WO2023199068A1 - Liants à base de mésothéline - Google Patents

Liants à base de mésothéline Download PDF

Info

Publication number
WO2023199068A1
WO2023199068A1 PCT/GB2023/050997 GB2023050997W WO2023199068A1 WO 2023199068 A1 WO2023199068 A1 WO 2023199068A1 GB 2023050997 W GB2023050997 W GB 2023050997W WO 2023199068 A1 WO2023199068 A1 WO 2023199068A1
Authority
WO
WIPO (PCT)
Prior art keywords
msln
cells
antibody
domain antibody
single domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2023/050997
Other languages
English (en)
Inventor
Colette JOHNSTON
Phil HAYES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Crescendo Biologics Ltd
Original Assignee
Crescendo Biologics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Crescendo Biologics Ltd filed Critical Crescendo Biologics Ltd
Publication of WO2023199068A1 publication Critical patent/WO2023199068A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/001168—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4274—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • A61K40/4276—Prostate specific membrane antigen [PSMA]
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/35—Valency
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to an isolated nucleic acid encoding a VH single domain antibody as described herein.
  • FIG. 1 Human antibody VH domain-based CAR targeting PSMA is expressed and signals in T cells.
  • A Schematic diagram of J591 and PSMA-VH constructs.
  • B, C Representative flow cytometry plots (B) and summary (C) illustrating J591 and PSMA-VH expression in T cells.
  • the CD19-specific CAR (CD19) and non-transduced T cells (NT) were used as positive and negative controls, respectively.
  • T cells expressing the human antibody VH domain-based CAR targeting PSMA are functional in vitro.
  • A Representative flow cytometry plots showing the expression of PSMA in C4-2, PC3 and PC3 cells engineered with a retroviral vector to express PSMA.
  • VH or “variable domain” refers to immunoglobulin variable domains defined by Kabat et al., as referenced above.
  • a VH domain is the smallest antigen binding fragment.
  • antibody broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.
  • mesothelin High mRNA expression of mesothelin is found in mesothelioma, lung, ovarian, breast and pancreatic adenocarcinomas.
  • Mesothelin over-expression has also been noted in some other human cancers, including squamous cell carcinomas of different sites such as cervix, lung and head and neck carcinomas, endometrial adenocarcinomas, colorectal, gastric, and esophageal cancers (Morello et al Mesothelin-Targeted cars: driving T cells to solid tumors.
  • Isoform 2 The sequence of this isoform differs from the canonical sequence as follows: Residues 409-416: Missing.
  • Isoform 3 Also known as: SMRP.
  • the sequence of this isoform differs from the canonical sequence as follows: Residues 409-416: Missing. 601-630:
  • Isoform 4 The sequence of this isoform differs from the canonical sequence as follows: Residues 44-44: Missing. Residues 409-416: Missing.
  • MSLN binding molecule/protein/polypeptide/agent/moiety refers to a molecule capable of specifically binding to the human MSLN antigen.
  • the binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity. Binding is to human MSLN unless otherwise defined.
  • human MSLN is one that binds the antigen with sufficient affinity such that the CAR with the single domain antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen MSLN as described herein. Binding is to the extracellular domain of MSLN.
  • the VH single domain antibody comprises or consists of SEQ ID NO. 3 or a variant thereof. In one embodiment, the VH single domain antibody may be a variant of SEQ ID NO. 3 having one or more amino acid substitutions, deletions, insertions or other modifications.
  • the variant (VH1.2) has a substitution of S to N in CDR3 and the CDR3 sequence is: SEQ ID NO. 7: YNTSSETAFDI
  • Amino acid substitutions in variants as described herein can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Substitutions, insertions, additions or deletions in the framework region may optionally be in the range of about 1 to 25 or 1 to 50, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20 amino acids, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • the modification is a conservative sequence modification.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones 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.
  • amino acids with 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, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • amino acid residues within the CDR regions of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e. antigen binding) using the functional assays described herein.
  • these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen.
  • single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity.
  • substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity.
  • Table 1 Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 1 below.
  • modifications can be made to decrease the immunogenicity of the single domain antibody.
  • one approach is to revert one or more framework residues to the corresponding human germline sequence.
  • a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived. In one embodiment, all framework sequences are germline sequence.
  • the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.
  • glycosylation is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for the antigen.
  • the one or more substitution is in the CDR1 , 2 or 3 region provided that the CDRs as follows: there may be 1 , 2, 3 or more amino acid substitutions in the CDR1 , 2 or 3. In another example, there may be 1 , 2, 3 amino acid deletions or addition.
  • the one or more substitution, addition or deletion is in the framework region.
  • Variants can also be defined by reference to sequence identity.
  • Sequence identity as defined herein can be at least 40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
  • a variant of SEQ ID NO. 3 has at least 75%, 80%, 90% or 95% sequence identity to SEQ ID NO. 3 provided that the CDRs are as defined above.
  • one or more non-germline residue in SEQ ID NO. 3 is replaced with a germline residue.
  • the residue at position 1 , 34, 60, 65, 70 and/or 103 is replaced with the germline residue as shown below. In one embodiment, all of these residues are replaced with the germline residue.
  • sequence “homology” or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity.
  • percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known.
  • the percent identity between two amino acid sequences can be determined using well known mathematical algorithms.
  • Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, for example with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST, FASTA, the Smith-Waterman algorithm, or the TBLASTN program. In particular, the psi-Blast algorithm may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments can be performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g, antibody or antigen -binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).
  • Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA).
  • KD is calculated from the quotient of koff/kon
  • KA is calculated from the quotient of kon/koff.
  • Kon refers to the association rate constant of, e.g, an antibody or antigenbinding fragment thereof to an antigen
  • koff refers to the dissociation of, e.g, an antibody or antigen-binding fragment thereof from an antigen.
  • the association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art.
  • the kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.
  • binding or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or lower.
  • the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • VH affinity for MSLN as a single VH (monomer) recombinant protein is in the nanomolar range, e.g. 20 to 40 nM, e.g. 28-34nM. Measurement may be by Biacore.
  • antigen(s) and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor.
  • the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components.
  • Epitopes can be recognized by antibodies in solution, e.g. free from other molecules.
  • Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.
  • epitopes or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes.
  • Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • epitope mapping are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment.
  • Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. The degree of competition can be expressed as a percentage of the reduction in binding.
  • Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance), HTRF; flow cytometry; fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.
  • a real time, label-free bio-layer interferometry assay e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance), HTRF; flow cytometry; fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassay
  • the VH single domain antibody as described above has one or more of the following properties:
  • (c) binds human MSLN with a ka of about 1.15-1.6 x 10 6 , a kd of 3.93-4.49 x 10' 2 and a KD of about 2.8-3.4 x 10' 8 (28-34 nM) as measured by BiaCore;
  • (e) has an EC50 of about 1 .2 nM as measured using an FMAT assay and/or
  • (f) is 96.7% monomer after incubation at 4°C for about 17 hours and 98.1 % monomer after incubation at 40°C for about 17 hours.
  • the VH single domain antibody as described herein binds human MSLN in the nanomolar range, e.g. 20 to 40 nM, e.g 28-34 nM as measured by BiaCore.
  • Such binding affinity can be particularly useful in applications such as chimeric antigen receptors it enables specific binding to MSLN expressed on the surface of cells and subsequent activation of the CAR-T cells.
  • the VH single domain antibody as described herein also binds cyno MSLN. This is advantageous because of the utility of cynomolgous macaques as the non-human primate species of choice for IND enabling activities.
  • a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN as described herein and at least a second moiety that binds to a second antigen, for example a tumor specific antigen.
  • the terms binding agent and binding molecule are used interchangeably herein to refer to such multispecific molecule.
  • the binding molecule may be a fusion protein.
  • the first target and the second target are not the same, i.e. are different targets, e.g., proteins; both may be present on a cell surface.
  • a multispecific, e.g. bispecific binding molecule as described herein can selectively and specifically bind to a cell that expresses (or displays on its cell surface) the first target MSLN and the second target.
  • a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target binding sites respectively.
  • the at least second moiety is a binding molecule that binds to a target of interest, for example selected from an antibody or antibody fragment (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or single domain antibody, for example a VH or VHH domain) or antibody mimetic protein.
  • a target of interest for example selected from an antibody or antibody fragment (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or single domain antibody, for example a VH or VHH domain) or antibody mimetic protein.
  • the single domain antibody of the invention can be linked to an antibody Fc region or fragment thereof, comprising one or both of CH2 and CH3 domains, and optionally a hinge region.
  • the at least second moiety is a VH domain.
  • the binding molecule is bispecific.
  • the invention relates to a bispecific molecule comprising a single domain antibody described herein linked to a second functional moiety having a different binding specificity than said single domain antibody.
  • the bispecific binding molecule has the following formula: VH (A)- L-VH (B) wherein A or B is MSLN.
  • V H (A) is conjugated to V H (B), i.e. linked to VH (B), for example with a peptide linker.
  • L denotes a linker, for example a polypeptide linker.
  • Each VH comprises CDR and FR regions.
  • the binding molecule may have the following formula: FR1 (A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)-L-FR1 (B)-CDR1(B)- FR2(B)-CDR2(BA)-FR3(B)-CDR3(B)-FR4(B).
  • the order of the single V H domains A and B is not particularly limited, so that, within a polypeptide of the invention, single variable domain A may be located N-terminally and single variable domain B may be located C-terminally, or vice versa wherein A or B is MSLN.
  • peptide linker refers to a peptide comprising one or more amino acids.
  • a peptide linker comprises 1 to 44 amino acids, more particularly 2 to 20 amino acids.
  • Peptide linkers are known in the art or are described herein.
  • Suitable, non-immunogenic linker peptides are, for example, linkers that include G and/or S residues, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n” is generally a number between 1 and 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the peptide is for example selected from GGGGS (SEQ ID NO: 8), GGGGSGGGGS (SEQ ID NO: 9), SGGGGSGGGG (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GSGSGS (SEQ ID NO: 12), GGSGSGSG (SEQ ID NO: 13), GGSGSG (SEQ ID NO: 14), GGSG (SEQ ID NO: 15) and GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16).
  • the one or more single VH domain antibody "binds" or is “capable of binding” an antigen of interest, i.e. targets, antigen with sufficient affinity useful in therapy in targeting a cell or tissue expressing the antigen.
  • the term "target” refers to a biological molecule (e.g., antigen, peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain which has a binding site can selectively bind.
  • the target can be, for example, an intracellular target (such as an intracellular protein target) or a cell-surface target (such as a membrane protein, e.g., a receptor protein).
  • a target is a cell-surface target, such as a cell-surface protein.
  • the target is a tumor specific antigen.
  • the target antigen as used herein may be selected from a list including, but not limited to PSMA, Her2, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF CECAM6, CAXII, CD24, CEA, cMet, TAG72, MUC1 , MUC16, STEAP, Ephvlll, FAP, GD2, IL- 13Ra2, L1-CAM, PSCA, GPC3, Her3, gpA33, 5T4 and ROR1 , CD3, CDE28, CD27, CD40, GITTA, 0X40, CD80, CD86, ICOS.
  • PSMA Her2, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF CECAM6, CAXII, CD24, CEA, cMet, TAG72, MUC1 , MUC16, STEAP, Ephvlll, FAP
  • the binding molecule binds MSLN and PSMA.
  • the antigen binding domain includes a VH single domain antibody that binds MSLN as described herein and a VH single domain antibody that binds specifically PSMA.
  • binding to PSMA is to wild type human PSMA (accession NO. Q04609).
  • the sequence for the wild type human PSMA monomer is shown below (SEQ ID NO. 17).
  • the antigen binding domain includes a VH single domain antibody that bind PSMA which with the following sequence or a variant thereof.
  • SEQ ID NO. 18 (termed 2.1) full length sequence, CDRs underlined EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ GTMVTVSS
  • VH single domain antibodies that bind PSMA are described in WO 2017/122017, W02019/012260 and WO2017/191476, both incorporated herein by reference.
  • the variant may have one or more amino acid modification, i.e. a substitution, deletion, addition or addition.
  • the VH single domain antibody comprises a CDR1 comprising SEQ NO. 19 or a sequence with 1 , 2 or 3 amino acid modification, a CDR2 comprising SEQ NO. 20 or a sequence with 1 , 2 or 3 amino acid modification and a CDR3 comprising SEQ NO. 21 or a sequence with 1 , 2 or 3 amino acid modification.
  • the one or more modification is in the CDR1 , 2 or 3 region.
  • there may be 1 , 2, 3 or more amino acid modification is in the CDR1 , 2 or 3.
  • the one or more modification is in the framework region.
  • the VH single domain antibody that binds PSMA has at least 60%, 70%, 80% or 90% homology thereto, for example 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO. 18.
  • said sequence homology or identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the VH single domain antibody that binds PSMA is selected from one of the VH single domain antibody as shown in Table 2 shown below.
  • Table 2b Family 2 PSMA binders Note that the PSMA binder identified in SEQ ID 18 belongs to this family.
  • a linker such as polypeptide linker (e.g. (G4S)n) may be used to link the VH single domain antibody that binds MSLN with the VH single domain antibody that binds PSMA.
  • G4S polypeptide linker
  • the affinity of bispecific antigen binding domain to huPSMA may be about 100 to about 250pm, for example 116-213pM.
  • the affinity of the bispecific antigen binding domain to MSLN may be about 16 to 49nM.
  • the single domain antibody or binding agent described above comprises a further moiety to prolong the half-life of the binding molecule.
  • the further moiety may comprise a protein, for example an antibody, or part thereof that binds a serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA).
  • the further moiety may comprise a VH domain that binds serum albumin, e.g., human serum albumin (HSA) ora variant thereof such as HSA C34S or mouse serum albumin (MSA).
  • Increased half life can also be conferred by conjugating the molecule to an antibody fragment, for example a VH domain that increases half life as disclosed in W02020/099871 or WO2020/229842).
  • half-life refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.
  • Half-life may be increased by at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding VH single domain antibodies of the invention.
  • increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding VH single domain antibodies or fusion protein of the invention.
  • the in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Half life can for example be expressed using parameters such as the t1/2-alpha t1/2-beta and the area under the curve (AUG).
  • the anti-MSLN single domain antibody or multivalent binding molecule is labelled with a detectable or functional label.
  • a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.
  • the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
  • the anti-MSLN single VH domain antibody or multivalent binding molecule is coupled to at least one therapeutic moiety, such as a drug, an enzyme or a toxin.
  • the anti-MSLN single domain antibody or multivalent binding molecule is modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome.
  • linker for example a polypeptide linker.
  • a VH single domain antibody as described herein is generated from human heavy chain only antibody produced in a transgenic rodent that expresses human heavy chain loci.
  • One aspect also relates to a method for producing a human heavy chain only antibodies capable of binding human MSLN said method comprising a) immunising a transgenic rodent, e.g. mouse with an MSLN antigen wherein said rodent expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains, b) isolating human heavy chain only antibodies.
  • Further steps can include isolating a VH domain from said heavy chain only antibody, for example by generating a library of sequences comprising VH domain sequences from said rodent, e.g. mouse and isolating sequences comprising VH domain sequences from said libraries.
  • Another aspect also relates to a method for producing a single VH domain antibody capable of binding human MSLN said method comprising a) immunising a transgenic rodent with an MSLN antigen wherein said rodent, e.g. mouse, expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains, b) generating a library of sequences comprising VH domain sequences from said rodent, e.g. mouse and c) isolating sequences comprising VH domain sequences from said libraries.
  • Further steps may include identifying a single VH domain antibody or heavy chain only antibody that binds to human MSLN, for example by using functional assays as shown in the examples.
  • Methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries can comprise the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; and b) screening said set, collection or library for amino acid sequences that can bind to I have affinity for MSLN and c) isolating the amino acid sequence(s) that can bind to I have affinity for MSLN.
  • the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
  • suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art (see for example Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (October 28, 1996) Brian K. Kay, Jill Winter, John McCafferty).
  • Libraries for example phage libraries, are generated by isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody, cloning the sequence encoding the VH domain(s) from mRNA derived from the isolated cell or tissue and displaying the encoded protein using a library.
  • the VH domain(s) can be expressed in bacterial, yeast or other expression systems.
  • Another aspect also relates to an isolated VH single domain antibody or an isolated heavy chain only antibody comprising a VH domain binding to MSLN comprising an amino acid product of or derived from a human VH germline sequence.
  • the heavy chain only antibody may be fully human or comprise mouse sequences.
  • the transgenic rodent for example a mouse or rat, may have a reduced capacity to express endogenous antibody genes.
  • the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes.
  • the rodent may therefore comprise modifications to disrupt expression of endogenous light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced.
  • the rodent is a mouse.
  • the mouse may comprise a non-functional endogenous lambda light chain locus.
  • the mouse does not make a functional endogenous lambda light chain.
  • the lambda light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing.
  • at least the constant region genes C1 , C2 and C3 may be deleted or rendered non-functional through insertion or other modification as described above.
  • the locus is functionally silenced so that the mouse does not make a functional lambda light chain.
  • the mouse may comprise a non-functional endogenous kappa light chain locus.
  • the mouse does not make a functional endogenous kappa light chain.
  • the kappa light chain locus is deleted in part or completely or rendered non- functional through insertion, inversion, a recombination event, gene editing or gene silencing.
  • the locus is functionally silenced so that the mouse does not make a functional kappa light chain.
  • the mouse having functionally silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO 2003/000737, which is hereby incorporated by reference in its entirety.
  • the mouse may comprise a non-functional endogenous heavy chain locus.
  • the heavy chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing.
  • the locus is functionally silenced so that the mouse does not make a functional heavy chain.
  • all 8 endogenous heavy chain constant region immunoglobulin genes are absent in the mouse, or partially absent to the extent that they are nonfunctional, or genes 5, y3, y1 , y2a, y2b and s are absent and the flanking genes p and a are partially absent to the extent that they are rendered non-functional, or genes p, 5, y3, y1 , y2a, y2b and s are absent and a is partially absent to the extent that it is rendered non-functional, or 5, y3, y1 , y2a, y2b, s and a are absent and p is partially absent to the extent that it is rendered non-functional.
  • deletion in part is meant that the endogenous locus gene sequence has been deleted or disrupted, for example by an insertion, to the extent that no functional endogenous gene product is encoded by the locus, i.e., that no functional product is expressed from the locus.
  • the locus is functionally silenced.
  • the mouse comprises a non-functional endogenous heavy chain locus, a nonfunctional endogenous lambda light chain locus and a non-functional endogenous kappa light chain locus.
  • the mouse therefore does not produce any functional endogenous light or heavy chains.
  • the mouse is a triple knockout (TKO) mouse.
  • the transgenic mouse may comprise a vector, for example a Yeast Artificial Chromosome (YAC) for expressing a heterologous heavy chain locus.
  • YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast.
  • ARS autonomously replicating sequence
  • CEN centromere
  • TEL telomere
  • the YAC may comprise multiple human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions.
  • Transgenic mice can be created according to standard techniques as illustrated in the examples. The two most characterised routes for creating transgenic mice are via pronuclear microinjection of genetic material into freshly fertilised oocytes or via the introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos. Regardless of how the genetic material is introduced, the manipulated embryos are transferred to pseudopregnant female recipients where pregnancy continues and candidate transgenic pups are born.
  • ES clones can be screened extensively before their use to create a transgenic animal.
  • pronuclear microinjection relies on the genetic material integrating to the host genome after its introduction and, generally speaking, the successful incorporation of the transgene cannot be confirmed until after pups are born.
  • the invention also relates to an anti-human MSLN single VH domain antibody or an anti-human MSLN heavy chain only antibody comprising a human VH domain or obtained or obtainable from a rodent, preferably a mouse, immunised with a human MSLN antigen and which expresses a human heavy chain locus.
  • a rodent preferably a mouse
  • said rodent is not capable of making functional endogenous kappa and lambda light and/or heavy chains.
  • the human heavy chain locus is located on a transgene which can be as described above.
  • SEQ ID NO. 427 (encodes VH domain 1.20) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
  • SEQ ID NO. 429 (encodes VH domain 2.4) CAGGTCACCTTGAAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAAA
  • SEQ ID NO. 438 (encodes VH domain 2.13)
  • SEQ ID NO. 444 (encodes VH domain 2.19) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 447 (encodes VH domain 2.22) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 448 (encodes VH domain 2.23)
  • SEQ ID NO. 450 (encodes VH domain 2.25) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 451 (encodes VH domain 3.1) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 458 (encodes VH domain 3.8)
  • SEQ ID NO. 463 (encodes VH domain 3.13)
  • SEQ ID NO. 466 (encodes VH domain 3.16)
  • SEQ ID NO. 469 (encodes VH domain 3.19) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 472 (encodes VH domain 3.22) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 473 (encodes VH domain 3.23)
  • SEQ ID NO. 475 (encodes VH domain 4.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCTTGAGA
  • SEQ ID NO. 478 (encodes VH domain 4.4)
  • SEQ ID NO. 479 (encodes VH domain 5.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
  • SEQ ID NO. 487 (encodes VH domain 6.7) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA.
  • RNA includes in vitro transcribed RNA, synthetic RNA or mRNA sequence.
  • the nucleic acid construct may further comprise a suicide gene.
  • the construct may be in the form of a plasmid, vector, transcription or expression cassette.
  • the invention in another aspect, relates to an isolated nucleic acid construct comprising a nucleic acid as defined above.
  • the construct may be in the form of a plasmid, vector, transcription or expression cassette.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the vectors can be suitable for replication and integration eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013).
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • retroviruses such as adenovirus vectors can be used.
  • a lentivirus vector is used.
  • the invention also relates to an isolated cell or cell population comprising one or more nucleic acid construct or vector as described above.
  • the cell is an isolated recombinant host cell comprising one or more nucleic acid construct as described above.
  • the host cell may be a bacterial, viral, plant, mammalian or other suitable host cell. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, H EK-293 cells and a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.
  • Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae,
  • Pichia thermotolerans Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella.
  • compositions comprising single domain antibody or binding molecule according to the present invention and optionally a pharmaceutically acceptable carrier.
  • the genetically modified cells or pharmaceutical composition of the present invention can be administered by any convenient route, including parenteral administration.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration.
  • Compositions can take the form of one or more dosage units.
  • the composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension.
  • the liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously.
  • the liquid compositions of the invention can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin,
  • a composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.
  • the amount of the pharmaceutical composition of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • compositions of the invention comprise an effective amount of a binding molecule of the present invention such that a suitable dosage will be obtained.
  • the correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.
  • this amount is at least about 0.01 % of a binding molecule of the present invention by weight of the composition.
  • compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present invention.
  • the composition can comprise from typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.
  • compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • compositions can be prepared using methodology well known in the pharmaceutical art.
  • a composition intended to be administered by injection can be prepared by combining a binding molecule of the present invention with water so as to form a solution.
  • a surfactant can be added to facilitate the formation of a homogeneous solution or suspension.
  • composition of the invention can be co-administered with other therapeutics, for example anti-cancer agents.
  • other therapeutics for example anti-cancer agents.
  • Exemplary combinations with other agents for example anti-cancer agents.
  • a therapeutic agent is a compound or molecule which is useful in the treatment of a disease.
  • therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes.
  • An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a VH domain, or antibody mimetic protein.
  • the single variable heavy chain domain antibody that binds to MSLN, a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN or pharmaceutical composition described herein is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy.
  • the invention also relates to a combination therapy comprising administration of a single variable heavy chain domain antibody that binds to MSLN, a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN or pharmaceutical composition described herein and an anti-cancer therapy.
  • the anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs.
  • therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g.
  • modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens including EGFR antagonists
  • an anti-inflammatory agent including a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy.
  • the single domain antibody is used in combination with surgery.
  • the single variable heavy chain domain antibody that binds to MSLN, a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN or a pharmaceutical composition described herein is administered concurrently with a chemotherapeutic agent or with radiation therapy.
  • the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.
  • the single variable heavy chain domain antibody that binds to MSLN, a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN or pharmaceutical composition described herein may be administered with two or more therapeutic agents.
  • the binding agents of the invention may be administered with two or more therapeutic agents.
  • the single variable heavy chain domain antibody that binds to MSLN, a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN or a pharmaceutical composition as described herein may be administered at the same time or at a different time as the other therapy or therapeutic compound or therapy, e.g., simultaneously, separately or sequentially.
  • MSLN is expressed on the surface of tumour cells and high expression levels of soluble MSLN have been correlated with poor prognosis in several cancers.
  • Anti- MSLN antibodies have been investigated as anti-cancer therapeutics. These anti-MSLN antibodies either induce direct cell killing through their ADCC activity or are used in the form of ADCs. The molecules and cells described herein are therefore expected to find application in the treatment of disease, in particular cancer.
  • the disease is a disease associated with expression of mesothelin.
  • the molecules of the invention may preferentially bind to MSLN present on the surface of a cancer cell as compared to soluble MSLN.
  • the cancer to be treated using an antibody molecule of the invention therefore preferably expresses, or has been determined to express, MSLN. More preferably, cells of the cancer to be treated comprise, or have been determined to comprise, MSLN at their cell surface, i.e. to comprise cell-surface bound MSLN.
  • Methods for determining the presence of an antigen on a cell surface are known in the art and include, for example, flow cytometry.
  • the disease is cancer and the invention thus relates to methods for the prevention and/or treatment of cancer, comprising administering to a subject a cell or cell population comprising a single domain antibody as described herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a cell and/or of a pharmaceutical composition of the invention.
  • the invention also relates to a single domain antibody as described herein for use in therapy.
  • the invention also relates to a single domain antibody as described herein for use in the treatment of cancer.
  • the invention also relates to the use of a single domain antibody as described herein in the manufacture of a medicament for the treatment of cancer.
  • cancer refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of the histopathologic type or stage of invasiveness. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
  • the cancer may be a primary or a secondary cancer.
  • an antibody molecule as described herein may be for use in a method of treating cancer in an individual, wherein the cancer is a primary tumour and/or a tumour metastasis.
  • the cancer to be treated using an antibody molecule of the invention may be a solid cancer.
  • various cancers include, but are not limited to, mesothelioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • disease associated with expression of mesothelin includes, but is not limited to, a disease associated with expression of mesothelin or condition associated with cells which express mesothelin including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a mesothelial hyperplasia; or a noncancer related indication associated with cells which express mesothelin.
  • proliferative diseases such as a cancer or malignancy or a precancerous condition such as a mesothelial hyperplasia
  • a noncancer related indication associated with cells which express mesothelin include but are not limited to, mesothelioma, lung cancer, ovarian cancer, pancreatic cancer, and the like.
  • the cancer is selected from a haematological cancer or malignancy or a solid tumor.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas.
  • the cancer is metastatic.
  • Cancers that may be treated by methods, uses and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the therapy can be used in combination with existing therapies.
  • the binding agent is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy.
  • the invention also relates to a combination therapy comprising administration of the binding agent or a pharmaceutical composition of the invention and an anti-cancer therapy.
  • the anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs.
  • therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g.
  • the binding agent or pharmaceutical composition of the invention is used in combination with surgery.
  • the binding agent or pharmaceutical composition of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.
  • an immune checkpoint inhibitor is also administered with the cell or cell population or pharmaceutical composition.
  • the immune checkpoint inhibitor may be an anti- PD1 , anti PDL-1 , anti PDL-2, anti CTL-4, anti-TIM-3 or anti LAG-3 antibody.
  • the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, avelumab, durvalumab, or atezolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Pidilizumab, Toripalimab, Ipilimumab or Tremelimumab.
  • the immune checkpoint inhibitor is an interfering nucleic acid molecule, a small molecule or a PROteolysis TArgeting Chimera (PROTAC).
  • the immune checkpoint inhibitor is administered before, after or at the same time as the cell or cell population.
  • an immunoconjugate comprising a single variable heavy chain domain antibody that binds to MSLN or a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN described herein conjugated to at least one therapeutic and/or diagnostic agent.
  • the invention also relates to the use of a single variable heavy chain domain antibody that binds to MSLN or a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN described herein for use a diagnostic agent.
  • the invention also relates to the use of a single variable heavy chain domain antibody that binds to MSLN or a binding molecule comprising a single variable heavy chain domain antibody that binds to MSLN described herein conjugated to a label.
  • the invention provides a kit for detecting cancer, treatment, prognosis or monitoring comprising a genetically modified cell or pharmaceutical composition of the invention.
  • the kit may also comprise instructions for use.
  • the single domain antibody or pharmaceutical composition comprises a label and one or more compounds for detecting the label.
  • the invention in another aspect provides a binding molecule of the invention packaged in lyophilized form, or packaged in an aqueous medium.
  • a single variable heavy chain domain antibody that binds to MSLN described herein is used for non-therapeutic purposes, such as diagnostic tests and assays.
  • a method for detecting the presence of human MSLN in a test sample comprises contacting said sample with a single domain antibody described herein and at least one detectable label and detecting binding of said single domain antibody to human MSLN.
  • Modifications of antibodies for diagnostic purposes are well known in the art.
  • antibodies may be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme.
  • Compounds of the invention can be used for diagnostic purposes and e.g. labelled using conventional techniques. Suitable detectable labels include but are not limited to fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners.
  • mice carrying a heavy-chain antibody transgenic locus in germline configuration within a background that is silenced for endogenous heavy and light chain antibody expression were created as previously described (W02004/076618 and W02003/000737, Ren et al., Genomics, 84, 686, 2004; Zou et al., J. Immunol., 170, 1354, 2003, Teng et al 30 all incorporated herein by reference).
  • transgenic mice were derived following pronuclear microinjection of freshly fertilised oocytes with a yeast artificial chromosome (YAC) comprising multiple human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions.
  • yeast artificial chromosomes YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast.
  • YACs As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells.
  • ARS autonomously replicating sequence
  • CEN centromere
  • TEL telomeres
  • the YAC used comprised multiple human heavy chain V genes, human heavy chain D and J genes. It lacks the CH1 exon.
  • the transgenic founder mice were back crossed with animals that lacked endogenous immunoglobulin expression to create the Tg/TKO lines used for immunisation with recombinant MSLN antigen.
  • MSLN is synthesized as a 71-kD precursor protein, then cleaved by the endoprotease furin into:
  • MPF megakaryocyte potentiating factor
  • mice Eight Tg/TKO mice aged 8-12 weeks of age at the initiation of immunisation each received eight weekly doses of MSLN plasmid DNA delivered via Genegun.
  • sera from immunised animals were tested by flow cytometry using mammalian cells transiently transfected with the target cDNA cloned into an Aldevron proprietary expression vector containing a N-terminal tag-sequence.
  • a negative control non-transfected cells were used that does not express the antigen of interest. Reactivity of the immune sera against cells transiently transfected with the test construct could be detected in the immunized animals when compared to negative control cells.
  • terminal bleeds were collected from all animals, processed to serum and assayed for the presence of heavy-chain antibody responses to the immunogen by ELISA.
  • Multiwell plates were coated with a His-tagged human or cyno mesothelin recombinant protein then washed with PBS.
  • Non-specific protein interactions were blocked with 3% (w/v) skimmed milk powder (Marvel®) in PBS.
  • Dilutions of serum in 3% Marvel TM/PBS were incubated for one hour at room temperature then transferred to the blocked ELISA plate for at least one hour. Unbound protein was removed by repetitive washes with PBS/Tween20 followed by PBS.
  • Biotin-conjugated, goat anti-mouse IgG, Fcgamma subclass 1 specific antibody prepared in PBS/3% Marvel was added to each well and incubated at room temperature for one hour, then washed as above.
  • Neutravidin-HRP solution in 3% Marvel/PBS was added to the ELISA plates for 30 minutes, then washed as above and developed using TMB substrate. The reaction was stopped after 10 minutes by the addition of 0.5M sulphuric acid solution. Absorbances were determined by reading at an optical density of 450nm.
  • EXAMPLE 4 Generation of Libraries from Immunised Mice a) processing tissues, RNA extraction and cDNA synthesis
  • RNAIater® Inguinal and axilliary lymph nodes and spleens were collected from each immunised animal into RNAIater®.
  • tissues were removed from the RNALater and placed in a microtube with a stainless steel bead and Qiazol Lysis Reagent.
  • Tissues were lysed and homogenised by physical disruption via shaking for 3 mins at 1600 rpm in a MPBio FastPrep96 homogeniser. The lysate was cleared by centrifugation, chloroform added, mixed by shaking, then separated into phases by chilled centrifugation at 4700rpm for 20 minutes. The aqueous phase was collected in a semi-automated way using the QIAcube robot.
  • RNA was prepared using RNeasy 96 QIAcube kit and QIAcube HT plastic ware, based on the manufacturer’s protocol with minor modifications.
  • RNA quality was assessed using the QIAxcel electrophoresis automated DNA and RNA analysis system, running the RNA alongside the QX RNA 15nt alignment marker. RNA extracted from all spleen and lymph node tissues was found to be of high quality. cDNA was synthesised using Superscript III RT-PCR high-fidelity kit (Invitrogen), following the manufacturer’s guidelines. Five RT-PCR reactions were performed on each RNA sample, using a common reverse primer in combination with forward primers designed to specifically amplify VH from the specific frameworks present in the Crescendo mouse. cDNA products of the correct size were confirmed by analysis on the QIAxcel.
  • cDNAs derived from lymph nodes from a single mouse were pooled together as were cDNAs from the spleen, then each pool of cDNAs was purified with the GeneJet PCR purification kit.
  • b) Cloning into phagemid vector cDNA pools from spleen and lymph nodes were cloned into clean, linear phagemid vector pUCG3 using a PCR-based method.
  • 800ng linearised pUCG3 was mixed with 200ng VH cDNA in a final volume of 50pl, including 1.5pl DMSO and 25pl Phusion GC 2x mix.
  • Vn/phagemid PCR products were pooled, combining material from spleen and lymph nodes, in order to create one phage library per immunised animal. Material was purified using Fermentas PCR purification kit. Phagemid DNA as above was mixed with 160pl of TG1 E. coli, split between two BioRad 0.2cm cuvettes and transformed by electroporation at 2500V, 25m F, 200W. Electroporated cells were recovered in 10ml media for 1 hour at 37°C with shaking. A 10-fold dilution series of an aliquot of the transformed culture was plated onto Ampicillin agar plates and used to estimate library size.
  • Round 1 panning selections were carried out on 10mg/mL human mesothelin recombinant protein (296-580) with C terminal His tag.
  • Round 2 panning selections were carried out on 10mg/mL cyno mesothelin recombinant protein (296-580) with C terminal His tag.
  • E. coli colonies were picked into liquid culture and grown overnight. Phage rescue was carried out by adding M13 K07, releasing phage displaying VH into the supernatant. ELISA plates were coated with the same antigens used for phage selection, then blocked as described in Example 3. Pre-incubated phage were added to the plate and allowed to bind antigen before multiple washes. Phage were bound by a secondary antibody, anti-M13-HRP, then detected with TMB as previously described and absorbance at 450nm was measured.
  • EXAMPLE 8 Design, cloning and small scale purification of clones that bind MSLN
  • Clone VH1.2 was engineered in silico to remove a potential N-glycosylation site by substituting a serine residue (S) for the asparagine residue (N) at position 96 using Kabat numbering to create clone VH1.1.
  • S serine residue
  • N asparagine residue
  • the amino acid sequence of VH1.1 is shown below, with the engineered amino acid underlined.
  • VH1.1 was synthesised by TWIST Bioscience www.twistbioscience.com, custom cloned into E. coli expression vector pJex401 and sequence verified. For ease of purification, a C terminal 6-His tag was included.
  • Plasmid DNA was transformed into E. coli TG1 strain, cultured in a 96 deep well plate at 37°C in TB medium with 50mg/mL kanamycin, with shaking, to an ODeoo of 0.5-1 . Protein expression was induced with IPTG at reduced temperature for approximately 16 hours.
  • Bacterial cells were pelleted by centrifugation at 4500rpm for 40 minutes and the supernatant recovered and filtered using a 0.45um membrane. Protein was bound to nickel resin in a sodium phosphate/sodium chloride buffer with 20mM imidazole, removing the flow through by applying a vacuum pressures of -5 kPa via a multi-well plate vacuum manifold, then washed.
  • Protein was eluted by increasing addition of buffer with 200mM imidazole and centrifugation at 500g for 2 minutes, then buffer exchanged into PBS to a final concentration of 10mM.
  • VH1.1 protein 1 OO
  • the SEC column (Waters ACQUITY) was run isocratically in SEC Buffer (5% 1-propanol, 200mM NaCI, 100mM sodium phosphate, pH 7.4) at 0.4 mL/min for 6 minutes per sample. Data was collected using a PDA detector at 280 nm. VH1.1 protein was found to be 96.7% monomer after incubation at 4°C and 98.1 % monomer after incubation at 40°C.
  • Binding kinetics to human mesothelin recombinant protein Human mesothelin recombinant protein with a C-terminal His tag at 2 mg/mL was immobilised by amine coupling onto a CM5 sensor chip for 90 s at 25°C. The chip was quality controlled using dilutions of a MSLN binding V Vn1.1from 0.1 - 1000 nM. Protein samples prepared as described in Example 8 were normalised to 10mM, then analysed at 3 nM, 15 nM, 75 nM and 375 nM. Association and dissociation times were 180 s and 400 s respectively and the chip was regenerated for 20 s.
  • Cyno mesothelin recombinant protein with a C-terminal His tag at 2 mg/mL was immobilised by amine coupling onto a CM5 sensor chip for 150 s at 25°C.
  • the chip was quality controlled essentially as described above.
  • Protein samples prepared as described in Example 8 were normalised to 10mM, then analysed at 33.3 nM, 100 nM, 300 nM and 900 nM. Association and dissociation times were 180 s and 600 s respectively and the chip was regenerated for 2 x 30 s.
  • amino acid sequence of cyno MSLN is shown below (SEQ ID NO 507).
  • VH1.1 proteins purified as described in Example 8 were assayed using Fluorescence Microvolume Assay Technology (FMAT), a fluorescence-based platform that detects fluorescence localized to beads or cells settled at the bottom of microwells (Dietz et al., Cytometry 23:177-186 (1996), Miraglia et al., J. Biomol. Screening 4:193-204 (1999).
  • FMAT Fluorescence Microvolume Assay Technology
  • CHO TREX cell lines were generated in-house using full-length human MSLN using standard procedures. Parent CHO cells (no modification; not expressing human MSLN) were used as a negative control.
  • the EC50 of VH1.1 was found to be 1 .2 nM
  • MSLN VH1.1 was cloned into pJex401 E. coli expression plasmid with a C terminal 6xHis tag spaced with a cleavable TEV protease recognition site, then sequence verified. Plasmid DNA was transformed into TG1 E. coli cells and grown in 900ml TB medium with 50mg/mL kanamycin, shaking, to an ODeoo of 0.5-1. Protein expression was induced with IPTG at reduced temperature for approximately 16 hours.
  • Ni Sepharose Excel affinity resin was equilibrated from storage buffer into PBS in a 200 mL Econo-Column, and the volume adjusted to a 50% slurry. An appropriate volume of slurry was added to the culture supernatants which were left to mix on a rolling bed (33 rpm) for >1 hour. The supernatant/resin mixtures were poured into clean 25 mL Econo-Columns fitted with 250 mL funnels to collect the resin and bound protein.
  • Preparative SEC was performed using a HiLoad 26/600 Superdex 75 pg column running isocratically in PBS pH 7.4 on an Akta system.
  • the elution samples from Ni-affinity chromatography were loaded via a sample pump with a maximum injection volume of 13 mL per run and eluted with 1.2 CV of PBS (pH 7.4) with a flow rate of 2.6 mL/min.
  • the peak collection threshold was set at 20 mAU and 2 mL fractions were collected using a fraction collector.
  • A280 was measured throughout the run using a UV detector. Fractions containing pure samples of protein were pooled and concentrated to 2mg/mL in a Amicon Ultra-15, 3,000 MWCO RC centrifugal filter unit, centrifuged at max. 4,000 xg.
  • the expression titre was 32.0 mg/L and 24.6mg of high purity protein was recovered.
  • Human mesothelin recombinant protein with a C-terminal His tag at 2 mg/mL was immobilised by amine coupling as described in Example 9. Association and dissociation times were 180 s and 400 s respectively and the chip was regenerated for 20 s.
  • VH1.1 prepared as described in Example 11 was run using multi cycle kinetics using a 6-point dilution series ranging from 1000nM to 4.12 nM. Each sample was run twice.
  • antigen-binding moieties were used: scFv derived from the J591 Ab specific for PSMA; human VH domain specific for PSMA (PSMA-VH); SCFV derived from a MSLN-specific Ab Amatuximab; human VH domain specific for MSLN (MSLN-VH). All ligands were assembled with the CD8a hinge and transmembrane domain, the CD28 costimulatory domain and CD3 intracellular signaling domain and cloned into the SFG retroviral vector. 24 A FLAG-tag was incorporated after the antigen ligand to detect the expression of CARs by an anti-FLAG Ab.
  • Dual specific (PSMA and MSLN) CARs were also generated by linking the two VH domains.
  • the linkers used are described in more detail below.
  • the corresponding CARs were called J591 , PSMA-VH, MSLN scFv, MSLN-VH and PSMA-VH/MSLN-VH.
  • Retroviral supernatants were produced by transfection of 293 T cells with the retroviral vectors, the RD114 envelope from RDF plasmid and the MoMLV gag-pol from PegPam3-e plasmid. Supernatants were collected 48 hours and 72 hours after the transfection and filtered with 0.45 pm filter.24
  • the polypeptide sequences of the VH domains used were as follows: MSLN-VH SEQ ID NO. 3
  • PC-3, C4-2 (prostate cancer) and Aspc-1 pancreatic cancer were purchased from ATCC (American Type Culture Collection). All tumor cell lines were cultured with RPMI- 1640 (Gibco) supplemented with 10% Fetal bovine serum (Sigma), 2 mM GlutaMax (Gibco) and penicillin (100 units/mL) and streptomycin (100 pg/mL; Gibco). All cells were cultured at 37°C with 5% CO2.
  • PC-3 cell line was transduced with retroviral vectors encoding PSMA or MSLN to make PC-3-PSMA and PC-3-MSLN.
  • PC-3-PSMA, PC-3-MSLN and Aspc-1 were transduced with retroviral vectors encoding Firefly-Luciferase-eGFP (FFIuc-eGFP) gene.
  • FFIuc-eGFP Firefly-Luciferase-eGFP
  • CAR-T cell generation Buffy coats from healthy donors (Gulf Coast Regional Blood Center) were processed with Lymphoprep density separation (Fresenius Kabi Norge) to isolate peripheral blood mononuclear cells, which were then activated on plates coated with 1 pg/mL CD3 (Miltenyi Biotec) and 1 pg/mL CD28 (BD Biosciences) monoclonal Abs (mAbs). Two days later, activated T cells were transduced with retroviral supernatants on 24-well plates coated with retronectin (Takara Bio).
  • T cells were collected 3 days after transduction and expanded in 40% RPMI-1640(Gibco) and 40% Click’s medium (Irvine Scientific), 10% HyClone FBS (GE healthcare), 2 mM GlutaMAX(Gibco), 100 unit/mL of Penicillin and 100 mg/mL of streptomycin (Gibco) with 10 ng/mL IL-7 (PeproTech) and 5 ng/mL IL-15 (PeproTech). T cells were collected for functional assays 12-14 days after activation. 2526
  • Flow cytometry mAbs for human CD3 (APC-H7; SK7; 560176), CD4 (BV711 ; SK3; 563028), CD8 (APC; SK1 ; 340584), CD45RA (PE; HI100; 555489), CD45RO (BV786; UCHL1 ; 564290), CD69 (FITC; L78; 347823), CCR7 (FITC; 150503; FAB197F-100), PD-1 (PE-Cy7; EH12.1 ;561272), Lag3(PE;T47-530;565616), FLAG (APC; L5; 637308), Granzyme-B (PE;GB11 ;561142) from BD biosciences and BioLegend were used. Samples were acquired with BD FACSCanto II or BD LSRFortessa. A minimum of 10000 events were acquired for each sample and were analyzed using FlowJo 10 (FlowJo).
  • T cells were labeled with 1.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) and plated with tumor cells at an effector to target (E:T) ratio of 1 :1.
  • CFSE signal dilution from gated T cells on day 5 was measured using flow cytometry.
  • In vitro cytotoxicity assay Tumor cells were seeded in 24-well plates at a concentration of 2.5x10 5 cells/well overnight. CAR-T cells were added to the plate at an E:T of 1 :5 without exogenous cytokines. Cocultures were analyzed 5-7 days following coculture to measure residual tumor cells and T cells by flow cytometry. Dead cells were recognized by Zombie Aqua Dye (Biolegend) staining while CAR- T cells were identified by CD3 staining and tumor cells by GFP. 26 CD69, PD-1 and Lag3 expression was measured by flow cytometry from day 0 to day 5 each day after coculture of CAR-T cells with tumor cells.
  • Golgi protein inhibitor (BD Biosciences) was added on day 1 of coculture for 6 hours. Cocultures were then first stained with Zombie Aqua Dye (Biolegend) and CD3 mAb, followed by fixation/permeabilization solution (BD Biosciences). Intracellular staining of granzyme-B was then conducted.
  • CAR-T cells (1 xio 5 cells) were cocultured with 2.5x10 5 tumor cells in 24-well plates without exogenous cytokines. Supernatant was collected after 24 hours, and cytokines (interferon-y (IFN-y) and IL-2) were measured by using ELISA kits (R&D, Research And Development system) in duplicates following manufacturer’s instructions.
  • a panel of recombinant proteins was produced, comprizing bispecific (2VH) proteins that bind both PSMA and MSLN, monospecific VH protein binding PSMA, monospecific VH protein binding MSLN and a control scFv protein based on Amatuximab.
  • Bispecific protein was made in two formats, one with a short flexible linker (G4S)s, aother one with a long flexible linker (G4S)e.
  • Bispecific proteins were expressed in mammalian cells and purified by protein A binding. Monospecific proteins were His tagged at the C terminus, expressed in Escherichia coli and purified by His trap and size exclusion chromatography.
  • Binding analyses were performed at 25°C using BIAcore 8K system. The instrument was run on 1 x HBS-EP + (BR100669) buffer and the data were analyzed using Biacore Insight Evaluation software. Recombinant human MSLN was diluted to 2 ug/mL in 10 mM sodium acetate buffer pH4.0 and immobilized on a CM5 sensor chip (contact time 120 s) using amine- coupling kit with accordance to the manufacturer’s instructions. Humabody VH samples were tested for binding at 5 concentrations 3.7 nM, 11.1 nM, 33.3 nM, 100 nM and 300 nM using multicycle kinetics method.
  • Each sample was injected for 100 s at the flow rate 35 pL/min and dissociated for 100 s.
  • the antigen surface was regenerated by 20 s injection of 10 mM glycine pH 2.0.
  • Recombinant human PSMA antigen with a human Fc tag was captured on a Protein G sensor.
  • Humabody VH samples were tested in Single-cycle kinetics mode at increasing concentrations of 2.22 nM, 6.67 nM, 20 nM and 60 nM with 90 s association and 600 s dissociation time at the flow rate of 30 pL/min. Buffer injections were made to allow for doublereference subtraction.
  • the sensor surface was regenerated with 10 mM glycine pH1.5 (GE Healthcare BR100354).
  • PSMA antigen surface was captured as above.
  • Bispecific PSMA-MSLN Humabody constructs were captured on the PSMA surface by injecting 100 nM of each sample for 100 s at 35 pL/min flow rate. The capture was immediately followed by an injection of 300 nM recombinant human MSLN with 100 s contact time and 100 s dissociation.
  • a PSMA-specific Humabody construct without a MSLN-binding arm was used as a control.
  • Xenograft murine models NSG (NOD scid gamma mouse) mice (6-8 weeks old) were injected intravenously through tail vein with either PC-3-PSMA-FFIuc-eGFP, or PC-3-PSMA-FFIuc-eGFP and PC-3-MSLN- FFIuc-eGFP mixed at 1 to 1 ratio, or Aspc-1-FFIuc-eGFP tumor cells of 1 xio 6 cells per mice.
  • CAR-T cells were injected intravenously through tail vein.
  • For the high dose treatment 4x10 6 CAR-T cells per mice were injected, while for the low dose treatment, 1 xio 6 CAR-T cells per mice were injected.
  • mice were infused 1 xio 6 tumor cells per mice on clearance of the previous tumor. Tumor growth was monitored by bioluminescence using IVIS (In Vivo Imaging Systems)-Kinetics Optical in vivo imaging system (PerkinElmer) (PSMA-VH and MSLN-VH part) or AMI (AMI Medical Imaging) Optical in vivo imaging system (Spectral instruments imaging) (PSMA-VH/MSLN-VH part).
  • Human VH domain-based CAR targeting PSMA is expressed and signals in T cells
  • PSMA-specific CARs using the scFv from the J591 mAb (J591) and the PSMA binding human VH domain (PSMA-VH) joined to the CD8a stalk, CD28 costimulatory domain and CD3 intracellular domain.
  • a flag-based tag was incorporated into the cassettes to detect CAR expression by flow cytometry (figure 1A).
  • Activated T cells were successfully transduced and expressed the CARs equally (figure 1 B,C).
  • the CD19-specific CAR (CD19) and non-transduced (NT) T cells were used as controls.
  • J591-T cells and PSMA-VH-T cells showed similar expansion in vitro when exposed to IL-15 and IL-7 cytokines, which was similar to CD19-T cells and NT-T cells (figure 1 D). Furthermore, no differences were observed in T cell composition as assessed by flow cytometry at day 12-14 of culture (figure 1 E).
  • proximal signaling of CAR-T cells before and after CAR cross-linking mediated by an anti-Flag Ab. Phosphorylation of the CAR-associated CD3 as well as phosphorylation of Akt and ERK were equal in J591-T cells and PSMA-VH-T cells (figure 1 F). Therefore, a VH domain-based CAR is expressed and signals in T cells on cross-linking as observed for scFv-based CAR-T cells.
  • PSMA-specific VH domain-based CAR-T cells are functional in vitro and in vivo

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Reproductive Health (AREA)
  • Pregnancy & Childbirth (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gynecology & Obstetrics (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps à domaine unique isolés qui se lient à la mésothéline, des molécules multispécifiques associées, des compositions pharmaceutiques et des méthodes de traitement.
PCT/GB2023/050997 2022-04-14 2023-04-13 Liants à base de mésothéline Ceased WO2023199068A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2205589.1 2022-04-14
GBGB2205589.1A GB202205589D0 (en) 2022-04-14 2022-04-14 Mesothelin binders

Publications (1)

Publication Number Publication Date
WO2023199068A1 true WO2023199068A1 (fr) 2023-10-19

Family

ID=81753166

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2023/050997 Ceased WO2023199068A1 (fr) 2022-04-14 2023-04-13 Liants à base de mésothéline

Country Status (2)

Country Link
GB (1) GB202205589D0 (fr)
WO (1) WO2023199068A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025143287A1 (fr) * 2023-12-27 2025-07-03 서울대학교산학협력단 Anticorps spécifique de la mésothéline et ses utilisations

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000737A2 (fr) 2001-06-21 2003-01-03 The Babraham Institute Locus de la chaine legere $g(l) de souris
WO2004076618A2 (fr) 2003-02-26 2004-09-10 Babraham Institute Mammiferes non humains et cellules non humaines genetiquement modifies
WO2016062990A1 (fr) 2014-10-22 2016-04-28 Crescendo Biologics Limited Souris transgéniques
WO2016113556A1 (fr) 2015-01-12 2016-07-21 Crescendo Biologics Limited Procédés de production de molécules thérapeutiques optimisées
WO2017122017A1 (fr) 2016-01-12 2017-07-20 Crescendo Biologics Limited Molécules thérapeutiques
WO2017191476A1 (fr) 2016-05-06 2017-11-09 Crescendo Biologics Limited Récepteur antigénique chimérique à anticorps à domaine unique
WO2018127709A1 (fr) * 2017-01-06 2018-07-12 Crescendo Biologics Limited Anticorps à domaine unique dirigés contre la mort cellulaire programmée (pd-1)
WO2019012260A1 (fr) 2017-07-10 2019-01-17 Crescendo Biologics Limited Molécules thérapeutiques se liant à psma
WO2020099871A1 (fr) 2018-11-13 2020-05-22 Crescendo Biologics Limited Anticorps à domaine unique se liant à l'albumine sérique humaine
WO2020229842A1 (fr) 2019-05-15 2020-11-19 Crescendo Biologics Limited Molécules de liaison
WO2023064876A1 (fr) * 2021-10-14 2023-04-20 Teneobio, Inc. Protéines de liaison à la mésothéline et leurs utilisations

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000737A2 (fr) 2001-06-21 2003-01-03 The Babraham Institute Locus de la chaine legere $g(l) de souris
WO2004076618A2 (fr) 2003-02-26 2004-09-10 Babraham Institute Mammiferes non humains et cellules non humaines genetiquement modifies
WO2016062990A1 (fr) 2014-10-22 2016-04-28 Crescendo Biologics Limited Souris transgéniques
WO2016113556A1 (fr) 2015-01-12 2016-07-21 Crescendo Biologics Limited Procédés de production de molécules thérapeutiques optimisées
WO2017122017A1 (fr) 2016-01-12 2017-07-20 Crescendo Biologics Limited Molécules thérapeutiques
WO2017191476A1 (fr) 2016-05-06 2017-11-09 Crescendo Biologics Limited Récepteur antigénique chimérique à anticorps à domaine unique
WO2018127709A1 (fr) * 2017-01-06 2018-07-12 Crescendo Biologics Limited Anticorps à domaine unique dirigés contre la mort cellulaire programmée (pd-1)
WO2019012260A1 (fr) 2017-07-10 2019-01-17 Crescendo Biologics Limited Molécules thérapeutiques se liant à psma
WO2020099871A1 (fr) 2018-11-13 2020-05-22 Crescendo Biologics Limited Anticorps à domaine unique se liant à l'albumine sérique humaine
WO2020229842A1 (fr) 2019-05-15 2020-11-19 Crescendo Biologics Limited Molécules de liaison
WO2023064876A1 (fr) * 2021-10-14 2023-04-20 Teneobio, Inc. Protéines de liaison à la mésothéline et leurs utilisations

Non-Patent Citations (52)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. AAH03512.1
AHMAD ZAYEAP SKALI AM ET AL.: "scFv antibody: principles and clinical application", CLIN DEV IMMUNOL, vol. 2012, 2012, pages 1 - 15, XP055549405, DOI: 10.1155/2012/980250
AHN SLI JSUN C ET AL.: "Cancer immunotherapy with T cells carrying bispecific receptors that mimic antibodies", CANCER IMMUNOL RES, vol. 7, 2019, pages 773 - 83, XP055772069, DOI: 10.1158/2326-6066.CIR-18-0636
BARBER AZHANG TDEMARS LR ET AL.: "Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer", CANCER RES, vol. 67, 2007, pages 5003 - 8
BOLLARD CMTRIPIC TCRUZ CR ET AL.: "Tumor-Specific T-cells engineered to overcome tumor immune evasion induce clinical responses in patients with relapsed Hodgkin lymphoma", J CLIN ONCOL, vol. 36, 2018, pages 1128 - 39, XP009528705, DOI: 10.1200/JCO.2017.74.3179
BRIAN K. KAYJILL WINTERJOHN MCCAFFERTY: "Phage Display of Peptides and Proteins: A Laboratory Manual", 28 October 1996, ACADEMIC PRESS
BROWN CEALIZADEH DSTARR R ET AL.: "Regression of glioblastoma after chimeric antigen receptor T-cell therapy", N ENGL J MED, vol. 375, 2016, pages 2561 - 9, XP055564981, DOI: 10.1056/NEJMoa1610497
BRUSCHI, C.V.GJURACIC, K.: "Encyclopedia of Life Sciences", 2002, MACMILLAN PUBLISHERS LTD., NATURE PUBLISHING GROUP, article "Yeast Artificial Chromosomes"
CHANG SS: "Overview of prostate-specific membrane antigen", REV UROL, vol. 6, no. 10, 2004, pages 313 - 18
DIACONU IBALLARD BZHANG M ET AL.: "Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells", MOL THER, vol. 25, 2017, pages 580 - 92, XP055692323, DOI: 10.1016/j.ymthe.2017.01.011
DIETZ ET AL., CYTOMETRY, vol. 23, 1996, pages 177 - 186
DOTTI GGOTTSCHALK SSAVOLDO B ET AL.: "Design and development of therapies using chimeric antigen receptor-expressing T cells", IMMUNOL REV, vol. 257, 2014, pages 107 - 26, XP055552726, DOI: 10.1111/imr.12131
DU HHIRABAYASHI KAHN S ET AL.: "Antitumor responses in the absence of toxicity in solid tumors by targeting B7-H3 via chimeric antigen receptor T cells", CANCER CELL, vol. 35, 2019, pages 221 - 37
ESHHAR ZWAKS TGROSS G ET AL.: "Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors", PROC NATL ACAD SCI U S A, vol. 90, 1993, pages 720 - 4, XP002009770, DOI: 10.1073/pnas.90.2.720
FINNEY HMLAWSON ADBEBBINGTON CR ET AL.: "Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.", J IMMUNOL, vol. 161, 1998, pages 2791 - 7
FUCA GREPPEL LLANDONI E ET AL.: "Enhancing chimeric antigen receptor T-cell efficacy in solid tumors", CLIN CANCER RES, vol. 26, 2020, pages 2444 - 51, XP055967810, DOI: 10.1158/1078-0432.CCR-19-1835
HAMERS-CASTERMAN CATARHOUCH TMUYLDERMANS S ET AL.: "Naturally occurring antibodies devoid of light chains", NATURE, vol. 363, 1993, pages 446 - 8, XP002535892, DOI: 10.1038/363446a0
HECZEY ALOUIS CUSAVOLDO B ET AL.: "Car T cells administered in combination with Lymphodepletion and PD-1 inhibition to patients with neuroblastoma", MOL THER, vol. 25, 2017, pages 2214 - 24, XP093008964, DOI: 10.1016/j.ymthe.2017.05.012
HOLLIGER PHUDSON PJ: "Engineered antibody fragments and the rise of single domains", NAT BIOTECHNOL, vol. 23, 2005, pages 1126 - 36, XP008076746, DOI: 10.1038/nbt1142
HONG LKCHEN YSMITH CC ET AL.: "CD30-Redirected Chimeric Antigen Receptor T Cells Target CD30+ and CD30- Embryonal Carcinoma via Antigen-Dependent and Fas/FasL Interactions", CANCER IMMUNOL RES, vol. 6, 2018, pages 1274 - 87
IMAI CMIHARA KANDREANSKY M ET AL.: "Chimeric receptors with 4-1 BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.", LEUKEMIA, vol. 18, 2004, pages 676 - 176
JAMNANI FRRAHBARIZADEH FSHOKRGOZAR MA ET AL.: "T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy", BIOCHIM BIOPHYS ACTA, vol. 1840, 2014, pages 378 - 86, XP055108962, DOI: 10.1016/j.bbagen.2013.09.029
KABAT ET AL., ANN. NY ACAD. SCI., vol. 190, 1971, pages 382 - 391
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, NIH PUBLICATION
LAM NTRINKLEIN NOBUELOW B ET AL.: "Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains", NAT COMMUN, vol. 11, 2020, pages 283
LANDONI EFUC6 GWANG J ET AL.: "Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling", CANCER IMMUNOL RES, 2021
LV JZHAO RWU D ET AL.: "Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer", J HEMATOL ONCOL, vol. 12, 2019, pages 18, XP055727450, DOI: 10.1186/s13045-019-0704-y
MA XSHOU PSMITH C ET AL.: "Interleukin-23 engineering improves CAR T cell function in solid tumors", NAT BIOTECHNOL, vol. 38, 2020, pages 448 - 59, XP037086863, DOI: 10.1038/s41587-019-0398-2
MILLER BRDEMAREST SJLUGOVSKOY A ET AL.: "Stability engineering of scFvs for the development of bispecific and multivalent antibodies", PROTEIN ENG DES SEL, vol. 23, 2010, pages 549 - 57, XP002690428, DOI: 10.1093/protein/gzq028
MIRAGLIA ET AL., J. BIOMOL. SCREENING, vol. 4, 1999, pages 193 - 204
MORELLO ASADELAIN MADUSUMILLI PS: "Mesothelin-Targeted cars: driving T cells to solid tumors", CANCER DISCOV, vol. 6, 2016, pages 133 - 46
O'ROURKE DMNASRALLAH MPDESAI A ET AL.: "A single dose of peripherally infused EGFRvlll-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma", SCI TRANSL MED, vol. 2017, no. 9, pages eaaa0984
PARK SSHEVLIN EVEDVYAS Y ET AL.: "Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity", SCI REP, vol. 7, 2017, pages 14366, XP055656078, DOI: 10.1038/s41598-017-14749-3
RAJABZADEH ARAHBARIZADEH FAHMADVAND D ET AL.: "A VHH-Based anti-MUC1 chimeric antigen receptor for specific retargeting of human primary T cells to MUC1-positive cancer cells", CELL J, vol. 22, 2021, pages 502 - 13
RAMOS CAGROVER NSBEAVEN AW ET AL.: "Anti-Cd30 CAR-T cell therapy in relapsed and refractory Hodgkin lymphoma", J CLIN ONCOL, vol. 38, 2020, pages 3794 - 804
REN ET AL., GENOMICS, vol. 84, 2004, pages 686
RICHMAN SANUNEZ-CRUZ SMOGHIMI B ET AL.: "High-Affinity GD2-Specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model", CANCER IMMUNOL RES, vol. 6, 2018, pages 36 - 46, XP055891296, DOI: 10.1158/2326-6066.CIR-17-0211
SADELAIN MBRENTJENS RRIVIERE I: "The basic principles of chimeric antigen receptor design", CANCER DISCOV, vol. 3, 2013, pages 388 - 98, XP055287277, DOI: 10.1158/2159-8290.CD-12-0548
SAMPSON JHGUNN MDFECCI PE ET AL.: "Brain immunology and immunotherapy in brain tumours", NAT REV CANCER, vol. 20, 2020, pages 12 - 25, XP036974944, DOI: 10.1038/s41568-019-0224-7
SHAFFER DRSAVOLDO BYI Z ET AL.: "T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies", BLOOD, vol. 117, 2011, pages 4304 - 14, XP002738263, DOI: 10.1182/blood-2010-04-278218
SHAH NNFRY TJ: "Mechanisms of resistance to CAR T cell therapy", NAT REV CLIN ONCOL, vol. 3, 2019, pages 372 - 85, XP036789414, DOI: 10.1038/s41571-019-0184-6
SHARIFZADEH ZRAHBARIZADEH FSHOKRGOZAR MA ET AL.: "Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents", CANCER LETT, vol. 334, 2013, pages 237 - 44, XP028555132, DOI: 10.1016/j.canlet.2012.08.010
STANFIELD RLDOOLEY HFLAJNIK MF ET AL.: "Crystal structure of a shark single-domain antibody V region in complex with lysozyme", SCIENCE, vol. 305, 2004, pages 1770 - 3, XP002989770, DOI: 10.1126/science.1101148
TENG YYOUNG JLEDWARDS B ET AL.: "Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse", N BIOTECHNOL, vol. 55, 2020, pages 65 - 76, XP086034492, DOI: 10.1016/j.nbt.2019.10.003
WANG DSHAO YZHANG X ET AL.: "11-23 and PSMA-targeted duo-CAR T cells in prostate cancer eradication in a preclinical model", J TRANSL MED, vol. 18, 2020, pages 23
WANG GUANMENG ET AL: "Fully human antibody V H domains to generate mono and bispecific CAR to target solid tumors", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 9, no. 4, 1 April 2021 (2021-04-01), pages e002173, XP093054299, DOI: 10.1136/jitc-2020-002173 *
WANG QCHEN YPARK J ET AL.: "Design and production of bispecific antibodies", ANTIBODIES, vol. 8, 2019, pages 43
WATANABE KTERAKURA SUCHIYAMA S ET AL.: "Excessively high-affinity single-chain fragment variable region in a chimeric antigen receptor can counteract T-cell proliferation", BLOOD, vol. 124, 2014, pages 4799, XP086740002, DOI: 10.1182/blood.V124.21.4799.4799
XIONG WCHEN YKANG X ET AL.: "Immunological synapse predicts effectiveness of chimeric antigen receptor cells", MOL THER, vol. 26, 2018, pages 963 - 75, XP055525745, DOI: 10.1016/j.ymthe.2018.01.020
YU HPAN JGUO Z ET AL.: "Cart cell therapy for prostate cancer: status and promise", ONCO TARGETS THER, vol. 12, 2019, pages 391 - 5
ZHANG ZJIANG DYANG H ET AL.: "Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor", CELL DEATH DIS, vol. 10, 2019, pages 476, XP055727451, DOI: 10.1038/s41419-019-1711-1
ZOU ET AL., J. IMMUNOL., vol. 170, 2003, pages 1354

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025143287A1 (fr) * 2023-12-27 2025-07-03 서울대학교산학협력단 Anticorps spécifique de la mésothéline et ses utilisations

Also Published As

Publication number Publication date
GB202205589D0 (en) 2022-06-01

Similar Documents

Publication Publication Date Title
CN109641948B (zh) 具有单结构域抗体的嵌合抗原受体
CN112703013B (zh) Cd3抗原结合片段及其应用
CN112794909A (zh) 一种抗tigit单克隆抗体及其应用
CN111909271B (zh) 一种基于单域抗体的bcma嵌合抗原受体及其应用
CN116199781B (zh) 一种靶向cd22的单域抗体、嵌合抗原受体及其应用
CN116903757B (zh) Cd70纳米抗体和双靶向嵌合抗原受体
WO2021052307A1 (fr) Anticorps anti-b7-h3 et son application
CN104098698A (zh) 一种抗cd3抗体及其制法和应用
CN113631184A (zh) 通过抑制ebag9增强溶细胞性t细胞活性
CN120005019A (zh) 一种靶向gprc5d的单域抗体、嵌合抗原受体及其应用
EP4257617A1 (fr) Protéine de liaison à l'antigène ciblant cd70 et son utilisation
EP3969473A1 (fr) Molécules de liaison
CN118580359B (zh) 抗her2单克隆抗体及其基因以及制备方法和应用
EP4458956A1 (fr) Récepteur antigénique chimérique à nanocorps anti-mésothéline et son utilisation
WO2023273762A1 (fr) Épitope conformationnel spatial induisant une rétention efficace de cd3 dans des cellules et son utilisation
WO2023199069A1 (fr) Récepteur antigénique chimérique qui se lie à la mésothéline
CN116284389A (zh) 抗afp/hla02 tcr样抗体及其用途
WO2022268196A1 (fr) Protéine de liaison à un antigène ciblant gpc3
CN118206661B (zh) 一种靶向间皮素的单域抗体、嵌合抗原受体及其应用
WO2025202653A1 (fr) Thérapie par lymphocytes t ciblant la calréticuline mutante
HK40128378A (zh) 对δ样配体3(DLL3)具有特异性的抗体和嵌合抗原受体
CN120775047A (zh) 靶向Claudin18.2的抗体及其应用
JP2025542097A (ja) 抗ａｆｐ／ｈｌａ０２ ｔｃｒ様抗体及びその使用
CN121969647A (zh) 一种靶向cd79b的单域抗体和嵌合抗原受体及其应用
CN118271440A (zh) 靶向IL13Rα2的抗体以及靶向IL13Rα2的免疫效应细胞

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23719869

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23719869

Country of ref document: EP

Kind code of ref document: A1