by Surface Plasmon Resonance (SPR).

In one or more embodiments, the anti-MUC 1 single domain antibody comprises FR1, FR2, FR3, and FR4. The FR region may be derived from a mutation that is a wild-type single domain antibody or a wild-type single domain antibody.

In one or more embodiments, FR1 is shown as SEQ ID NO 26 or 27, FR2 is shown as SEQ ID NO 28, FR3 is shown as SEQ ID NO 29 or 30, and FR4 is shown as SEQ ID NO 31.

In one or more embodiments, the MUC1 single domain antibody has an amino acid sequence as set forth in any one of SEQ ID NOS.33-49, preferably as set forth in SEQ ID NOS.33, 34, 40 or 45.

The MUC1 binding molecules described herein may be monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, antibodies or antigen-binding fragments thereof comprising one, two or more anti-MUC 1 single domain antibodies described herein.

In some embodiments, the MUC1 binding molecules herein comprise an anti-MUC 1 single domain antibody and an immunoglobulin Fc region. The Fc regions useful in the present invention may be from immunoglobulins of different subtypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM. In some embodiments, the immunoglobulin Fc region is an IgG4 Fc, which has the amino acid sequence shown in SEQ ID NO. 50.

In some embodiments, the MUC1 binding molecules described herein are heavy chain antibodies. The heavy chain antibody further comprises a heavy chain constant region, for example a constant region of a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

The invention also includes the antibody derivatives and analogs. "derivatives" and "analogs" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The derivative or analogue of the invention may be (i) a polypeptide having a substituent in one or more amino acid residues, or (ii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, for example polyethylene glycol, or (iii) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader or secretory sequence or a sequence for purification of the polypeptide or a pro-protein sequence, or a fusion protein with a 6His tag). These derivatives and analogs fall within the scope of the teachings herein, as known to those skilled in the art.

One skilled in the art can alter the sequences of the invention by one or more (e.g., 1,2, 3,4, 5, 6,7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequences thereof without substantially affecting the activity of the antibody. Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. Conservative substitutions with amino acids of similar or similar properties generally do not alter the function of the protein in the art. Amino acids having similar properties are substituted, for example, in the FR and/or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. They are all considered to be included within the scope of the present invention.

Variant forms of the antibodies described herein include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions to the DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

In some embodiments, the sequences of the variants of the invention may have at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which they were derived. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The invention also includes those molecules having antibody heavy chain variable regions with CDRs, provided that the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

Antibodies of the invention can be prepared using methods conventional in the art, such as hybridoma techniques well known in the art. The heavy chain antibodies of the invention may be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or heavy chain antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells may be transformed with sequences encoding antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotides in liposomes, and direct microinjection of DNA into the nucleus, etc. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hepG 2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial MUC1 binding properties.

Nucleic acid

The invention also provides polynucleotides encoding the antibodies or fragments thereof. Provided herein are polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and CDRs. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids may be made, all of which encode an antibody or antigen binding fragment thereof of the invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. As used herein, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

Thus, the invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more nucleotide sequences including a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acid encoding an antibody to be expressed, and selectable marker elements.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Therapeutic uses and pharmaceutical compositions

By constructing nanobody libraries, the inventors discovered and expressed purified a plurality of nanobodies that can bind to MUC1 protein. All aspects of the antibodies described herein are useful in the preparation of medicaments to prevent or treat various conditions and diseases described herein, particularly diseases or conditions in which the conditions are associated with MUC1 expressing cells. In some embodiments, the conditions and diseases are cancers, including but not limited to breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gall bladder cancer, esophageal cancer, prostate cancer, or glioblastoma.

The pharmaceutical compositions herein comprise a binding molecule as described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. The adjuvant is preferably non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In the case of lyophilization of a composition, this method may be used to sterilize the composition either before or after lyophilization and reconstitution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle. Or alternatively the composition may be for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted (e.g., lyophilized) prior to administration. The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides a method of treating a patient, particularly a MUC 1-associated disease in a patient, by administering a binding molecule according to any of the embodiments of the invention or a pharmaceutical composition thereof. The terms "patient," "subject," "individual," "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). The treatment regimen effective to treat a patient can vary depending on a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject by therapy.

The therapeutically effective amount of the pharmaceutical composition comprising the binding molecule of the invention to be employed will depend, for example, on the degree of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect. Such as from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

The frequency of administration will depend on the pharmacokinetic parameters of the binding molecule in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is according to known methods, for example by oral, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional route, by sustained release systems or by implantation devices.

Diagnostic, detection and kit

The binding molecules of the invention may be used in assays, for example binding assays, to detect and/or quantify MUC1 expressed in a tissue or cell due to their high affinity for MUC1. Binding molecules such as single domain antibodies may be used in studies to further investigate the role of MUC1 in disease. The method for detecting MUC1 is generally described as obtaining a cell and/or tissue sample and detecting the level of MUC1 in the sample.

The MUC1 binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with MUC 1. The present invention provides for the detection of the presence of MUC1 in a sample using classical immunohistological methods known to those skilled in the art. The detection of MUC1 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of MUC1 include ELISA, FACS, RIA and the like.

For diagnostic applications, binding molecules such as single domain antibodies are typically labeled with a detectable label group. Suitable labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for a secondary antibody, metal binding domains, epitope tags), MRI (magnetic resonance imaging), or CT (electronic computer tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used to carry out the present invention.

Another aspect of the invention provides a method of detecting the presence of a test molecule competing with an antibody of the invention for binding to MUC1. An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of MUC1 in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind to MUC 1) would indicate that the test molecule is able to compete with the antibody for binding to MUC1. In one embodiment, the antibody is labeled with a labeling group. Or labeling the test molecule and monitoring the amount of free test molecule in the presence or absence of antibody.

The invention also provides a detection kit for detecting MUC1 level, which comprises an antibody for recognizing MUC1 protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, and the like. The detection kit may be an in vitro diagnostic device.

The invention will be illustrated by way of specific examples. It is to be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

EXAMPLE 1 purification of MUC1 nanobody expression

The full-length nucleic acid sequence is synthesized according to the sequence of the MUC1 nano antibody, the sequence of the MUC1 nano antibody is shown in table 1, and the antibodies except NBL502-1-C4 are all mutants of NBL 502-1-C4.

TABLE 1MUC1 nanobody amino acid sequence

The eukaryotic expression vector for constructing pCDNA3.4 (Thermo) plasmid is synthesized by Suzhou gold and only company, nanobody is constructed on the pCDNA3.4-IgG4 vector to construct VHH-IgG4 form, then expressed by EXpiCHOTM (Thermo Fisher) expression system, and after one week of expression, the supernatant is collected for Protein A (GE) purification. Protein quality was then checked using Nanodrop and protein purity was checked by HPLC. The purity and yield of the obtained protein meet the requirements of subsequent experiments.

Example 2 characterization of candidate antibodies

(1) Protein level ELISA detection:

The affinity of the antibodies to the human MUC1-His antigen was determined using enzyme-linked immunosorbent assay (ELISA). MUC1 antigen was diluted to 2. Mu.g/mL with coating solution, 100. Mu.L/well was added to 96-well strips and incubated at 37℃for 2 hours. Washing 3 times. After washing, 100. Mu.L of 3% BSA blocking solution was added to each well and incubated at 37℃for 2 hours. Washing 3 times. mu.L/well MUC1 antibody was added and the initial concentration of antibody dilution was 4. Mu.g/mL, 4-fold over 8 gradients, and incubated at 37℃for 1 hour. Each plate was washed 3 times. Each well was incubated with 100. Mu.L of the second enzyme-labeled antibody for 30min. Washing 3 times. 100. Mu.L TMB was added to each well and developed for 5min. The reaction was stopped by adding 100. Mu.L of hydrochloric acid stop solution to each well, and the plate was read at a wavelength of 450 nm. The detection results are shown in fig. 1-2, and the mutated MUC1 antibody has binding activity with MUC1 antigen.

(2) Protein level affinity assay:

The binding kinetics and affinity of antibodies to human MUC1-His antigen were determined using Surface Plasmon Resonance (SPR). Purified antibodies were passed through a sensor chip to which protein a was immobilized in advance, the antibodies were captured by protein a, and then 5 different concentrations of muc1.His proteins were used as mobile phases, with binding and dissociation times of 30min and 60min, respectively. Binding rate (ka), dissociation rate (KD) and equilibrium constant (KD) were analyzed using Biacore Evaluation Software 2.0.0 (GE). BMK1 is an IgG4 antibody in the form of an scFv (VH-VL) prepared according to the antibody sequence SEQ ID NO:73 of the GENUS oncology company US2016/0340442 A1 patent. The results of protein level affinity assays are shown in table 2 below.

TABLE 2 detection results of different antibody affinities

(3) Cell level binding Activity assay

Tumor cells MB468, H226, RPMI8226 and SKOV3 expressing MUC1 protein are paved in a 96-well plate, each well is 3 multiplied by 10 ⁵ cells, then gradient diluted candidate antibodies and MB468 cells are incubated at 2-8 ℃, after half an hour, detection antibodies anti-human IgG PE (Jackson Immuno Research, code: 109-117-008) are added after washing for incubation, and then CytoFLEX flow cytometry is used for detection. Isotype is a isotype control (negative control, sequence derived from SEQ ID NO:1 of patent CN 106146653A). The results of the candidate antibodies are shown in FIGS. 3-6, where the candidate antibodies have different binding activities to different tumor cells.

Example 3 VHH antibody immune group assay

Normal tissues and 9 tumor tissues are selected to be frozen and sliced, and the normal tissues and 9 tumor tissues are fixed by acetone after being dried at normal temperature. Blocking was performed using reagents a and B of the endogenous biotin blocking kit (manufacturer, E674001), the biotin-labeled antibody sample was incubated for 30min, washed and then incubated for 15min with horseradish peroxidase-labeled streptavidin (Abcam, ab 7403). DAB color development and hematoxylin counterstain are used, a neutral plastic sealing piece is used, and after natural air drying, microscopic examination is carried out. The 2 positive controls were Anti-MUC1 Anti [ SM3] (ab 22711 from Abcam) and BMK1, respectively, and the negative control was an IgG4 isotype control.

The results are shown in tables 3-5, which indicate that the mutated MUC1 binding molecules are not stained in normal tissue, but exhibit good staining in tumor tissue, while 1C4 is stained in normal tissue, potentially resulting in damage to normal tissue.

Claims (14)

1. A MUC1 binding molecule comprising a MUC1 single domain antibody having one or more amino acid mutations compared to a wild-type single domain antibody having a wt-CDR1 as set forth in SEQ ID No. 1, a wt-CDR2 as set forth in SEQ ID No. 10, and a wt-CDR3 as set forth in SEQ ID No. 17.

2. The MUC 1-binding molecule of claim 1, wherein the amino acid mutation is located in a CDR region of a wild-type single domain antibody;

Preferably, the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, the MUC1 single domain antibody having one or more of the following features (1) - (3):

(1) The CDR1 contains one or more mutations compared to SEQ ID NO 1 of T3Q, R D, R5H, R5K, R5E, R5Y, R6E, R6H, R6K, R6D, R Y, preferably one or more mutations selected from the group consisting of T3Q, R5D, R6E, R H and R6H, R K and R6K, R5D and R6D, R5E and R6E, R Y and R6Y,

(2) The CDR2 contains one or more mutations compared to SEQ ID NO 10 of I1G, T3Q, T3W, F4D, D6A, D6G, D7S, T Q, preferably one or more mutations selected from the group consisting of I1G, T3W, D6G, F D and D6A, T3Q and T8Q, D G and D7S,

(3) The CDR3 comprises one or more mutations compared to SEQ ID NO 17 of T1Q, I3G, Y6H, Q8E, Q8Y, L9G, L9Y, L9S, S10N, D Q, preferably one or more mutations selected from the group consisting of T1Q, Q8E, Q8Y, L9G, I G and L9G, L9Y, L9S, 6H and S10N and D12Q;

more preferably, the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, said CDR1, CDR2, CDR3 comprising the mutations shown in any one of the following groups A1 to a 17:

3. the MUC 1-binding molecule of claim 2, wherein the MUC1 single domain antibody has one or more of the following features (1) - (3):

(1) The CDR1 is shown in any one of SEQ ID NO 1-9,

(2) The CDR2 is shown in any one of SEQ ID NO 10-16,

(3) The CDR3 is shown in any one of SEQ ID NO 17-25;

And the CDR1, the CDR2 and the CDR3 are not SEQ ID NO 1, SEQ ID NO 10 and SEQ ID NO 17 respectively;

Preferably, the MUC1 single domain antibody contains CDR1, CDR2 and CDR3 as shown in any one of the following sets a1 to a17 of SEQ ID NOs:

4. The MUC 1-binding molecule of any of claims 1-3, wherein the MUC1 single domain antibody has reduced affinity for MUC1 as compared to the wild-type single domain antibody, preferably the anti-MUC 1 single domain antibody binds MUC1 with a K _D of about 1 x 10 ^-8～5×10^{- 7} M.

5. The MUC1 binding molecule according to claim 1 to 4, wherein,

The anti-MUC 1 single domain antibody comprises FR1, FR2, FR3 and FR4, wherein FR1 is shown as SEQ ID NO 26 or 27, FR2 is shown as SEQ ID NO 28, FR3 is shown as SEQ ID NO 29 or 30, and FR4 is shown as SEQ ID NO 31, preferably, the amino acid sequence of the MUC1 single domain antibody is shown as any one of SEQ ID NO 33-49, and/or

The MUC1 binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment thereof comprising one, two, or more of the single domain antibodies.

6. A polynucleotide selected from the group consisting of:

(1) A coding sequence for a MUC1 binding molecule according to any one of claims 1 to 5;

(2) The complement of (1);

(3) A fragment of 5-50bp of any one of the sequences (1) or (2).

7. A nucleic acid construct comprising the polynucleotide of claim 6, preferably a recombinant vector or an expression vector.

8. A host cell, wherein the host cell:

(1) Expression of a MUC1 binding molecule according to any of claims 1 to 5, and/or

(2) Comprising the polynucleotide of claim 6, and/or

(3) Comprising the nucleic acid construct of claim 7.

9. A method of producing a MUC1 binding molecule comprising culturing the host cell of claim 8 under conditions suitable for production of the MUC1 binding molecule, and optionally purifying the MUC1 binding molecule from the culture.

10. A pharmaceutical composition comprising the MUC 1-binding molecule of any one of claims 1-5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, or the host cell of claim 8, and a pharmaceutically acceptable adjuvant, preferably for use in the treatment of cancer.

11. Use of a MUC1 binding molecule according to any one of claims 1 to 5 in the manufacture of a medicament for the prophylaxis or treatment of cancer, preferably wherein the cancer is a MUC 1-associated cancer.

12. A kit for detecting MUC1 for assessing the efficacy of a drug treatment or diagnosing cancer, said kit comprising a MUC1 binding molecule according to any one of claims 1 to 5, a polynucleotide according to claim 6, a nucleic acid construct according to claim 7, or a host cell according to claim 8;

preferably, the kit further comprises reagents for detecting the binding of MUC1 to a single domain antibody, antibody or antigen binding fragment thereof;

more preferably, the reagent is a reagent that detects the binding by an enzyme-linked immunosorbent assay.

13. A non-diagnostic method of detecting the presence of MUC1 in a sample, the method comprising incubating the sample with a MUC1 binding molecule according to any one of claims 1 to 5, and detecting the binding of MUC1 to a single domain antibody, antibody or antigen binding fragment thereof, thereby determining the presence of MUC1 in the sample.

14. Use of a MUC 1-binding molecule according to any one of claims 1 to 5 in the manufacture of a kit for detecting MUC1 in a sample, assessing the effect of a drug treatment or diagnosing cancer.