WO2023198011A1 - Anticorps anti-nectine-4 à domaine unique - Google Patents

Anticorps anti-nectine-4 à domaine unique Download PDF

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WO2023198011A1
WO2023198011A1 PCT/CN2023/087399 CN2023087399W WO2023198011A1 WO 2023198011 A1 WO2023198011 A1 WO 2023198011A1 CN 2023087399 W CN2023087399 W CN 2023087399W WO 2023198011 A1 WO2023198011 A1 WO 2023198011A1
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antibody
seq
nos
antigen
amino acid
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PCT/CN2023/087399
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Zhong Wang
Haizhou Zhang
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Bj Bioscience Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the Nectin family is the Ca 2+ -independent immunoglobulin-like molecules consisting of four members, Nectin-1, -2, -3, and -4.
  • the Nectin proteins play a role in cell-cell adhesion. They bind afadin, an actin filament (F-actin) -binding protein, through their cytoplasmic tails and associate with the actin cytoskeleton, and regulate many other cellular activities such as movement, differentiation, polarization, and the entry of viruses, in cooperation with other cell adhesion molecules and cell surface membrane receptors.
  • F-actin actin filament
  • Nectin-4 also known as poliovirus receptor-related protein 4 (PVRL4) , is a single pass type I transmembrane protein of about 52 kDa in size.
  • the extracellular domain of Nectin-4 has three Ig-like subdomains designated as V, C1 and C2.
  • Nectins 1, 2 and 3 are widely expressed in adult tissues, but Nectin-4 is expressed specifically in the embryo and placenta. However, it has been shown that Nectin-4 can be expressed in various cancer cells, making it a suitable target for cancer therapy.
  • the present disclosure provides single-domain antibodies having binding specificity to the human Nectin-4 protein. These antibodies can cross-react to cynomolgus Nectin-4. With the excellent binding affinity and the small size, these antibodies can suitably be used to generate bispecific antibodies, such as those that also target an immune cell.
  • a single-domain antibody or antigen-binding fragment thereof having specificity to a human Nectin-4 protein comprising a CDR1, a CDR2 and a CDR3, wherein the CDR1, CDR2 and CDR3, respectively, comprise the CDR1, CDR2 and CDR3 sequences of any one 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.
  • These example antibodies have amino acid sequences as set forth in SEQ ID NOs: 1-14.
  • the CDR1, CDR2 and CDR3, respectively comprise the amino acid sequences of 15-17, SEQ ID NOs: 18-20, SEQ ID NOs: 21-23, SEQ ID NOs: 24-26, SEQ ID NOs: 27-29, SEQ ID NOs: 30-32, SEQ ID NOs: 33-35, SEQ ID NOs: 36-38, SEQ ID NOs: 39-41, SEQ ID NOs: 42-44, SEQ ID NOs: 45-47, SEQ ID NOs: 48-50, SEQ ID NOs: 51-53, or SEQ ID NOs: 54-56.
  • the single-domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14.
  • bispecific antibody comprising the single-domain antibody or antigen-binding fragment thereof disclosed herein and a second antibody or antigen-binding fragment having specificity to an antigen different from Nectin-4.
  • the antigen is human CD3.
  • the bispecific antibody comprises 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 having specificity to the human CD3.
  • each single-chain domain antibody is fused to the VH or VL through a peptide linker.
  • the peptide linker has a length that is longer than 7 amino acids. In some embodiments, the peptide linker has a length that is shorter than 50 amino acids.
  • Methods are also provided, for using the disclosed antibodies or fragment for treating diseases such as cancer.
  • FIG. 1 shows SDS-PAGE images confirming the expression of the antibodies.
  • FIG. 2 shows the results of the ELISA-based antibody affinity testing.
  • FIG. 3 shows that all anti-human Nectin-4 VHH antibodies cross-reacted to cynomolgus Nectin-4.
  • FIG. 4 illustrates two formats (Format A and Format B) of tested bispecific antibodies.
  • FIG. 5 shows the results of T cell activation assays in the presence of Nectin-4-expressing cells.
  • FIG. 6-7 show the results of T cell killing of target Nectin-4-expressing cells (FIG. 6: MCF-7 cells; FIG. 7: T-47D cells) .
  • a or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.
  • the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides, ” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds) .
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • polypeptides dipeptides, tripeptides, oligopeptides, “protein, ” “amino acid chain, ” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide, ” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
  • nucleic acids such as DNA or RNA
  • isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
  • 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 chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides is meant to encompass both purified and recombinant polypeptides.
  • the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25%identity, with one of the sequences of the present disclosure.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 %or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example 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.
  • Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • an equivalent nucleic acid or polynucleotide refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof.
  • a homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.
  • an equivalent polypeptide refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide.
  • the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.
  • the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide.
  • 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 conditions of different “stringency” .
  • a low stringency hybridization reaction is carried out at about 40 °C in about 10 x SSC or a solution of equivalent ionic strength/temperature.
  • a moderate stringency hybridization is typically performed at about 50 °C in about 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in about 1 x SSC.
  • Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art.
  • a non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg 2+ normally found in a cell.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A) ; cytosine (C) ; guanine (G) ; thymine (T) ; and uracil (U) for thymine when the polynucleotide is RNA.
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • polymorphism refers to the coexistence of more than one form of a gene or portion thereof.
  • a polymorphic region can be a single nucleotide, the identity of which differs in different alleles.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag) , 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.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double-and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
  • the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
  • CDR complementarity determining region
  • 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 and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
  • antibody fragment includes aptamers, spiegelmers, and diabodies.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins.
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V H with the C-terminus of the V L , or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ l- ⁇ 4) . It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses isotypes) e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 , etc.
  • immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
  • the four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • an antibody By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, 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 (whether partial or total) , whether detectable or undetectable.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as “to a patient in need of treatment” or “asubject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • Nectin-4 is a single pass type I transmembrane protein of about 52 kDa in size. Unlike Nectins 1, 2 and 3 which are widely expressed in adult tissues, Nectin-4 is expressed specifically in the embryo and placenta. However, Nectin-4 can also be expressed in various cancer cells, making it a suitable target for cancer therapy.
  • a single-domain antibody also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain.
  • the earliest single-domain antibodies were engineered from heavy-chain antibodies found in camelids which are also referred to as VHH fragments.
  • each VHH includes three CDRs, CDR1, CDR2 and CDR3.
  • a VHH can further include constant domains such as CH2 and CH3.
  • VHH antibodies As shown in FIG. 2, all of the identified VHH antibodies, from CMB7-6 through CMB7-23, exhibited strong binding to the human Nectin-4 protein. Most of these antibodies, meanwhile, also have strong affinity the counterpart cynomolgus protein (FIG. 3) , allowing them to be tested in cynomolgus monkeys as a preclinical model.
  • VHH 36 CMB7-6 (VHH 36)
  • FIG. 5-7 demonstrate that bispecific antibodies of Format B exhibited excellent T cell activation and T cell killing activities.
  • these VHH antibodies can be suitably used for treating diseases such as cancer.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of CDR1, CDR2, and CDR3 of antibody 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.
  • the sequences of these antibodies are provided in Table 1, as SEQ ID NOs: 1-14.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 15-17. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 18-20. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 21-23.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 24-26.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 27-29.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 30-32. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 33-35. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 36-38.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 39-41.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 42-44.
  • an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 45-47. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 48-50. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 51-53. In one embodiment, provided is an antibody or antigen-binding fragment that includes a CDR1, a CDR2, and a CDR3, respectively having the amino acid sequences of SEQ ID NOs: 54-56.
  • an antibody or antigen-binding fragment that includes 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 to any one of SEQ ID NOs: 1-14.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NOs: 1-14 retains the CDR sequences of the corresponding reference antibody.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 1.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 1 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 15, 16 and 17.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 2.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 2 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 18, 19 and 20.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 3.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 3 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 21, 22 and 23.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 4.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 4 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 24, 25 and 26.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 5.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 5 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 27, 28 and 29.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 6.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 6 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 30, 31 and 32.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 7.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 7 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 33, 34 and 35.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 8.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 8 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 36, 37 and 38.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 9.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 9 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 39, 40 and 41.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 10.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 10 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 42, 43 and 44.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 11.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 11 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 45, 46 and 47.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 12.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 12 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 48, 49 and 50.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 13.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 13 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 51, 52 and 53.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 14.
  • the amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence to any one of SEQ ID NO: 14 retains the CDR sequences of the corresponding reference antibody, such as SEQ ID NO: 54, 55, and 56.
  • an antibody or antigen-binding fragment that includes the amino acid sequence of any one of SEQ ID NO: 1-14, optionally with 1, 2, 3, 4, or 5 amino acid additions, deletions and/or substitutions.
  • the substitutions are conservative substitutions.
  • the additions, deletions and/or substitutions are within the framework regions.
  • the antibody or fragment further includes constant domains, such as CH2 and/or CH3.
  • the CH2 and/or CH3 are from human IgG1, IgG2, IgG3 or IgG4 sequences.
  • substitutions are conservative substitutions.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalan
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.
  • the antibody or fragment is of class IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody or fragment is antibody-dependent cellular cytotoxicity (ADCC) -competent. In some embodiments, the antibody or fragment is not ADCC-competent.
  • ADCC antibody-dependent cellular cytotoxicity
  • these newly identified anti-Nectin-4 VHH antibodies are suitable for inclusion in a bispecific antibody.
  • Format B which is quadruple-valent with respect to Nectin-4, has strong binding to the cells. Further, when incubated with T cells (targeted by binding to CD3) and Nectin-4-expressing tumor cells, these bispecific antibodies exhibited strong T cell activation, and T cell mediated killing of the tumor cells.
  • a bispecific antibody that includes any of the VHH antibody of the present disclosure and a second antibody or antigen-binding fragment that binds another antigen.
  • the second antigen is a protein expressed on an immune cell.
  • Proteins on immune cells that can be targeted include, without limitation, CD3, CD47, PD1, PD-L1, 4-1BB, OX40, SIRPA, CD16, CD28, CTLA4, and CD27.
  • the immune cell surface protein is CD3.
  • a 4: 2 VHH: Fab format (Format B) has excellent therapeutic activity as compared to the other format tested.
  • the 4: 2 format is illustrated in FIG. 4B, which includes an antibody (e.g., anti-CD3) in the conventional Fab format, and four VHH unit specific to Nectin-4.
  • Each VHH is fused to the N-terminus of a variable region of the Fab, through 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) .
  • the length of the peptide linker has significant effect on the bispecific antibody’s activity in binding to the Nectin-4 on a cell surface. Therefore, in some embodiments, the peptide linker may have a length that is at least 2 amino acids, or at least 5, 7, 8, 9, 10, 12, 15, 17, 20, 22, or 25 amino acids. In some embodiments, the length is not longer than 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • Example linkers include multiple glycine (G) and serine (S) .
  • the linker includes at least 50%, 60%, 70% or 80% glycine.
  • Example linker sequences include GS (GGGGS) (SEQ ID NO: 57) , GS (GGGGS) 3 (SEQ ID NO: 58) , and GS (GGGGS) 6 (SEQ ID NO: 59) , without limitation.
  • the present disclosure provides a bispecific antibody having specificity to an immune cell (e.g., targeting CD3) and Nectin-4.
  • the bispecific antibody includes a conventional antibody specific to the human CD3 complex.
  • the bispecific antibody includes multiple (e.g., 2 and 4) VHH targeting Nectin-4.
  • a bispecific antibody comprising a first portion and a second portion, wherein the first portion comprises two pairs of VH and VL each capable of binding a human CD3 complex, the second portion comprises four single-domain antibody (VHH) fragments as disclosed herein, wherein each VHH fragment is fused, through a peptide linker, to the N-terminus of each of the VH and VL of the first portion.
  • VHH single-domain antibody
  • the bispecific antibody further includes constant domains, such as CH1 and CL, and CH2 and/or CH3.
  • the constant regions are from human IgG1, IgG2, IgG3 or IgG4 sequences.
  • antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.
  • the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below.
  • an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label) .
  • Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope.
  • the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.
  • the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • the antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • a therapeutic agent which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • the antibodies can 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 presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the antibodies can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure.
  • the polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.
  • both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human.
  • Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.
  • the prepared antibodies will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
  • antigen-binding polypeptides, variants, or derivatives thereof of the disclosure are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.
  • CDRs complementarity determining regions
  • De-immunization can also be used to decrease the immunogenicity of an antibody.
  • the term “de-immunization” includes alteration of an antibody to modify T-cell epitopes (see, e.g., International Application Publication Nos.: WO/9852976 A1 and WO/0034317 A2) .
  • variable heavy chain and variable light chain sequences from the starting antibody are analyzed and a human T-cell epitope “map” from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence is created.
  • CDRs complementarity-determining regions
  • T-cell epitopes from the T-cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody.
  • a range of alternative variable heavy and variable light sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides.
  • 12 and 24 variant antibodies are generated and tested for binding and/or function.
  • Complete heavy and light chain genes comprising modified variable and human constant regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody.
  • the antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
  • binding specificity of antigen-binding polypeptides of the present disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) .
  • in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) .
  • the antibodies, variants or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.
  • the present disclosure is further directed to antibody-based therapies which involve administering the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein.
  • Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein) .
  • the antibodies of the disclosure can also be used to treat or inhibit cancer.
  • Nectin-4 is overexpressed in tumor cells.
  • at least one of the cancer cells (e.g., stromal cells) in the patient expresses, over-express, or is induced to express the tumor antigen. Induction of a gene expression, for instance, can be done by administration of a tumor vaccine or radiotherapy.
  • Tumors that can be suitably treated include those of bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell cancer, Merkel cell carcinoma, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Accordingly, the presently disclosed antibodies can be used for treating any one or more such cancers.
  • Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) ) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease) , multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sar
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
  • the amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • Methods of administration of the antibodies, variants or 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, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents.
  • compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , bucally, or as an oral or nasal spray.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.
  • Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the antibodies polypeptides or compositions of the disclosure may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the protein does not absorb.
  • compositions comprise an effective amount of an antibody, and an acceptable carrier.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and 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 if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the disclosure can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • This example shows how anti-human-Nectin-4 single domain antibodies were generated using immunization of alpaca followed by phage library construction and selection.
  • Recombinant human Nectin-4/hFc fusion proteins were used as the immunogen to raise anti-human Nectin-4 antibodies.
  • Alpaca PBMCs were collected, and an antibody cDNA library was generated by RNA isolation and PCR amplification and cloning into a phage display vector. The libraries were then subjected for one round of liquid phase selection and one round of solid phase selection.
  • the binders were amplified from antigen positive phages by PCR and sequenced.
  • the expressed proteins were confirmed with SDS-PAGE (FIG. 1) . Sequences of the unique antibodies and their CDR regions are provided in the table below.
  • the antibodies were subjected to an ELISA-based binding assay.
  • the same concentration (0.5 ⁇ g/mL) of human Nectin-4 was coated on 96 well enzyme plate.
  • 2 ⁇ g/mL of each antibody and different concentration (0.2 ⁇ g/mL and 1 ⁇ g/mL) of Goat anti-human Nectin-4 antibody (as control) were added after blocking.
  • Goat anti-human IgG Fc cross adsorbed antibody or Rabbit anti-goat IgG antibody
  • HRP can react with substrate 3, 3’, 5, 5’-tetramethylbenzidine (TMB) to produce colored products.
  • the binding affinity of the CMB7 with 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 combined with antigen. Therefore, ELISA was used to detect the binding affinity of CMB7 to human Nectin-4.
  • TMB Substrate Solution
  • the ELISA testing results are shown in FIG. 2 and Table 3. As shown, all of the antibodies exhibited potent binding affinity to the human Nectin-4 protein.
  • This exampled measured the kinetic binding affinity of the 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.
  • the HFC (Anti-HIgG FC) Probes (Probelife) was pre-wetted in Kinetics Buffer (Probelife) for 5 mins at 30°C in Crimson 96 MAX 96-well reaction plate (ET Healthcare, 06-0098) . Probes were then dipped into wells containing the antibody at 4 ⁇ g/mL in Kinetics Buffer in the Greiner black microplate.
  • the analyte (Human Nectin-4) with different concentration gradient dilution (starting from 200 nM, with step-wise 2-fold dilutions, a total of 5 concentration gradients) were used a 5-min association step then followed by a 16-min dissociation step in Kinetics Buffer.
  • the data were analyzed by subtracting reference sample and fit to a 1: 1 K binding model for Structured Data Method of affinity constants using Data Analysis Software 1.7.2.0609 (Gator) .
  • the final slope should preferably not be higher than 0.02 nm/min.
  • the probes dissociate in K buffer for 16 mins, and the antigen dissociate from the probes in the column 8/9/10/11/12 at the Greiner 96-well polypropylene microplate.
  • the probes Run Regeneration procedure (The probes are regenerated for 5 s in R Buffer in the column 11 Crimson 96 MAX 96-well reaction plate, followed by neutralization for 5s in Q Buffer in the column 12 Crimson 96 MAX 96-well reaction plate, this process is repeated 3 times) .
  • This example tested the binding affinity of the antibodies to cynomolgus Nectin-4.
  • the binding affinity of the antibodies to cynomolgus Nectin-4 were detected by ELISA.
  • the same concentration (0.5 ⁇ g/mL) of cynomolgus Nectin-4 was coated on 96 well enzyme plate.
  • the antibodies with different concentration gradient dilution starting from 2 ⁇ g/mL, with step-wise 3-fold dilutions, a total of 7 concentration gradients) were added after blocking. After washing off the excess samples, Goat anti-human IgG Fc cross adsorbed antibody coupled with horseradish peroxidase (HRP) was added. HRP can react with substrate 3, 3’ , 5, 5’ -tetramethylbenzidine (TMB) to produce colored products.
  • HRP horseradish peroxidase
  • the binding affinity of the CMB7 with cynomolgus 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 combined with antigen. Therefore, ELISA was used to detect the binding affinity of CMB7 to cynomolgus Nectin-4.
  • TMB Substrate Solution
  • FIG. 3 shows that all of the tested antibodies cross-reacted to cynomolgus Nectin-4, and most of them exhibited potent binding affinity. This suggests that cynomolgus can be a suitable preclinical model for testing these antibodies.
  • This example detected binding affinity of the antibodies to human breast cancer cells expressing Nectin-4, by Fluorescence Activating Cell Sorter (FACS) .
  • FACS Fluorescence Activating Cell Sorter
  • the FACS results show that, at each test concentration, each antibody bound to the human breast cancer cell line MCF-7.
  • VHH single domain antibody
  • VHH 36 The single domain antibody (VHH 36” ) was used to construct bispecific antibodies that also target the human CD3. Two different formats of bispecific antibodies were used, as illustrated in FIG. 4.
  • FIG. 4A illustrates a format (Format A) in which a single VHH and a single VH/VL pair from an anti-CD3 antibody are fused to a Fc fragment.
  • Format A is asymmetric, therefore, and has a 1: 1 valent against CD3 and Nectin-4.
  • each of 4 VHH is fused to the N-terminus of a variant domain of a full anti-CD3 antibody.
  • This format is therefore is symmetric, and has a 2: 4 valent against CD3 and Nectin-4.
  • the bispecific configurations and each chain’s structure are shown in Tables 5-6.
  • This example tested the bispecific antibodies’ ability to activate T cells in presence of Nectin-4-expressing MCF-7 cells or T-47D cells.
  • FIG. 5 shows the T cell activation results for all bispecific antibodies.
  • Format A BJ182/12L1/BJ183-36
  • BJ192-36/BJ196-36 and BJ194-36/BJ198-36 exhibited dose-dependent strong activities.
  • the aim of this study was to detect the cytotoxicity of anti-Nectin-4 to MCF-7 and T-47D cells.
  • MCF-7 and T-47D cells were the target cells and primary human T cells were the Effector cell.
  • Primary human T cells were isolated from human PBMC cells and were frozen in liquid nitrogen.
  • Target cell and Effector cells were added in the ratio of 1: 2 (MCF-7 /T-47D cells were 3x10 4 and primary human T cells were 6x10 4 ) in each well of the 96 well plate with 100 nM of anti-Nectin-4. The images were scanned after 40 hours of co-incubation.
  • Digestion was terminated with growth medium, gently blow the cells to remove them completely. Centrifuge 300 g for 5 min. Remove the supernatant and add 1 ml medium to blow away. Transfer the vial contents to a 100 mm dish containing 10 mL of growth medium. Place the culture at 37 °C in 5% CO 2 .
  • T cells (about 6*10 4 cells per well) at 6*10 5 /mL in test medium.
  • the antibody should be diluted to 100 nM as the sample concentration.
  • test medium 2 times Wash the target cells with test medium 2 times. Add 80 ⁇ L test medium to each well. Add 20 ⁇ L antibody solution to the plate. Add 100 ⁇ L T cell suspension (about 6*10 ⁇ 4 cells per well) to the plate.
  • FIG. 6 MCF-7 cells
  • FIG. 7 shows the T cell killing results for all bispecific antibodies. Larger and dark particles indicate target cell death. Consistent with the T cell activation results in Example 7, treatment with any antibody of Format A did not result in T cell killing, and treatment with most of the Format B bispecific antibodies caused cell death of the target cells (MCF-7 or T-47D cells) , demonstrating the efficacy of these antibodies.

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Abstract

L'invention concerne des anticorps à domaine unique dirigés contre la protéine nectine-4 humaine qui ont une excellente affinité de liaison. Ces anticorps sont particulièrement appropriés pour être inclus dans un anticorps bispécifique, tel qu'un anticorps bispécifique qui cible également un antigène sur une cellule immunitaire.
PCT/CN2023/087399 2022-04-11 2023-04-10 Anticorps anti-nectine-4 à domaine unique WO2023198011A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180243434A1 (en) * 2015-09-09 2018-08-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity to nectin-4 and uses thereof
CN110392697A (zh) * 2017-03-02 2019-10-29 国家医疗保健研究所 对nectin-4具有特异性的抗体及其用途
CN111675761A (zh) * 2017-06-05 2020-09-18 艾更斯司股份有限公司 柄蛋白-4结合蛋白及其使用方法
CN112088167A (zh) * 2018-05-09 2020-12-15 耶路撒冷希伯来大学伊森姆研究发展有限公司 特异性针对人类连接蛋白4的抗体
WO2021069508A1 (fr) * 2019-10-07 2021-04-15 Université D'aix-Marseille Anticorps ayant une spécificité pour la nectine -4 et leurs utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180243434A1 (en) * 2015-09-09 2018-08-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibodies having specificity to nectin-4 and uses thereof
CN110392697A (zh) * 2017-03-02 2019-10-29 国家医疗保健研究所 对nectin-4具有特异性的抗体及其用途
CN111675761A (zh) * 2017-06-05 2020-09-18 艾更斯司股份有限公司 柄蛋白-4结合蛋白及其使用方法
CN112088167A (zh) * 2018-05-09 2020-12-15 耶路撒冷希伯来大学伊森姆研究发展有限公司 特异性针对人类连接蛋白4的抗体
WO2021069508A1 (fr) * 2019-10-07 2021-04-15 Université D'aix-Marseille Anticorps ayant une spécificité pour la nectine -4 et leurs utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PIA M CHALLITA-EID: "Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models", CANCER RES., vol. 76, no. 10, 15 May 2016 (2016-05-15), XP055385700, DOI: 10.1158/0008-5472.CAN-15-1313 *
WANG ZHE: "Research Progress on the Application of Nectin-4 Target in Tumor Therapy", CHINESE AND FOREIGN MEDICAL RESEARCH, vol. 18, no. 36, 25 December 2020 (2020-12-25), XP093098562, DOI: 10.14033/j.cnki.cfmr.2020.36.073 *
WONG JEFFREY L., ROSENBERG JONATHAN E.: "Targeting nectin-4 by antibody-drug conjugates for the treatment of urothelial carcinoma", EXPERT OPINION ON BIOLOGICAL THERAPY, INFORMA HEALTHCARE, vol. 21, no. 7, 3 July 2021 (2021-07-03), pages 863 - 873, XP093099420, ISSN: 1471-2598, DOI: 10.1080/14712598.2021.1929168 *

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