WO2023199068A1 - Mesothelin binders - Google Patents

Mesothelin binders 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
Application number
PCT/GB2023/050997
Other languages
French (fr)
Inventor
Colette JOHNSTON
Phil HAYES
Original Assignee
Crescendo Biologics Limited
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 Limited filed Critical Crescendo Biologics Limited
Publication of WO2023199068A1 publication Critical patent/WO2023199068A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—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]
    • A61K39/464495—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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], 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 A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 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)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Developmental Biology & Embryology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to isolated single domain antibodies that bind mesothelin, related multispecific molecules, pharmaceutical compositions and methods of treatment.

Description

Mesothelin binders
Field of the invention
The invention relates to single domain antibodies that bind mesothelin and can be used in treating cancer.
Introduction
Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is however highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian, endometrial and lung adenocarcinoma. In the context of cancer, high expression levels of MSLN have been correlated with poor prognosis in ovarian cancer, cholangiocarcinoma, lung adenocarcinoma and triple-negative breast cancer. The limited expression of mesothelin on normal human tissues and high expression in several human cancers makes mesothelin an attractive candidate for cancer therapy. Several therapeutic strategies have been designed for targeting mesothelin on tumor cells including tumor vaccine strategy, antibody-based therapies and adoptive CAR T-cell therapy. These therapies are being evaluated in phase I and/or phase II clinical trials.
Heavy-chain-only antibodies (HcAbs) without light chains have been reported in camelids and camelids and cartilaginous fish, and shown to exhibit strong and specific antigen binding (Holliger P and Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126-36).
Summary of the Invention
In a first aspect, the invention relates to an isolated VH single domain antibody that specifically binds to human mesothelin (MSLN), wherein the VH single domain antibody comprising a CDR1 comprising SEQ NO. 4 or a sequence with 1 , 2 or 3 amino acid modifications, CDR2 comprising SEQ NO. 5 or a sequence with 1 , 2, 3 or 4 amino acid modifications and a CDR3 comprising SEQ NO. 6 or a sequence with 1 , 2 or 3 amino acid modifications.
In another aspect, the invention relates to an isolated nucleic acid encoding a VH single domain antibody as described herein.
In another aspect, the invention relates to a vector comprising a nucleic acid as described herein. In another aspect, the invention relates to a host cell comprising a nucleic acid or a vector as described herein.
In another aspect, the invention relates to a binding molecule comprising an isolated VH single domain antibody as described herein.
In another aspect, the invention relates to a use of a single domain antibody as described herein in a multispecific or multivalent binding agent.
In another aspect, the invention relates to a pharmaceutical composition comprising a VH single domain antibody or a binding molecule as described herein and a pharmaceutical acceptable carrier, excipient or diluent. In another aspect, the invention relates to a method for treating a cancer comprising administering a VH single domain antibody or a binding molecule as described herein.
In another aspect, the invention relates to a VH single domain antibody, a pharmaceutical composition or a binding molecule as described herein for use in the treatment of cancer.
In another aspect, the invention relates to an immunoconjugate comprising a single domain antibody according or a binding molecule as described herein linked to a therapeutic agent.
In another aspect, the invention relates to a method for producing a VH single domain antibody as described herein comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.
In another aspect, the invention relates to a kit comprising a single domain antibody or a binding molecule, or a pharmaceutical composition as described herein and optionally instructions for use.
In another aspect, the invention relates to a method for detecting the presence of human MSLN in a test sample or detecting or diagnosing a cancer in a subject comprising contacting said sample with a single domain antibody as described herein and at least one detectable label and detecting binding of said single domain antibody to human MSLN.
In another aspect, the invention relates to a method for producing a VH single domain antibody that binds to human MSLN comprising a) immunising a transgenic animal that expresses a nucleic acid construct comprising human heavy chain V genes and that is not capable of making functional endogenous light or heavy chains with an MSLN antigen, b) generating a library from said animal c) isolating single VH domain antibodies from said libraries and d) identifying a single VH domain antibody that binds to human MSLN and e) isolating said antibody.
In another aspect, the invention relates to a single VH domain antibody obtained or obtainable by the method above.
In another aspect, the invention relates to an isolated heavy chain only antibody comprising a VH domain that binds to human MSLN.
In another aspect, the invention relates to a heavy chain only antibody comprising a VH domain that binds to human MSLN obtained or obtainable from a transgenic mouse which expresses human V, D and J loci and does not produce functional endogenous lambda and kappa light chains and heavy chain.
In another aspect, the invention relates to an antibody drug conjugate comprising a VH single domain antibody or a binding molecule as described herein.
Figures
The invention is further described in the following non-limiting figures.
Figure 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. ****p < 0.0001 , One-way ANOVA. (D) In vitro expansion of CD19, J591 , PSMA-VH and NT T cells; error bars represent SD, (n = 4). p >0.05 by One-way ANOVA. (E) T cell subset composition based on CD45RA and CCR7 expression in CD19, J591 , PSMA-VH and NT T cells at day 14 of culture; error bars represent SD, (n = 4). p >0.05 by One-way ANOVA. (F) Western blots detecting phosphorylation of CAR- CD3 , Akt and ERK in J591 and PSMA-VH T cells activated via CAR cross-linking with an anti- FLAG Ab followed by incubation with a secondary Ab to induce the aggregation of CAR molecules. Total CAR. CD3 and endogenous CD3 were used as loading controls. Data are representative of 2 experiments. Figure 2. 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. (B, C) Representative flow cytometry plots (B) and summary (C) illustrating Granzyme-B expression of T cells expressing either J591 or PSMA-VH cocultured overnight with a tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2; error bars represent SD, (n = 4). p >0.05 by t-test. (D, E) Representative flow cytometry plots (D) and summary (E) illustrating the kinetics of CD69 expression of T cells expressing either J591 or PSMA-VH and cocultured overnight with a tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2. Data are representative of 4 experiments. ***p < 0.001 Two-way ANOVA. (F) Representative flow cytometry plots showing coculture of CD19, J591 and PSMA-VH T cells with C4-2-eGFP, PC3-eGFP and PC3-PSMA-eGFP. T cells were cocultured with tumor cells at an E:T ratio of 1 :5 for 6 days. At day 6, all cells were collected and analyzed by flow cytometry to quantify tumor cells (GFP) and T cells (CD3), respectively. (G) Summary of coculture of CD19, J591 and PSMA-VH T cells with tumor cells in (F); error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (H, I) IFN-y (H) and IL-2 (I) were detected by ELISA in the coculture supernatant of cocultures of CD19, J591 and PSMA-VH T cells with tumor cells illustrated in (F); error bars represent SD, (n = 5). *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , Two-way ANOVA. (J) Representative flow cytometry plots showing the proliferation of J591 and PSMA-VH T cells in response to tumor cells as assed by CFSE dilution. Data are representative of 4 experiments.
Figure 3. T cells expressing the human antibody VH domain-based CAR targeting PSMA are functional in vivo. (A) Schematic of the metastatic prostate cancer model using PC3- PSMA-FFIuc-eGFP tumor cells in NSG mice and treatment with CD19, J591 and PSMA-VH T cells (n = 5 mice per group). (B) Representative images of tumor bioluminescence (BLI) at selected time points post T cell injections. (C) Kinetics of tumor BLI post T cell injections. (D) Schematic of the metastatic prostate cancer model using PC3-PSMA-FFIuc-eGFP tumor cells in NSG mice and treatment with low dose of CD19, J591 and PSMA-VH T cells (n 4 mice per group). (E) Representative images of tumor BLI at selected time points post T cell injections for low dose of T cells. (F) Kinetics of tumor BLI post T cell injections low dose of T cells. (G, H) Percentage of T cells in the gate of live cells (G) and total cell numbers (H) in blood, spleen, and bone marrow from PC3-PSMA-bearing mice treated with low doses of CAR-T cells. Mice were euthanized at day 58 after CAR-T cells infusion and T cells were identified as CD45+CD3+ cells by flow cytometry. J594 group (n = 5) PSMA-VH group (n = 4). p >0.05 by t-test.
Figure 4. T cells expressing the human antibody VH domain-based CAR targeting MSLN demonstrate antitumor activity. (A) Schematic diagram of MSLN-scFv and MSLN-heavy- chain-only (MSLN-VH) CAR constructs. (B) Summary of coculture of CD19, MSLN.scFv and MSLN-VH T cells with Aspc-1-eGFP (MSLN+) and PC3-eGFP (MSLN-) tumor cell lines. T cells were cocultured with tumor cells at an E:T ratio of 1 :5 for 6 days. At day 6, all cells were collected and analyzed by flow cytometry to quantify tumor cells and T cells, respectively. Error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (C) IFN-y (upper panel) and IL- 2 (lower panel) detected in the supernatants of the cocultures illustrated in (B) as measured by ELISA; error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (D) Representative flow cytometry plots showing the proliferation of MSLN.scFv and MSLN-VH T cells in response to tumor cells as assessed by CFSE dilution. Data are representative of 3 experiments. (E) Schematic of the metastatic pancreatic cancer model using Aspc-1-FFIuc- eGFP tumor cells in NSG mice. (F, G) Representative tumor BLI (F) and bioluminescence kinetics (G) of Aspc-1-FFIuc-eGFP tumor growth at the representative time points post T cell injections, (n = 5 mice per group). (H) Kaplan-Meier survival curve of mice in (E) (n= 5 mice per group). Data are representative of two experiments. (I) Frequency of human CD45+CD3+ cells in blood at 22 days (left) post T-cell infusion and at euthanasia (right) of MSLN-scFv and MSLN-VH T cells, respectively. Data are shown as individual values and the mean (n = 5 mice per group), p >0.05 by t-test.
Figure 5. In vitro analysis of monospecific and bispecific Humabody VH binding. (A) Schematic representation of monospecific (single VH) or bispecific (double VH) proteins. (B) Single cycle Biacore kinetic analysis of PSMA binding. (C) Biacore kinetic analysis of MSLN binding, 3-fold dilution series starting at 300nM, except the control scFv protein which started at 33.3nM. Data are representative of two experiments.
Figure 6. T cells expressing two human antibody VH domain-based CARs demonstrate dual specificity in vitro. (A) Schematic diagram of PSMA-VH, MSLN-VH, and PSMA/MSLN- VH CAR constructs. (B, C) Representative flow cytometry plots (B) and summary (C) illustrating CAR expression in T cells. The CD19-specific CAR (CD19) was used as negative controls. p >0.05 by One-way ANOVA. (D) Representative flow cytometry plots showing PC3-PSMA- eGFP (PSMA target), PC3-MSLN-eGFP (MSLN target), and mixture of PC3-PSMA-eGFP and PC3-MSLN-eGFP (1 :1 ratio) cotultured with CD19.CAR, PSMA-VH.CAR, MSLN-VH. CAR and PSMA/MSLN-VH.CAR T cells at the E: T ratio of 1 :5 for 6 days. T umor cells and T cells were quantified at day 6 by flow cytometry. (E) Summary of coculture experiments illustrated in (D); error bars represent SD, (n = 5). *p < 0.05, **p < 0.01 , ****p < 0.0001 , Two-way ANOVA. (F, G) IFN-y (F) and IL-2 (G) detected in the coculture supernatant of the coculture experiments described in (D) as measured by ELISA; error bars represent SD, (n = 3) *p < 0.05, **p < 0.01 ,
***p < 0.001 , ****p < 0.0001 , Two-way ANOVA. Figure 7. T cells expressing two human antibody VH domain-based CARs demonstrate dual specificity in vivo. (A) Schematic of the xenograft mouse model in which NSG mice were systemically engrafted with mixed FFIuc-eGFP labeled PC3-PSMA (5 x 105 cells) and PC3-MSLN (5 x 105 cells) cells at 1 :1 ratio, and treated with two doses of CAR-T cells at day 0 and day 7, respectively (6 x 106 cells each dose, n = 5 mice per group). (B, C) Representative tumor BLI images (B) and bioluminescence kinetics (C) at selected time points post T cell injections. (D) Number of human CD45+CD3+ cells in the peripheral blood collected at day 21 post second T-cell infusion in mice treated as described in (A). Data are shown as individual values and the mean (n = 5 mice per group) and are representative of two experiments, p >0.05 by one-way ANOVA. (E) Representative antigen expression pattern in the tumor cells isolated from the mice with relapsed tumor in mice treated as described in (A).
Figure 8. Heavy-chain-only-based CAR-T cells express LAG3 and PD-1 upon activation as scFv-based CAR-T cells. (A) Representative flow cytometry plots illustrating Granzyme-B expression in T cells expressing either J591 or PSMA-VH without co-culture with tumor cells (rest condition). (B-E) Representative flow plots and summary illustrating the kinetics of LAG3 (B,C) and PD-1 (D,E) expression in T cells expressing either J591 or PSMA-VH cocultured overnight with the tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2. Data are representative of 4 experiments. **p < 0.01 Two-way ANOVA.
Figure 9. Transduction efficiency of MSLN-scFv and MSLN-VH CARS. (A,B) Representative flow pots (A) and summary (B) illustrating MSLN-scfv and MSLN-VH expression in T cells. The CD19-specific CAR (CD19) and non-transduced T cells (NT) were used as positive and negative controls, respectively. ****p < 0.0001 , One-way ANOVA.
Figure 10. Expression of MSLN in Aspc-1, PC3 and engineered PC3 cells. Representative flow cytometry histograms showing the expression of MSLN in Aspc-1 , PC3 and PC3 cells engineered with retroviral vector to express MSLN.
Figure 11. Bispecific heavy-chain-only-based CAR-T cells demonstrate dual specificity.
(A) Representative flow cytometry plots showing coculture of PC3-PSMA-eGFP (PSMA target) and Aspc-1 -eGFP (MSLN target) tumor cells with CD19.CAR, PSMA-VH. CAR, MSLN-VH. CAR and PSMA-VH/MSLN-VH.CAR T cells at E:T ratio of 1 :5 for 6 days. At the end of co-culture, cells were collected to enumerate T cells (CD3) and tumor cells (GFP), respectively by flow cytometry. (B) Summary of coculture experiments illustrated in (A); error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (C,D) IFN-y (C) and IL-2 (D) released in the coculture supernatant of the experiments illustrated in (A) as measured by ELISA; error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. Figure 12. Phenotypic characterization of T cells in the peripheral blood of mice in the PC3-PSMA and PC3-MSLN Mixed tumor model. CAR-T cells in the peripheral blood at day 21 post second T-cell infusion were identified by the expression of CD45 and CD3 by flow cytometry. PD1 (A), TIM3 (B), CD45RA and CCR7 expression (C) were examined, error bars represent SD, (n = 5). p >0.05 by One-way ANOVA.
Figure 13: A)-O) Sequences of VH single domain antibodies that bind PSMA grouped according to family based on sequence similarity. This figure shows the full length VH sequence. CDR1 , CDR2 and CDR3 are highlighted in bold.
Detailed description The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2013)). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, immunology, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. The term VH domain as used herein refers to an isolated single human VH domain antibody which is also termed VH sdAb. A VH domain is referred to as Humabody® herein. Humabody® is a registered trademark of Crescendo Biologies Ltd. These terms are thus used interchangeably. The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to a single domain antibody that is substantially free of other single domain antibodies, antibodies or antibody fragments. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.
The terms “single domain antibody”, “single variable domain antibody”, “single variable heavy chain domain antibody”, “single VH domain antibody”, “immunoglobulin single variable domain (ISV)”, “immunoglobulin single variable domain antibody”, “VH single domain antibody”, “single heavy chain domain”, “single variable heavy chain domain”, “single VH domain” or “VH domain” are all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen.
A single variable “heavy chain domain antibody, single variable heavy chain domain, immunoglobulin single heavy chain variable domain (ISV), human VH single domain” etc as used herein therefore does not comprise any other parts of a full antibody, but the antigen binding VH domain only; e.g. it only includes the VH domain and does not comprise constant heavy chain domains and does not comprise a light chain. A single variable heavy chain domain antibody is capable of specific binding to an antigen in the absence of light chain or other antibody fragments.
VH domains are small molecules of 12-14 kDa which can be combined into different formats (formatted Humabody®) to give multivalent or multispecific antigen binding domains. VH domains are robust and are characterised by high affinity and stability in serum30.
Each single VH domain antibody comprises three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (VH) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
The term "CDR" refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The numbering system described by Kabat is used herein.
The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (/.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
As used herein, the VH domain is a human VH domain. The term “a human VH domain” includes a VH domain that is derived from or based on a human VH domain amino acid or nucleic acid sequence. Thus, the term includes variable heavy chain regions derived from human germline immunoglobulin sequences. The VH domain can be produced using known methods in the art. For example, the term “human VH domain” includes VH domains that are isolated from transgenic mice expressing human immunoglobulin V genes, in particular in response to an immunisation with an antigen of interest, for example as described in WO 2016/062990. Such domains are preferably fully human. In one embodiment, a human VH domain can also include a VH domain that is derived from or based on a human VH domain amino acid or nucleic acid sequence encoding such VH domain. Thus, the term includes variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences. A substantially human VH domain or VH domain that is derived from or based on a human VH domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo). The term “human VH domain” therefore also includes a substantially human VH domain wherein one or more amino acid residue has been modified. For example, a substantially human VH domain the VH domain may include up to 10, for example 1 , 2, 3, 4 or 5 amino acid modifications compared to a fully human sequence.
However, the term "human VH domain" or "substantially human VH domain", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Preferably, the term "human VH domain", as used herein, is also not intended to include camelized VH domains, that is human VH domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the VH domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid VHH domain.
As used herein, the term 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. The term "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. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and
VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2) or subclass.
An antibody fragment as used herein is a portion of an antibody, for example as F(ab')2, Fab (Fragment, antibody), scFv (single chain variable chain fragments), single domain antibodies (dAbs), Fv, sFv, and the like. Functional fragments of a full-length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments have been used to develop therapeutics as an alternative to therapeutics based on mAbs. scFv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domains are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).
MSLN is a glycoprotein anchored to the plasma membrane by a glycophosphatidyl inositol (GPI) domain. It is initially synthesized as a 69 kDa cell-surface protein. After cleavage of the amino terminus by the furin protease, a 40-kDa C-terminal fragment remains attached to the membrane and a soluble 32-kDa N-terminal fragment, named MPF (megakaryocyte potentiating factor), is released. A soluble form of MSLN has also been detected in the sera of patients with solid tumors, which is referred to as soluble MSLN-related protein (SMRP).
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.
Cancer Discov 2016).
As used herein, the terms mesothelin or MSLN refer to the 40-kDa protein, mesothelin, which is anchored at the cell membrane by a glycosylphosphatidyl inositol (GPI) linkage and its soluble form that circulates in the serum of cancer patients. MSLN contains N-glycosylation sites. For example, the term refers to a human mesothelin of GenBank accession number AAH03512.1.
The human mesothelin protein sequence and nucleic acid sequences are shown below.
SEQ ID NO. 1 human MSLN amino acid sequence MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLS PRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLL FLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRAL GGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVST MDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGK KAREI DESLI FYKKWELEACVDAALLATQM DRVN Al PFTYEQLDVLKH KLDELYPQGYPESVI QHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGR GQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAF QNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGP
HVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGP GPVLTVLALLLASTLA
SEQ ID NO. 2 human MSLN nucleic acid sequence
ATGGCCTTGCCAACGGCTCGACCCCTGTTGGGGTCCTGTGGGACCCCCGCCCTCGGC AGCCTCCTGTTCCTGCTCTTCAGCCTCGGATGGGTGCAGCCCTCGAGGACCCTGGCTG GAGAGACAGGGCAGGAGGCTGCGCCCCTGGACGGAGTCCTGGCCAACCCACCTAACA TTTCCAGCCTCTCCCCTCGCCAACTCCTTGGCTTCCCGTGTGCGGAGGTGTCCGGCCT GAGCACGGAGCGTGTCCGGGAGCTGGCTGTGGCCTTGGCACAGAAGAATGTCAAGCT CTCAACAGAGCAGCTGCGCTGTCTGGCTCACCGGCTCTCTGAGCCCCCCGAGGACCTG GACGCCCTCCCATTGGACCTGCTGCTATTCCTCAACCCAGATGCGTTCTCGGGGCCCC
AGGCCTGCACCCGTTTCTTCTCCCGCATCACGAAGGCCAATGTGGACCTGCTCCCGAG GGGGGCTCCCGAGCGACAGCGGCTGCTGCCTGCGGCTCTGGCCTGCTGGGGTGTGC GGGGGTCTCTGCTGAGCGAGGCTGATGTGCGGGCTCTGGGAGGCCTGGCTTGCGACC TGCCTGGGCGCTTTGTGGCCGAGTCGGCCGAAGTGCTGCTACCCCGGCTGGTGAGCT GCCCGGGACCCCTGGACCAGGACCAGCAGGAGGCAGCCAGGGCGGCTCTGCAGGGC GGGGGACCCCCCTACGGCCCCCCGTCGACATGGTCTGTCTCCACGATGGACGCTCTG CGGGGCCTGCTGCCCGTGCTGGGCCAGCCCATCATCCGCAGCATCCCGCAGGGCATC GTGGCCGCGTGGCGGCAACGCTCCTCTCGGGACCCATCCTGGCGGCAGCCTGAACGG ACCATCCTCCGGCCGCGGTTCCGGCGGGAAGTGGAGAAGACAGCCTGTCCTTCAGGC AAGAAGGCCCGCGAGATAGACGAGAGCCTCATCTTCTACAAGAAGTGGGAGCTGGAAG CCTGCGTGGATGCGGCCCTGCTGGCCACCCAGATGGACCGCGTGAACGCCATCCCCT TCACCTACGAGCAGCTGGACGTCCTAAAGCATAAACTGGATGAGCTCTACCCACAAGGT TACCCCGAGTCTGTGATCCAGCACCTGGGCTACCTCTTCCTCAAGATGAGCCCTGAGG ACATTCGCAAGTGGAATGTGACGTCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCAAC AAAGGGCACGAAATGAGTCCTCAGGCTCCTCGGCGGCCCCTCCCACAGGTGGCCACC CTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACACCC TGACCGCCTTCTACCCTGGGTACCTGTGCTCCCTCAGCCCCGAGGAGCTGAGCTCCGT GCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTGTGACCCAAG GCAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGTCC GAATACTTCGTGAAGATCCAGTCCTTCCTGGGTGGGGCCCCCACGGAGGATTTGAAGG CGCTCAGTCAGCAGAATGTGAGCATGGACTTGGCCACGTTCATGAAGCTGCGGACGGA TGCGGTGCTGCCGTTGACTGTGGCTGAGGTGCAGAAACTTCTGGGACCCCACGTGGAG GGCCTGAAGGCGGAGGAGCGGCACCGCCCGGTGCGGGACTGGATCCTACGGCAGCG GCAGGACGACCTGGACACGCTGGGGCTGGGGCTACAGGGCGGCATCCCCAACGGCTA CCTGGTCCTAGACCTCAGCATGCAAGAGGCCCTCTCGGGGACGCCCTGCCTCCTAGGA
CCTGGACCTGTTCTCACCGTCCTGGCACTGCTCCTAGCCTCCACCCTGGCCTGA
Variants of the sequences above are also included. The term variant is define elsewhere herein but includes biologically active variants with at least 90% sequence identity to the sequences shown above. Different isoforms of MSLN are also included. There are 4 different isoforms of mesothelin which are produced by alternative splicing. SEQ ID NO. 1 is isoform 1 which is the canonical sequence.
Isoforms 2, 3 and 4 differ from isoform 1 as follows:
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:
MQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO. 508) VQGGRGGQARAGGRAGGVEVGALSHPSLCRGPLGDALPPRTWTCSHRPGTAPSLHPGLR APLPC (SEQ ID NO. 509) Isoform 4: The sequence of this isoform differs from the canonical sequence as follows: Residues 44-44: Missing. Residues 409-416: Missing.
The terms "MSLN binding molecule/protein/polypeptide/agent/moiety”, "MSLN antigen binding molecule molecule/protein/polypeptide/agent/moiety”, “anti- MSLN single domain antibody”, “anti- MSLN single immunoglobulin variable domain”, “anti- MSLN heavy chain only antibody” or “anti- MSLN antibody” all refer 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. A single domain antibody as described herein, "which binds" or is “capable of binding” an antigen of interest, e.g. 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.
In one embodiment, the invention relates to an isolated CAR comprising a VH single domain antibody that specifically binds to human MSLN wherein the VH single domain antibody comprises a CDR1 comprising SEQ NO. 4 or a sequence with 1 , 2 or 3 amino acid modifications, a CDR2 comprising SEQ NO. 5 or a sequence with 1 , 2, 3 or 4 amino acid modifications and a CDR3 comprising SEQ NO. 6 or a sequence with 1 , 2 or 3 amino acid modifications.
SEQ ID NO. 3 VH single domain antibody full length sequence (VH 1.1 , also termed MSLN VH 1.1) (CDRs underlined)
EITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWNDDKRYR PSLKNRLTIAKDTSKNQVVLTMTNMDPVDTARYYCAHYSTSSETAFDI RGQGTMVTVSS
The CDRs of SEQ ID NO. 3 are as follows:
CDR1 SEQ ID NO. 4 : TSGLGVG
CDR2 SEQ ID NO. 5 : LIYWNDDKRYRPSLKN
CDR3 SEQ ID NO. 6 : YNTSSETAFDI
In one embodiment, 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.
In one embodiment, the variant (VH1.2) has a substitution of S to N in CDR3 and the CDR3 sequence is: SEQ ID NO. 7: YNTSSETAFDI
A variant as used herein retains a biological function of the single domain antibody, that is binding to the target antigen (e.g. MSLN) and, thus, the variant antibody or antibody fragment thereof can be sequence engineered. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence provided that the CDRs are as defined above.
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.
In one embodiment, the modification (amino acid substitutions, insertion, addition or deletion) is in HCDR1 , HCDR2 and or HCDR3 as defined above.
In one embodiment, variations are only in the framework sequences and the VH single domain antibody comprises CDR1 , 2 and 3 (e.g. SEQ ID Nos 4, 5 and 6 or 4, 5 and 7).
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.
In one embodiment, the modification is a conservative sequence modification. As used herein, the term "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. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more 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. Thus, 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. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. 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.
Table 1. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions
Figure imgf000016_0001
Figure imgf000017_0001
A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including yeast display and in vitro and in vivo expression libraries. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and I or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the antibodies described herein.
In one embodiment, modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence. More specifically, 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.
To return one or more of the amino acid residues in the framework region sequences to their germline configuration, 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.
In still another embodiment, glycosylation is modified. For example, 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. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, 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. In one embodiment, 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.
In one embodiment, the one or more substitution, addition or deletion is in the framework region. For example, there may be 1 to 20, e.g. 1 to 10 or more amino acid substitutions in the framework regions.
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. For example, 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.
In one embodiment, one or more non-germline residue in SEQ ID NO. 3 is replaced with a germline residue. Thus, 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.
Position (Kabat) residue in SEQ ID NO. 3 residue in germline (VH2-05)
1 E Q
34 L V 60 R S
65 N S
70 A T
103 R W
As used herein, the terms 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. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither 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). The KD is calculated from the quotient of koff/kon, whereas 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, and 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.
The term "specific 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. In one embodiment, 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.
The 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.
The terms “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. As used herein, 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.
The term “epitope” 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. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e. , 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.
In one aspect, the VH single domain antibody as described above has one or more of the following properties:
(a) binds cyno MSLN;
(b) binds human MSLN with a ka of about 1 .45 x 106, a kd of about 4.49 x 10'2 and a KD of about 3.09 x 10'8 (30.9 nM) using a surface plasmon resonance (SPR) assay on Biacore 8K using immobilised cyno MSLN recombinant protein assay;
(c) binds human MSLN with a ka of about 1.15-1.6 x 106, 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;
(d) binds cyno MSLN with a ka of about 1.07 x 106, a kd of about 4.68 x 10'2 and a KD of about 4.38 x 10'8 (43.8 nM) using a surface plasmon resonance (SPR) assay on Biacore 8K using immobilised cyno MSLN recombinant protein assay;
(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.
Multispecific binding molecules
In one aspect, there is provided 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. Accordingly, 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. In one embodiment, 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.
In one embodiment, 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. In one embodiment, 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. In one embodiment, the at least second moiety is a VH domain.
In one embodiment, the binding molecule is bispecific. Thus, in one aspect, 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.
In one embodiment, 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. Thus, 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 VH 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.
The term "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. In one embodiment, 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), GSGSGSGS (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.
As used herein, 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). Preferably, a target is a cell-surface target, such as a cell-surface protein. In one embodiment, 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.
In one embodiment, the binding molecule binds MSLN and PSMA. In one embodiment, 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).
1 MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEAT NITPKHNMKA
61 FLDELKAENI KKFLYNFTQI PHLAGTEQNF QLAKQIQSQW KEFGLDSVEL AHYDVLLSYP
121 NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDIVPP FSAFSPQGMP EGDLVYVNYA
181 RTEDFFKLER DMKINCSGKI VIARYGKVFR GNKVKNAQLA GAKGVILYSD PADYFAPGVK
241 SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGIAEAVGLP SIPVHPIGY
301 DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTN EVTRIYNVIG
361 TLRGAVEPDR YVILGGHRDS VWFGGIDPQS GAAVVHEIVR SFGTLKKEGW RPRRTILFAS 421 WDAEEFGLLG STEWAEENSR LLQERGVAYI NADSSIEGNY TLRVDCTPLM YSLVHNLTKE
481 LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGI ASGRARYTKN 541 WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANS IVLPFDCRDY 601 AVVLRKYADK IYSISMKHPQ EMKTYSVSFD SLFSAVKNFT EIASKFSERL QDFDKSNPIV 661 LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGIYD ALFDIESKVD 721 PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA
In one embodiment, 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
The CDRs of SEQ ID NO. 18 are shown below
CDR1 SEQ ID NO. 19: GYGMH
CDR2 SEQ ID NO. 20: YISYDGSNKYYADSVKG
CDR3 SEQ ID NO. 21 : DPAWGLRLGESSSYDFDI
Suitable 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. In one embodiment, 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.
In one embodiment, the one or more modification is in the CDR1 , 2 or 3 region. For example, there may be 1 , 2, 3 or more amino acid modification is in the CDR1 , 2 or 3.
In one embodiment, the one or more modification is in the framework region. For example, there may be 1 to 20, e.g. 1 to 10 or more amino acid substitutions framework regions.
In one embodiment, 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. In one embodiment, 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%.
In one embodiment, 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 2a Family 1 PSMA binders
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Table 2b Family 2 PSMA binders. Note that the PSMA binder identified in SEQ ID 18 belongs to this family.
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Table 2c Family 3 PSMA binders
Figure imgf000031_0002
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Table 2d Family 4 PSMA binders
Figure imgf000035_0002
Figure imgf000036_0003
Table 2e Family 5 PSMA binders
Figure imgf000036_0001
Table 2f Family 6 PSMA binders
Figure imgf000036_0002
Figure imgf000037_0001
Table 2g Family 7 PSMA binders
Figure imgf000037_0002
Figure imgf000038_0001
Figure imgf000039_0001
Table 2h Family 8 PSMA binders
Figure imgf000039_0002
Table 2i Family 9 PSMA binders
Figure imgf000039_0003
Table 2j Family 10 PSMA binders
Figure imgf000040_0001
Table 2kFamily 11 PSMA binders
Figure imgf000040_0002
Table 2I Family 12 PSMA binders
Figure imgf000040_0003
Figure imgf000041_0001
Table 2m Family 13 PSMA binders
Figure imgf000041_0002
Table 2n Family 14 PSMA binders
Figure imgf000041_0003
Table 2o Family 15 PSMA binders
Figure imgf000042_0001
As described above, 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.
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. In one embodiment, 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).
The term "half-life" as used herein 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. For example, 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).
Exemplary modifications
In one embodiment, 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. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
In still other embodiments, 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.
In one embodiment, the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin.
In another aspect, 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.
To generate multivalent binding agents and fusion proteins as described above, two or more polypeptides can be connected by a linker, for example a polypeptide linker. Suitable linkers include for example a linker with GS residues such as (Gly4Ser)n, where n=from 1 to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. Exemplary methods for making the VH single domain antibody
In one embodiment, 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.
In the above method, 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. Thus, in one embodiment, 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.
For example, the rodent is a mouse. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, the mouse does not make a functional endogenous lambda light chain. In one embodiment, 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. For example, 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. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional lambda light chain.
Furthermore, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, the mouse does not make a functional endogenous kappa light chain. In one embodiment, 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.
In one embodiment, 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. Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus. Thus, the mouse does not make a functional endogenous heavy chain. In one embodiment, 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. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional heavy chain.
For example, as described in WO 2004/076618 (hereby incorporated by reference in its entirety), all 8 endogenous heavy chain constant region immunoglobulin genes (p, 5, y3, y1 , y2a, y2b, s and a) 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. By 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. In another embodiment, the locus is functionally silenced.
For example, 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. Thus, 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. As well as comprising all three cisacting 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.
For example, 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.
Alternative methods known in the art may be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of 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.
The main differences between these broad methods are that ES clones can be screened extensively before their use to create a transgenic animal. In contrast, 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.
There are many methods known in the art to both assist with and determine whether successful integration of transgenes occurs. Transgenic animals can be generated by multiple means including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are various tools and techniques that can be used to both drive and select for transgene integration and subsequent modification including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques, and nucleases to improve the efficiency of recombination. Most of these methods are commonly used in the modification of ES cells.
However, some of the techniques may have utility for enhancing transgenesis mediated via pronuclear injection.
Further refinements can be used to give more efficient generation of the transgenic line within the desired background. As described above, in preferred embodiments, the endogenous mouse immunoglobulin expression is silenced to permit sole use of the introduced transgene for the expression of the heavy-chain only repertoire that can be exploited for drug discovery. Genetically manipulated mice, for example TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain and mouse lambda chain) can be used as described above. The transfer of any introduced transgene to this TKO background can be achieved via breeding, (either conventional or with the inclusion of an IVF step to give efficient scaling of the process). However, it is also possible to include the TKO background during the transgenesis procedure. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for use in transgenesis. Triple knock-out mice into which transgenes have been introduced are referred to herein as TKO/Tg. In one embodiment, the mouse is as described in WO 2016/062990. The transgenic rodent described above produces human variable heavy chains which can be isolated and used for the generation of human VH domains, for example as described in WO 2016/062990, WO2016113556 and Teng et al. Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse. N Biotechnol 2020;55:65-76.
In one embodiment, the mouse is as described in WO2016/062990.
Thus the invention also relates to a genetically modified rodent, e.g. a mouse or a rat which expresses a human heavy chain locus and which has been immunized with a MSLN antigen. The rodent may have the features set out above. The invention also relates to a rodent as described above, e.g. a mouse or a rat which expresses a heavy chain only antibody comprising a human VH domain that binds to human MSLN. Preferably, 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.
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. Preferably, 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.
Polynucleotides and cells In another aspect, the invention relates to an isolated nucleic acid molecule comprising at least one nucleic acid encoding a single domain antibody as defined above. This includes therefore the nucleic acid encoding a VH domain that targets MSLN as described herein. The nucleic acid sequence encoding SEQ ID NO. 3 is shown below.
GAGATCACCT TGAAGGAGTC TGGTCCTACG CTGGTGAAAC CCACACAGAC CCTCAC GCTG ACCTGTACCT TCTCTGGCTT CTCACTCAGC ACTAGTGGAC TGGGTGTGGG CT GGATCCGT CAGCCCCCAG GAAAGGCCCT AGAATGGCTT GCACTCATTT ATTGGAATG A TGATAAACGC TACAGACCAT CTCTGAAGAA CAGGCTCACC ATCGCCAAGG ACACC TCCAA AAACCAGGTG GTCCTTACAA TGACCAACAT GGACCCTGTG GACACAGCCA G ATATTACTG TGCACATTAT AGCACCTCGT CCGAGACTGC TTTTGATATC CGGGGCCA AG GGACAATGGT CACCGTCTCC TCA
SEQ ID NO. 22
In one embodiment, the variant has a substitution and the C underlined above at position 117 is replaced with an A.
The nucleic acid sequence encoding SEQ ID NO. 18 is shown below. GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGGAAGTAATAAAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG GCCAAGGGACAATGGTCACCGTCTCCTCA
SEQ ID NO. 23
The nucleic acid sequence encoding SEQ ID NO. 18 may be as shown below or a variant thereof.
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGGAAGTAATAAAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG GCCAAGGGACAATGGTCACCGTCTCCTCA
SEQ ID NO. 23
Nucleic acids encoding other PSMA VH domains as described herein can also be included. For example, the nucleic acid may encode one of the VH amino acid sequences shown in Table
2. Exemplary sequences are listed below.
Family 1
SEQ ID NO. 408 (encodes VH domain 1.1)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCATGAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATGATGGTACCACA
GACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAGTAT
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO. 409 (encodes VH domain 1.2)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATAATAATAATAGCACA
GAGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCA
CGCTGTATCTGCAAATGAACAGCCTGAGCGCCGAGGACACGGCCGTATATTACTGTGT
GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA
SEQ ID NO. 410 (encodes VH domain 1.3) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGACTGGAGTGGGTCTCAAGTATTGGTGATAATAATAATAGCACA
GACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGTA
CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGT
GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA
SEQ ID NO. 411 (encodes VH domain 1.4)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATGGAACCACATACTAC
GCAGACTCCGTGAAGGGCCGTTTCACCATCTCCAGAGACAATTCCAAGAGCACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAT
GGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA
SEQ ID NO. 412 (encodes VH domain 1.5)
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCACTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAAAATGATCGAACCACA
TACTACGTAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA
SEQ ID NO. 413 (encodes VH domain 1.6)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATAATAATAGAACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCA
CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGC
GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 414 (encodes VH domain 1.7)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATGGAACCACATACTAC
GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAT
GGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO. 415 (encodes VH domain 1.8)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCATGAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATGATGGTACCACA
GACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 416 (encodes VH domain 1.9)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCACA
GACTACGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 417 (encodes VH domain 1.10)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCACA
GACTACGCAGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 418 (encodes VH domain 1.11)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCACA
GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATA
CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG
AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 419 (encodes VH domain 1.12) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCACA
GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATA CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG
AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 420 (encodes VH domain 1.13)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCACA
GACTACGCAGCCGACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATA
CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG
AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO. 421 (encodes VH domain 1.14)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCACA
GACTACGCAGACGTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 422 (encodes VH domain 1.15)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCACA
GACTACGCAGCCTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 423 (encodes VH domain 1.16)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCACA
GACTACGCAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 424 (encodes VH domain 1.17)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCACA
GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATA
CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG
AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 425 (encodes VH domain 1.18)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCACA
GACTACGCAGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 426 (encodes VH domain 1.19)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCACA
GACTACGCAGCCTACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 427 (encodes VH domain 1.20) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCACA
GACTACGCAGCCACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGA
AAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO. 428 (encodes VH domain 2.3)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGCTTCAGTGGCTATGGCATGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACATATATCATATGATGGAAGTAATAG ATACTATGCAGAATCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAATTCCAAGAACA
CGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC
GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGACATTT
GGGGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 429 (encodes VH domain 2.4) CAGGTCACCTTGAAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAAA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCG
AGAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGAAATCTG
GGGCCAAGGGACAATGGTCACCGTCTCCTCA
SEQ ID NO. 430 (encodes VH domain 2.5)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
ACTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGAAATTTGG
GGCCAAGGGACAATGGTCACCGTCTCTTCA
SEQ ID NO. 431 (encodes VH domain 2.6)
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGGAACTATCGTCCTATAAATTTGAAATCTGG
GGCCAAGGGACAATGGTCACCGTCTCTTCA
SEQ ID NO. 432 (encodes VH domain 2.7)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGTAATAAAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA AGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGATATCTGGG
GCCAAGGGACAATGGTCACCGTCTCCTCA
SEQ ID NO. 433 (encodes VH domain 2.8)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGTCAGTTATATCATATGATGGAAGTAATAAAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGAAATCTGGG
GCCAAGGTACAATGGTCACCGTCTCCTCA
SEQ ID NO. 434 (encodes VH domain 2.9)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGAAATCCGG
GGCCAGGGGACAACGGTCACCGTCTCTTCA
SEQ ID NO. 435 (encodes VH domain 2.10)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATATATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAGAC
GCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCG AAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCATATGATTTTGATATCTG
GGGCCAAGGGACAATGGTCACCGTCTCCTCA
SEQ ID NO. 436 (encodes VH domain 2.11)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAATAAAT
ACTATGCACCCAGCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACCGTCTCCTCA SEQ ID NO. 437 (encodes VH domain 2.12)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAATAAAT
ACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 438 (encodes VH domain 2.13)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATAAAT
ACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 439 (encodes VH domain 2.14)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCGTGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATAAA
TACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGG
GGCCAAGGGACAATGGTCACTGTCTCTTCA SEQ ID NO. 440 (encodes VH domain 2.15)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAATAAAT
ACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 441 (encodes VH domain 2.16)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCGCGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAATAAA
TACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA AAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGG
GGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 442 (encodes VH domain 2.17)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATAAAT
ACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 443 (encodes VH domain 2.18)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCAGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATAAA
TACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGG
GGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 444 (encodes VH domain 2.19) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCTTCCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATAAAT
ACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 445 (encodes VH domain 2.20)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAGCTTCAGTGGCTATGGCATGCACTGGGTCCGCC AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTAATAG
ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA
CGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC
GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGAAATTT
GGGGCCAAGGGACAATGGTCACCGTCTCCTCA SEQ ID NO. 446 (encodes VH domain 2.21)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAAA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTCTCTACAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGATCCGGCCTGGGGATTACGTTTGGGGAAATTATCGTCCTATGATTTTGAAATCTGG
GGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 447 (encodes VH domain 2.22) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCACGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGGGAGTAATAA
ATACTATGCAGCCCCGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA
CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC
GAAAGACGCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCT
GGGGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 448 (encodes VH domain 2.23)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCACGCACTGGGTCCGCC AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAATAAA
TACTATGCATCCAGCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGACCGGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGG
GGCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 449 (encodes VH domain 2.24)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAATAAAT
ACTATGCAAGGCTGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGACACGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGGG
GCCAAGGGACAATGGTCACTGTCTCTTCA
SEQ ID NO. 450 (encodes VH domain 2.25) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACCTGAGTAATAAAT
ACTATGCAAGGGGGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGACGTGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCTGG
GGCCAAGGGACAATGGTCACTGTCTCCTCA family 3
SEQ ID NO. 451 (encodes VH domain 3.1) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGACATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGATGAGGACACGGCTCTATATTACTGTGCGA
GAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCA
SEQ ID NO. 452 (encodes VH domain 3.2)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATATCATATGATGGAAGTAATAAAT
ATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAA
AGATCGTATAGTGGGAGCCAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 453 (encodes VH domain 3.3)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCCTCATTAGCTATGGCATGAACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATATCATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATAGTGGGAGCTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 454 (encodes VH domain 3.4) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGCGGTCCAGCCTGGGAGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCC
AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG
ATATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA
CGCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCG
AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA
CAATGGTCACCGTCTCCTCA
SEQ ID NO. 455 (encodes VH domain 3.5)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 456 (encodes VH domain 3.6)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 457 (encodes VH domain 3.7)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGGACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA
ATGGTCACTGTCTCTTCA
SEQ ID NO. 458 (encodes VH domain 3.8)
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAGGGAACC CTGGTCACTGTCTCCTCA
SEQ ID NO. 459 (encodes VH domain 3.9)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATATCATATGATGGAAGTAATAGAT ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACCGTCTCTTCA
SEQ ID NO. 460 (encodes VH domain 3.10)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACTGTCTCCTCA
SEQ ID NO. 461 (encodes VH domain 3.11)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACTGTCTCCTCA SEQ ID NO. 462 (encodes VH domain 3.12)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 463 (encodes VH domain 3.13)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACTGTCTCCTCA
SEQ ID NO. 464 (encodes VH domain 3.14)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACTGTCTCCTCA SEQ ID NO. 465 (encodes VH domain 3.15)
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACCGTCTCCTCA
SEQ ID NO. 466 (encodes VH domain 3.16)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAA AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCCTCA
SEQ ID NO. 467 (encodes VH domain 3.17)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACCGTCTCCTCA
SEQ ID NO. 468 (encodes VH domain 3.18)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGAT
ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTTTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCTGTATATTACTGTGCGAA
AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACA ATGGTCACCGTCTCCTCA
SEQ ID NO. 469 (encodes VH domain 3.19) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGACATATGATGGAAGTAATAGA
TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
GAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGAC AATGGTCACCGTCTCTTCA
SEQ ID NO. 470 (encodes VH domain 3.20)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGGCAGTAATAGA
TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
GAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGAC AATGGTCACCGTCTCTTCA SEQ ID NO. 471 (encodes VH domain 3.21)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGGCAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
GAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCA
SEQ ID NO. 472 (encodes VH domain 3.22) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGCCAGTAATAGA
TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
GAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCA
SEQ ID NO. 473 (encodes VH domain 3.23)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATAACATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGA
AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC
AATGGTCACTGTCTCCTCA
SEQ ID NO. 474 (encodes VH domain 3.24)
AGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGAC
TCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCAG
GCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATAACATATGATGGAAGTAATAGATA
CTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC TTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAA
GATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGACAAT
GGTCACTGTCTCCTCA family 4
SEQ ID NO. 475 (encodes VH domain 4.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCTTGAGA
CTCTCCTGTGTAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCGGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTATTGTGCGA
AAGAGAGGATTTTTGGAGTGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGGACA
ACGGTCACCGTCTCCTCA
SEQ ID NO. 476 (encodes VH domain 4.2)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGAGAGGATTTTTGGAGTGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGGACA
ACGGTCACTGTCTCCTCA
SEQ ID NO. 477 (encodes VH domain 4.3)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAGCTAATAGA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTATTGTGCGA
AAGAGAGGATTTTTGGCGTGCTTACCCCTGATGATTTTGAAATCTGGGGCCAAGGGACA
ACGGTCACCGTCTCCTCA
SEQ ID NO. 478 (encodes VH domain 4.4)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCACTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTGCGA AAGAGAGGATTTTTGGAGCGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGGACA
ACGGTCACCGTCTCTTCA family 5
SEQ ID NO. 479 (encodes VH domain 5.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAATACTAAAT
ATTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAATAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGAA AGGTTTATGGCCTTCGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA
SEQ ID NO. 480 (encodes VH domain 5.2)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACTATGGCATGCACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAATAGTAAAT
ATTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG
CTGTATCTGCAAATGAATAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGAA AGGTTTATGGCCTTCGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA family 6
SEQ ID NO. 481 (encodes VH domain 6.1)
CAGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCC
TCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGGTC
CGCCAGCACCCAGGGAAGGACCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGCA
TCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAAC CAGTTCTCCCTGAAAATGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGC
GAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA
SEQ ID NO. 482 (encodes VH domain 6.2)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGAT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC
ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAA
CCAGTTCTCCCTGAAAATGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTG
CGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA
SEQ ID NO. 483 (encodes VH domain 6.3)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGGT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAA
CCAGTTCTCCCTGAAACTGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTG
CGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA
SEQ ID NO. 484 (encodes VH domain 6.4)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGAT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC
ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAA CCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT
GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA
SEQ ID NO. 485 (encodes VH domain 6.5)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGGT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC
ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAA
CCAGTTCTCCCTGAAAATGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTG
CGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA
SEQ ID NO. 486 (encodes VH domain 6.6)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGGT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC
ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAA
CCAGTTCTCCCTGAAACTGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTG
CGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA
SEQ ID NO. 487 (encodes VH domain 6.7) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC
CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGGGT
CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAGC
ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAA
CCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCA family 7
SEQ ID NO. 488 (encodes VH domain 7.1)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA
SEQ ID NO. 489 (encodes VH domain 7.2)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC
GAGAGATAACCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA SEQ ID NO. 490 (encodes VH domain 7.3)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGGGGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACT
SEQ ID NO. 491 (encodes VH domain 7.4)
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACCAGGGAAGTGAGAA ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC
CGTCTCCTCA
SEQ ID NO. 492 (encodes VH domain 7.5)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACCCCGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC
GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA
SEQ ID NO. 493 (encodes VH domain 7.6)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGAGGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC
GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA
SEQ ID NO. 494 (encodes VH domain 7.7) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACATCGGAAGTGAGAAA
TACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC
ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGCG
AGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA
SEQ ID NO. 495 (encodes VH domain 7.8)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC
GAGAGATACCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA family 8
SEQ ID NO. 496
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCC
CTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCA
GCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAAC
TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTT
CTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGA GGCCCCATACCAGCCACTGCTATACCCGATGCTTTTGATATCTGGGGCCAAGGGACAAT GGTCACTGTCTCCTCA family 9
SEQ ID NO. 497
GAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCC
CTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTCACTACTGGAGCTGGATCCGCC
AGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGACATAAATCATAGTGGAAGCACCAA
CTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAATCAGT
TCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGTGAG
AGACTACGGTGACTCCCGTAGCCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCTTCA family 10 SEQ ID NO. 498
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCA
GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGTCATATGATGGCAGTAATAAA
TACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
AAGGCGATTACGATTTTTGGAGTGGTTACCCCGACTACGATATGGACGTCTGGGGCCAA GGGACCACGGTCACCGTCTCCTCA family 11
SEQ ID NO. 499 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCAACTTGATTAGCTATGGCATGTACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAA AACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATAC GCTGTTTCTGCAAATGAACAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGA
AAGGGGGGAATGCCTTGTATAGCAGTGGCTGGCCCGATGATGGTTTTGATATCAGGGG
CCAAGGGACAATGGTCACTGTCTCCTCA family 12
SEQ ID NO. 500
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTTTGGCATGCACTGGGCCCGCCA
GGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTAATATCATATGATGGAAATAGTAAAT
ACTATGCAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGGAAATGAACAGCCTGAGAGCTGATGACACGGCTGTGTATTACTGTGCGAA
AGGCCTATGGCCCCCAATGGACGTCAGGGGCCAAGGGACCACGGTCACCGTCTCCTC A family 13
SEQ ID NO. 501
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTCCAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTGACTATTGGATGACCTGGGTCCGCCA
GGTTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAA
ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT
CACTATATCTGCAAATGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCG AGAGATCGAGGAGGAGCAGTGGCCCTTTATCACAACGGTATGGACATGGGGGGCCAAG
GGACCACGGTCACTGTCTCTTCA family 14
SEQ ID NO. 502
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT
CTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGG
CCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACACAGG
CTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCACA
GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGA
GAGGGAACGGGCCCGGTATAACTGGAACTACTGACTACTGGGGCCAGGGAACCCTGG TCACTGTCTCTTCA family 15
SEQ ID NO. 503 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCA AGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATCGTACC GGTTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC CCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGGGA GAGAGAATGTTATAGTACCAGCTGCTACCTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA
The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" 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.
In another aspect, the invention 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. For example, 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.
In one embodiment, 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. For example, retroviruses such as adenovirus vectors can be used. In one embodiment, a lentivirus vector is used. In a further aspect, the invention also relates to an isolated cell or cell population comprising one or more nucleic acid construct or vector as described above. In one embodiment, 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). 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.
Pharmaceutical compositions
In another aspect of the present invention, there is provided a pharmaceutical composition 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, whether they are solutions, suspensions or other like form, 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. 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.
The 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.
Typically, this amount is at least about 0.01 % of a binding molecule of the present invention by weight of the composition.
Preferred 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. For intravenous administration, 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.
The present compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, 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.
The pharmaceutical composition of the invention can be co-administered with other therapeutics, for example anti-cancer agents. Exemplary combinations with other agents
The molecules or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of 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. In one embodiment, 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. Thus, in another aspect, 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. Examples of other 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, 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. In one embodiment, the single domain antibody is used in combination with surgery.
In a specific embodiment 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 a pharmaceutical composition described herein is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, 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.
In some embodiments, 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. In some embodiments, 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.
Methods of treatment
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.
In one embodiment, 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. In one embodiment, 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.
The term "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. Thus, 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. Examples of various cancers are described further herein and 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.
The phrase "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. Examples of various cancers that express mesothelin include but are not limited to, mesothelioma, lung cancer, ovarian cancer, pancreatic cancer, and the like.
In one embodiment, 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.
In one embodiment, 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. In addition, 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; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In one aspect, where the binding agent binds MSLN and PSMA, the binding agent is used to target PSMA and treat prostate cancer.
In therapies of prostatic disorders, e.g., prostate cancer, the therapy can be used in combination with existing therapies. In one embodiment, the binding agent is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, 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. Examples of other 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, 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. In one embodiment, the 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.
In one embodiment, 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. In another embodiment, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, avelumab, durvalumab, or atezolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Pidilizumab, Toripalimab, Ipilimumab or Tremelimumab. In another embodiment, 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.
Immunoconjugates and other agents
In another aspect, there is provided 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.
Kits and methods
In another aspect, 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. In one embodiment, 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.
Exemplary non- therapeutic applications
In another aspect, 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. For example, 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.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents and sequence database identifiers mentioned in this specification are incorporated herein by reference in their entirety.
"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
The invention is further described in the non-limiting examples.
Examples Materials and methods
Construction of Tg/TKO mice
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 (triple knock-out, or TKO) 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 al30 all incorporated herein by reference). Briefly, 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. 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. The construction and use of YACs is well known in the art (e.g., Bruschi, C.V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002, Macmillan Publishers Ltd., Nature Publishing Group / www.els.net).
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.
Generation of VH domains
EXAMPLE 2. Immunisation Protocol to generate MSLN binders DNA immunisations were carried out at Aldevron www.aldevron.com
Full length mesothelin cDNA was cloned into a proprietary expression vector with an expression tag. Plasmid DNA was transiently transfected into cells and surface expression confirmed by flow cytometry, detecting the tag.
MSLN is synthesized as a 71-kD precursor protein, then cleaved by the endoprotease furin into:
• secreted N-terminal region (a. a.1-295), called megakaryocyte potentiating factor (MPF)
• 41 -kD mature MSLN (296-606) cell surface protein, which remains tethered to the membrane via a GPI link
• The full length protein is 622 amino acids, 607-622 are lost during processing of the GPI anchor
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. On day 59, 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. As 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.
For immunisations, isolation and characterisation of PSMA binders see WO 2017/122017 incorporated herein by reference.
EXAMPLE 3. Serum ELISA
At the end of the immunisation, 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 ™/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.
ELISA results showed that all animals had generated an immune response to human mesothelin. In addition, all animals showed cross reactivity to cyno mesothelin.
EXAMPLE 4. Generation of Libraries from Immunised Mice a) processing tissues, RNA extraction and cDNA synthesis
Inguinal and axilliary lymph nodes and spleens were collected from each immunised animal into RNAIater®. At the point of processing, 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.
PCR was performed as follows:
98°C 30sec 98°C 10sec 'I
58°C 20sec j 2 cycles
72°C 2min
72°C 5min
Hold at 10°C
PCR products (cloned VH) were analysed on a 1 % (w/v) agarose gel. c) Creation of phagemid libraries in E. coli
Cloned 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. The remainder was plated on large format Bioassay dishes containing 2xTY agar supplemented with 2% (w/v) glucose and 100pg/ml ampicillin. All agar plates were incubated overnight at 30°C. Colonies were scraped into 10 ml of 2xTY broth; aliquots were stored at minus 80°C in cryovials after addition of 50% (v/v) glycerol solution.
All library sizes were in excess of 107 (range 2.6 x 107 to 4.9 x 107).
EXAMPLE 6. Selection strategies for isolation of MSLN binders
Preparation of library phage stocks and phage display selections were performed essentially according to published methods (Antibody Engineering, edited by Benny Lo, chapter 8, p161 - 176, 2004).
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.
A number of colonies from R2 outputs were assayed for binding to human and cyno mesothelin by phage ELISA (40-48 colonies per output). Binding to human 5T4-his (negative control) was also assayed.
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.
All outputs contained clones binding to both human and cyno mesothelin. There was no binding to human 5T4-His, as expected.
EXAMPLE 7. Sequencing MSLN binders from Round 2 outputs and identification of clones
All clones found to cross-bind human and cyno mesothelin were sequenced. 76 clones returned full length high quality sequence. One of these clones was MSLN VH1.2, from which the final clone MSLN VH1.1 was derived.
Amino acid sequence of MSLN VH1.2 SEQ ID NO. 504 EITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWNDDKRYR PSLKNRLTIAKDTSKNQWLTMTNMDPVDTARYYCAHYNTSSETAFDIRGQGTMVTVSS
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. The amino acid sequence of VH1.1 is shown below, with the engineered amino acid underlined.
Amino acid sequence of VH1.1 SEQ ID NO. 3
EITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWNDDKRYR PSLKNRLTIAKDTSKNQWLTMTNMDPVDTARYYCAHYSTSSETAFDIRGQGTMVTVSS
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.
EXAMPLE 9. Stability and binding kinetics of MLN094-B09
Overnight stability
1 OO|JL aliquots of VH1.1 protein were dispensed into two identical 96 well plates and sealed with foil seals. One plate was incubated at 4°C and the other at 40°C for approximately 17 hours. Both samples were analysed by SE-UPLC with positive and negative controls. 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.
VH1.1 was run using single cycle kinetics and a 1 :1 binding model measured the binding kinetics to human mesothelin as: ka = 1.45 x 106 kd = 4.49 x 1 O'2
KD = 3.09 x 10'8 (30.9 nM)
Binding kinetics to cyno mesothelin recombinant protein
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.
VH1.1 was run using single cycle kinetics and a 1 :1 binding model measured the binding kinetics to human cyno mesothelin as: ka = 1.07 x 106 kd = 4.68 x 10’2
KD = 4.38 x 10'8 (43.8 nM)
The amino acid sequence of cyno MSLN is shown below (SEQ ID NO 507).
MALPMARPLSGSCGTPALGSLLFLLFSLGWVQPSRVLAGETRQEAAPLDGILTNAPDIASLS PRQLLGFTCVEVSGLSTELVQELAVALGQKNVKLSAEQLRCLAHRLSEPPEDLDALPLDLLL FLNPDAFSGPQACTHFFSRVAKANVDLLPRGAPERQRLLPAALTCWGVRGSLLSEADVRAL GGLACDLPGRFVAESAEWLPRLVRCLGPLDQDQQEAARAALQRGGPPYGPPSTWSISTL DDLQSLLPVLGQPVIHSIPQGILAAWRQRSSRDPSWQQPEQTVLRPRFRRDVERTTCPPEK EVHEIDESLIFYKKRELEACVDAALLAAQMDRVDAIPFTYEQLDVLKHKLDELYPQGYPESVI
RHLGHLFLKMSPEDIRKWNVTSLETLKALLKVSKGHEMSAQVATLIDRVWGRGQLDKDTA DTLTAFCPGCLCSLSPERLSSVPPSIIGAVRPQDLDTCGPRQLDVLYPKARLAFQNMSGSEY FVKIRPFLGGAPTEDLKALSQQNVSMDLATFMKLRREAVLPLSVAEVQKLLGPHVEGLKVEE QHSPVRDWILKQRQDDLDTLGLGLQGGIPNGYLILDLSVREALSGTPCLLGPGPVLTVLALLL
ASTLA
Human and cyno MSLN have 88.42% identity across the full length sequence (622 amino acids).
EXAMPLE 10. FMAT Direct cell Binding Assay to test MSLN binders
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). 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.
Cells were resuspended at 1 x 105 cells/ml in FMAT assay buffer (pH 7.4) with the addition of 120nM DRAQ5. VH proteins were diluted as a 10-point series ranging from 3.33 x 10'8 M to 1.69 x 10'12 M. 10pl per well was transferred into 384 well black clear-bottomed assay plates with 10pl of 6nM mouse Anti-His and 10pl of 12nM Goat Anti-Mouse Alexa Fluor-488. DRAQ5 stained cells (20pl per well) were added and the assay plates incubated in the dark for 2 hours at room temperature. Plates were read in the FL2 (502nm-537nm) and FL5 (677-800nm) channels on the TTP Mirrorball plate reader following excitation at 488nm and 640nm. Data was gated on FL5 perimeter and peak intensity and the FL2 median mean fluorescence intensity of the gated data used for determination of VH binding.
The EC50 of VH1.1 was found to be 1 .2 nM
EXAMPLE 11. Scale up purification of VH1.1
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. Collected resin was washed with 20 resin volumes of 20 mM sodium phosphate, 500mM NaCI, 20 mM imidazole, pH 7.4 before the bound protein was eluted in fractions of 20 mM sodium phosphate, 500mM NaCI, 500 mM imidazole, pH 7.4. The absorbance at 280 nm of each elution fraction was measured using a nanodrop.
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.
EXAMPLE 12. Kinetic and affinity assessment by BiaCore of MSLN binders
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.
1 :1 binding model measured the binding kinetics to human mesothelin as: ka = 1.15-1.6 x 106 kd = 3.93-4.49 x 10'2
KD = 2.8-3.4 x 10’8 (28-34 nM)
Figure imgf000089_0001
Example 16 CAR
CAR construction
The following 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
PSMA-VH SEQ ID NO. 8
PSMA-VH- (G4S)3 -MSLN-VH SEQ ID NO. 505
EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ GTMVTVSSGGGGSGGGGSGGGGSEITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVG WIRQPPGKALEWLALIYWNDDKRYRPSLKNRLTIAKDTSKNQVVLTMTNMDPVDTARYYCA HYSTSSETAFDI RGQGTMVTVSS
PSMA-VH- (G4S)6 -MSLN-VH SEQ ID NO. 506
EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ
GTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEITLKESGPTLVKPTQTLTL TCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWNDDKRYRPSLKNRLTIAKDTSKNQVVL TMTNM DPVDTARYYCAHYSTSSETAFDI RGQGTMVTVSS
Cell lines
Tumor cell lines 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.
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).
Western blot CAR-T cells were incubated with 2 pg anti-FLAG Ab in 100 pL PBS for 20 mins on ice and then with 2 pg goat antimouse secondary Ab for another 20 mins on ice. Cells were then incubated in the 37°C water bath for the selected time points and then lysed with 2 x Laemelli buffer for 10 mins. Cell lysates were then separated in 4% to 15% 10 well SDS-PAGE gels and transferred to polyvinylidene difluoride membranes at 75V for 120 mins (Bio-Rad). Blots were examined for human CD3 (Santa Cruz Biotechnology), p-Y142 CD3 (Abeam), pan-ERK (BD Biosciences), and pan-Akt, p-S473 Akt, and p-T202/Y204 MAPK (Cell Signaling Technology) with 1 :1000 dilution in 5% TBS-Tween milk. Membranes were incubated with HRP-conjugated secondary goat anti-mouse or goat anti-rabbit IgG (Santa Cruz) at a dilution of 1 :3000 and imaged with the ECL substrate kit (Thermofisher) on the ChemiDoc MP System (Bio-Rad) according to the manufacturer’s instructions.26
Proliferation assay
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. 26
In vitro cytotoxicity assay Tumor cells were seeded in 24-well plates at a concentration of 2.5x105 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. For the granzyme-B staining, 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.
Cytokine analysis
CAR-T cells (1 xio5 cells) were cocultured with 2.5x105 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. 26
Expression and purification of recombinant proteins
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 and kinetic analysis
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). To detect dual binding to MSLN and PSMA, human 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 xio6 cells per mice. Fourteen days later, CAR-T cells were injected intravenously through tail vein. For the high dose treatment, 4x106 CAR-T cells per mice were injected, while for the low dose treatment, 1 xio6 CAR-T cells per mice were injected. In the rechallenge experiments, mice were infused 1 xio6 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). Statistics
All data was calculated and represented as mean with SD. One-way analysis of variance (ANOVA) or two-way ANOVA analyses were performed to compare multiple groups. Two- tailed t-test was used to compare two groups. P value of less than 0.05 was significant. All calculations and figures were achieved by GraphPad Prism V.7 (La Jolla, California, USA).
Results
Human VH domain-based CAR targeting PSMA is expressed and signals in T cells
We constructed the 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. On transduction, 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). We examined 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
To compare the antitumor effect of PSMA-VH-T cells and J591-T cells in vitro, we engineered the PC3 cells to express PSMA antigen (figure 2A), and we observed that J591-T cells and PSMA-VH-T cells showed comparable granzyme-B expression when cultured with or without tumor cells (figure 2B, C and figure 8A). Similarly, both PSMA-VH-T cells and J591-T cells showed equal upregulation and subsequent down regulation of CD69 as a marker of T cell activation (figure 2D). Similar upregulation on antigen stimulation and down regulation after antigen removal were observed for PD-1 and Lag-3 (figure 8B-E). We then cocultured PSMA- VH-T cells and J591-T cells in vitro with tumor cells (either PSMA-positive or PSMA-negative), and measured the remaining tumor cells after 5 days of coculture. CD19-T cells did not eliminate tumor cells, while PSMA-VH-T cells specifically eliminated PSMA-positive target cells (C4-2 and PC3-PSMA) to the same extent as conventional J591-T cells, and did not demonstrate off-target effect on PSMA-negative cells (PC3) (figure 2F,G). We also measured the secretion of IFNy and IL-2 after 24 hours of coculture with tumor cells. When the PSMA- VH-T and J591-T cells target PSMA-positive C4-2 and PC3-PSMA cells, both of them secreted high amount of IFNy and IL-2 compared with control CD19-CAR-T cells (figure 2H,I). Furthermore, PSMA-VH-T cells and J591-T cells proliferated similarly on encounter with tumor cells as shown by CFSE dilution (figure 2J). To investigate the antitumor effects of Humabody VH CAR-T cells in vivo, NSG mice engrafted with PSMA-positive tumor cells labeled with Firefly luciferase were treated with a high doses (4x106 cells/mouse) of CAR-T cells (figure 3A). CAR- T cell treatment showed tumor control as measured by tumor bioluminescence without differences in mice treated with PSMA-VH-T cells or J591-T cells (figure 3B,C). To further assess differences between PSMA-VH-T cells and J591-T cells, we used low doses of T cells (1 xio6 cells/mouse) in tumor-bearing mice (figure 3D). We observed that PSMA-VH-T cells still eliminated tumor cells in vivo as J591-T cells (figure 3E,F). In addition, we also observed similar VH CAR-T cell persistence in the peripheral blood, spleen and bone marrow compared with traditional scFv-based CAR-T cells at day 58 at the time of euthanasia (figure 3G,H). Therefore, Humabody VH CAR-T cells demonstrated comparable antitumor effects to scFv- based CAR-T cells in vitro and in vivo.
MSLN-specific VH domain-based CAR-T cells demonstrate antitumor activity To further assess the reproducibility of VH domain-based CARs, we tested a MSLN-specific Humabody VH. We constructed the conventional MSLN scFv CAR (MSLN-scFv) and VH domain CAR (MSLN-VH) using the same backbone developed for PSMA-specific CARs (figure 4A). MSLN-scFv and MSLN-VH were equally expressed in T cells (figure 9A, B). In a similar fashion, we examined the antitumor activity of MSLN-scFv-T cells and MSLN-VH-T cells in vitro through coculture experiments with tumor cells, cytokine release assay, proliferation assay. MSLN-scFv-T cells and MSLN-VH-T cells selectively eliminated Aspc-1 tumor cells that express MSLN, while spared PC3 cells that do not express MSLN (figure 4B and figure 10). They released similar amount of IFNy and IL-2 (figure 4C) and proliferated on encounter with tumor cells (figure 4D). In the xenotransplant model in NSG mice engrafted with Aspc-1 cells labeled with Firefly luciferase (figure 4E), MSLN-VH-T cells showed even more profound antitumor effects as compared with mice treated with MSLN-scFv CAR-T cells (figure 4F,G), which translated in prolonged survival of the mice (figure 4H). However, we observed similar T cells expansion/persistence between MSLN-VH and MSLN-ScFv (figure 4I). Thus VH domainbased CARs can reproducibility redirect antitumor activity of engineered T cells.
In vitro analysis of monovalent and bivalent VH domain recombinant proteins
To test whether the VH domains are suitable to construct bispecific CARs, two VH domains in tandem recombinant proteins linking PSMA-specific and MSLN-specific VH were generated (figure 5A). To test whether the linkers had any effect on the target binding affinity, two different linkers were used: the (G4S)s linker (‘short flexible linker’) and a longer linker (G4S)e with 6 copies of the (G4S) repeat (‘long flexible linker’). Monomer VH proteins and a MSLN binding scFv were made as controls (figure 5A). Analysis of binding to PSMA recombinant protein by surface plasmon resonance (SPR) Biacore assay showed that the affinity of the PSMA-VH remained the same when the PSMA-VH was formatted with the MSLN-VH domain using either flexible linkers (figure 5B). Similarly, analysis of binding to MSLN recombinant protein by SPR Biacore assay showed that the affinity of the MSLN-VH domain was not altered when the PSMA-VH was formatted with the MSLN-VH using either flexible linkers (figure 5C). In summary, these data show that VH modules in bispecific format are capable of binding their specific target with the same affinity as their monovalent counterparts. Bispecific VH domain-based CAR-T cells demonstrate dual specificity
We constructed a bispecific VH domain CAR to facilitate CAR-T cells to specifically recognize two antigens simultaneously. We used the MSLN-VH and PSMA-VH domains fused with the short (G4S)s linker to generate the bispecific PSMA-VH/MSLN-VH CAR (figure 6A). The PSMA- VH/MSLN-VH CAR was expressed in T cells (figure 6B,C). PSMA-VH-T cells, MSLN-VH-T cells and PSMA-VH/MSLN-VH-T cells were cocultured with tumor the cell line Aspc-1 , which express MSLN, and the PC3-PSMA cell line. We observed the PSMA-VH/MSLN-VH-T can eliminate both tumor cell lines compared with single CAR-T cells, which only eliminate tumor cells expressing the targeted antigen (figure 11A, B). In addition, we also observed the expected cytokine release profile (figure 11C, D). Next, we confirmed that PSMA-VH/MSLN-VH-T cells displayed specific cytotoxicity toward the same cell line PC3 expressing either MSLN or PSMA similar to MSLN-VH-T cells and PSMA-VH-T cells without off-target effect (figure 6D,E). Importantly, when PC3-PSMA and PC3-MSLN were plated as 1 :1 ratio mixture in coculture experiments, only PSMA-VH/MSLN-VH-T cells fully eliminated the tumor cells, although PSMA- VH-T cells and MSLN-VH-T cells showed some bystander killing effect as previously observed27 (figure 6D,E). The in vitro antitumor effect was corroborated by release of IFN-y and IL-2 (figure 6F,G). To evaluate if bispecific VH domain CAR-T cells can eradicate tumors with mixed antigen expression in vivo, we established a metastatic xenograft mouse model by infusing PC3-PSMA cells and PC3-MSLN cells at 1 :1 ratio into NSG mice by intravenous injection. Mice were then treated with CAR19-T, PSMA-VH-T, MSLN-VH-T and PSMA-VH/MSLN-VH-T cells (figure 7A). Dual targeting PSMA-VH/MSLN-VH-T cells controlled the tumor growth more effectively than either single targeting PSMA-VH-T or MSLN-VH-T cells (figure 7B,C). CAR-T cells were detectable in the peripheral blood of these mice up to 4 weeks after infusion (figure 7D). We also observed that T cells expressing bispecific CAR showed similar phenotypic profile as single CAR targeting T cells for exhaustion and memory markers (figure 12A-C). Analyses of antigen expression in tumor cells in vivo showed that tumor cells growing in mice receiving either PSMA-VH-T or MSLN-VH-T cells were predominantly MSLN and PSMA expressing cells, respectively (figure 7E). These results indicate that bispecific CARs generated by joining two human Ab VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.
Discussion
CARs approved by the Food and Drug Administration and those in clinical studies are mostly based on scFv-binding moieties. Here we demonstrated that monospecific human VH domainbased CAR-T cells achieved comparable antitumor effects both in vitro and in vivo as scFv- based CAR-T cells. Furthermore, VH domains combined in tandem to create bispecific molecules allowed the generation of effective CAR-T cells targeting two antigens. Redirected T cell based on single-domain Abs have been recently proposed.17 28 29 However, most of them are obtained from llamas or camelid-derived libraries. Biological therapeutic molecules with non-human sequence can cause immune responses.18 28 Transgenic mouse technology has enabled the generation of biophysically robust fully human VH domains known as Humabody VH or Humabodies30 which have the potential for use in CAR constructs while mitigating immunogenicity risk. Despite the remarkable clinical activity of CAR-T cells in hematological malignancies, objective responses in patients with solid tumors are modest.10 26 31-33 Heterogeneity of antigen expression is one of the main reasons causing tumor escape in solid tumors after targeted therapies.10 1920 Furthermore, murine-based scFv may cause immune responses especially in solid tumor patients who are usually less immunosuppressed compared with patients with liquid tumors. Targeting multiple TAAs and using human binding moiety in CAR molecules may improve the outcome of CAR-T cells in solid tumors.10 Here, we demonstrated that human VH domains generated from a transgenic mouse might solve both issues of immunogenicity and tumor heterogeneity since bispecific CAR-T cells can be efficiently generated using two human VH domains in tandem.
In addition to the issue of heterogeneity in antigen expression, the complex inhibitory pathways of the tumor microenvironment in solid tumors mean that additional genetic modification of T cells would likely be required to enhance T cell trafficking and functions.531 34-36 Generation of vector cassettes encoding multiple genes requires a significant optimization of the engineering strategies since the size of the entire cassette is limited. VH domains are a good alternative to scFv since they are approximately half the size.
Here, we have used two target antigens, PSMA and MSLN, that are currently under evaluation to treat mesothelioma, lung cancer, breast cancer, pancreatic cancer and prostate cancer via scFv-based CAR-T cells.37-39 Our preclinical experiments validate the potential use of bispecific human VH domains targeting both PSMA and MSLN in these difficult to treat malignancies. It remains to be validated if dual or multiple targeting with VH domain-based CARs can be broadly applicable, and if targeting multiple antigens in solid tumors leads to increased potential for toxicity.
Additionally, we observed that VH domain-based CAR-T cells have comparable cytotoxicity and proliferative capacity as traditional scFv-based CAR-T cells. MSLN-VH-T cells showed even more profound antitumor effects as compared with mice treated with MSLN-scFv CAR-T cells. Interestingly, MSLN-VH showed lower affinity than MSLN-scFv (28 nM compared with 79pM) recapitulating what has been observed for other scFvs that very high affinity is not necessarily optimal for CAR-based targeting for some targets.40-42 However, we cannot exclude that the observed superior antitumor activity of the MSLN-Vn-based CAR-T cells can be associated with the recognition of a different epitope rather than to different affinity. In summary, we have demonstrated that VH domain CAR-T cells in monospecific format achieved comparable antitumor response compared with traditional scFv-based CAR-T cells both in vitro and in vivo. Furthermore, bispecific VH domain CAR-T cells delivered potent anti-tumor effects demonstrating the potential to target solid tumors with heterogeneous antigen expression. These proof-of-concept experiments lay the foundation for further development of human VH domain-based CAR-T cells in clinical trials.
References
1 . Eshhar Z , Waks T , Gross 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 1993;90:720-4.doi:10.1073/pnas.90.2.720 pmid:http://www.ncbi. nlm.nih.gov/pubmed/8421711
2. Finney HM , Lawson AD , Bebbington CR , et al . Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol
1998; 161 :2791-7.pmid:http://www.ncbi. nlm.nih.gov/pubmed/9743337
3. Imai C , Mihara K , Andreansky M , et al . Chimeric receptors with 4-1 BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004; 18:676-84. doi: 10.1038/sj.leu.2403302 pmid:http://www. ncbi.nlm.nih.gov/pubmed/14961035
4. Sadelain M , Brentjens R , Riviere I . The basic principles of chimeric antigen receptor design. Cancer Discov 2013;3:388-98.doi:10.1158/2159-8290.CD-12-0548 pmid:http://www.ncbi.nlm.nih.gov/pubmed/23550147
5. Dotti G , Gottschalk S , Savoldo B , et al . Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev 2014;257:107-
26.doi:10.1111/imr.12131 pmid:http://www.ncbi. nlm.nih.gov/pubmed/24329793
6. Xiong W , Chen Y , Kang X , et al . Immunological synapse predicts effectiveness of chimeric antigen receptor cells. Mol Ther 2018;26:963-
75.doi:10.1016/j.ymthe.2018.01.020 pmid:http://www.ncbi.nlm.nih.gov/pubmed/29503199
7. Barber A , Zhang T , DeMars LR , et al . Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer. Cancer Res 2007;67:5003-8. doi: 10.1158/0008- 5472. CAN-06-4047 pmid:http://www. ncbi.nlm.nih.gov/pubmed/17510432
8. Shaffer DR , Savoldo B , Yi Z , et al . T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies. Blood 2011 ;117:4304-
14. doi: 10.1182/blood-2010-04-278218 pmid:http://www.ncbi.nlm.nih.gov/pubmed/21304103 Brown CE , Alizadeh D , Starr R , et al . Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med 2016;375:2561-
9.doi:10.1056/NEJMoa1610497 pmid:http://www.ncbi.nlm.nih.gov/pubmed/28029927 Ahn S , Li J , Sun C , et al . Cancer immunotherapy with T cells carrying bispecific receptors that mimic antibodies. Cancer Immunol Res 2019;7:773- 83. doi : 10.1158/2326-6066. Cl R- 18-0636 pmid:http://www.ncbi.nlm.nih.gov/pubmed/30842091 Miller BR , Demarest SJ , Lugovskoy A , et al . Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sei 2010;23:549-57.doi: 10.1093/protein/gzq028 pmid:http://www.ncbi.nlm.nih.gov/pubmed/20457695 Landoni E , Fuca G , Wang J , et al . Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling. Cancer Immunol Res 2021. doi:doi:10.1158/2326-6066. Cl R-20-0451. [Epub ahead of print: 05 Feb
2021].pmid:http://www. ncbi.nlm.nih.gov/pubmed/33547226 Holliger P , Hudson PJ . Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126-36. doi:10.1038/nbt1142 pmid:http://www. ncbi.nlm.nih.gov/pubmed/16151406 Ahmad ZA , Yeap SK , Ali AM , et al . scFv antibody: principles and clinical application. Clin Dev Immunol 2012;2012:1-15.doi:10.1155/2012/980250 pmid:http://www. ncbi.nlm.nih.gov/pubmed/22474489 Hamers-Casterman C , Atarhouch T , Muyldermans S , et al . Naturally occurring antibodies devoid of light chains. Nature 1993;363:446-8. doi: 10.1038/363446a0 pmid:http://www.ncbi.nlm.nih.gov/pubmed/8502296 Stanfield RL , Dooley H , Flajnik MF , et al . Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science 2004;305:1770- 3.doi:10.1126/science.1101148 pmid:http://www.ncbi. nlm.nih.gov/pubmed/15319492 Jamnani FR , Rahbarizadeh F , Shokrgozar MA , et al . T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy. Biochim Biophys Acta 2014;1840:378-86. doi:10.1016/j.bbagen.2013.09.029 pmid:http://www.ncbi.nlm.nih.gov/pubmed/24076235 Lam N , Trinklein ND , Buelow B , et al . Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains. Nat Commun 2020; 11 :283. doi : 10.1038/S41467-019-14119-9 pmid:http://www. ncbi.nlm.nih.gov/pubmed/31941907 O'Rourke DM , Nasrallah MP , Desai A , et al . A single dose of peripherally infused EGFRvI Il-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med 2017;9:eaaa0984. doi:10.1126/scitranslmed.aaa0984 pmid:http://www. ncbi.nlm.nih.gov/pubmed/28724573 Sampson JH , Gunn MD , Fecci PE , et al . Brain immunology and immunotherapy in brain tumours. Nat Rev Cancer 2020;20: 12-25. doi: 10.1038/s41568-019-0224-7 pmid:http://www.ncbi.nlm.nih.gov/pubmed/31806885 Chang SS . Overview of prostate-specific membrane antigen. Rev Urol 2004;6 Suppl 10:S13-18. pmid:http://www. ncbi.nlm.nih.gov/pubmed/16985927 Yu H , Pan J , Guo Z , et al . Cart cell therapy for prostate cancer: status and promise. Onco Targets Ther 2019;12:391-5.doi:10.2147/OTT.S185556 pmid:http://www.ncbi.nlm.nih.gov/pubmed/30655675 Morello A , Sadelain M , Adusumilli PS . Mesothelin-Targeted cars: driving T cells to solid tumors. Cancer Discov 2016;6:133-46. doi:10.1158/2159-8290. CD-15-0583 pmid:http://www.ncbi.nlm.nih.gov/pubmed/26503962 Hong LK , Chen Y , Smith 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 2018;6:1274-87.doi:10.1158/2326- 6066.CIR-18-0065 pmid:http://www.ncbi.nlm.nih.gov/pubmed/30087115 Wang Q , Chen Y , Park J , et al . Design and production of bispecific antibodies. Antibodies 2019;8:43.doi:10.3390/antib8030043 Du H , Hirabayashi K , Ahn 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 2019;35:221-37. doi:10.1016/j.ccell.2019.01.002 pmid:http://www.ncbi.nlm.nih.gov/pubmed/30753824 Diaconu I , Ballard B , Zhang M , et al . Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Mol Ther
2017;25:580-92. doi:10.1016/j.ymthe.2017.01 .011 pmid:http://www. ncbi.nlm.nih.gov/pubmed/28187946 28. Rajabzadeh A , Rahbarizadeh F , Ahmadvand 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 2021 ;22:502-13.doi:10.22074/cellj.2021.6917 pmid:http://www.ncbi.nlm.nih.gov/pubmed/32347044
29. Sharifzadeh Z , Rahbarizadeh F , Shokrgozar MA , et al . Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents. Cancer Lett 2013;334:237- 44.doi:10.1016/j.canlet.2012.08.010 pmid:http://www.ncbi.nlm.nih.gov/pubmed/22902507 30. Teng Y , Young JL , Edwards B , et al . Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse. N Biotechnol 2020;55:65- 76.doi:10.1016/j.nbt.2019.10.003 pmid:http://www.ncbi.nlm.nih.gov/pubmed/31600579
31. Fuca G , Reppel L , Landoni E , et al . Enhancing chimeric antigen receptor T-cell efficacy in solid tumors. Clin Cancer Res 2020;26:2444-51. doi:10.1158/1078- 0432.CCR-19-1835 pmid:http://www.ncbi.nlm.nih.gov/pubmed/32015021
32. Shah NN , Fry TJ . Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol 2019;3:372-85.doi: 10.1038/s41571-019-0184-6
33. Ramos CA , Grover NS , Beaven AW , et al . Anti-Cd30 CAR-T cell therapy in relapsed and refractory Hodgkin lymphoma. J Clin Oncol 2020;38:3794-
804.doi:10.1200/JC0.20.01342 pmid:http://www.ncbi.nlm.nih.gov/pubmed/32701411
34. Ma X , Shou P , Smith C , et al . Interleukin-23 engineering improves CAR T cell function in solid tumors. Nat Biotechnol 2020;38:448-59.doi:10.1038/s41587-019-0398-2 pmid:http://www.ncbi.nlm.nih.gov/pubmed/32015548
35. Bollard CM , Tripic T , Cruz 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 2018;36:1128-39.doi:10.1200/JC0.2017.74.3179 pmid:http://www. ncbi.nlm.nih.gov/pubmed/29315015
36. Heczey A , Louis CU , Savoldo B , et al . Car T cells administered in combination with Lymphodepletion and PD-1 inhibition to patients with neuroblastoma. Mol Ther
2017;25:2214-24. doi:10.1016/j.ymthe.2017.05.012 pmid:http://www.ncbi.nlm.nih.gov/pubmed/28602436
37. Wang D , Shao Y , Zhang X , et al . II-23 and PSMA-targeted duo-CAR T cells in prostate cancer eradication in a preclinical model. J Transl Med 2020; 18:23. doi: 10.1186/s 12967-019-02206-w pmid:http://www. ncbi.nlm.nih.gov/pubmed/31937346
38. Lv J , Zhao R , Wu D , et al . Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer. J Hematol Oncol 2019;12:18.doi:10.1186/s 13045-019- 0704-y pmid:http://www.ncbi.nlm.nih.gov/pubmed/30777106
39. Zhang Z , Jiang D , Yang H , et al . Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor. Cell Death Dis 2019;10:476.doi:10.1038/s41419-019-1711-1 pmid:http://www.ncbi.nlm.nih.gov/pubmed/31209210 40. Watanabe K , Terakura S , Uchiyama S , et al . Excessively high-affinity single-chain fragment variable region in a chimeric antigen receptor can counteract T-cell proliferation. Blood 2014;124:4799. doi:10.1182/blood.V124.21.4799.4799
41. Richman SA , Nunez-Cruz S , Moghimi B , et al . High-Affinity GD2-Specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model. Cancer Immunol Res 2018;6:36-46.doi: 10.1158/2326-6066.CI R-17-0211 pmid:http://www. ncbi.nlm.nih.gov/pubmed/29180536
42. Park S , Shevlin E , Vedvyas Y , et al . Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity. Sci Rep 2017;7:14366. doi:10.1038/s41598-017-14749-3 pmid:http://www.ncbi.nlm.nih.gov/pubmed/29085043

Claims

1. An isolated VH single domain antibody that specifically binds to human mesothelin (MSLN), wherein the VH single domain antibody comprising a CDR1 comprising SEQ NO. 4 or a sequence with 1 , 2 or 3 amino acid modifications, CDR2 comprising SEQ NO. 5 or a sequence with 1 , 2, 3 or 4 amino acid modifications and a CDR3 comprising SEQ ID NO. 6 or a sequence with 1 , 2 or 3 amino acid modifications.
2. The isolated VH single domain antibody according to claim 1 wherein the VH single domain antibody comprises SEQ ID NO. 3 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto.
3. The isolated VH single domain antibody according to claim 1 or 2 wherein the antibody molecule is capable of activating an immune cell in the presence of MSLN.
4. The isolated VH single domain antibody according to claim 3 wherein the immune cell is a T cell, B cell, natural killer (NK) cell, natural killer T (NKT) cell, or dendritic cell (DC).
5. The isolated VH single domain antibody according to a preceding claim wherein the VH single domain antibody binds to human MSLN with an affinity of 20 to 40 nM.
6. The isolated VH single domain antibody according to a preceding claim wherein the VH single domain antibody binds to cyno MSLN.
7. An isolated nucleic acid encoding a VH single domain antibody according to any of claims 1 to 6.
8. A vector comprising a nucleic acid according to claim 7.
9. The vector according to claim 8 which is a retroviral vector, a DNA vector, a plasmid, a RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.
10. A host cell comprising a nucleic acid according to claim 7 or a vector according to claim 8 or 9.
11 . The host cell of claim 10 wherein said host cell is a bacterial, yeast, insect, plant, viral or mammalian cell.
12. A binding molecule comprising an isolated VH single domain antibody according to any of claims 1 to 6.
13. The binding molecule according to claim 12 wherein the VH single domain antibody is linked to a second antigen binding molecule that does not bind to MSLN. The binding molecule according to claim 13 wherein said second antigen binding domain is an antibody of fragment thereof. The binding molecule according to claim 14 wherein said fragment is a Fab, Fab', F(ab')2, dAb, Fd, Fv, or a single chain Fv fragment, a human heavy chain variable domain (VH), or an isolated CDR. The binding molecule according to any of claims 13 to 15 wherein said antigen is a tumor specific antigen, optionally PSMA. The binding molecule according to any of claims 13 to 14 wherein said antigen is 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. The binding molecule according to any of claims 13 to 15 wherein said antigen is a checkpoint target. The binding molecule according to claim 18 wherein said antigen binding domain binds to an immune checkpoint inhibitor. The binding molecule according to claim 19 wherein said immune checkpoint inhibitor is an anti-PD1 , anti PDL-1 , anti PDL-2, anti CTL-4, anti -TIM-3 or anti LAG-3 antibody The binding molecule according to any of claims 12 to 20 conjugated to a toxin, enzyme, radioisotope, half-life extending moiety, therapeutic molecule, label or other chemical moiety. The isolated binding molecule according to claim 21 wherein said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and an albumin binding peptide or single domain antibody that binds to human serum albumin. The use of a single domain antibody according to any of claims 1 to 6 in a multispecific or multivalent binding agent. A pharmaceutical composition comprising a VH single domain antibody according to claim 1 to 6 or a binding molecule according to any of claims 12 to 22 and a pharmaceutical acceptable carrier, excipient or diluent.
25. A method for treating a cancer comprising administering a VH single domain antibody according to any of claims 1 to 6 or a binding molecule according to any of claims 11 to 22.
26. The VH single domain antibody according to claim any of claims 1 to 6, a pharmaceutical composition of claim 24 or a binding molecule according to any of claims 12 to 22 for use in the treatment of cancer.
27. The method according to claim 25, a VH single domain, binding molecule or pharmaceutical composition according to claim 26 wherein the cancer is selected from a haematological cancer or solid cancer. 28. The method, VH single domain antibody, binding molecule, pharmaceutical composition of claim 27 wherein the cancer is oesophageal cancer, breast cancer, gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, and endometrial cancer.
29. The method according to any of claims 25, 27 or 28, a VH single domain antibody, the binding molecule or a pharmaceutical composition according to any of claims 26, 27 or 28 further comprising the step of measuring mesothelin expression in a sample from said subject.
30. The method according to any of claims 25, 27, 28 or 29, a VH single domain antibody, the binding molecule or a pharmaceutical composition according to any of claims 26, 27, 28 or 29 wherein the cancer is MSLN-positive.
31. The method according to any of claims 25, 27, 28 to 30, a VH single domain antibody, the binding molecule or a pharmaceutical composition according to any of claims 26, 27, 28 to 30 further comprising the step of administering an immune checkpoint inhibitor or other therapeutic moiety.
32. The method, the cell, cell population, pharmaceutical composition or use according to claim 31 wherein said immune checkpoint inhibitor is an anti-PD1 , anti PDL-1 , anti PDL- 2, anti CTL-4, anti TIM-3 or anti LAG-3 antibody.
33. An immunoconjugate comprising a single domain antibody according to any of claims 1 to 6 or a binding molecule according to any of claims 12 to 22 linked to a therapeutic agent.
34. A method for producing a VH single domain antibody according to any one of claims 1 to 6 comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.
35. A kit comprising a single domain antibody according to any of claims 1 to 6 or a binding molecule according to any of claims 12 to 22, or a pharmaceutical composition according to claim 24 and optionally instructions for use..
36. A method for detecting the presence of human MSLN in a test sample or detecting or diagnosing a cancer in a subject comprising contacting said sample with a single domain antibody according to any of claims 1 to 6 and at least one detectable label and detecting binding of said single domain antibody to human MSLN.
37. The method according to claim 36 wherein the biological sample is a tissue sample or a fluid sample. 38. A method for producing a single VH domain antibody that binds to human MSLN comprising a) immunising a transgenic animal that expresses a nucleic acid construct comprising human heavy chain V genes and that is not capable of making functional endogenous light or heavy chains with an MSLN antigen, b) generating a library from said animal c) isolating single VH domain antibodies from said libraries and d) identifying a single VH domain antibody that binds to human MSLN and e) isolating said antibody.
39. A single VH domain antibody obtained or obtainable by the method of claim 38. 40. An isolated heavy chain only antibody comprising a VH domain that binds to human
MSLN.
41 . A transgenic rodent that produces a heavy chain only antibody of claim 40.
42. A heavy chain only antibody comprising a VH domain that binds to human MSLN obtained or obtainable from a transgenic mouse which expresses human V, D and J loci and does not produce functional endogenous lambda and kappa light chains and heavy chain.
43. An antibody drug conjugate comprising a VH single domain antibody according to any of any of claims 1 to 5 or a binding molecule according to any of claims 11 to 21 .
PCT/GB2023/050997 2022-04-14 2023-04-13 Mesothelin binders WO2023199068A1 (en)

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 (en) 2023-10-19

Family

ID=81753166

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2023/050997 WO2023199068A1 (en) 2022-04-14 2023-04-13 Mesothelin binders

Country Status (2)

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

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000737A2 (en) 2001-06-21 2003-01-03 The Babraham Institute MOUSE μ LIGHT CHAIN LOCUS
WO2004076618A2 (en) 2003-02-26 2004-09-10 Babraham Institute Genetically modified non-human mammals and cells
WO2016062990A1 (en) 2014-10-22 2016-04-28 Crescendo Biologics Limited Transgenic mice
WO2016113556A1 (en) 2015-01-12 2016-07-21 Crescendo Biologics Limited Methods for producing optimised therapeutic molecules
WO2017122017A1 (en) 2016-01-12 2017-07-20 Crescendo Biologics Limited Therapeutic molecules
WO2017191476A1 (en) 2016-05-06 2017-11-09 Crescendo Biologics Limited Chimeric antigen receptor with single domain antibody
WO2018127709A1 (en) * 2017-01-06 2018-07-12 Crescendo Biologics Limited Single domain antibodies to programmed cell death (pd-1)
WO2019012260A1 (en) 2017-07-10 2019-01-17 Crescendo Biologics Limited Therapeutic molecules binding psma
WO2020099871A1 (en) 2018-11-13 2020-05-22 Crescendo Biologics Limited Single domain antibodies that bind human serum albumin
WO2020229842A1 (en) 2019-05-15 2020-11-19 Crescendo Biologics Limited Binding molecules
WO2023064876A1 (en) * 2021-10-14 2023-04-20 Teneobio, Inc. Mesothelin binding proteins and uses thereof

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000737A2 (en) 2001-06-21 2003-01-03 The Babraham Institute MOUSE μ LIGHT CHAIN LOCUS
WO2004076618A2 (en) 2003-02-26 2004-09-10 Babraham Institute Genetically modified non-human mammals and cells
WO2016062990A1 (en) 2014-10-22 2016-04-28 Crescendo Biologics Limited Transgenic mice
WO2016113556A1 (en) 2015-01-12 2016-07-21 Crescendo Biologics Limited Methods for producing optimised therapeutic molecules
WO2017122017A1 (en) 2016-01-12 2017-07-20 Crescendo Biologics Limited Therapeutic molecules
WO2017191476A1 (en) 2016-05-06 2017-11-09 Crescendo Biologics Limited Chimeric antigen receptor with single domain antibody
WO2018127709A1 (en) * 2017-01-06 2018-07-12 Crescendo Biologics Limited Single domain antibodies to programmed cell death (pd-1)
WO2019012260A1 (en) 2017-07-10 2019-01-17 Crescendo Biologics Limited Therapeutic molecules binding psma
WO2020099871A1 (en) 2018-11-13 2020-05-22 Crescendo Biologics Limited Single domain antibodies that bind human serum albumin
WO2020229842A1 (en) 2019-05-15 2020-11-19 Crescendo Biologics Limited Binding molecules
WO2023064876A1 (en) * 2021-10-14 2023-04-20 Teneobio, Inc. Mesothelin binding proteins and uses thereof

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

Also Published As

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

Similar Documents

Publication Publication Date Title
US11866510B2 (en) Chimeric antigen receptor with single domain antibody
US20220218746A1 (en) Bcma chimeric antigen receptor based on single domain antibody and use thereof
CN112794909B (en) anti-TIGIT monoclonal antibody and application thereof
JP2021502104A (en) Single domain antibody that binds to CD137
CN107660213A (en) The anti-MUC1* antibody of humanization
US20230241104A1 (en) Anti-human msln antibody and msln-targeting immune effector cell
CN111848809A (en) CAR molecule targeting Claudin18.2, immune cell modified by same and application
WO2021052307A1 (en) Anti-b7-h3 antibody and application thereof
CN111909271B (en) BCMA chimeric antigen receptor based on single domain antibody and application thereof
CN108864307A (en) The Chimeric antigen receptor of signal peptide optimization targeting CD19, the T cell and preparation method and application for expressing the Chimeric antigen receptor
JP2023106392A (en) Cd3 antigen binding fragment and application thereof
CN112601761A (en) Polypeptides that bind OX40 and uses thereof
CN116710112A (en) CD 5-targeting fully human single domain tandem Chimeric Antigen Receptor (CAR) and application thereof
WO2019085102A1 (en) Bcma-specific chimeric antigen receptor t cell and application thereof
EP4257617A1 (en) Antigen-binding protein targeting cd70 and use thereof
CN117229407A (en) Single-domain antibody targeting GPRC5D, chimeric antigen receptor and application thereof
CN113631184A (en) Enhancement of cytolytic T cell activity by inhibition of EBAG9
CN115916833B (en) Antigen binding polypeptide targeting B7H3 and application thereof
WO2023199068A1 (en) Mesothelin binders
CN115386007A (en) anti-GPRC 5D antibody, preparation method and application thereof
KR20230154235A (en) Antibodies against NKp46 and their applications
WO2023199069A1 (en) Chimeric antigen receptor that binds mesothelin
CN117164712B (en) CD 22-targeted single domain antibody, chimeric antigen receptor and application thereof
WO2023273762A1 (en) Spatial conformational epitope mediating efficient retention of cd3 within cells and application thereof
CN116419970B (en) Low toxicity anti-OX 40 antibodies, pharmaceutical compositions and uses thereof

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