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

Abstract

The present disclosure provides a single domain antibody having 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 with specificity to human Nectin-4 protein and application thereof.

Background

The Nectin family is Ca consisting of four members of Nectin-1, -2, -3 and-4 ²⁺ Independent immunoglobulin-like molecules. The Nectin protein plays a role in cell adhesion. They bind the actin filament (F-actin) binding protein, afadin, via their cytoplasmic tail and in association with the actin cytoskeleton, and coordinate with other cell adhesion molecules and cell surface membrane receptors, regulating many other cellular activities such as migration, differentiation, polarization, and viral entry.

Nectin-4, also known as poliovirus receptor associated protein 4 (PVRL 4), is a type I single-pass transmembrane protein of approximately 52 kDa in size. The extracellular domain of Nectin-4 has three Ig-like subdomains, 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, it has been demonstrated that Nectin-4 can be expressed in a variety of cancer cells, making it a suitable target for cancer therapy.

Disclosure of Invention

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

Thus, according to one embodiment of the present disclosure, there is provided a single domain antibody or antigen binding fragment thereof specific for a human Nectin-4 protein comprising a CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the CDR1, CDR2 and CDR3 sequences of any one of the antibodies CMB7-1, CMB7-2, CMB7-3, CMB7-4 or CMB7-5, respectively. These exemplary antibodies have the amino acid sequences shown in SEQ ID NOS 1-5.

In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOS: 6-8, SEQ ID NOS: 9-11, SEQ ID NOS: 12-14, SEQ ID NOS: 15-17, or SEQ ID NOS: 18-20, respectively.

In some embodiments, the single domain antibody comprises an amino acid sequence selected from SEQ ID NOs 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 (VL) of a whole Fab antibody specific for human CD3. In some embodiments, each single chain domain antibody is fused to a VH or VL via a peptide linker.

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

The disclosure also provides polynucleotides encoding any of the antibodies or fragments.

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

Drawings

FIG. 1 shows SDS-PAGE images confirming antibody expression.

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

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

Figure 4 illustrates two formats (format a and format B) of bispecific antibody tested.

FIG. 5 shows the results of T cell activation assays performed in the presence of Nectin-4 expressing cells.

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

Detailed Description

Definition of

It is noted that the term "a" or "an" entity refers to one or more of the entity; for example, "an antibody" is understood to represent one or more antibodies. Thus, 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 both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and not to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms. The term "polypeptide" also means the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

The term "isolated" as used herein with respect to cells, nucleic acids (e.g., DNA or RNA) refers to molecules that are separated from other DNA or RNA, respectively, that are present in the natural source of the macromolecule. The term "isolated" as used herein also refers to nucleic acids or peptides that are 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. In addition, "isolated nucleic acid" refers to a nucleic acid fragment that does not occur naturally as a fragment and is not found in nature. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides.

As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that does not occur naturally, a non-limiting example of which may be created by combining polynucleotides or polypeptides that do not normally occur simultaneously.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which 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, to one of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, 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. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff =60; desired value =10; matrix =62; =50 sequences are described; the sorting mode = high score; database = non-redundant, genBank + EMBL + DDBJ + PDB + GenBank-CDS-transitions + SwissProtein + SPupdate + PIR. A bioequivalent polynucleotide refers to a polynucleotide having the above specified percent homology and encoding a polypeptide having the same or similar biological activity.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. Homologues of a double-stranded nucleic acid are intended to include nucleic acids having a nucleotide sequence with some degree of homology to their complement. In one aspect, a homolog of the nucleic acid is capable of hybridizing to the nucleic acid or its complement. Likewise, an "equivalent polypeptide" refers to a polypeptide that has a degree of homology or sequence identity to 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, as compared to a reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

The hybridization reaction can be performed under different "stringent" conditions. Generally, low stringency hybridization reactions are performed in about 10 XSSC or solutions of equivalent ionic strength/temperature at about 40 ℃. Medium stringency hybridization is typically performed at about 50 ℃ in about 6x SSC, while high stringency hybridization is typically performed at about 60 ℃ in about 1x SSC. The hybridization reaction can also be carried out 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 typically present in cells.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (a); cytosine (C); guanine (G); thymine; when the polynucleotide is RNA, uracil (U) represents thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one gene form or part thereof. A portion of a gene having at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of the gene". The polymorphic region may be a single nucleotide, the identity of which differs 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. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polynucleotide assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any polynucleotide embodiment of the present disclosure comprises a double-stranded form and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce an mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of this 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. The antibody may be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein-or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Such examples include, but are not limited to, complementarity Determining Regions (CDRs) of a heavy or light chain or ligand binding portion thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions or any portion thereof, 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, e.g., F (ab') ₂ 、F(ab) ₂ Fab', fab, fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen that is recognized by an intact antibody. The term "antibody fragment" includes aptamers, mirror images, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a particular antigen to form a complex.

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

The term antibody includes a wide variety of biologically distinguishable classes of polypeptides. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, epsilon, including some subclasses (e.g., gamma 1-gamma 4). It is the nature of this chain that determines the "class" of antibodies to be IgG, igM, igA, igG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igG5, and the like, are well characterized and are known to have functional specificity. In view of this disclosure, modified versions of each of these classes and isoforms are readily identified by those skilled in the art and are therefore within the scope of this disclosure. All classes of immunoglobulins are clearly within the scope of the present disclosure, and 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 having a molecular weight of about 23000 daltons and two identical heavy chain polypeptides having a molecular weight of 53000-70000 daltons. These four chains are typically linked by disulfide bonds in a "Y" shaped structure, where the light chain surrounds the heavy chain starting at the "Y" mouth and continuing through the variable region.

"specifically binds" or "has specificity" usually means that an antibody binds to an epitope through its antigen binding domain, and such binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, when an antibody binds to an epitope through its antigen binding domain, it "specifically binds" to the epitope more readily than a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity of a particular antibody for binding to a particular epitope. For example, antibody "a" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "a" may be considered to bind epitope "C" with a higher specificity than the relevant epitope "D".

As used herein, the terms "treatment" or "therapy" refer to both medical treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, remission, diminishment of extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an increase in survival compared to the expected survival without treatment. Persons in need of treatment include persons already suffering from the disease or disorder, as well as persons susceptible to suffering from the disease or disorder, or persons in need of prevention of the disease or disorder.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, in need of diagnosis, prognosis or treatment. Mammalian subjects include humans, domestic animals, farm animals, zoo animals, sport animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc.

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

Single domain anti-Nectin-4 antibodies

The Nectin protein plays an important role in cell adhesion. Nectin-4 is a type I single-pass transmembrane protein of approximately 52 kDa in size. 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 may also be expressed in a variety of 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 consisting of a single monomeric variable antibody domain. The earliest single domain antibodies were engineered from heavy chain antibodies found in camelids, also referred to as VHH fragments. As with the VH of conventional antibodies, each VHH includes three CDRs, CDR1, CDR2 and CDR3.VHH may further comprise constant domains, such as CH2 and CH3.

As shown in FIG. 2, all the 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 have a strong affinity for the corresponding cynomolgus monkey proteins (fig. 3), making it possible to test them in cynomolgus monkeys as a preclinical model.

One of the 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 FIGS. 5-7 show that the B form of bispecific antibody exhibits excellent T cell activation and T cell killing activity. Therefore, these VHH antibodies can be suitably used for the treatment of diseases such as cancer.

In one embodiment, an antibody or antigen-binding fragment is provided that includes a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 of an antibody CMB7-1, CMB7-2, CMB7-3, CMB7-4, or CMB7-5, respectively. The sequences of these antibodies are provided in Table 1, as shown in SEQ ID NOS: 1-5.

In one embodiment, an antibody or antigen-binding fragment is provided that includes CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOS: 6-8, respectively. In one embodiment, an antibody or antigen binding fragment is provided that includes CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOS: 9-11, respectively. In one embodiment, an antibody or antigen binding fragment is provided that includes CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOS 12-14, respectively. In one embodiment, an antibody or antigen-binding fragment is provided that includes CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 15-17, respectively. In one embodiment, an antibody or antigen-binding fragment is provided that includes CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 18-20, respectively.

In one embodiment, an antibody or antigen-binding fragment is provided that includes the amino acid sequence of any one of SEQ ID NOs 1-5, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs 1-5. In some embodiments, an 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, an antibody or antigen-binding fragment is provided that includes the amino acid sequence of SEQ ID No. 1, or an amino acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 1. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 1 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID NOs 6, 7, and 8.

In one embodiment, an antibody or antigen binding fragment is provided that includes the amino acid sequence of SEQ ID No. 2, or an amino acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 2. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 2 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID nos. 9, 10, and 11.

In one embodiment, an antibody or antigen-binding fragment is provided that includes the amino acid sequence of SEQ ID No. 3, or an amino acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 3. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 3 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID NOs 12, 13, and 14.

In one embodiment, an antibody or antigen binding fragment is provided that includes the amino acid sequence of SEQ ID No. 4, or an amino acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 4. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 4 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID NOs 15, 16, and 17.

In one embodiment, an antibody or antigen-binding fragment is provided that includes the amino acid sequence of SEQ ID No. 5, or an amino acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 5. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 5 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID nos. 18, 19, and 20.

In one embodiment, an antibody or antigen binding fragment is provided comprising the amino acid sequence of any one of SEQ ID NOs 1-5, optionally with 1, 2, 3, 4 or 5 amino acid additions, deletions and/or substitutions. In some embodiments, the substitution is a conservative substitution. In some embodiments, the additions, deletions, and/or substitutions are within a framework region.

In some embodiments, the antibody or fragment further comprises a constant domain, e.g., CH2 and/or CH3. In some embodiments, the CH2 and/or CH3 is from a human IgG1, igG2, igG3, or IgG4 sequence.

In some embodiments, the substitution is a conservative substitution. "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, 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 non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid string may be substituted with a structurally similar string that differs in the order and/or composition of the side chain family members.

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

Table a. Amino acid similarity matrix

Table b. Conservative amino acid substitutions

Amino acids	Is substituted by
		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 belongs to the IgG1, 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 antibodies

As mentioned above, these newly identified anti-Nectin-4 VHH antibodies are suitably included in bispecific antibodies. Both forms (form a and form B) were tested, but form a did not bind well to cell surface expressed Nectin-4. Form B is tetravalent relative to Nectin-4 and binds strongly to cells. Furthermore, these bispecific antibodies exhibit strong T cell activation and T cell-mediated killing of tumor cells when incubated with T cells (targeted by binding to CD 3) and tumor cells expressing Nectin-4.

Thus, 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 an immune cell.

Proteins that can be targeted on immune cells 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 the data show, the 4. The 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 in binding to cell surface Nectin-4. 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, no more than 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Exemplary linkers include a plurality of 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 disclosure provides a bispecific antibody specific for immune cells (e.g., targeting CD 3) and Nectin-4. In some embodiments, the bispecific antibody comprises a conventional antibody specific for a human CD3 complex. In some embodiments, the bispecific antibody comprises a plurality (e.g., 2 and 4) of the Nectin-4-targeting VHHs.

Thus, in one embodiment, there is provided a bispecific antibody comprising a first part comprising two pairs of VH and VL, each pair being capable of binding to a human CD3 complex, and a second part comprising four single domain antibody (VHH) fragments as 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 a constant domain, e.g., CH1 and CL, and CH2 and/or CH3. In some embodiments, the constant region is from a human IgG1, igG2, igG3, or IgG4 sequence.

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

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

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

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

The antibody may be conjugated or fused to a therapeutic agent, which may include a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or toxin), an ultrasound enhancing agent, a non-radioactive label, combinations thereof with other such agents known in the art.

The antibody may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-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 ester, imidazole, acridinium salt and oxalate ester.

The antibody may also be labeled with a fluorescence emitting metal (e.g., 152 Eu) or other lanthanide. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques For binding different moieties to Antibodies are well known, see, for example, 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 (2 nd Ed.), robinson et al, ' (eds.), marcel Dekker, inc., pp. 623-53 (1987);" inorganic 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)," inorganic resources, solutions, and inorganic functional pathogenic Of The Therapeutic Use Of radioactive inorganic Antibody In Cancer Therapy ", in Monoclonal Antibodies For Cancer Detection Therapy, baldwin et al (eds.)," inorganic predictive reagents For Cancer Detection Therapy, academatic Press, 303-16 (1985), and "inorganic precursors Of Cytotoxic reagents 119", tojJ.52 (1985)).

Polynucleotides encoding antibodies and methods of making antibodies

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

Methods of making antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen binding polypeptides of the present disclosure are both 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 prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous site has been nullified. Exemplary techniques that can be used to make such antibodies are described in U.S. patent nos. 6,150,584, 6,458,592, 6,420,140, which is incorporated herein by reference in its entirety.

In certain embodiments, the antibodies produced do not elicit an adverse immune response in the animal to be treated (e.g., in a human). In one embodiment, the antigen binding polypeptides of the present disclosure, variants or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibody may be humanized, primatized, deimmunized, or a chimeric antibody may be prepared. These types of antibodies are derived from non-human antibodies, typically 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 by a variety of methods, including (a) grafting the entire non-human variable domain to a human constant region to produce a chimeric antibody; (b) Grafting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a human framework and constant region, with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains but "masking" them with human-like portions by replacing surface residues. Such methods are described in Morrison et al, proc. Natl. Acad. Sci. USA 57, 6851-6855 (1984), morrison et al, adv. Immunol. 44 (1988); verhoeyen et al, science 239.

Deimmunization may also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see, e.g., international application publication Nos.: WO/9852976 A1 and WO/0034317 A2). For example, variable heavy and variable light chain sequences from the starting antibody are analyzed and a human T cell epitope "map" of each V region is created showing the epitope positions relative to the Complementarity Determining Regions (CDRs) and other key residues within the sequence. Individual T cell epitopes in the T cell epitope map are analyzed to identify alternative amino acid substitutions with a low risk of altering the final antibody activity. A series of optionally variable heavy and light sequences, including combinations of amino acid substitutions, were designed and these sequences were 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 the human constant regions are then cloned into an expression vector, and the subsequent plasmids are introduced into a cell line to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological assays and the best variant is determined.

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

Cancer treatment

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

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

The antibodies of the present disclosure may also be used to treat or inhibit cancer. In some embodiments, nectin-4 is overexpressed in tumor cells. Thus, 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 present disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) of the patient expresses, overexpresses, or is induced to express a tumor antigen. For example, induction of gene expression can be achieved by administration of tumor vaccines or radiation therapy.

Tumors that may be suitably treated include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urinary tract cancer, colorectal cancer, head and neck cancer, squamous cell cancer, merkel cell cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. Thus, the current antibodies can be used to treat any one or more of these cancers.

Other diseases or disorders associated with increased cell survival that can be treated, prevented, diagnosed and/or predicted by the antibodies or variants or derivatives thereof of the present disclosure include, but are not limited to, the progression and/or metastasis of malignancies and related diseases, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblasts, promyelocytes, granulocytes, monocytes and erythrocytic leukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., hodgkin's disease and non-hodgkin's disease), multiple myeloma, fahrenheit macroglobulinemia, heavy chain diseases and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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 cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonic cancer, wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, and combinations thereof, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

The specific dosage and treatment regimen for any particular patient will depend upon a variety of factors including the specific antibody used, its variant or derivative, the age, body weight, general health, sex, and diet of the patient, as well as the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical personnel is within the ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the effect desired. The amount can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of administration of the antibodies, variants or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition 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 administered with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally (intracisternal), intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray.

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

Administration can be systemic or local. Furthermore, it may be desirable to introduce the 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, for example connected to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, as well as the use of aerosol formulations.

It may be desirable to administer the antibody polypeptides or compositions of the present disclosure topically to the area in need of treatment; this may be by way of example and not limitation, by local infusion during surgery, local administration, e.g., in conjunction with a post-operative wound dressing, by injection, by catheter, by suppository, or by way of an implant that is porous, non-porous, or a gel material (including membranes such as sialid 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.

Composition comprising a metal oxide and a metal oxide

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

In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In addition, a "pharmaceutically acceptable carrier" is typically a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or auxiliary agent of any type.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which a 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, such as 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 may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates, if desired. Antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; furthermore, agents for adjusting tonicity, such as sodium chloride or glucose, are also contemplated.

These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be made into suppository with conventional binder and carrier (such as triglyceride). Oral formulations may 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 in e.w. martin Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, and an appropriate amount of carrier, so as to provide the patient with a form suitable for administration. The formulation should be suitable for the mode of administration. The parent preparation may be contained in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated in accordance with conventional procedures as a pharmaceutical composition suitable for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a lytic agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a 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 ampule of sterile water for injection or physiological saline may be provided so that it can be mixed prior to administration.

The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Examples

Example 1 preparation of anti-human Nectin-4 Single Domain antibodies

This example demonstrates how anti-human Nectin-4 single domain antibodies can be generated by immunisation with alpaca followed by construction and selection of phage pools.

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

The binders were amplified from the antigen-positive phage by PCR and sequenced. The expressed protein was confirmed by SDS-PAGE (FIG. 1). The following table provides the sequences of the unique antibodies and their CDR regions.

TABLE 1 antibody sequences

TABLE 2 CDR sequences

Example 2 ELISA binding assay

In this example, an ELISA-based binding assay was performed on the antibody. Human Nectin-4 was coated at the same concentration (0.5. Mu.g/mL) onto 96-well enzyme plates. After blocking, 2. Mu.g/mL of each antibody and varying concentrations (0.2. Mu.g/mL and 1. Mu.g/mL) of goat anti-human Nectin-4 antibody (as a control) were added. After washing away the excess sample, goat anti-human IgG Fc cross-adsorbed antibody conjugated to horseradish peroxidase (HRP) (or rabbit anti-goat IgG antibody) was added. HRP reacts with a substrate, 3', 5' -Tetramethylbenzidine (TMB) to form a colored product. The binding affinity of CMB7 to human Nectin-4 can be determined by reading the OD of the reaction solution ₄₅₀ The value was calculated because the absorbance of the reaction solution was positively correlated with the content of the antigen-bound antibody. Therefore, the binding affinity of CMB7 to human Nectin-4 was tested by ELISA.

The test steps are as follows:

coated antigen formulations

The coating antigen (human Nectin-4) was diluted to the working concentration (0.5. Mu.g/mL) in PBS. The 96-well plate was immediately coated with 100. Mu.L of diluted coated antigen per well. The plates were sealed and incubated overnight at 4 ℃.

Sealing of

All microwells were aspirated and washed 5 times by microplate washer with 300 μ L/well wash buffer. The washing effect can be improved by leaving a soaking time (about 1 minute) in each washing step. The microplate is then dehydrated with a microplate dehydrator to remove any residual buffer. The microwells were sealed with 200 μ L of blocking buffer. Incubate for 1 hour in a water bath at 37 ℃.

Sample preparation

The washing/dewatering was repeated. The samples were diluted to working concentration (2. Mu.g/mL) in assay buffer. Add 100 μ L/well of pre-diluted sample to appropriate well. The plates were sealed and incubated in a water bath at 37 ℃ for 1 hour.

Secondary antibody incubation

The washing/dehydration was repeated. The secondary antibody was diluted in assay buffer at 1. To each well was added 100 μ L/well of diluted secondary antibody (1. The plate was incubated in a water bath at 37 ℃ for 1 hour in the absence of light.

Signal detection

The washing/dewatering was repeated. To each well was added 100 μ L/well of matrix solution (TMB). Plates were incubated for 3 min at room temperature in the dark. A stop solution of 50 μ L/well was added. Plates were read at 450 nm and the data analyzed.

As a result:

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

TABLE 3 ELISA affinity test results

Example 3 kinetic binding assay

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

And detecting the kinetic binding affinity of the antibody and the human Nectin-4 by using a biolayer interferometry. The HFC (anti-HIgG FC) probe (Probelife) was prewetted in a Crimson 96 MAX 96 well reaction plate (ET Healthcare, 06-0098) in kinetic buffer (Probelife) at 30 ℃ for 5 minutes. The probes were then immersed in wells of a Greiner black plate containing 4. Mu.g/mL of antibody in kinetic buffer. The analyte (human Nectin-4) was subjected to a binding step for 5 minutes using different concentration gradient dilutions (2-fold stepwise dilution starting from 200 nM for a total of 5 concentration gradients) followed by a dissociation step in kinetic buffer for 16 minutes. The data were analyzed by subtracting the reference samples and fitted to the 1K binding model of the structured data method of affinity constants using data analysis software 1.7.2.0609 (Gator).

The test process comprises the following steps:

probe balancing

The temperature of the plate was set to 30 ℃, the collection rate was set to standard kinetics (5.0 HZ), 260 μ L/well of pre-wetting buffer (kbuffer) was added per well in column 1 of the Crimson 96 MAX 96 well reaction plate, the probe was placed in the buffer, and the probe was pre-wetted for 5 min,1000 rpm.

Base line 1

In column 1 of Greiner 96-well polypropylene microplates, a baseline test was performed on 6 probes in 200 μ L/well K buffer at 1000 rpm for 2 minutes. As much as possible, the final slope is preferably not higher than 0.02 nm/min.

Loading of antibodies onto probes

The antibody was diluted to the working concentration with K buffer (4. Mu.g/mL, 200. Mu.L/well) and loaded onto the probe at 1000 rpm on a 2/3/4/5/6 column of Greiner 96-well polypropylene microwell plates for 5 minutes.

Base line 2

The probes were subjected to a baseline test in 200 μ L/well K buffer at 1000 rpm for 2 minutes in the 8/9/10/11/12 column of a Greiner 96 well polypropylene microplate. To remove unbound mAb from the biosensor, non-specific binding should be minimized or the bias of buffering effects should be reduced.

Bonding of

Antigen (human Nectin-4) was diluted to 6 concentrations (200, 100, 50, 25, 12.5, 0nM, 200 μ L/well) with K buffer and then this series of antigens was added to Greiner 96 well polypropylene microwell plates in columns 7, rows A to F. In column 7, the antibody on the probe binds to the antigen at different concentration gradients for 5 minutes at 1000 rpm.

Dissociation

The probes were dissociated for 16 minutes in K-buffer and the antigen was dissociated from the probes in column 8/9/10/11/12 on Greiner 96-well polypropylene microwell plates.

Regeneration

The probe was run through a regeneration program (probe was regenerated for 5s in R buffer in column 11 of a Crimson 96 MAX 96 well reaction plate, then neutralized for 5s in Q buffer in column 12 of a Crimson 96 MAX 96 well reaction plate, and the process was repeated 3 times).

As a result:

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

TABLE 4 affinity measurement of anti-Nectin 4 VHH-Fc

Antibodies	KD (M)	Response to
			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-reaction with Macaca fascicularis Nectin-4

This example tests the binding affinity of antibodies to cynomolgus monkey Nectin-4.

The binding affinity of the antibody to cynomolgus monkey Nectin-4 was tested by ELISA. The same concentration (0.5. Mu.g/mL) of cynomolgus monkey Nectin-4 was coated onto 96-well enzyme plates. After blocking, diluted antibodies of different concentration gradients were added (starting from 2. Mu.g/mL, 3-fold dilutions were performed step by step, for a total of 7 concentration gradients). After washing away the excess sample, goat anti-human IgG Fc cross-adsorbed antibodies conjugated to horseradish peroxidase (HRP) were added. HRP reacts with the substrate 3,3', 5' -Tetramethylbenzidine (TMB) to form a colored product. The binding affinity of CMB7 to cynomolgus monkey Nectin-4 can be determined by reading the OD of the reaction solution ₄₅₀ The value was calculated because the absorbance of the reaction solution was positively correlated with the content of the antigen-bound antibody. Thus, the binding affinity of CMB7 to cynomolgus monkey Nectin-4 was tested by ELISA.

The test process comprises the following steps:

coated antigen formulations

The coating antigen (cynomolgus monkey Nectin-4) was diluted to working concentration (0.5. Mu.g/mL) in PBS. Antigen-coated 96-well microplates were immediately diluted at 100 μ L per well. The plates were sealed and incubated overnight at 4 ℃.

Sealing of

All microwells were aspirated and washed 5 times by microplate washer with 300 μ L/well wash buffer. The washing effect can be improved by leaving a soaking time (about 1 minute) in each washing step. The microplate was then dehydrated with a microplate dehydrator to remove any residual buffer. The microwells were sealed with 200 μ L of blocking buffer. Incubate for 1 hour in a water bath at 37 ℃.

Sample preparation

The washing/dewatering was repeated. Samples were diluted to working concentration (2 μ g/mL) in assay buffer and 3-fold serial dilutions were performed to make a total of 7 point curves. Add 100 μ L/well of pre-diluted sample to the appropriate well. The plates were sealed and incubated in a water bath at 37 ℃ for 1 hour.

Secondary antibody incubation

The washing/dewatering was repeated. The secondary antibody was diluted to 1. To each well was added 100 μ L/well of diluted secondary antibody (1. The plates were incubated in a water bath at 37 ℃ for 1 hour in the absence of light.

Signal detection

The washing/dehydration was repeated. To each well was added 100 μ L/well of matrix solution (TMB). Plates were incubated for 3 min at room temperature in the dark. A stop solution of 50 μ L/well was added. Plates were read at 450 nm and the data analyzed.

As a result:

the results of the tests are shown in figure 3, which indicates that all antibodies tested cross-react with cynomolgus monkey Nectin-4, most of which show strong binding affinity. This suggests that cynomolgus monkeys may be a suitable preclinical model for testing these antibodies.

Example 5 flow cytometry analysis of binding to human Breast cancer cells

This example detects the binding affinity of antibodies to human breast cancer cells expressing Nectin-4 by Fluorescence Activated Cell Sorting (FACS).

The test steps are as follows:

cell preparation

1. After the cells reached 80% confluence, the cells were harvested from 100 mm culture dishes.

2. Wash with 2mL PBS. 1mL of 0.25% trypsin EDTA was added and cultured at 37 ℃ until the cells were detached from the plate. 5mL of warm medium was added. Cells were collected and transferred to a 15mL conical tube.

4. Cells were collected and transferred to 15ml conical tubes.

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

Cell plating

Washed with 5mL PBS-0.2% BSA and centrifuged at 300g,4 ℃ for 5 min. This was repeated twice. The supernatant was discarded and resuspended in 2mL PBS-0.2% BSA. Count cells and add 1 × 10 to each well ⁵ -2×10 ⁵ Individual cells (100 μ L).

Primary antibody incubation

Protein (10 μ g/mL or 5 nM) was added to each well and incubated at 4 ℃ for 1 hour.

Washing machine

To each well 200 μ L of ice-cold PBS-0.2% BSA was added and centrifuged at 300g,4 ℃ for 5 min. Repeat 3 times. Add 100 μ L PBS-0.2% BSA to resuspend the cells.

Secondary antibody incubation

Alexa Fluor 488 goat anti-human IgG (H + L) (1.

Washing machine

To each well was added 200 μ L of ice-cold PBS-0.2% BSA, and centrifuged at 300g at 4 ℃ for 5 min. Repeat 3 times. Add 200 μ L PBS-0.2% BSA to resuspend the cells.

Is applied to flow cytometry.

As a result:

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

Example 6 preparation of anti-Nectin-4/anti-CD 3 bispecific antibody

Single domain antibodies (VHH) CMB7-1 ("VHH 35") were used to construct bispecific antibodies that also target human CD3. As shown in figure 4, two different forms of bispecific antibodies were used.

Figure 4A shows form a in which a single VHH and a single VH/VL pair from an anti-CD 3 antibody are fused to an Fc fragment. Thus, form A is asymmetric, with titers of 1.

In the format of fig. 4B (format B), each of the 4 VHHs is fused to the N-terminus of the variant domain of a full anti-CD 3 antibody. Thus, this form is symmetrical, with titers of 2. The bispecific configuration and the structure of each chain are shown in tables 5-6.

TABLE 5 Structure of bispecific antibodies and related chains

Antibodies	Form(s) of	Joint (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 of the product
		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 prepare the antibodies.

Example 7T cell activation of bispecific antibodies

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

Figure 5 shows the results of T cell activation for all bispecific antibodies. Form A (BJ 182/12L1/BJ 183-35) contains only one VHH and shows no observable T cell activation activity. Interestingly, BJ192-35/BJ196-35 also showed low activity. This is probably due to the linker (GS (GGGGS) ₁ (SEQ ID NO: 21)) is too short. In contrast, BJ193-35/BJ197-35 (GS (GGGGS) as linker) ₃ (SEQ ID NO: 22)) and BJ194-35/BJ198-35 (GS (GGGGS) as linker) ₆ (SEQ ID NO: 23)) showed strong activity in a dose-dependent manner.

Example 8 cytotoxic Activity of bispecific antibodies

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

Cytotoxicity of anti-Nectin-4 antibodies against MCF-7 and T-47D cells was examined by an 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 in a ratio of 1 ⁴ Primary human T cells were 6x10 ⁴ ) Added to each well of a 96-well plate containing 100 nM of anti-Nectin-4 antibody. Images were scanned after 40 hours of co-incubation.

The test process comprises the following steps:

cell culture

T cells

Vials containing T cells were thawed in a water bath at 37 ℃ with gentle agitation. To reduce the possibility of contamination, the O-ring and the lid are kept away from the water. The thawing must be rapid. Immediately after the contents had thawed, the vial was removed from the water bath and purged by immersion or spraying with 70% ethanol. Note: all steps from this step should be performed under strict aseptic conditions. Transferring the cells toIn larger vials containing 15ml of pre-warmed growth medium. The vial was centrifuged at 400 g for 5 minutes. The supernatant containing the cryoprotectant was removed and the cells were resuspended in 1ml of T cell growth medium. The contents of the vial were transferred to a T75 cell culture flask containing 15ml of T cell growth medium. The cells were incubated at 37 ℃ with 5% CO ₂ In (1).

MCF-7/T-47D cells

Vials containing MCF-7/T-47D cells were thawed in a 37 ℃ water bath with gentle agitation. To reduce the possibility of contamination, the O-ring and the lid are kept away from the water. The thawing must be rapid. Immediately after the contents had thawed, the vial was removed from the water bath and purged by immersion or spraying with 70% ethanol. All steps from this step should be performed under strictly sterile conditions. The cells were transferred to 100 mm dishes containing 10 ml of pre-warmed medium. When cells grew to 80-90%, the cell supernatant was removed, washed 1-2 times with PBS, and digested with 1mL of 0.25% trypsin EDTA (1X), phenol red. Digestion was stopped with growth medium and the cells were removed completely by gentle blowing. 300g, centrifuging for 5 min. The supernatant was removed and 1ml of medium was added to blow away. The vial contents were transferred to a 100 mm petri dish containing 10 ml of growth medium. The culture was incubated at 37 ℃ with 5% CO ₂ In (1).

Target cell spreading (day 1)

Target cells are prepared in a test medium. Add 200. Mu.L of cell suspension to the culture dish (about 3x10 per well) ⁴ One cell). The cells were incubated at 37 ℃ with 5% CO ₂ In (3), cells are made to adhere.

T cell preparation (day 2)

6x10 in test medium ⁵ Preparation of sufficient T cells/mL (about 6X10 per well) ⁴ One cell).

Antibody dilution (day 2)

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

Target cells were mixed with antibody and T cells (day 2)

Target cells were washed 2 times with test medium. Add 80 μ L of test medium to each well. Add 20. Mu.L of antibody solution to the petri dish. Add 100 μ L of T cell suspension (about 6x 10) to the culture dish ⁴ Individual cells/well).

Imaging

A live cell imager (BioTek, rotation 5) was set up. Pictures were scanned after 40 hours of co-incubation.

As a result:

FIG. 6 (MCF-7 cells) and FIG. 7 (T-47D cells) show the results of T cell killing by 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 the A form did not result in T cell killing, and treatment with most bispecific antibodies in the B form resulted in cell death of the target cells (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 embodiments, which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods that are functionally equivalent are 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 science and technology (Hangzhou) Co., ltd

<120> anti-Nectin-4 antibody and bispecific antibody

<130> 193541-22D-CNP

<160> 23

<170> PatentIn version 3.5

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<211> 118

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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

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

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<223> CMB7-2

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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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 3

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<223> CMB7-3

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

1 5 10 15

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

35 40 45

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

50 55 60

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 Thr Ala Val Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 4

<211> 118

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<223> CMB7-4

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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 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

50 55 60

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

85 90 95

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

100 105 110

Gln 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

1 5 10 15

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

50 55 60

Gly Arg Ser 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 Thr Ala Val Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

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Ile Asn Ala Met Gly

1 5

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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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<212> PRT

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Gly Val Gly Val Pro Phe Glu Tyr Asp Tyr

1 5 10

<210> 9

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<220>

<223> CDR of CMB7-2

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Phe Asn Ala Met Ala

1 5

<210> 10

<211> 16

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

1 5 10 15

<210> 11

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Gly Val Gly Glu Pro Phe Glu Tyr Glu Tyr

1 5 10

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<223> CDR of CMB7-3

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Ile Asn Ala Met Gly

1 5

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<211> 16

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

1 5 10 15

<210> 14

<211> 10

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Ala Arg Gly Glu Pro Phe Glu Tyr Asp Thr

1 5 10

<210> 15

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<223> CDR of CMB7-4

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Tyr Asn Ala Met Gly

1 5

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

1 5 10 15

<210> 17

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Met Asp Arg Thr Thr Leu Glu Tyr Asp Tyr

1 5 10

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<211> 5

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<223> CDR of CMB7-5

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Ile Ala Ala Met Gly

1 5

<210> 19

<211> 16

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<220>

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<400> 19

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

1 5 10 15

<210> 20

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Ala Arg Gly Glu Pro Phe Glu Tyr Asp Tyr

1 5 10

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<220>

<223> joint

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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

<210> 23

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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 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 for a human Nectin-4 protein comprising a CDR1, CDR2, CDR3, wherein the CDR1, CDR2 and CDR3 are selected from any one of the following combinations:

(1) The amino acid sequence of the CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 7, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 8;

(2) The amino acid sequence of the CDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 10, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 11;

(3) The amino acid sequence of the CDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 13, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 14;

(4) The amino acid sequence of the CDR1 is shown as SEQ ID NO. 15, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 16, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 17;

(5) The amino acid sequence of the CDR1 is shown as SEQ ID NO. 18, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 19, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 20.

2. The single domain antibody or antigen binding fragment thereof of claim 1, consisting of an amino acid sequence selected from any one of SEQ ID NOs 1-5.

3. The single domain antibody or antigen-binding fragment thereof of claim 1, wherein said CDR1, CDR2, and CDR3 consist of the amino acid sequences of SEQ ID NOs 6, 7, and 8, respectively.

4. The single domain antibody or antigen binding fragment thereof of claim 3, consisting 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 for human CD3 antigen, wherein said bispecific antibody comprises four of said single domain antibodies, each fused to the heavy chain variable region VH or light chain variable region VL of a full Fab antibody specific for human CD3, each fused to the heavy chain variable region VH or light chain variable region VL by a peptide linker having a length longer than 7 amino acids.

6. The bispecific antibody of claim 5, wherein the peptide linker has a length of less than 50 amino acids.

7. A polynucleotide encoding the single domain antibody or antigen-binding fragment thereof of any one of claims 1-4 or the bispecific antibody of any one of claims 5-6.

8. A cell comprising the polynucleotide of claim 7.

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