CN114702588B - anti-Nectin-4 antibodies and bispecific antibodies - Google Patents

Abstract

The present disclosure provides a single domain antibody with excellent binding affinity to human Nectin-4 protein. These antibodies are particularly suitable for inclusion in bispecific antibodies, such as antibodies that also target antigens on immune cells.

Description

Anti-Nectin-4 Antibodies and Bispecific Antibodies

technical field

The invention belongs to the field of cellular immunology, and relates to a single-domain antibody specific to human Nectin-4 protein and its application.

Background technique

The Nectin family is a Ca ²⁺ -independent immunoglobulin-like molecule composed of four members, Nectin-1, -2, -3 and -4. Nectin proteins play a role in cell-cell adhesion. They bind the actin filament (F-actin) binding protein afadin through their cytoplasmic tails and associate with the actin cytoskeleton, cooperate with other cell adhesion molecules and cell surface membrane receptors, and regulate many other cellular activities such as Mobility, differentiation, polarization and entry of viruses.

Nectin-4, also known as poliovirus receptor-related protein 4 (PVRL4), is a type I single transmembrane protein of approximately 52 kDa in size. The extracellular domain of nectin-4 has three Ig-like subdomains, namely V, C1 and C2.

Nectin 1, 2, and 3 are widely expressed in adult tissues, but Nectin-4 is specifically expressed in embryos and placenta. However, Nectin-4 has been shown to be expressed in various cancer cells, making it a suitable target for cancer therapy.

Contents of the invention

In various embodiments, the present disclosure provides single domain antibodies having binding specificity for human Nectin-4 protein. These antibodies can cross-react with cynomolgus monkey Nectin-4. With their excellent binding affinity and small size, these antibodies can be suitably used to generate bispecific antibodies, such as antibodies that also target immune cells.

Therefore, according to one embodiment of the present disclosure, there is provided a single domain antibody or an antigen-binding fragment thereof specific to human Nectin-4 protein, which comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are respectively Comprising the CDR1, CDR2 and CDR3 sequences of any of the antibodies CMB7-1, CMB7-2, CMB7-3, CMB7-4 or CMB7-5. These exemplary antibodies have the amino acid sequences set forth in SEQ ID NO: 1-5.

In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 6-8, SEQ ID NO: 9-11, SEQ ID NO: 12-14, SEQ ID NO: 15-17 or SEQ ID NO : The amino acid sequence of 18-20.

In some embodiments, the single domain antibody comprises an amino acid sequence selected from SEQ ID NO: 1-5.

The present disclosure also provides a bispecific antibody comprising a single domain antibody or antigen-binding fragment thereof disclosed herein and a second antibody or antigen-binding fragment specific for an antigen other than Nectin-4. In some embodiments, the antigen is human CD3.

In some embodiments, the bispecific antibody comprises four single domain antibodies, each fused to the heavy chain variable region (VH) or light chain variable region (VH) of a full Fab antibody specific for human CD3. Variable region (VL). In some embodiments, each single chain domain antibody is fused to VH or VL via a peptide linker.

In some embodiments, the peptide linker has a length greater than 7 amino acids. In some embodiments, the peptide linker has a length of less than 50 amino acids.

The present disclosure also provides polynucleotides encoding any such antibodies or fragments.

The present disclosure also provides methods of treating diseases (eg, cancer) with the disclosed antibodies or fragments.

Description of drawings

Figure 1 shows an SDS-PAGE image demonstrating antibody expression.

Figure 2 shows the results of ELISA-based antibody affinity testing.

Figure 3 shows that all anti-human Nectin-4 VHH antibodies cross-react with cynomolgus monkey Nectin-4.

Figure 4 illustrates the two formats of the bispecific antibody tested (Form A and Form B).

Fig. 5 shows the results of T cell activation assay in the presence of Nectin-4 expressing cells.

Figure 6-7 shows the results of T cell killing of target Nectin-4 expressing cells (Figure 6: MCF-7 cells; Figure 7: T-47D cells).

Detailed ways

definition

It is to be noted that the term "an" entity refers to one or more of said entities; for example, "an antibody" is understood to represent one or more antibodies. Accordingly, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

As used herein, the term "polypeptide" is intended to encompass the singular as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide" and the term "polypeptide" ” can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" also means the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or by non-natural Amino acid modifications present. A polypeptide may be from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis.

The term "isolated" as used herein with reference to cells, nucleic acids (eg, DNA or RNA) refers to a molecule that is separated from other DNA or RNA, respectively, present in the macromolecule's natural source. As used herein, the term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized nucleic acids or peptides. Furthermore, an "isolated nucleic acid" refers to a fragment of a nucleic acid that does not naturally occur in fragmented form and is not found in its natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides.

As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a non-naturally occurring form of a polypeptide or polynucleotide, non-limiting examples of which may be created by combining polynucleotides or polypeptides that would not ordinarily occur together.

"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each sequence, which positions may be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity, but preferably less than 25% identity with one of the sequences of the disclosure.

A polynucleotide or a region of a polynucleotide (or a polypeptide or a region of a polypeptide) is a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% to another sequence) % or 99%) "sequence identity", which means that, when aligned, the percentage of bases (or amino acids) is the same when comparing the two sequences. Such alignments and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, with default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; Filter = None; Shares = both; Cutoff = 60; Expected = 10; Matrix = 62; Description = 50 sequences; Order by = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank-CDS-translations+SwissProtein+SPupdate+PIR. A bioequivalent polynucleotide refers to a polynucleotide having the above-specified percentage homology and encoding a polypeptide having the same or similar biological activity.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid that has a nucleotide sequence that has a degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. A homologue of a double-stranded nucleic acid is intended to include a nucleic acid having some degree of nucleotide sequence homology to its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Likewise, an "equivalent polypeptide" refers to a polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to a reference polypeptide or polynucleotide. In some aspects, equivalent sequences retain the activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.

Hybridization reactions can be performed under conditions of varying "stringency". Generally, low stringency hybridization reactions are performed at about 40°C in about 10 x SSC or a solution of equivalent ionic strength/temperature. Medium stringent hybridizations are typically performed at about 50°C in about 6 x SSC, while high stringency hybridization reactions are usually performed at about 60°C in about 1 x SSC. Hybridization reactions can also be performed under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are temperature, ionic strength, pH and Mg2+ concentration normally present in cells.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine; and when the polynucleotide is RNA, uracil (U) stands for thymine. Accordingly, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one gene form or parts thereof. A part of a gene with at least two different forms, that is, two different nucleotide sequences, is called a "gene polymorphic region". A polymorphic region can be a single nucleotide, the identity of which varies in different alleles.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (such as probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, nuclear Enzymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made either before or after polynucleotide assembly. Nucleotide sequences can be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of the present disclosure encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term "encoding" for a polynucleotide refers to a polynucleotide which, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce said polypeptide and/or A fragment of the mRNA is said to "encode" a polypeptide. The antisense strand is the complement of the nucleic acid from which the coding sequence can be deduced.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding an antigen. Examples of such include, but are not limited to, complementarity determining regions (CDRs) of heavy or light chains or ligand binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions, or Any portion, or at least a portion of a binding protein.

The term "antibody fragment" or "antigen-binding fragment", as used herein, is a portion of an antibody, eg, F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of structure, antibody fragments bind to the same antigens recognized by intact antibodies. The term "antibody fragment" includes aptamers, mirroromers and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are joined with short linkers of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. scFv molecules are known and described in the art, for example in US patent 5,892,019.

The term antibody includes a broad class of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, epsilon, including some subclasses (eg, gamma1-gamma4). It is the nature of this chain that determines the antibody's "class" as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and known to have functional specificity. Modified versions of each of these classes and isoforms are readily recognized by those skilled in the art in view of the present disclosure and are thus within the scope of the present disclosure. While all immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion is generally directed to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are usually linked by disulfide bonds in a "Y" configuration, where the light chain surrounds the heavy chain starting at the mouth of the "Y" and continuing through the variable region.

"Specifically binds" or "has specificity" generally means that an antibody binds an epitope through its antigen-binding domain, and that binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, when an antibody binds an epitope via its antigen-binding domain, it "specifically binds" to that epitope more readily than to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be said to be more specific for a given epitope than antibody "B", or antibody "A" may be said to bind epitope "C" more specifically than a related epitope "D". "higher.

As used herein, the terms "treatment" or "therapy" refer to both medical treatment and prophylactic or preventive measures, wherein the purpose is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented.

"Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, especially a mammalian subject, in need of diagnosis, prognosis or treatment. Mammalian subjects include humans, livestock, farm animals, zoo animals, sport animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

As used herein, phrases such as "to a patient in need of treatment" or "subject in need of treatment" include subjects who would benefit from administration of an antibody or composition of the present disclosure for detection, diagnostic procedures and/or treatment, For example a mammal object.

Single Domain Anti-Nectin-4 Antibody

Nectin protein plays an important role in cell-cell adhesion. Nectin-4 is a type I single transmembrane protein of about 52 kDa. Unlike Nectin 1, 2, and 3, which are widely expressed in adult tissues, Nectin-4 is specifically expressed in embryos and placenta. However, Nectin-4 can also be expressed in various cancer cells, making it a suitable target for cancer therapy.

The present disclosure provides anti-Nectin-4 antibodies in the form of single domain antibodies. Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. The earliest single-domain antibodies were engineered from heavy-chain antibodies found in camelids, also known as VHH fragments. Like the VH of conventional antibodies, each VHH includes three CDRs, CDR1, CDR2 and CDR3. A VHH may further comprise constant domains, such as CH2 and CH3.

As shown in Figure 2, all identified VHH antibodies from CMB7-1 to CMB7-5 showed strong binding to human Nectin-4 protein. At the same time, all these antibodies also had strong affinities to the corresponding cynomolgus monkey proteins (Fig. 3), allowing them to be tested in cynomolgus monkeys as a preclinical model.

One of these antibodies, CMB7-1 (VHH 35), was tested as a bispecific antibody in two different formats (Fig. 4), which also targets the human CD3 complex. The results in Figures 5-7 show that the bispecific antibody of Form B exhibits excellent T cell activation and T cell killing activities. Therefore, these VHH antibodies can be suitably used for the treatment of diseases such as cancer.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3, respectively having CDR1, CDR2 of antibody CMB7-1, CMB7-2, CMB7-3, CMB7-4 or CMB7-5 and the amino acid sequence of CDR3. The sequences of these antibodies are provided in Table 1 as shown in SEQ ID NO: 1-5.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NO: 6-8, respectively. In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NO: 9-11, respectively. In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NO: 12-14, respectively. In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NO: 15-17, respectively. In one embodiment, there is provided an antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NO: 18-20, respectively.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of any one of SEQ ID NOs: 1-5, or at least 85% identical to any one of SEQ ID NOs: 1-5 , 90%, 95%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-24 retains the CDR sequences of the corresponding reference antibody.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1, or at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 1 Amino acid sequences with sequence identity. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO: 6, 7 and 8.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2, or having at least 85%, 90%, 95%, 98% or 99% of the amino acid sequence of SEQ ID NO: 2 Amino acid sequences with sequence identity. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO: 9, 10 and 11.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO:3, or at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:3 Amino acid sequences with sequence identity. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO: 12, 13 and 14.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 4, or at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 4 Amino acid sequences with sequence identity. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO: 15, 16 and 17.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 5, or at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 5 Amino acid sequences with sequence identity. In some embodiments, the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO: 18, 19 and 20.

In one embodiment, there is provided an antibody or antigen-binding fragment comprising the amino acid sequence of any one of SEQ ID NO: 1-5, optionally with the addition of 1, 2, 3, 4 or 5 amino acids , deletion and/or substitution. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the additions, deletions and/or substitutions are within the framework regions.

In some embodiments, the antibody or fragment further comprises constant domains, such as CH2 and/or CH3. In some embodiments, the CH2 and/or CH3 is from a human IgGl, IgG2, IgG3 or IgG4 sequence.

In some embodiments, the substitutions are conservative substitutions. "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g. threonine, valine, isoleucine) and Aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably substituted with another amino acid residue from the same side chain family. In another embodiment, amino acid strings may be substituted with structurally similar strings that differ in the order and/or composition of side chain family members.

The following table provides non-limiting examples of conservative amino acid substitutions, where a similarity score of 0 or higher indicates a conservative substitution between two amino acids.

Table A. Amino Acid Similarity Matrix

Table B. Conservative Amino Acid Substitutions

amino acid replace with Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp glutamic acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp proline D-Pro serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

In some embodiments, the antibody or fragment is of the IgGl, IgG2, IgG3 or IgG4 class. In some embodiments, the antibody or fragment has antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, the antibody or fragment does not have ADCC activity.

bispecific antibody

As mentioned above, these newly identified anti-Nectin-4 VHH antibodies are suitable for inclusion in bispecific antibodies. Two forms (form A and form B) were tested, but form A did not bind sufficiently to cell surface expressed Nectin-4. Form B is tetravalent relative to Nectin-4 and binds cells strongly. Furthermore, these bispecific antibodies exhibited robust T cell activation and T cell-mediated tumor cell killing when incubated with T cells (targeted by binding CD3) and Nectin-4-expressing tumor cells.

Therefore, according to one embodiment of the present disclosure, there is provided a bispecific antibody comprising any of the VHH antibodies of the present disclosure and a second antibody or antigen-binding fragment that binds another antigen. In some embodiments, the second antigen is a protein expressed on immune cells.

Proteins on immune cells that can be targeted include, but are not limited to, CD3, CD47, PD1, PD-L1, 4-1BB, OX40, SIRPA, CD16, CD28, CTLA4, and CD27. In some embodiments, the immune cell surface protein is CD3.

As shown by the data, the 4:2 VHH:Fab format (Form B) had excellent therapeutic activity compared to the other formats tested. The 4:2 format is shown in Figure 4B, which includes a conventional Fab format of an antibody (eg, anti-CD3) and four VHH units specific for Nectin-4. Each VHH is fused to the N-terminus of the Fab variable region via a peptide linker such as GS(GGGGS) (SEQ ID NO: 21), GS(GGGGS) ₃ (SEQ ID NO: 22) and GS(GGGGS) ₆ (SEQ ID NO: 23).

For certain VHHs, an interesting finding was that the length of the peptide linker had a significant effect on the activity of the bispecific antibody binding to Nectin-4 on the cell surface. Thus, in some embodiments, the peptide linker may be at least 2 amino acids, or at least 5, 7, 8, 9, 10, 12, 15, 17, 20, 22, or 25 amino acids in length. In some embodiments, the length is no more than 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.

Exemplary linkers include multiple glycines (G) and serines (S). In some embodiments, the linker comprises at least 50%, 60%, 70%, or 80% glycine. Exemplary linker sequences include, but are not limited to, GS(GGGGS) (SEQ ID NO: 21 ), GS(GGGGS) ₃ (SEQ ID NO: 22), and GS(GGGGS) ₆ (SEQ ID NO: 23).

Thus, in one embodiment, the present disclosure provides a bispecific antibody specific for immune cells (eg, targeting CD3) and Nectin-4. In some embodiments, the bispecific antibody comprises a conventional antibody specific for the human CD3 complex. In some embodiments, the bispecific antibody comprises a plurality (eg, 2 and 4) of VHHs targeting Nectin-4.

Therefore, in one embodiment, there is provided a bispecific antibody comprising a first part and a second part, wherein the first part comprises two pairs of VH and VL, each pair is capable of binding a human CD3 complex, and the second part comprises such as Four single domain antibody (VHH) fragments disclosed herein, wherein each VHH fragment is fused to the N-terminus of each VH and VL of the first part by a peptide linker.

In some embodiments, the bispecific antibody further comprises constant domains, such as CH1 and CL, and CH2 and/or CH3. In some embodiments, the constant region is from a human IgGl, IgG2, IgG3 or IgG4 sequence.

Those of ordinary skill in the art will also understand that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides derived therefrom. For example, a polypeptide or amino acid sequence derived from a given protein may be similar, e.g., have a certain percentage identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85% identical to the starting sequence %, 90%, 95%, 98% or 99% the same.

In certain embodiments, an antibody comprises an amino acid sequence or one or more portions that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, antibodies of the disclosure can comprise flexible linker sequences, or can be modified to add functional moieties (eg, PEG, drugs, toxins, or labels).

Antibodies of the present disclosure, variants or derivatives thereof (including modified derivatives), are obtained by covalently linking any type of molecule to the antibody such that the covalent link does not prevent the antibody from binding to the epitope. For example, but not limited to, antibodies may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Linkages to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more non-canonical amino acids.

In some embodiments, an antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

Antibodies can be conjugated or fused to therapeutic agents, which can include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive labels, combinations thereof with other such agents known in the art.

Antibodies can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalates.

Antibodies can also be labeled with fluorescent emitting metals such as 152Eu or other lanthanides. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating different moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243 -56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc. , pp. 623- 53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 ( 1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).

Antibody-encoding polynucleotides and methods for making antibodies

The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the disclosed antibodies, variants or derivatives thereof. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen binding polypeptides of the disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and described herein. For example, fully human antibodies to a particular antigen can be produced by administering the antigen to transgenic animals that have been modified to produce such antibodies in response to antigen challenge but whose endogenous sites have been nullified. Exemplary techniques that can be used to make such antibodies are described in U.S. Patents 6,150,584; 6,458,592; 6,420,140, which are incorporated herein by reference in their entirety.

In certain embodiments, antibodies are produced that do not elicit a deleterious immune response in the animal to be treated (eg, in a human). In one embodiment, an antigen-binding polypeptide of the disclosure, a variant or derivative thereof, is modified to reduce its immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved in a number of ways, including (a) grafting entire non-human variable domains into human constant regions to generate chimeric antibodies; (b) incorporating one or more non-human complementarity determining regions (CDRs) Grafting at least partly into the human framework and constant regions, with or without key framework residues; or (c) grafting the entire non-human variable domain, but replacing surface residues with human-like portions" cover" them. Such methods are exemplified by Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos.: 5,585,089, 5,693,761 , 5,693,762 and 6,190,370, which are incorporated herein by reference in their entirety.

Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes altering antibodies to modify T cell epitopes (eg, see International Application Publication Nos: WO/9852976 Al and WO/0034317 A2). For example, the variable heavy and variable light chain sequences from a starting antibody are analyzed and a "map" of human T cell epitopes for each V region is created, showing interactions with complementarity determining regions (CDRs) and other key residues within the sequence. base-related epitope positions. Individual T-cell epitopes in the T-cell epitope map are analyzed to identify alternative amino acid substitutions with low risk of altering the activity of the final antibody. A series of alternative variable heavy and variable light sequences are designed, including combinations of amino acid substitutions, and these sequences are subsequently incorporated into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are generated and tested for binding and/or function. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into expression vectors, and the subsequent plasmids are introduced into cell lines to produce complete antibodies. Antibodies are then compared in appropriate biochemical and biological assays and the best variant identified.

The binding specificity of the antigen-binding polypeptides of the present disclosure can be determined by in vitro tests, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

cancer treatment

As described herein, the disclosed antibodies, variants or derivatives are useful in certain therapeutic and diagnostic methods.

The present disclosure further relates to antibody-based therapies involving the administration of the disclosed antibodies to patients (eg, animals, mammals, and humans) to treat one or more of the diseases or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, the antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies of the disclosure (including variants and derivatives thereof as described herein). derivative).

Antibodies of the disclosure can also be used to treat or inhibit cancer. In some embodiments, Nectin-4 is overexpressed in tumor cells. Accordingly, in some embodiments, methods for treating cancer in a patient in need thereof are provided. In one embodiment, the method entails administering to the patient an effective amount of an antibody of the disclosure. In some embodiments, at least one cancer cell (eg, a stromal cell) of the patient expresses, overexpresses, or is induced to express a tumor antigen. Induction of gene expression can be achieved, for example, by administering tumor vaccines or radiation therapy.

Appropriately treatable tumors include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer , ovarian cancer, kidney cancer, and small cell lung cancer. Therefore, the current antibodies can be used to treat any one or more of these cancers.

Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and/or predicted by the disclosed antibodies or variants or derivatives thereof include, but are not limited to, progression and/or metastasis of malignant tumors and related diseases, such as Leukemia (including acute leukemia (eg, acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythrocytic leukemia)) and chronic leukemia (eg, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and Solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, Mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma Carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma Carcinoma, Wilms tumor, Cervical cancer, Testicular tumor, Lung cancer, Small cell lung cancer, Bladder cancer, Epithelial cancer, Glioma, Astrocytoma, Medulloblastoma, Craniopharyngioma, Ependymoma, Pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody, variant or derivative thereof used, the patient's age, weight, general health, sex and diet, and the time of administration, rate of excretion , drug combination, and severity of the specific disease being treated. The judgment of medical personnel on such factors belongs to the ordinary skill in the art. Dosage also depends on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the desired effect. The amount used can be determined by principles of pharmacology and pharmacokinetics well known in the art.

Methods of administration of the antibody, variant or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be combined with other biologically active agents Administer together. Accordingly, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (such as by powder, ointment, drops, or a transdermal patch), via Apply buccally or as an oral or nasal spray.

As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Administration can be systemic or localized. In addition, it may be desirable to introduce antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, eg, connected to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example by use of an inhaler or nebulizer, and by use of aerosol formulations.

It may be desirable to administer an antibody polypeptide or composition of the present disclosure locally to the area in need of treatment; this may be by, for example, without limitation, local infusion during surgery, local application, e.g., in conjunction with a post-surgical wound dressing, by injection , by a catheter, by a suppository, or by an implant of porous, non-porous, or gel material (including membranes, such as sialic acid membranes, or fibers). Preferably, when administering proteins of the present disclosure, including antibodies, care must be taken to use materials that are not absorbed by the protein.

combination

The present disclosure also provides pharmaceutical compositions. Such compositions include an effective amount of antibody and an acceptable carrier.

In particular embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the US Pharmacopoeia or other recognized pharmacopoeia for use in animals, especially in humans. Furthermore, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or auxiliary formulation of any type.

The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol , propylene, ethylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; in addition, agents for tonicity, such as chlorinated sodium or glucose.

These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described by E.W. Martin in Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of an antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of carrier, so as to provide the patient with a form suitable for administration. The formulation should suit the mode of administration. The parent preparation can be presented in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a dissolving agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, these ingredients are presented separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or physiological saline can be provided so as to be mixed prior to administration.

The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide , isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and other salts.

Example

Example 1. Preparation of anti-human Nectin-4 single domain antibody

This example shows how to generate anti-human Nectin-4 single domain antibody by immunizing alpaca, then constructing and selecting a phage library.

Anti-human Nectin-4 antibody was produced by using recombinant human Nectin-4/hFc fusion protein as immunogen. Alpaca PBMCs were collected and antibody cDNA libraries were generated by RNA isolation, PCR amplification and cloning into phage display vectors. These pools were then subjected to one round of liquid-phase selection and one round of solid-phase selection.

Binders were amplified by PCR from antigen-positive phage and sequenced. The expressed proteins were confirmed by SDS-PAGE (Figure 1). The table below provides the sequences of unique antibodies and their CDR regions.

Table 1. Antibody sequences

Table 2. CDR sequences

Example 2. ELISA Binding Test

In this example, antibodies were subjected to an ELISA-based binding test. Human Nectin-4 at the same concentration (0.5 μg/mL) was coated on a 96-well enzyme plate. Add 2 μg/mL of each antibody and different concentrations (0.2 μg/mL and 1 μg/mL) of goat anti-human Nectin-4 antibody (as a control) after blocking. After washing away excess sample, horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc cross-adsorbed antibody (or rabbit anti-goat IgG antibody) was added. HRP can react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce colored products. The binding affinity between CMB7 and human Nectin-4 can be calculated by reading the OD ₄₅₀ value of the reaction solution, because the absorbance of the reaction solution is positively correlated with the content of the antibody bound to the antigen. Therefore, ELISA was used to test the binding affinity of CMB7 to human Nectin-4.

experiment procedure:

coated antigen preparation

Dilute the coating antigen (human Nectin-4) to the working concentration (0.5 μg/mL) in PBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted coating antigen. Seal the microplate and incubate overnight at 4 °C.

closed

Aspirate all microwells and wash 5 times with 300 µL/well wash buffer through a microplate washer. Allowing soaking time (approximately 1 minute) in each wash step improves wash performance. The microplate is then dehydrated with a microplate dryer to remove any residual buffer. Block the microwells with 200 µL of blocking buffer. Incubate for 1 hr in a 37 °C water bath.

Sample Preparation

Repeat wash/spin. Dilute the samples to the working concentration (2 µg/mL) in assay buffer. Add 100 µL/well of the pre-diluted sample to the appropriate wells. Seal the microplate and incubate for 1 hr in a 37°C water bath.

Secondary antibody incubation

Repeat wash/spin. Secondary antibodies were diluted 1:2000 in assay buffer. Add 100 µL/well of diluted secondary antibody (1:2000) to each well. Plates were incubated for 1 hour at 37°C in a water bath protected from light.

Signal Detection

Repeat wash/spin. Add 100 µL/well of matrix solution (TMB) to each well. Incubate the plate in the dark for 3 minutes at room temperature. Add 50 µL/well of stop solution. Plates were read at 450 nm and data analyzed.

result:

The results of the ELISA test are shown in Figure 2 and Table 3. As shown in the results, all antibodies showed strong binding affinity to human Nectin-4 protein.

Table 3. ELISA affinity test results

Example 3. Kinetic Binding Test

This example measures the kinetic binding affinity of an antibody (fused to human Fc, VHH Fc) to human Nectin-4.

The kinetic binding affinity of the antibody to human Nectin-4 was detected by biolayer interferometry. The HFC (anti-HIgGFC) probe (Probelife) was pre-wetted in kinetic buffer (Probelife) at 30 °C for 5 min in a Crimson 96 MAX 96-well reaction plate (ET Healthcare, 06-0098). Probes were then dipped into wells of a Greiner black microplate containing 4 μg/mL antibody in kinetic buffer. A 5-minute binding step of the analyte (human Nectin-4) was performed at different concentrations (starting at 200 nM, followed by 2-fold dilutions for a total of 5 concentration gradients), followed by a 16-minute solution in kinetic buffer. step away. Data were analyzed by subtracting reference samples and fitting a 1:1 K binding model of the structured data method for affinity constants using the data analysis software 1.7.2.0609 (Gator).

Experimental procedure:

probe balance

Set the temperature of the plate to 30°C, set the acquisition rate to standard kinetics (5.0 HZ), add 260 μL/well pre-wet buffer (K buffer) to each well in column 1 of the Crimson 96MAX 96-well reaction plate, Place the probe in the buffer and pre-wet the probe for 5 min at 1000 rpm.

Baseline 1

In column 1 of a Greiner 96-well polypropylene microplate, perform a baseline assay for 6 probes in 200 µL/well K buffer at 1000 rpm for 2 min. As balanced as possible, the final slope is preferably no higher than 0.02 nm/min.

Loading antibodies onto probes

Dilute the antibody to the working concentration (4 μg/mL, 200 µL/well) with K buffer, and then load onto the probe at 1000 rpm on columns 2/3/4/5/6 of a Greiner 96-well polypropylene microplate. Needle for 5 minutes.

Baseline 2

Baseline assay the probes in 200 µL/well K buffer at 1000 rpm for 2 min in columns 8/9/10/11/12 of a Greiner 96-well polypropylene microplate. To remove unbound mAb from biosensors, non-specific binding should be minimized or biased by buffering effects should be minimized.

combine

Dilute the antigen (human Nectin-4) to 6 concentrations (200, 100, 50, 25, 12.5, 0 nM, 200 µL/well) with K buffer, and then add this series of antigens to Greiner 96-well polypropylene microplates Wells in row A to row F of column 7 of the orifice plate. In column 7, antibodies on the probes were combined with different concentration gradients of antigens for 5 minutes at 1000 rpm.

dissociate

Probes were dissociated in K buffer for 16 min and antigens were dissociated from probes in columns 8/9/10/11/12 on Greiner 96-well polypropylene microplates.

regeneration

Run the regeneration program for the probe (the probe is regenerated in the R buffer of the 11th column of the Crimson 96 MAX 96 well reaction plate for 5s, and then neutralized in the Q buffer of the 12th column of the Crimson 96 MAX 96 well reaction plate for 5s, and the process is repeated 3 times Second-rate).

result:

Table 4 below summarizes the test results. All antibodies have strong affinity for human Nectin-14 protein.

Table 4. Affinity Measurements for Anti-Nectin 4 VHH-Fc

Antibody KD (M) response CMB7-1 6.23E-21 0.712 CMB7-2 1.19E-20 0.817 CMB7-3 6.85E-21 0.913 CMB7-4 4.41E-21 0.945 CMB7-5 5.97E-21 0.893

Example 4. Cross-reactivity with Cynomolgus Nectin-4

In this example, the binding affinity of the antibody to cynomolgus monkey Nectin-4 was tested.

The binding affinity of the antibody to cynomolgus monkey Nectin-4 was tested by ELISA. Cynomolgus monkey Nectin-4 at the same concentration (0.5 μg/mL) was coated on a 96-well enzyme plate. After blocking, dilute antibodies with different concentration gradients were added (starting from 2 μg/mL, gradually diluted 3 times, a total of 7 concentration gradients). After washing away excess sample, a goat anti-human IgG Fc cross-adsorbed antibody conjugated to horseradish peroxidase (HRP) was added. HRP can react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce colored products. The binding affinity between CMB7 and cynomolgus monkey Nectin-4 can be calculated by reading the OD ₄₅₀ value of the reaction solution, because the absorbance of the reaction solution is positively correlated with the content of the antibody bound to the antigen. Therefore, ELISA was used to test the binding affinity of CMB7 to cynomolgus monkey Nectin-4.

Experimental procedure:

coated antigen preparation

Dilute the coating antigen (cynomolgus Nectin-4) to a working concentration (0.5 μg/mL) in PBS. Immediately coat 96-well microplates with 100 μL of the diluted coating antigen per well. Seal the microplate and incubate overnight at 4 °C.

closed

Aspirate all microwells and wash 5 times with 300 µL/well wash buffer through a microplate washer. Allowing soaking time (approximately 1 minute) in each wash step improves wash performance. The microplate is then dehydrated with a microplate dryer to remove any residual buffer. Block the microwells with 200 µL of blocking buffer. Incubate for 1 hr in a 37 °C water bath.

sample preparation

Repeat wash/spin. Dilute the samples to the working concentration (2 µg/mL) in assay buffer and perform 3-fold serial dilutions to make a curve with a total of 7 points. Add 100 µL/well of the pre-diluted sample to the appropriate wells. Seal the microplate and incubate for 1 hr in a 37°C water bath.

Secondary antibody incubation

Repeat wash/spin. Secondary antibodies were diluted to 1:2000 in assay buffer. Add 100 µL/well of diluted secondary antibody (1:2000) to each well. Plates were incubated for 1 hour at 37°C in a water bath protected from light.

Signal Detection

Repeat wash/spin. Add 100 µL/well of matrix solution (TMB) to each well. Plates were incubated for 3 minutes at room temperature in the dark. Add 50 µL/well of stop solution. Plates were read at 450 nm and data analyzed.

result:

The test results are shown in Figure 3, which indicated that all tested antibodies cross-reacted with cynomolgus monkey Nectin-4, and most of them showed strong binding affinity. This suggests that cynomolgus monkeys could be a suitable preclinical model for testing these antibodies.

Example 5. Flow Cytometry Analysis of Binding to Human Breast Cancer Cells

In this example, the binding affinity of the antibody to human breast cancer cells expressing Nectin-4 was detected by fluorescence-activated cell sorting (FACS).

experiment procedure:

cell preparation

1. After the cells reach 80% confluency, harvest the cells from the 100 mm culture dish.

2. Wash with 2 mL PBS. Add 1 mL of 0.25% trypsin EDTA and incubate at 37°C until the cells dissociate from the plate. Add 5 mL of warm medium. Collect cells and transfer to a 15 mL conical tube.

4. Collect the cells and transfer to a 15ml conical tube.

5. Centrifuge at 300 g for 5 minutes at room temperature.

cell plating

Wash with 5mL PBS-0.2%BSA, centrifuge at 300g, 4°C for 5 min. Repeat twice. Discard the supernatant and resuspend in 2 mL of LPBS-0.2% BSA. Count the cells and add 1 x ¹⁰⁵ - 2 x ¹⁰⁵ cells (100 µL) to each well.

Primary antibody incubation

Add protein (10 µg/mL or 5 nM) to each well and incubate at 4 °C for 1 h.

washing

Add 200 µL of ice-cold PBS-0.2% BSA to each well, and centrifuge at 300 g, 4°C for 5 min. Repeat 3 times. Add 100 µL of PBS-0.2% BSA to resuspend the cells.

Secondary antibody incubation

Add Alexa Fluor 488 goat anti-human IgG (H+L) (1:500 dilution) to each well in ice-cold PBS-0.2% BSA and incubate at 4°C for 1 hr.

washing

Add 200 µL of ice-cold PBS-0.2% BSA to each well, and centrifuge at 300 g, 4°C for 5 min. Repeat 3 times. Add 200 µL of PBS-0.2% BSA to resuspend the cells.

Applied to flow cytometry.

result:

FACS results showed that each antibody bound to the human breast cancer cell line MCF-7 at each concentration tested.

Example 6. Preparation of anti-Nectin-4/anti-CD3 bispecific antibody

The single domain antibody (VHH) CMB7-1 ("VHH 35") was used to construct a bispecific antibody also targeting human CD3. As shown in Figure 4, two different formats of bispecific antibodies were used.

Figure 4A shows the A format in which a single VHH and a single VH/VL pair from an anti-CD3 antibody are fused to the Fc fragment. Thus, the A form is asymmetric with a 1:1 potency for CD3 and Nectin-4.

In the format of Figure 4B (format B), each of the four VHHs is fused to the N-terminus of the variant domain of a complete anti-CD3 antibody. Thus, this form is symmetric with a 2:4 potency for CD3 and Nectin-4. The bispecific configurations and structures of each chain are shown in Tables 5-6.

Table 5. Structures of bispecific antibodies and related chains

Antibody form Linker (SEQ ID NO:) BJ182/12L1/183-35 A - BJ192-35/BJ196-35 B GS(GGGGS)₁ (SEQ ID NO:21) BJ193-35/BJ197-35 B GS(GGGGS)₃ (SEQ ID NO: 22) BJ194-35/BJ198-35 B GS(GGGGS)₆ (SEQ ID NO: 23)

chain structure BJ192-35 Anti-Nectin VHH-linker 1-Anti-CD3-Fc (PGLALA) BJ193-35 Anti-Nectin VHH-linker 2-Anti-CD3-Fc (PGLALA) BJ194-35 Anti-Nectin VHH-linker 3-Anti-CD3-Fc (PGLALA) BJ196-35 Anti-Nectin VHH-linker 1-Anti-CD3-VL-CL BJ193-35 Anti-Nectin VHH-linker 2-Anti-CD3-VL-CL BJ198-35 Anti-Nectin VHH-linker 3-Anti-CD3-VL-CL

The cDNA sequences encoding these bispecific antibodies were synthesized and used to generate antibodies.

Example 7. T cell activation by bispecific antibodies

This example tests the ability of bispecific antibodies to activate T cells in the presence of Nectin-4 expressing MCF-7 cells or T-47D cells.

Figure 5 shows the T cell activation results for all bispecific antibodies. Form A (BJ182/12L1/BJ183-35) contained only one VHH and showed no observable T cell activation activity. Interestingly, BJ192-35/BJ196-35 also showed low activity. This may be because the linker (GS(GGGGS) ₁ (SEQ ID NO: 21)) is too short. In contrast, BJ193-35/BJ197-35 (the linker is GS(GGGGS) ₃ (SEQ ID NO: 22)) and BJ194-35/BJ198-35 (the linker is GS(GGGGS) ₆ (SEQ ID NO: 23 )) exhibited a strong dose-dependent activity.

Example 8. Cytotoxic activity of bispecific antibodies

The purpose of this study was to examine the cytotoxicity of anti-Nectin-4 antibodies on MCF-7 and T-47D cells.

The cytotoxicity of anti-Nectin-4 antibody on MCF-7 and T-47D cells was detected by image-based cell killing assay. In this study, MCF-7 and T-47D cells were target cells and primary human T cells were effector cells. Primary human T cells were isolated from human PBMC cells and frozen in liquid nitrogen. Target and effector cells were added at a ratio of 1:2 (3x10 ⁴ for MCF-7/T-47D cells, 6x10 ⁴ for primary human T cells) to each of 96-well plates containing 100 nM anti-Nectin-4 antibody in the hole. Scanned images after 40 hours of co-culture.

Experimental procedure:

cell culture

T cell

Thaw vials containing T cells in a 37 °C water bath with gentle agitation. To reduce the possibility of contamination, keep the o-ring and cap away from water. Thawing must be rapid. Immediately after the contents have thawed, the vials are removed from the water bath and decontaminated by immersion or spraying with 70% ethanol. NOTE: All steps from this step onwards should be performed under strict sterile conditions. Transfer the cells to a larger vial containing 15 ml of pre-warmed growth medium. Centrifuge the vial at 400 g for 5 minutes. Remove supernatant containing cryoprotectants and resuspend cells with 1 ml T cell growth medium. Transfer the contents of the vial to a T75 cell culture flask containing 15 ml of T cell growth medium. Place cells at 37°C, 5% _CO2 .

MCF-7/T-47D cells

Thaw vials containing MCF-7/T-47D cells in a 37°C water bath with gentle agitation. To reduce the possibility of contamination, keep the o-ring and cap away from water. Thawing must be rapid. Immediately after the contents have thawed, the vials are removed from the water bath and decontaminated by immersion or spraying with 70% ethanol. All steps from this one onwards should be performed under strict sterile conditions. Transfer the cells to a 100 mm Petri dish containing 10 ml of pre-warmed medium. When the cells grow to 80-90%, remove the cell supernatant, wash 1-2 times with PBS, and digest with 1 mL 0.25% trypsin EDTA (1X), phenol red. Terminate the digestion with growth medium and gently aspirate the cells to remove them completely. Centrifuge at 300 g for 5 min. Remove the supernatant and add 1 ml of medium to blow away. Transfer the contents of the vial to a 100 mm Petri dish containing 10 ml of growth medium. Place the culture at 37 °C, 5% _CO2 .

Target cell spread (Day 1)

Prepare target cells in test medium. Add 200 µL of cell suspension (approximately 3* ¹⁰⁴ cells per well) to the Petri dish. Place the cells at 37 °C, 5% _CO to allow the cells to adhere.

T cell preparation (Day 2)

Prepare a sufficient amount of T cells (approximately 6*10 ⁴ cells per well) at 6*10 ⁵ /mL in the test medium.

Antibody Dilution (Day 2)

Dilute BJ-009 and 12H3-1/12L1 to working concentrations (starting at 10 nM, 3-fold dilution, 6 points) in test medium. To achieve a working concentration of 10 nM, the antibody should be diluted to 100 nM as the sample concentration.

Mix target cells with antibodies and T cells (Day 2)

Target cells were washed twice with test medium. Add 80 µL of test medium to each well. Add 20 µL of antibody solution to the Petri dish. Add 100 µL of T cell suspension (approximately 6* ¹⁰ cells/well) to the Petri dish.

imaging

Set up the live cell imager (BioTek, Cytation5). Images were scanned after 40 hours of co-culture.

result:

Figure 6 (MCF-7 cells) and Figure 7 (T-47D cells) show the T cell killing results of all bispecific antibodies. Larger and darker particles indicate target cell death. Consistent with the T cell activation results in Example 7, treatment with any antibody in form A did not result in T cell killing, and treatment with most bispecific antibody forms in B resulted in target cell (MCF-7 or T -47D cells), demonstrating the efficacy of these antibodies.

The scope of the disclosure is not to be limited by the specific examples described, which are intended as single illustrations of various aspects of the disclosure, and any compositions or methods that are functionally equivalent are intended to be within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

sequence listing

<110> Boji Biomedical Technology (Hangzhou) Co., Ltd.

<120> Anti-Nectin-4 antibodies and bispecific antibodies

<130> 193541-22D-CNP

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<170> PatentIn version 3.5

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

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Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Ile Lys Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Thr Tyr Leu

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Ala Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Ala Gly Val Gly Val Pro Phe Glu Tyr Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Val Ser Gly Ser Ile Phe Ser Phe Asn

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Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Met Val

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Ala Gly Ile Thr Arg Gly Gly Ser Ile Ala Tyr Ala Asp Ser Val Lys

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

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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Ala Gly Val Gly Glu Pro Phe Glu Tyr Glu Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Phe Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

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Ala Ala Ile Thr Ser Gly Gly Ser Val Lys Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Ala Ala Arg Gly Glu Pro Phe Glu Tyr Asp Thr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

115

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Gly Leu Ser Cys Ala Ala Ser Glu Ser Ile Phe Ser Tyr Asn

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Ile Lys Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Arg Gly Val Tyr Tyr Cys Ala

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Val Met Asp Arg Thr Thr Leu Glu Tyr Asp Tyr Trp Gly Gln Gly Thr

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Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Thr Ile Ala

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Val Asn Tyr Ala Asp Ser Val Lys

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Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Ala Ala Arg Gly Glu Pro Phe Glu Tyr Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

115

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Ile Asn Ala Met Gly

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Ala Ile Thr Ser Gly Gly Ser Ile Lys Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

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Gly Val Gly Val Pro Phe Glu Tyr Asp Tyr

1 5 10

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<223> CDR of CMB7-2

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Phe Asn Ala Met Ala

1 5

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Gly Ile Thr Arg Gly Gly Ser Ile Ala Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

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Gly Val Gly Glu Pro Phe Glu Tyr Glu Tyr

1 5 10

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Ile Asn Ala Met Gly

1 5

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Ala Ile Thr Ser Gly Gly Ser Val Lys Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

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Ala Arg Gly Glu Pro Phe Glu Tyr Asp Thr

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Tyr Asn Ala Met Gly

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Ala Ile Thr Ser Gly Gly Ser Ile Lys Tyr Ala Asp Ser Val Lys Gly

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Met Asp Arg Thr Thr Leu Glu Tyr Asp Tyr

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Ile Ala Ala Met Gly

1 5

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Ala Ile Thr Ser Gly Gly Ser Val Asn Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

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Ala Arg Gly Glu Pro Phe Glu Tyr Asp Tyr

1 5 10

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Gly Ser Gly Gly Gly Gly Ser

1 5

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

Claims (8)

1. A single domain antibody or antigen-binding fragment thereof specific to human Nectin-4 protein, comprising CDR1, CDR2, CDR3, wherein said CDR1, CDR2 and CDR3 are selected from any group in the following combinations: (1) The amino acid sequence of the CDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 8; (2) The amino acid sequence of the CDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 11; (3) The amino acid sequence of the CDR1 is shown in SEQ ID NO: 12, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 13, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 14; (4) The amino acid sequence of the CDR1 is shown in SEQ ID NO: 15, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 17; (5) The amino acid sequence of CDR1 is shown in SEQ ID NO: 18, the amino acid sequence of CDR2 is shown in SEQ ID NO: 19, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 20. 2. The single domain antibody or antigen-binding fragment thereof according to claim 1, which consists of an amino acid sequence selected from any one of SEQ ID NO: 1-5. 3. The single domain antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR1, CDR2 and CDR3 consist of the amino acid sequences of SEQ ID NO: 6, 7 and 8, respectively. 4. The single domain antibody or antigen-binding fragment thereof according to claim 3, which consists of the amino acid sequence of SEQ ID NO: 1. 5. A bispecific antibody comprising the single domain antibody or antigen-binding fragment thereof of any one of claims 1-4 and a second antibody or antigen-binding fragment specific to human CD3 antigen, wherein the The bispecific antibody comprises four of said single domain antibodies, each fused to either the heavy chain variable region VH or the light chain variable region VL of a complete Fab antibody specific for human CD3, each single chain Domain antibodies are all fused to either the heavy chain variable region VH or the light chain variable region VL by a peptide linker having a length greater than 7 amino acids. 6. The bispecific antibody of claim 5, wherein the peptide linker has a length shorter than 50 amino acids. 7. A polynucleotide encoding the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-4 or the bispecific antibody according to any one of claims 5-6. 8. A cell comprising the polynucleotide of claim 7.

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