TW202342523A - 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

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

The Nectin family is a Ca ²⁺ -independent immunoglobulin protein molecule composed of four members, Nectin-1, -2, -3 and -4. Nectin protein plays a role in cell-cell adhesion. They bind to the actin filament (F-actin) binding protein afadin through their cytoplasmic tails and combine with the actin hemoglobin skeleton to coordinate with other cell adhesion molecules and cell surface membrane receptors to regulate many other cellular activities, such as Movement, differentiation, polarization and viral entry.

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

Nectin-1, -2, and -3 are widely expressed in adult tissues, but nectin-4 is specifically expressed in embryos and placentas. However, Nectin-4 has been shown to express itself in a variety of cancer cells, making it a suitable target for cancer therapy.

In various embodiments, the present invention provides single domain antibodies with binding specificity for human Nectin-4 protein. These antibodies can cross-react with cynomolgus nectin-4. by virtue of Due to their high binding affinity and small size, these antibodies can be suitably used to generate bispecific antibodies, such as those that also target immune cells.

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

In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO:6-8, SEQ ID NO:9-11, SEQ ID NO:12-14, SEQ ID NO:15-17 or SEQ ID NO: Amino acid sequence 18-20. In some embodiments, the single domain antibody comprises an amino acid sequence selected from SEQ ID NO: 1-5. The present invention also provides a bispecific antibody, which contains the single domain antibody or antigen-binding fragment thereof disclosed in the present invention and a second antibody or antigen-binding fragment specific for an antigen different from Nectin-4. In some embodiments, the antigen is human CD3.

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

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

The invention also provides polynucleotides encoding any such antibodies or fragments.

The present invention also provides the use of a composition for the preparation of a medicament for treating a disease such as cancer, wherein the composition includes an effective amount of the antibody or fragment of the present invention.

Figure 1 shows SDS-PAGE images confirming antibody performance. Abbreviation: NR refers to non-reducing (Non-reducing); R refers to reducing (reducing).

Figure 2 shows the results of ELISA-based antibody affinity testing. Abbreviation: ^2nd Ab refers to secondary antibody.

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

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

Figure 5 shows the results of T cell activation assay in the presence of nectin-4 expressing cells. Abbreviation: IF refers to immunofluorescence.

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

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

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

As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "Polypeptide" refers to a molecule composed of monomers (amino acids) linearly connected through amide bonds (also called peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, rather than to a specific length of the product. Therefore, "peptide", "dipeptide", "tripeptide", "oligopeptide", "protein", "amino acid chain" or any other term used to refer to two or more amino acid chains. Included within the definition of "polypeptide" and the term "polypeptide" may be used instead of, or interchanged with, any of these terms. The term "polypeptide" also means the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization through known protecting/blocking groups, proteolytic cleavage, or Non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including through chemical synthesis.

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

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

"Homology" or "identity" or "similarity" refers to two peptides or two nuclei Sequence similarity between acid molecules. Homology can be determined by comparing positions within 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, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity with one of the sequences of the invention, but preferably less than 25% identity.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) is identical to another sequence by a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) "sequence identity", which means that, when aligned, the percentage of bases (or amino acids) are identical when two sequences are compared. Such alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, which uses default parameters. In particular, the programs were BLASTN and BLASTP, using the following default parameters: genetic code = standard; screening program = none; shares = both; cutoff = 60; expected value = 10; matrix = 62; description = 50 sequences; sorting Method = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank-CDS-translations+SwissProtein+SPupdate+PIR. Bioequivalent polynucleotides are polynucleotides that have the percentage of homology specified above and encode a polypeptide with 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 certain degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. Homologs of a double-stranded nucleic acid are intended to include nucleic acids that share a degree of nucleotide sequence homology with their complement. In one aspect, homologs of the nucleic acid are capable of hybridizing to the nucleic acid or its complement. Likewise, "equivalent peptides" Refers to a polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference polypeptide or polynucleotide. In some aspects, equivalent sequences retain the activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.

Hybridization reactions can be performed under different "stringent" conditions. Generally, low stringency hybridization reactions are performed at approximately 40°C in approximately 10 x SSC or a solution with equivalent ionic strength/temperature. Medium stringency hybridization reactions are typically performed at approximately 50°C in approximately 6 x SSC, while high stringency hybridization reactions are typically performed at approximately 60°C in approximately 1 x SSC. The hybridization reaction can also be carried out under "physiological conditions" well known to those of ordinary skill in the art to which the present invention belongs. Non-limiting examples of physiological conditions are temperature, ionic strength, pH and Mg ²⁺ concentration typically found in cells.

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

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or the like. things. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (such as probes, primers, EST or SAGE tags), exons, introns, message RNA (mRNA), transfer RNA (tRNA), ribosomes RNA (rRNA), 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 include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after assembly of the polynucleotide. Nucleotide sequences can 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 stated or required, any polynucleotide embodiment of the invention encompasses 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 for a polynucleotide refers to a polynucleotide that, if in its native state or when manipulated by methods well known to one of ordinary skill in the art to which this invention belongs, can be transcribed and/or translated to produce the The mRNA for a polypeptide and/or its fragments is said to "encode" the polypeptide. 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. Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Such examples include, but are not limited to, complementarity determining regions (CDRs) of heavy or light chains or ligand binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework regions (FRs) ) or any part thereof, or at least part of a binding protein.

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

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

The term "antibodies" includes a broad class of polypeptides that can be biochemically distinguished. One of ordinary skill in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, epsilon, including some subclasses (eg, gamma 1-gamma 4). It is the nature of this chain that determines the "class" of the antibody, which is IgG, IgM, IgA, IgG or IgE. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and known to have functional specificity. In view of the present invention, one of ordinary skill in the art to which this invention pertains will readily recognize modified versions of each of these classes and isoforms, and are included within the scope of this invention. All immunoglobulin classes are obviously within the scope of the present invention, and the following discussion is generally directed to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical polypeptides with a molecular weight of 53,000-70,000 daltons. Heavy chain polypeptides. These four chains are usually connected in a "Y"-shaped structure through disulfide bonds, in which the light chain starts from the "Y"-shaped mouth and surrounds the heavy chain, and continues through the variable region.

"Specific binding" or "having specificity" generally means that the antibody binds to an epitope through its antigen-binding domain, and this 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 is more likely to "specifically bind" to that epitope than a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to be more specific for a given epitope than antibody "B", or antibody "A" may be considered to bind epitope "C" more specifically than a related epitope "D" "higher.

As used herein, the terms "treatment" or "therapy" refer to both medical treatment and prophylactic or prophylactic measures aimed at preventing or slowing down (mitigating) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stable disease status (i.e., no worsening), delayed or slowed disease progression, improved or reduced disease status, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared with expected survival without treatment. Those in need of treatment include those who already have the disease or disorder, as well as those who are susceptible to the disease or disorder, or who require prevention of the disease or disorder.

"Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis or treatment is required, in particular a mammalian subject. Mammalian subjects include humans, livestock, farm animals, zoo animals, sport animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.

As used herein, phrases such as "to a patient in need of treatment" or "a subject in need of treatment" include persons who will benefit from administration of an antibody or composition thereof of the invention for use in detection, diagnostic procedures, and/or treatment. Objects, such as mammalian objects.

Single domain anti-Nectin-4 antibody

Nectin protein plays an important role in 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 placentas. However, nectin-4 can also be expressed in a variety of cancer cells, making it a suitable target for cancer therapy.

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

As shown in Figure 2, all identified VHH antibodies from CMB7-1 to CMB7-5 showed strong binding to human Nectin-4 protein. At the same time, all these antibodies also have strong affinity to the corresponding cynomolgus monkey proteins (Figure 3), allowing them to be tested 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 Figures 5-7 show that the B-form bispecific antibody exhibits excellent T cell activation and T cell killing activities. Therefore, these VHH antibodies can be appropriately used to treat diseases such as cancer.

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

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 NO: 6-8, 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: 9-11, 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: 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 NO: 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 NO: 1-5, or has an amino acid sequence of at least 85% with any one of SEQ ID NO: 1-5 Amino acid sequences with %, 90%, 95%, 98% or 99% sequence identity. 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 CDRs of the corresponding reference antibody sequence.

In one embodiment, an antibody or antigen-binding fragment is provided that includes the amino acid sequence of SEQ ID NO: 1, or is at least 85%, 90%, 95%, 98%, or 99 identical to SEQ ID NO: 1 % sequence identity of the amino acid sequence. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1 retains the CDR sequence of the corresponding reference antibody, for example, SEQ ID NO: 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 is at least 85%, 90%, 95%, 98%, or 99 identical to SEQ ID NO: 2 % sequence identity of the amino acid sequence. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2 retains the CDR sequence of the corresponding reference antibody, such as SEQ ID NO: 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 is at least 85%, 90%, 95%, 98%, or 99 identical to SEQ ID NO: 3 % sequence identity of the amino acid sequence. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3 retains the CDR sequence of the corresponding reference antibody, such as SEQ ID NO: 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 is at least 85%, 90%, 95%, 98%, or 99 identical to SEQ ID NO: 4 % sequence identity of the amino acid sequence. In some embodiments, an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4 retains the CDR sequence of the corresponding reference antibody, such as SEQ ID NO: 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 is at least 85%, 90%, 95%, 98%, or 99 identical to SEQ ID NO: 5 % sequence identity of the amino acid sequence. 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 sequence of the corresponding reference antibody, such as SEQ ID NO: 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 having 1, 2, 3, 4 or 5 amino acids Addition, deletion and/or substitution of amino acids. In some embodiments, the substitution is a conservative substitution. In some embodiments, the additions, deletions and/or substitutions are within framework regions.

In some embodiments, the antibody or fragment further includes a constant domain, such as CH2 and/or CH3. In some embodiments, the CH2 and/or CH3 are from human IgGl, IgG2, IgG3 or IgG4 sequences.

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. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, amino acids), 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), β-branched side chains (e.g. threonine, valine, iso- Leucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, 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, an amino acid string can 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 higher indicates a conservative substitution between two amino acids.

Table A. Amino acid similarity matrix

Table B. Conservative amino acid substitutions

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

bispecific antibodies

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

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

Proteins 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 shown in the data, the 4:2 VHH:Fab form (Form B) had superior therapeutic activity compared to the other forms tested. The 4:2 format is shown in Figure 4B, which includes a conventional Fab format of an antibody (eg, anti-CD3) and four VHH units specific for nectin-4. Each VHH is fused to the N-terminus of the Fab variable region through a peptide linker. For example, the peptide linker is GS(GGGGS) (SEQ ID NO: 21), GS(GGGGS) ₃ (SEQ ID NO: 22) and GS ( GGGGS) ₆ (SEQ ID NO: 23).

An interesting finding for certain VHHs is that the length of the peptide linker has a significant impact on the activity of the bispecific antibody in binding to cell surface nectin-4. Thus, in some embodiments, the peptide linker can 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, the length is no more than 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

Exemplary linkers include multiple glycines (G) and serines (S). In some embodiments, the linker includes 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).

Accordingly, in one embodiment, the invention provides a bispecific antibody specific for immune cells (eg, targeting CD3) and Nectin-4. In some embodiments, the bispecific antibodies include conventional antibodies specific for human CD3 complexes. In some embodiments, the bispecific antibody includes multiple (eg, 2 and 4) VHHs targeting Nectin-4.

Accordingly, in one embodiment there is provided a bispecific antibody comprising a first part comprising two pairs of VH and VL, each pair capable of binding a human CD3 complex, and a second part comprising as described herein Four single domain antibody (VHH) fragments are disclosed, wherein each VHH fragment is fused to the N-terminus of each VH and VL of the first part through 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 human IgGl, IgG2, IgG3 or IgG4 sequences.

One of ordinary skill in the art will also appreciate that the antibodies disclosed herein can be modified so that they 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% identical to the starting sequence. , 85%, 90%, 95%, 98% or 99% the same.

In certain embodiments, an antibody contains an amino acid sequence or one or more moieties that do not normally bind to the antibody. Exemplary modifications are described in more detail below. For example, the anti- Bodies may contain flexible linker sequences, or may be modified to add functional moieties (eg, polyethylene glycol (PEG), drugs, toxins, or labels).

The antibodies of the invention, variants or derivatives thereof (including modified derivatives) are made 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 may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amide, derivatization by known protecting/blocking groups, proteolysis Cleavage, attachment to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, antibodies may contain 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, pharmaceutical agent, or PEG.

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

Antibodies can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide 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, imidazole, acridinium salts and oxalate esters.

Antibodies can also be labeled with fluorescent emitting metals (such as ¹⁵² Eu) or other lanthanide elements. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating 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 (2nd Ed.), Robinson et al., (eds.), Marcel Dekker , Inc., pp.623-53(1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp .475-506(1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52: 119-58 (1982)).

Polynucleotides encoding antibodies and methods of preparing antibodies

The invention also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies of the invention, variants or derivatives thereof. The polynucleotides of the invention may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, or a variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides of the invention 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 preparing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the invention are fully human. Fully human antibodies can be prepared using techniques described in the art and described herein. For example, fully human antibodies against a specific antigen can be produced by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but in which the endogenous locus has been nullified. Exemplary techniques that can be used to make such antibodies are described in US Patent Nos. 6,150,584; 6,458,592; and 6,420,140, which are incorporated herein by reference in their entirety.

In certain embodiments, antibodies are produced that do not elicit a deleterious immune response in the animal to be treated (eg, in humans). In one embodiment, the antigen-binding polypeptides of the invention, variants or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, that 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 into a human constant region to produce a chimeric antibody; (b) grafting at least part of one or more non-human complementarity-determining regions (CDRs) into the human framework and constant regions with or without retaining key framework residues; or (c) transplant the entire non-human variable domain but "mask" them with human-like parts by replacing surface residues. Such methods include Morrison et al., Proc. Natl. Acad. Sci. USA 57: 6851-6855 (1984); Morrison et al., Adv. Immunol. 44: 65-92 (1988); Verhoeyen et al., Science 239：1534-1536(1988)；Padlan,Molec.Immun.25：489-498(1991)；Padlan,Molec.Immun.31：169-217(1994)，及美國專利5,585,089；5,693,761；5,693,762和6,190,370 , which is incorporated herein by reference in its entirety.

Deimmunization can also be used to reduce the immunogenicity of antibodies. As used in this article, the term "to Immunization involves altering antibodies to modify T cell epitopes (see, for example, 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 is created for each V region, showing correlations with the complementarity determining regions (CDRs) and other key residues within the sequence. Relevant epitope locations. Analyze individual T cell epitopes in the T cell map to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A series of alternative variable heavy and variable light sequences were designed, including combinations of amino acid substitutions, 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 modified variable regions and human constant regions are then cloned into expression vectors, and the subsequent plasmids are introduced into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological assays and the best variants are identified.

The binding specificity of the antigen-binding polypeptide of the present invention can be determined through in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme binding immunosorbent assay (ELISA).

cancer treatment

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

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

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

Appropriately treatable tumors 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, stomach cancer, esophageal cancer, ovarian cancer carcinoma, renal cancer, and small cell lung cancer. Therefore, the current antibodies can be used to treat any one or more of these cancers.

Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and/or predicted by the antibodies of the invention or variants or derivatives thereof include, but are not limited to, progression and/or metastasis of malignant tumors and related diseases, Such as leukemia (including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyeloid, granular monocytic, monocytic and erythroid leukemia)) and chronic leukemia (such as chronic myelogenous leukemia) granulosa leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (such as Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disorders, and Solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovialoma, Mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma Carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, breast Capitate adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendrocyte Cytomas, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variants or derivatives thereof used, the patient's age, weight, general health, sex and diet, as well as the timing of administration, excretion rate, The combination of drugs and severity of the specific disease treated. The judgment of medical personnel on such factors belongs to the ordinary skills in this field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the properties of the compound used, the severity of the disease and the desired effect. The dosage 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 polypeptide or composition may be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be combined with other bioactive agents Administer together. Accordingly, pharmaceutical compositions containing the antigen-binding polypeptides of the invention can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (e.g., via powders, ointments, drops, or transdermal patches) , administered bucally or as an oral or nasal spray.

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

Administration can be systemic or local. Furthermore, it may be necessary to introduce the antibodies of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated through 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, and the use of aerosol formulations.

It may be desirable to apply an antibody polypeptide or composition of the invention locally to the area in need of treatment; this may be by, for example, but not limited to, local infusion during surgery, topical application, for example, in combination with a post-operative wound dressing, by injection, Through a catheter, through a suppository, or through an implant of porous, non-porous, or gel material (including membranes, such as sialic acid membranes, or fibers). Preferably, when administering proteins (including antibodies) of the invention, care must be taken to use materials that are not absorbable by the protein.

Composition

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

In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, especially humans. In addition, a "pharmaceutically acceptable carrier" is usually a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of auxiliary preparation.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the therapeutic agent is administered. Such pharmaceutical carriers may 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 solution, glucose aqueous solution and glycerin Aqueous oil solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, Glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates. Antimicrobial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; in addition, agents for tonicity adjustment, such as chlorination, are also envisaged sodium or glucose.

These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, and the like. The composition may be formulated into a suppository 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 saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin, which is incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The parent preparation may be supplied in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic buffer. If necessary, the composition may also include a dissolving agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Typically the ingredients are presented separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous 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 may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or physiological saline. When administered by injection this group For the compound, an ampoule of sterile water for injection or normal saline may be provided so that it can be mixed prior to administration.

The compounds of the invention can 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 , salts of isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

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

This example demonstrates how to generate anti-human Nectin-4 single domain antibodies through alpaca immunization, followed by construction and selection of a phage library.

Anti-human Nectin-4 antibodies were produced using recombinant human Nectin-4/hFc fusion protein as the immunogen. Alpaca peripheral blood mononuclear cells (PBMC) were collected and the antibody cDNA library was generated through 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.

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

Table 1. Antibody sequences

Table 2. CDR sequences

Example 2. ELISA binding test

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

Test steps:

coated antigen preparation

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

closed

Aspirate all microwells and wash 5 times with 300 μL/well wash buffer through a microplate washer. Allowing soaking time (about 1 minute) in each washing step can improve the washing effect. The microplate is then dehydrated using a microplate dehydrator to remove any residual buffer. Block the wells with 200 μL of blocking buffer. Incubate in a 37°C water bath for 1 hour.

Sample preparation

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

Secondary antibody incubation

Repeat wash/spin. Dilute the secondary antibody 1:2000 in assay buffer. Add 100 μL/well of diluted secondary antibody (1:2000) to each well. Incubate the plate in a 37°C water bath protected from light for 1 hour.

Signal detection

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

result:

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

Table 3. ELISA affinity test results

Example 3. Kinetic Binding Test

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

The kinetic binding affinity of antibodies to human Nectin-4 was detected by biolayer interferometry. The HFC (anti-HIgG FC) probe (Probelife) was prewetted in kinetic buffer (Probelife) at 30°C for 5 minutes in a Crimson 96 MAX 96-well reaction plate (ET Healthcare, 06-0098). The probe was then immersed into the wells of a Greiner black microplate containing 4 μg/mL antibody in kinetic buffer. A 5-minute binding step was performed using dilutions of the analyte (human Nectin-4) at different concentrations (starting at 200 nM and diluting 2-fold over 5 concentration gradients), followed by a 16-minute dissociation step in kinetic buffer. . The data were analyzed by subtracting the reference sample and a 1:1 K binding model of the structured data method of affinity constants was fitted using data analysis software 1.7.2.0609 (Gator).

Test process:

Probe balance

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

Baseline 1

Baseline the 6 probes in 200 μL/well K buffer in column 1 of a Greiner 96-well polypropylene microplate at 1000 rpm for 2 min. Be as balanced as possible, and the final slope should not be higher than 0.02nm/min.

Load antibodies onto probes

The antibody was diluted to working concentration (4 μg/mL, 200 μL/well) with K buffer and then loaded into the probe at 1000 rpm on columns 2/3/4/5/6 of a Greiner 96-well polypropylene microplate. Leave the needle on for 5 minutes.

Baseline 2

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

combine

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

dissociate

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

regeneration

The probe runs the regeneration program (the probe is regenerated in the R buffer in the 11th column of the Crimson 96 MAX 96 well reaction plate for 5 s, and then neutralized in the Q buffer in the 12th column of the Crimson 96 MAX 96 well reaction plate for 5 s. This process Repeat 3 times).

result:

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

Table 4. Affinity measurements of anti-Nectin 4 VHH-Fc

Example 4. Cross-reactivity with cynomolgus monkey Nectin-4

This example tested the binding affinity of the antibody to cynomolgus monkey Nectin-4.

The binding affinity of the antibody to cynomolgus monkey Nectin-4 was tested by ELISA. Cynomolgus monkey Nectin-4 at the same concentration (0.5 μg/mL) was coated on a 96-well enzyme plate. After blocking, add diluted antibodies of different concentration gradients (starting from 2 μg/mL, gradually diluting 3 times, a total of 7 concentration gradients). After washing away excess sample, goat anti-human IgG Fc cross-adsorbed antibody conjugated to horseradish peroxidase (HRP) was added. HRP can react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce colored products. The binding affinity of CMB7 to cynomolgus monkey Nectin-4 can be calculated by reading the OD450 value of the reaction solution, because the absorbance of the reaction solution is positively correlated with the content of the antibody bound to the antigen. Therefore, ELISA was used to test the binding affinity of CMB7 to cynomolgus monkey Nectin-4.

Test process:

coated antigen preparation

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

closed

Aspirate all microwells and wash 5 times with 300 μL/well wash buffer through a microplate washer. Allowing soaking time (about 1 minute) in each washing step can improve the washing effect. The microplate is then dehydrated using a microplate dehydrator to remove any residual buffer. Block the wells with 200 μL of blocking buffer. Incubate in a 37°C water bath for 1 hour.

Sample preparation

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

Secondary antibody incubation

Repeat wash/spin. Dilute the secondary antibody to 1:2000 in assay buffer. Add 100 μL/well of diluted secondary antibody (1:2000) to each well. Place the plate at 37°C Incubate in a water bath protected from light for 1 hour.

Signal detection

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

result:

The test results are shown in Figure 3, which shows that all tested antibodies cross-reacted with cynomolgus nectin-4, with most showing strong binding affinity. This suggests that cynomolgus monkeys could be a suitable preclinical model for testing these antibodies.

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

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

Test steps:

Cell preparation

1. After the cells reach 80% confluence, collect the cells from the 100mm culture dish.

2. Wash with 2mL PBS.

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

4. Collect cells and transfer to 15 mL conical tube.

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

cell plating

Wash with 5 mL PBS-0.2% BSA, and centrifuge at 300 g and 4°C for 5 min. Repeat twice. Discard the supernatant and resuspend in 2 mL PBS-0.2% BSA. Count cells and add 1*10 ⁵ -2*10 ⁵ cells (100 μL) to each well.

Primary antibody incubation

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

wash

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

Secondary antibody incubation

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

wash

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

Used in flow cytometry.

result:

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

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

The single domain antibody (VHH) CMB7-1 ("VHH 35") was used to construct a bispecific antibody that also targets 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-CD3 antibody are fused to the Fc fragment. Therefore, the A form is asymmetric and has a 1:1 potency against CD3 and Nectin-4.

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

Table 5. Structure of bispecific antibodies and associated chains

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

Example 7. T cell activation by bispecific antibodies

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

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

Example 8. Cytotoxic activity of bispecific antibodies

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

The cytotoxicity of anti-nectin-4 antibodies on MCF-7 and T-47D cells was detected by image-based cell killing assay. In this study, MCF-7 and T-47D cells were target cells and primary human T cells were effector cells. Primary human T cells were isolated from human peripheral blood mononuclear cells (PBMC) cells and frozen in liquid nitrogen. Target cells and effector cells were added to a 96-well plate containing 100 nM anti-Nectin-4 antibody at a ratio of 1:2 (3*10 ⁴ for MCF-7/T-47D cells and 6*10 ⁴ for primary human T cells). in each hole. Images were scanned after a total of 40 hours of culture.

Test process:

cell culture

T cells

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

MCF-7/T-47D cells

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

Target cell spread (Day 1)

Prepare target cells in test medium. Add 200 μL of cell suspension to the culture dish (approximately 3* ¹⁰ cells per well). Place the cells in 37 °C, 5% _CO to allow cells to adhere.

T cell preparation (day 2)

Prepare sufficient T cells (approximately 6* ¹⁰ cells per well) at 6*105/mL in the test medium.

Antibody dilution (Day 2)

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

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

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

imaging

Set up the live cell imager (BioTek, Cytation5). Pictures were scanned after a total of 40 hours of culture.

result:

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

The scope of the present invention is not limited by the specific embodiments, which are intended as single illustrations of various aspects of the invention, and any functionally equivalent compositions or methods are included within the scope of the invention. It will be apparent to those of ordinary skill in the art that various modifications and variations can be made in the methods and compositions of the invention without departing from the spirit or scope of the invention. This invention is therefore intended to cover the modifications and variations of this invention, provided they are 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 (14)

A single domain antibody or an antigen-binding fragment thereof that is specific for human Nectin-4 protein, comprising CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 1-5 CDR1, CDR2 and CDR3 sequences of single domain antibodies represented. The single domain antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR1, CDR2 and CDR3 further comprise SEQ ID NO: 6-8, SEQ ID NO: 9-11, SEQ ID NO: 12-14, respectively. The amino acid sequence of SEQ ID NO: 15-17 or SEQ ID NO: 18-20. The single domain antibody or antigen-binding fragment thereof according to claim 1, which comprises an amino acid sequence selected from SEQ ID NO: 1-5. The single domain antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR1, CDR2 and CDR3 are the amino acid sequences of SEQ ID NO: 6, 7 and 8 respectively. The single domain antibody or antigen-binding fragment thereof according to claim 4, which comprises the amino acid sequence of SEQ ID NO: 1. A bispecific antibody, comprising the single domain antibody or antigen-binding fragment thereof described in any one of claims 1-5 and a second antibody or antigen-binding fragment specific for an antigen different from Nectin-4. The bispecific antibody according to claim 6, wherein the pair of antigens different from Nectin-4 are human CD3. The bispecific antibody according to claim 7, further comprising four of the single domain antibodies, each single domain antibody fused to the heavy chain variable region of a complete antigen-binding fragment antibody specific for human CD3 ( VH) or light chain variable region (VL). The bispecific antibody according to claim 8, wherein each single domain antibody is fused to VH or VL through a peptide linker. The bispecific antibody according to claim 9, wherein the peptide linker has a length longer than 7 amino acids. The bispecific antibody according to claim 9 or 10, wherein the peptide linker has a length shorter than 50 amino acids. One or more genes encoding an antibody or fragment as described in any one of claims 1-11 Polynucleotides. A cell comprising one or more polynucleotides as described in claim 12. Use of a composition for preparing a medicament for treating cancer, wherein the composition includes an effective amount of the antibody or fragment described in any one of claims 1-11.

Images