CN116063498A

CN116063498A - Single domain anti-Nectin-4 antibodies

Info

Publication number: CN116063498A
Application number: CN202211034782.1A
Authority: CN
Inventors: 王�忠; 张海洲
Original assignee: Bo Bo Bio Pharmaceutical Technology Hangzhou Co ltd
Current assignee: Bo Bo Bio Pharmaceutical Technology Hangzhou Co ltd
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2023-05-05
Also published as: CN114671953B; TW202342541A; CN114671953A

Abstract

The invention provides a single domain antibody with excellent binding affinity to human Nectin-4 protein for the single domain anti-Nectin-4 antibody. These antibodies are particularly suitable for inclusion in bispecific antibodies, e.g. antibodies that also target antigens on immune cells.

Description

Single domain anti-Nectin-4 antibodies

The present application is a divisional application of the invention patent application with the application number of 202210459465.8 and the application date of 2022, 04, 27 and the name of "single domain anti-Nectin-4 antibody".

Technical Field

The invention belongs to the field of cell immunology, and relates to a single domain antibody with specificity to human Nectin-4 protein and application thereof.

Background

Nectin family is Ca consisting of four members of Nectin-1, -2, -3 and-4 ²⁺ A non-dependent immunoglobulin-like molecule. Nectin protein plays a role in cell adhesion. They bind actin filaments (F-actin) binding protein afadin through their cytoplasmic tail and associate with actin cytoskeleton, coordinating with other cell adhesion molecules and cell surface membrane receptors, regulating many other cellular activities such as movement, differentiation, polarization, and viral entry.

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

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 a variety of cancer cells, making it a suitable target for cancer treatment.

Disclosure of Invention

In various embodiments, the present disclosure provides single domain antibodies with binding specificity for human Nectin-4 protein. These antibodies can cross-react with cynomolgus monkey Nectin-4. By virtue of excellent binding affinity and small size, these antibodies can be suitably used to generate bispecific antibodies, such as 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 human Nectin-4 protein comprising CDR1, CDR2 and CDR3, wherein said CDR1, CDR2 and CDR3 comprise the CDR1, CDR2 and CDR3 sequences of any of antibodies CMB7-6, CMB7-7, CMB7-8, CMB7-9, CMB7-10, CMB7-11, CMB7-12, CMB7-17, CMB7-18, CMB7-19, CMB7-20, CMB7-21, CMB7-22, or CMB7-23, respectively. These exemplary antibodies have the amino acid sequences shown in SEQ ID NOS.1-14.

In some embodiments, the CDR1, CDR2, and CDR3 comprise amino acid sequences 5-17, SEQ ID NO:18-20, SEQ ID NO:21-23, SEQ ID NO:24-26, SEQ ID NO:27-29, SEQ ID NO:30-32, SEQ ID NO:33-35, SEQ ID NO:36-38, SEQ ID NO:39-41, SEQ ID NO:42-44, SEQ ID NO:45-47, SEQ ID NO:48-50, SEQ ID NO:51-53 or SEQ ID NO:54-56.

In some embodiments, the single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-14.

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 the light chain variable region (VL) of a full Fab antibody specific for human CD3. 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 of greater 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 demonstrating antibody expression.

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

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

Fig. 4 illustrates two forms of the bispecific antibody tested (form a and form B, wherein fig. 4A is form a and fig. 4B is form B).

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

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

Detailed Description

Definition of the definition

It is noted that the term "an" entity refers to one or more of the entities; 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" can be used interchangeably herein.

As used herein, the term "polypeptide" is intended to include both the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) that are 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 does not refer to a particular length of a 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 products of modification after expression of the 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 polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from the specified nucleic acid sequences. It can be produced in any manner, including by chemical synthesis.

The term "isolated" as used herein with respect to a cell, nucleic acid (e.g., DNA or RNA) refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" as used herein 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. Furthermore, "isolated nucleic acid" refers to a nucleic acid fragment that does not occur naturally in fragment form and that is not found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. An isolated polypeptide is intended to include both purified and recombinant polypeptides.

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

"homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology may be determined by comparing positions in each sequence, which may be aligned for comparison purposes. When a position in the comparison sequence 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 matched or homologous positions shared by the sequences. "unrelated" or "non-homologous" sequences have 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 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 the two sequences are compared. Such alignments and percent homology or sequence identity may 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, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cut-off value = 60; expected value = 10; matrix = 62; description = 50 sequences; ordering = high score; database = non-redundant, genBank + EMBL + DDBJ + PDB + GenBank-CDS-transmissions + SwissProtein + spl. Bioequivalent polynucleotides refer to polynucleotides that have the specified percentage homology described above and encode polypeptides having the same or similar biological activity.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having 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 double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence with a degree of homology to its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or complement thereof. Similarly, an "equivalent polypeptide" refers to a polypeptide that has some 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, the 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.

Hybridization reactions can be performed under different "stringent" conditions. Generally, the low stringency hybridization reaction is carried out at about 40℃in about 10 XSSC or a solution having the same ionic strength/temperature. Medium stringency hybridization reactions are typically carried out in about 6 x SSC at about 50 ℃, while high stringency hybridization reactions are typically carried out in about 1 x SSC at about 60 ℃. Hybridization reactions 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, which are typically present in cells.

Polynucleotides consist 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 alphabetically ordered representation of a polynucleotide molecule. Such alphabetically arranged representations may be entered into a database in a computer having a central processing unit and used for bioinformatic applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one genetic form or portion thereof. At least two different forms, i.e., two different nucleotide sequences, of a portion of a gene are referred to as "polymorphic regions of a gene. Polymorphic regions may be single nucleotides whose identity varies among 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. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, 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. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The nucleotide structure, if present, may be modified before or after assembly of the polynucleotide. 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 indicated or required, any polynucleotide embodiment of the present disclosure comprises a double stranded form and each of the two complementary single stranded forms known or predicted to constitute the double stranded form.

The term "encoding" as used with respect to a polynucleotide refers to a polynucleotide, which is referred to as "encoding" a polypeptide if it is transcribed and/or translated to produce an mRNA of the polypeptide and/or fragment thereof in its native state or when manipulated by methods well known to those skilled in the art. The antisense strand is the complement of such a nucleic acid from which the coding sequence can be deduced.

As used herein, "antibody" or "antigen binding polypeptide" refers to a polypeptide or complex of polypeptides that specifically recognizes and binds an antigen. The antibody may be an entire antibody and any antigen binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen. Examples of such 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, the antibody fragment binds to the same antigen as recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror image, 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 immunoglobulin heavy chain (VH) and light chain (VL) variable regions. In some aspects, these regions are linked to short linkers 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 to the C-terminus of VL, and vice versa. The protein retains the original immunoglobulin specificity despite removal of the constant region and introduction of the linker. 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 biochemically 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 as IgG, igM, igA, igG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., are well characterized and are known to have functional specificity. Modifications of each of these categories and isoforms are readily recognized by those skilled in the art in view of this disclosure, and are therefore within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion is generally directed to IgG classes 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. The four chains are typically linked by disulfide bonds in a "Y" structure, wherein the light chain surrounds the heavy chain starting at the "Y" mouth and continuing through the variable region.

"specifically bind" or "specific" generally means that an antibody binds to an epitope through its antigen binding domain, and that such binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is more likely to "specifically bind" to an epitope than a random, unrelated epitope when it binds to the epitope through its antigen binding domain. The term "specific" 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" more specifically than the relevant epitope "D".

As used herein, the term "treatment" or "therapy" refers to both medical treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the progress of an undesired physiological change or disorder, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (partial or total), whether detectable or undetectable. "treatment" may also mean an extension of survival compared to the expected survival without treatment. The person in need of treatment includes those already with the disease or disorder, as well as those prone to the disease or disorder, or those in need of prophylaxis of the disease or disorder.

"subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis or treatment. Mammalian subjects include humans, domestic animals, farm animals, zoo animals, sports 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 "subject in need of treatment" include subjects, e.g., mammalian subjects, that would benefit from administration of the antibodies or compositions of the present disclosure for detection, diagnostic procedures, and/or treatment.

Single domain anti-Nectin-4 antibodies

Nectin protein plays an important role in cell-cell adhesion. Nectin-4 is a type I single transmembrane protein of approximately 52 kDa. Unlike

Nectin

1, 2 and 3, which are widely expressed in adult tissues, nectin-4 is specifically expressed in embryos and placenta. Nectin-4, however, can also be expressed in a variety of cancer cells, making it a suitable target for cancer treatment.

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 known as VHH fragments. As with VH of conventional antibodies, each VHH includes three CDRs, CDR1, CDR2 and CDR3. The VHH may further comprise constant domains, such as CH2 and CH3.

As shown in FIG. 2, all of the identified VHH antibodies from CMB7-6 to CMB7-23 exhibited strong binding to human Nectin-4 protein. At the same time, most of these antibodies also have a strong affinity for the corresponding cynomolgus proteins (fig. 3), allowing them to be tested in cynomolgus monkeys as preclinical models.

One of the antibodies CMB7-6 (VHH 36) was tested as two different forms (fig. 4) of bispecific antibody, which also targets the human CD3 complex. The results in FIGS. 5-7 show that form B bispecific antibodies exhibit excellent T cell activation and T cell killing activity. Thus, 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 CDR1, CDR2 and CDR3, having the amino acid sequences of the antibodies CMB7-6, CMB7-7, CMB7-8, CMB7-9, CMB7-10, CMB7-11, CMB7-12, CMB7-17, CMB7-18, CMB7-19, CMB7-20, CMB7-21, CMB7-22 or CDR1, CDR2 and CDR3, respectively. The sequences of these antibodies are provided in Table 1 as shown in SEQ ID NOS.1-14.

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 CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOS: 21-23, 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 24-26, 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 27-29, respectively.

In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:30-32, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:33-35, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:36-38, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:39-41, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:42-44, CDR1, CDR2 and CDR3 of the amino acid sequence.

In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:45-47, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO: CDR1, CDR2 and CDR3 of the 48-50 amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:51-53, CDR1, CDR2 and CDR3 of the amino acid sequence. In one embodiment, an antibody or antigen binding fragment is provided comprising a polypeptide having the amino acid sequence of SEQ ID NO:54-56, CDR1, CDR2 and CDR3 of the amino acid sequence.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of any one of SEQ ID NOS.1-14, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOS.1-14. 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 1-14 retains the CDR sequences of the corresponding reference antibody.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 1, or an amino acid sequence having 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 a corresponding reference antibody, e.g., SEQ ID NO. 15, 16 and 17.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 2, or an amino acid sequence having 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 antibody, e.g., SEQ ID NO. 18, 19 and 20.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 3, or an amino acid sequence having 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 NO. 21, 22 and 23.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 4, or an amino acid sequence having 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 NO. 24, 25 and 26.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 5, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 5. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 5 retains the CDR sequences of a corresponding reference antibody, e.g., SEQ ID NO. 27, 28 and 29.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 6, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 6. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 6 retains the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO. 30, 31 and 32.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 7, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 7. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 7 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID NO. 33, 34 and 35.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 8, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 8. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 8 retains the CDR sequences of a corresponding reference antibody, e.g., SEQ ID NO. 36, 37 and 38.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 9, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 9. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO 9 retains the CDR sequences of a corresponding reference antibody, e.g., SEQ ID NO 39, 40 and 41.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 10, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 10. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO 10 retain the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO 42, 43 and 44.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 11, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 11. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 11 retain the CDR sequences of the corresponding reference antibody, e.g., SEQ ID NO. 45, 46 and 47.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 12, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 12. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 12 retains the CDR sequences of a corresponding reference antibody, e.g., SEQ ID NO. 48, 49 and 50.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 13, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 13. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 13 retains the CDR sequences of a corresponding reference antibody, e.g., SEQ ID NO. 51, 52 and 53.

In one embodiment, an antibody or antigen binding fragment is provided that comprises the amino acid sequence of SEQ ID NO. 14, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 14. In some embodiments, amino acid sequences having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 14 retain the CDR sequences of the corresponding reference antibodies, e.g., SEQ ID NO. 54, 55 and 56.

In one embodiment, an antibody or antigen binding fragment is provided comprising the amino acid sequence of any one of SEQ ID NOs 1-14, optionally with additions, deletions and/or substitutions of 1, 2, 3, 4 or 5 amino acids. In some embodiments, the substitution is a conservative substitution. In some embodiments, the additions, deletions and/or substitutions are within the framework regions.

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 substitution of an amino acid residue with an amino acid residue having a similar side chain. Amino acid residue families 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 substituted with another amino acid residue from the same side chain family. In another embodiment, the amino acid strings may be substituted with structurally similar strings that differ in the order and/or composition of the side chain family members.

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

TABLE A amino acid similarity matrix

TABLE B conservative amino acid substitutions

Amino acids	Is replaced by
		Alanine (Ala)	D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys
Arginine (Arg)	D-Arg, Lys, D-Lys, Orn D-Orn
		Asparagine derivatives	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 (S)	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 (Gly)	Ala, D-Ala, Pro, D-Pro, Aib, β-Ala
		Isoleucine (Ile)	D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine (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 (Phe)	D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp
Proline (proline)	D-Pro
		Serine (serine)	D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys
Threonine (Thr)	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 (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 described above, these newly identified anti-Nectin-4 VHH antibodies are suitable for inclusion in bispecific antibodies. Both 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 has strong binding to cells. In addition, these bispecific antibodies exhibit strong T cell activation and T cell mediated killing of tumor cells when incubated with T cells (targeted by binding 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 VHH antibody of the present disclosure and a second antibody or antigen-binding fragment that binds to 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, the 4:2 vhh: fab form (form B) has excellent therapeutic activity compared to the other forms tested. The 4:2 format is shown in FIG. 4B, which includes an antibody in the form of a conventional Fab (e.g., anti-CD 3) and four VHH units specific for Nectin-4. Each VHH is fused to the N-terminus of the Fab variable region by a peptide linker, such as GS (GGGGS) (SEQ ID NO: 57), GS (GGGGS) ₃ (SEQ ID NO: 58) and GS (GGGGS) ₆ （SEQ ID NO: 59）。

For some VHHs, one interesting finding is that the length of the peptide linker has a significant effect on the activity of the bispecific antibody to bind to Nectin-4 on the cell surface. Thus, in some embodiments, the peptide linker may be at least 2 amino acids in length, 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 glycine (G) and serine (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: 57), GS (GGGGS) ₃ (SEQ ID NO: 58) and GS (GGGGS) ₆ （SEQ ID NO: 59）。

Thus, in one embodiment, the present disclosure provides a bispecific antibody that is specific for immune cells (e.g., targeted to CD 3) 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 (e.g., 2 and 4) of VHHs that target Nectin-4.

Thus, in one embodiment, there is provided a bispecific antibody comprising a first moiety and a second moiety, wherein the first moiety comprises two pairs of VH and VL, each pair being capable of binding to a human CD3 complex, and the second moiety comprises 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 moiety 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 IgG1, igG2, igG3, or IgG4 sequence.

One of ordinary skill in the art will also appreciate that the antibodies disclosed herein may be modified to differ in amino acid sequence from the naturally occurring binding polypeptide 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 percentage 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 are not normally associated with the antibody. Exemplary modifications are described in more detail below. For example, antibodies of the disclosure may comprise flexible linker sequences, or may be modified to add functional moieties (e.g., PEG, drug, toxin, or label).

The antibodies, variants or derivatives (including modified derivatives) 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 an 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 performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, an antibody may comprise one or more non-classical amino acids.

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

The antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (possibly drugs or toxins), ultrasound enhancers, non-radioactive labels, 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-tagged antigen binding polypeptide is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts and oxalic esters.

The antibody may also be labeled with a fluorescent emitting metal (e.g., 152 Eu) or other lanthanide. These metals can be attached to the antibody using metal chelating groups such as diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA). Techniques for binding different moieties to antibodies are well known, for example, see 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. Lists, 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), thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies' 84: biological And Clinical Applications, picchera 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. -16 (1985), and Thorpe et al, the Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugs", immunol et al (1985), immunol Rev (52:119).

Polynucleotides encoding antibodies and methods of making antibodies

The disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies of the disclosure, 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 an isolated polynucleotide molecule. Furthermore, 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 are described herein. In certain embodiments, the variable and constant regions of the antigen binding polypeptides of the present disclosure are all human in origin. Fully human antibodies can be prepared using techniques described in the art and described herein. For example, fully human antibodies to a particular antigen may 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 disabled. Exemplary techniques that may be used to make such antibodies are described in U.S. patent 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 a detrimental immune response in the animal (e.g., in a human) to be treated. 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 chimeric antibodies 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 accomplished by a variety of methods, including (a) transplanting the entire non-human variable domain to a human constant region to produce a chimeric antibody; (b) Transplanting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a human framework and constant regions with or without the retention of critical framework residues; or (c) grafting the entire non-human variable domains, but "masking" them with human-like moieties by replacing surface residues. Such methods are described, for example, in 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, molecular Immun. 25:489-498 (1991), padlan, molecular 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.

De-immunization may also be used to reduce the immunogenicity of antibodies. The term "deimmunize" as used herein includes altering antibodies to modify T cell epitopes (see, e.g., international application publication Nos. WO/9852976 A1 and WO/0034317 A2). For example, the variable heavy and variable light chain sequences from the starting antibody are analyzed and a "map" of human T cell epitopes for each V region is created showing epitope positions associated with the Complementarity Determining Regions (CDRs) and other critical residues within the sequence. Single T cell epitopes in the T cell epitope map were analyzed to identify alternative amino acid substitutions with low risk of altering the final antibody activity. A series of optionally variable heavy and variable light sequences, including combinations of amino acid substitutions, were designed and subsequently incorporated into a series of binding polypeptides. Typically, between 12 and 24 variant antibodies are produced and tested for binding and/or function. The complete heavy and light chain genes comprising the modified variable and human constant regions are then cloned into an expression vector and the subsequent plasmids are introduced into a cell line to produce the complete antibody. Antibodies are then compared in appropriate biochemical and biological assays and the best variants are determined.

The binding specificity of the antigen binding polypeptides of the present disclosure can be determined by in vitro assays, such as immunoprecipitation, radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (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 that involve administering an antibody of the present disclosure to a patient (e.g., animal, mammal, and human) 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).

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 the tumor antigen. Induction of gene expression may be achieved, for example, 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 carcinoma, merck cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. Thus, current antibodies can be used to treat any one or more of such cancers.

Other diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed, and/or predicted by the antibodies or variants of the present disclosure or derivatives thereof include, but are not limited to, progression and/or metastasis of malignant tumors and related diseases, such as leukemias including acute leukemias (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia (including myeloblasts, promyelocytic, granulocytic, monocytic, and erythrocytic leukemias) and chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., hodgkin's disease and non-hodgkin's disease), multiple myelomas, fahrenheit macroglobulinemia, heavy chain diseases, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, 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 carcinoma, and renal cell carcinoma basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, cyst gland carcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngeal pipe tumor, and, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

The particular dosage and treatment regimen of any particular patient will depend on a variety of factors including the particular antibody used, the variant or derivative thereof, the age, 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. The 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 antibodies, variants or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may 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 (intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), bucally, or as an oral or nasal spray.

As used herein, the term "parenteral" refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

Administration may be systemic or topical. 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, through the use of an inhaler or nebulizer, as well as the use of aerosol formulations.

It may be desirable to administer an antibody polypeptide or composition of the present disclosure topically to an area in need of treatment; this may be by, for example, but not limited to, local infusion during surgery, local application, e.g., in combination with a wound dressing after surgery, by injection, by catheter, by suppository, or by way of an implant that is porous, non-porous, or a gel material (including a membrane, e.g., sialic acid membrane, or fiber). Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that are not absorbed by the protein.

Composition and method for producing the same

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

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

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. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions, aqueous dextrose solutions, and aqueous glycerol solutions can 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, for example, acetates, citrates or phosphates, if desired. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; in addition, agents for regulating tonicity, such as sodium chloride or dextrose, 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 formulated into suppositories with conventional binders and carriers such as triglycerides. 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 drug 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 adapted to the mode of administration. The parent formulation may be contained in an ampoule, disposable syringe or a multi-dose bottle made of glass or plastic.

In one embodiment, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a 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, these ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or pouch, indicating the amount of active agent. When the composition is administered by infusion, it may 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 may be provided so that it may be mixed prior to administration.

The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, 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 alpaca immunization followed by construction and selection of phage libraries.

An anti-human Nectin-4 antibody was generated using recombinant human Nectin-4/hFc fusion protein as an immunogen. 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 conjugate was amplified from the antigen positive phage by PCR and sequenced. Expressed proteins were confirmed by SDS-PAGE (FIG. 1). The following table provides the sequences of the unique antibodies and their CDR regions.

TABLE 1 antibody sequences

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TABLE 2 CDR sequences

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Example 2 ELISA binding test

In this example, antibodies were subjected to ELISA-based binding assays. The same concentration (0.5. Mu.g/mL) of human Nectin-4 was coated 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 were added (as a control). After washing off the excess sample, goat anti-human IgG Fc cross-adsorbed antibody (or rabbit anti-goat IgG antibody) conjugated with horseradish peroxidase (HRP) was added. HRP can react with the substrate 3,3', 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. Thus, CMB7 was tested for binding affinity to human Nectin-4 using ELISA.

The test steps are as follows:

coated antigen preparation

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

Closure

All microwells were aspirated and washed 5 times with 300 μl/well wash buffer through the microplate washer. The soaking time (about 1 minute) is set aside in each washing step, and the washing effect can be improved. The microplate is then dehydrated with a microplate dehydrator to remove any remaining buffer. The microwells were closed with 200 μl of closing buffer. Incubate in a 37℃water bath for 1 hour.

Sample preparation

The washing/dehydration was repeated. The samples were diluted to working concentration (2. Mu.g/mL) in assay buffer. A pre-diluted sample of 100 μl/well was added to the appropriate well. The microwell plates were sealed and incubated in a 37 ℃ water bath for 1 hour.

Secondary antibody incubation

The washing/dehydration was repeated. The secondary antibodies were diluted 1:2000 in assay buffer. 100 μl/well of diluted secondary antibody was added to each well (1:2000). The plates were incubated in a 37℃water bath in the absence of light for 1 hour.

Signal detection

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

Results:

ELISA test results 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.

The kinetic binding affinity of the antibodies to human Nectin-4 was detected by biolayer interferometry. HFC (anti-HIgG FC) probes (ProbeLife) were pre-wetted in kinetic buffer (Probelife) at 30℃for 5 min in Crimson 96 MAX 96 well reaction plates (ET Healthcare, 06-0098). The probes were then immersed in wells of a Greiner black microplate containing 4. Mu.g/mL antibody in kinetic buffer. Analyte (human Nectin-4) diluted with different concentration gradients (starting from 200 nM, 2-fold stepwise, total of 5 concentration gradients) was subjected to a 5-minute binding step followed by a 16-minute dissociation step in kinetic buffer. The data were analyzed by subtracting the reference sample and a 1:1K binding model of the structured data method for affinity constants was fitted 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 μl/Kong Yushi buffer (K buffer) was added per well in column 1 of the Crimson 96 MAX 96 well reaction plate, the probes were placed in the buffer, and the probes were pre-wetted for 5 min at 1000 rpm.

Baseline 1

In column 1 of Greiner 96-well polypropylene microwell plates, 6 probes were subjected to baseline testing in 200 μl/well K buffer at 1000 rpm for 2 minutes. As balanced as possible, the final slope is preferably not higher than 0.02 nm/min.

Antibody Loading onto probes

The antibodies were diluted to working concentration (4. Mu.g/mL, 200. Mu.L/well) with K buffer and then loaded onto probes on a 2/3/4/5/6 column of Greiner 96 well polypropylene microwell plate at 1000 rpm for 5 minutes.

Baseline 2

In column 8/9/10/11/12 of Greiner 96-well polypropylene microwell plates, probes were subjected to baseline testing in 200 μl/well K buffer at 1000 rpm for 2 minutes. To remove unbound mAb from the biosensor, non-specific binding should be minimized or bias in the buffer effect should be reduced.

Bonding of

Antigen (human Nectin-4) was diluted to 6 concentrations (200, 100, 50, 25, 12.5, 0 nM, 200 μl/well) with K buffer and this series of antigens was then added to wells of Greiner 96 well polypropylene microwell plates, columns 7, lines A to F. In column 7, the antibodies on the probes were bound to antigens at different concentration gradients for 5 minutes at 1000 rpm.

Dissociation of

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

Regeneration of

The probe was run in a regeneration procedure (the probe was regenerated 5s in R buffer in column 11 of Crimson 96 MAX 96 well plates and then neutralized 5s in Q buffer in column 12 of Crimson 96 MAX 96 well plates) and the procedure was repeated 3 times.

Results:

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

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

Antibodies to	KD (M)	Response to
			CMB7-6	6.94E-21	0.835
CMB7-7	4.74E-21	0.972
			CMB7-8	4.50E-21	0.915
CMB7-9	5.30E-10	0.891
			CMB7-10	9.58E-10	0.891
CMB7-11	4.06E-21	0.94
			CMB7-12	6.01E-21	0.97
CMB7-17	4.64E-21	0.833
			CMB7-18	8.10E-10	0.729
CMB7-19	4.08E-21	0.867
			CMB7-20	4.09E-21	0.783
CMB7-21	1.47E-11	0.809
			CMB7-22	4.78E-21	0.841
CMB7-23	7.98E-21	0.877

Example 4 Cross-reaction with cynomolgus Nectin-4

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

The binding affinity of the antibodies to cynomolgus monkey Nectin-4 was tested by ELISA. Cynomolgus monkey Nectin-4 at the same concentration (0.5. Mu.g/mL) was coated on 96-well enzyme plates. After blocking, diluted antibodies of different concentration gradients (starting from 2. Mu.g/mL, 3-fold stepwise dilution, total 7 concentration gradients) were added. After washing off excess sample, goat anti-human IgG Fc cross-adsorbed antibody conjugated with horseradish peroxidase (HRP) was added. HRP can react with the substrate 3,3', 5' -Tetramethylbenzidine (TMB) to form a colored product. The binding affinity of CMB7 to cynomolgus 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 Nectin-4 was tested by ELISA.

The test process comprises the following steps:

coated antigen preparation

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

Closure

Sample preparation

The washing/dehydration was repeated. Samples were diluted to working concentration (2. Mu.g/mL) in assay buffer and 3-fold serial dilutions were performed to make a total 7-point curve. A pre-diluted sample of 100 μl/well was added to the appropriate well. The microwell plates were sealed and incubated in a 37 ℃ water bath for 1 hour.

Secondary antibody incubation

The washing/dehydration was repeated. The secondary antibodies were diluted to 1:2000 in assay buffer. 100 μl/well of diluted secondary antibody was added to each well (1:2000). The plates were incubated in a 37℃water bath in the absence of light for 1 hour.

Signal detection

Results:

the test results are shown in figure 3, which shows 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 Sorter (FACS).

The test steps are as follows:

cell preparation

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

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

4. Cells were collected and transferred to a 15ml conical tube.

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

Cell plating

With 5mL PBS-0.2% BSA washing, centrifugation at 300g at 4℃for 5 min. The procedure 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 μl).

Primary antibody incubation

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

Washing

200 μl of ice-cold PBS-0.2% BSA was added to each well and centrifuged at 300g at 4deg.C for 5 min. Repeated 3 times. 100 μl PBS-0.2% BSA was added to re-suspend the cells.

Secondary antibody incubation

Alexa Fluor 488 goat anti-human IgG (H+L) (1:500 dilution) was added to each well in ice-cold PBS-0.2% BSA and incubated for 1 hour at 4 ℃.

Washing

200 μl of ice-cold PBS-0.2% BSA was added to each well and centrifuged at 300g at 4deg.C for 5 min. Repeated 3 times. 200 μl PBS-0.2% BSA was added to re-suspend the cells.

Is applied to flow cytometry.

Results:

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

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

The single domain antibody (VHH) CMB7-6 ("VHH 36") was used to construct bispecific antibodies that also target human CD 3. As shown in fig. 4, two different forms of bispecific antibodies were used.

FIG. 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, format A format is asymmetric, with a 1:1 potency for CD3 and Nectin-4.

In the format of fig. 4B (format B), each of the 4 VHHs is fused to the N-terminus of the variant domain of one full anti-CD 3 antibody. Thus, this form is symmetrical, with a 2:4 potency for CD3 and Nectin-4. The bispecific configuration and structure of each strand are shown in table 5.

TABLE 5 Structure of bispecific antibodies and related chains

Antibodies to	Form of the invention	Joint (SEQ ID NO:)
			BJ182/12L1/183-36	A	-
BJ192-36/BJ196-36	B	GS(GGGGS) ₁ (SEQ ID NO:57)
			BJ193-36/BJ197-36	B	GS(GGGGS) ₃ (SEQ ID NO:58)
BJ194-36/BJ198-36	B	GS(GGGGS) ₆ (SEQ ID NO:59)

Chain	Structure of the
		BJ192-36	Anti-Nectin VHH-linker 1-Anti-CD3-Fc (PGLALA)
BJ193-36	Anti-Nectin VHH-linker 2-Anti-CD3-Fc (PGLALA)
		BJ194-36	Anti-Nectin VHH-linker 3-Anti-CD3-Fc (PGLALA)
BJ196-36	Anti-Nectin VHH-linker 1-Anti-CD3-VL-CL
		BJ193-36	Anti-Nectin VHH-linker 2-Anti-CD3-VL-CL
BJ198-36	Anti-Nectin VHH-linker 3-Anti-CD3-VL-CL

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

EXAMPLE 7T cell activation of bispecific antibodies

Bispecific antibodies were tested in this example for their ability to activate T cells in the presence of Nectin-4 expressing MCF-7 cells or T-47D cells.

Figure 5 shows T cell activation results for all bispecific antibodies. Form A (BJ 182/12L1/BJ 183-36) contained only one VHH, and did not show observable T cell activating activity. Interestingly, the two bispecific antibodies of format B (BJ 192-36/BJ196-36 and BJ194-36/BJ 198-36) exhibited strong dose-dependent activity.

EXAMPLE 8 cytotoxic Activity of bispecific antibodies

The aim of this study was to detect 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 detected 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 cells and effector cells were present in a 1:2 ratio (MCF-7/T-47D cells 3X 10) ⁴ Primary human T cells 6x10 ⁴ ) To each well of a 96-well plate containing 100 nM anti-Nectin-4 antibody. Images were scanned after 40 hours of co-incubation.

The test process comprises the following steps:

cell culture

T cell

The vials containing T cells were thawed in a 37 ℃ water bath with gentle agitation. To reduce the possibility of contamination, the O-ring and cap are kept away from the water. Thawing must be rapid. Immediately after thawing the contents, the vials were removed from the water bath and purged by dipping or spraying with 70% ethanol. And (3) injection: all steps from this step should be performed under strictly sterile conditions. Cells were transferred to larger vials containing 15 ml pre-warmed growth medium. The vials were centrifuged at 400 g for 5 minutes. The supernatant containing the cryoprotectant was removed and the cells resuspended in 1 ml T cell growth medium. The contents of the vial were transferred to a T75 cell culture flask containing 15 ml of T cell growth medium. The cells were exposed to 5% CO at 37 ℃ ₂ Is a kind of medium.

MCF-7/T-47D cells

The 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 cap are kept away from the water. Thawing must be rapid. Immediately after thawing the contents, the vials were removed from the water bath and purged by dipping or spraying with 70% ethanol. All steps from this step should be performed under strictly sterile conditions. Cells were transferred to 100 mm dishes containing 10 ml pre-warmed medium. When the cells are grown to 80-90%,cell supernatants were removed, washed 1-2 times with PBS and digested with 1mL of 0.25% trypsin EDTA (1X), phenol red. The digestion was stopped with growth medium, and the cells were gently blown and removed completely. 300 g, centrifuging for 5 min. The supernatant was removed and 1ml medium was added to blow off. The vial contents were transferred to a 100 mm petri dish containing 10 ml growth medium. The culture was subjected to 5% CO at 37 ℃ ₂ Is a kind of medium.

Target cell diffusion (day 1)

Target cells are prepared in a test medium. 200. Mu.L of the cell suspension (about 3X 10 per well) was added to the petri dish ⁴ Individual cells). Cells were placed in 5% CO2 at 37℃to adhere the cells.

T cell preparation (day 2)

At 6X 10 in test medium ⁵ Preparation of sufficient T cells per mL (about 6X 10 per well) ⁴ Individual cells).

Antibody dilution (day 2)

BJ-009 and 12H3-1/12L1 were diluted to working concentration in test medium (starting from 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.

Mixing target cells with antibodies and T cells (day 2)

The target cells were washed 2 times with the test medium. 80 μl of test medium was added to each well. 20 mu L of antibody solution is added into the culture dish. 100 [ mu ] L T cell suspension (about 6X 10) was added to the petri dish ⁴ Individual cells/well).

Imaging system

A living cell imager (BioTek, cystation 5) was set. Pictures were scanned after 40 hours of co-cultivation.

Results:

the T cell killing results for all bispecific antibodies are shown in FIG. 6 (MCF-7 cells) and FIG. 7 (T-47D cells). Larger and darker particles indicate target cell death. Consistent with the T cell activation results in example 7, treatment with any of the antibodies in form a did not result in T cell killing, and treatment with most of the bispecific antibodies in form B resulted in cell death of the target cells (MCF-7 or T-47D cells), demonstrating the efficacy of these antibodies.

The scope of the present disclosure is not to be limited by the specific embodiments described, which are intended as a single illustration of the various 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. Accordingly, 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.

Claims

1. A single domain antibody or antigen binding fragment thereof specific for human Nectin-4 protein comprising CDR1, CDR2 and CDR3, wherein said CDR1, CDR2 and CDR3 consist of the CDR1, CDR2 and CDR3 sequences, respectively, of a single domain antibody represented by an amino acid sequence selected from the group consisting of SEQ ID NOs 2-14.

2. The single domain antibody or antigen binding fragment thereof according to claim 1, wherein said CDR1, CDR2 and CDR3 consist of the amino acid sequences of SEQ ID NOs 18-20, 21-23, 24-26, 27-29, 30-32, 33-35, 36-38, 39-41, 42-44, 45-47, 48-50, 51-53 or 54-56, respectively.

3. The single domain antibody or antigen binding fragment thereof according to claim 1, consisting of an amino acid sequence selected from any one of SEQ ID NOs 2-14.

4. A bispecific antibody comprising the single domain antibody or antigen-binding fragment thereof of any one of claims 1-3 and a second antibody or antigen-binding fragment specific for an antigen other than Nectin-4.

5. The bispecific antibody of claim 4, wherein the antigen is human CD3.

6. The bispecific antibody of claim 5, comprising four of the single domain antibodies, each fused to a heavy chain variable region VH or a light chain variable region VL of a full Fab antibody specific for human CD3.

7. The bispecific antibody of claim 6, wherein each single chain domain antibody is fused to VH or VL via a peptide linker.

8. The bispecific antibody of claim 7, wherein the peptide linker has a length of longer than 7 amino acids.

9. The bispecific antibody of claim 7 or 8, wherein the peptide linker has a length of less than 50 amino acids.

10. A polynucleotide encoding the single domain antibody or antigen binding fragment thereof of any one of claims 1-3 or the bispecific antibody of any one of claims 4-9.

11. A cell comprising the polynucleotide of claim 10.

12. Use of the single domain antibody or antigen-binding fragment thereof of any one of claims 1-3 or the bispecific antibody of any one of claims 4-9 in the manufacture of a medicament for treating cancer, comprising administering an effective amount of the single domain antibody or antigen-binding fragment thereof or bispecific antibody to a cancer patient.