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WO2016022939A1 - Anticorps monoclonaux humains spécifiques de 5t4 et leurs procédés d'utilisation - Google Patents

Anticorps monoclonaux humains spécifiques de 5t4 et leurs procédés d'utilisation Download PDF

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Publication number
WO2016022939A1
WO2016022939A1 PCT/US2015/044253 US2015044253W WO2016022939A1 WO 2016022939 A1 WO2016022939 A1 WO 2016022939A1 US 2015044253 W US2015044253 W US 2015044253W WO 2016022939 A1 WO2016022939 A1 WO 2016022939A1
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antibody
antigen
seq
binding fragment
domain
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PCT/US2015/044253
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WO2016022939A8 (fr
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Dimiter S. Dimitrov
Tianlei YING
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The United States Of America, As Represented By The Secretary, Department Of Health & Human Services
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Publication of WO2016022939A8 publication Critical patent/WO2016022939A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This disclosure concerns 5T4-specific human monoclonal antibodies and use of the monoclonal antibodies for detection and treatment of 5T4-positive cancers.
  • 5T4 is a 72 kDa N-glycosylated transmembrane glycoprotein containing seven leucine- rich repeat regions. This protein is often referred to as an oncofetal antigen, due to its expression in fetal trophoblast (where it was first discovered), or trophoblast glycoprotein
  • TPBG tumor necrosis factor-associated tumors
  • 5T4 is expressed in a number of carcinoma tumors, including in colorectal, ovarian, renal, non-small cell lung and gastric tumors. It has very limited expression in normal adult tissue, but is widespread in malignant tumors throughout their development.
  • 5T4 The confined expression of 5T4 gives this antigen the potential to be useful in cancer immunotherapy.
  • mAb5T4 high-affinity murine monoclonal antibody
  • 5T4 is also the target of the cancer vaccine TROVAXTM, which is in clinical trials for the treatment of a range of different solid tumor types.
  • Fully human monoclonal antibodies that bind 5T4 are disclosed.
  • Antibody-drug conjugates (ADCs), chimeric antigen receptors (CARs), immunoconjugates, bispecific antibodies and compositions that include the fully human 5T4-specific monoclonal antibodies are also disclosed.
  • the disclosed antibodies and compositions can be used, for example, in the diagnosis and treatment of 5T4-positive cancers.
  • the amino acid sequence of the VL domain and/or the VH domain of the monoclonal antibody includes at least one (such as all three) complementarity determining regions (CDRs) of the VL domain and/or the VH domain of clone mlOOl or clone ml002 disclosed herein.
  • the monoclonal antibody is an antibody fragment (an antigen-binding fragment), such as an Fab fragment, an Fab' fragment, an F(ab)' 2 fragment, a single chain variable fragment (scFv) or a disulfide stabilized variable fragment (dsFv).
  • the antibody is an IgG.
  • ADCs antibody drug conjugates
  • CARs Chimeric antigen receptors
  • Immunoconjugates that include a 5T4-specific monoclonal antibody, or an antigen-binding fragment thereof, and an effector molecule (such as a toxin or a detectable label) are further provided by the present disclosure.
  • bispecific antibodies that include a 5T4-specific monoclonal antibody or antigen-binding fragment.
  • compositions that include a 5T4-specific monoclonal antibody or antigen-binding fragment, ADC, CAR, bispecific antibody or immunoconjugate and a pharmaceutically acceptable carrier.
  • Nucleic acid molecules and vectors encoding the disclosed 5T4-specific monoclonal antibodies (including antigen-binding fragments), ADCs, CARs, bispecific antibodies and immunoconjugates are also provided herein.
  • immunoconjugate bispecific antibody or composition.
  • FIG. 1 is a graph demonstrating binding of Fabs HI, H3, H4, H5, CI, C2 and C5 to 5T4, as measured by ELISA. Serially diluted Fabs were added to 5T4-coated wells. HRP-conjugated goat anti-FLAG antibody was used for detection.
  • FIGS. 2A-2C are flow cytometry plots showing binding of IgG Is to 5T4-expressing cells.
  • FIG. 2A The expression of 5T4 in A498 cells. A498 cells were incubated with serially diluted IgGl C5 (FIG. 2B) and A498 cells were incubated with serially diluted IgGl H4 (FIG. 2C). FITC-conjugated goat F(ab')2 anti-human IgG (Fc-specific) antibody was used for detection.
  • SEQ ID NO: 1 is the amino acid sequence of the mlOOl VL domain.
  • SEQ ID NO: 2 is the amino acid sequence of the mlOOl VH domain.
  • SEQ ID NO: 3 is the amino acid sequence of the ml002 VL domain.
  • SEQ ID NO: 4 is the amino acid sequence of the ml002 VH domain.
  • SEQ ID NO: 5 is the amino acid sequence of Pseudomonas exotoxin (PE).
  • SEQ ID NO: 6 is the amino acid sequence of PE38.
  • SEQ ID NO: 7 is the amino acid sequence of PE-LR.
  • SEQ ID NO: 8 is the amino acid sequence of PE-LR/6X.
  • SEQ ID NO: 9 is the amino acid sequence of PE with reduced immunogenicity.
  • SEQ ID NO: 10 is the amino acid sequence of PE-LR/8M.
  • Antibody A polypeptide ligand comprising at least a light chain and/or heavy chain immunoglobulin variable region which recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen, such as 5T4, or a fragment thereof.
  • Immunoglobulin molecules are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V H ) region and the variable light (V L ) region.
  • V H region and the V L region are responsible for binding the antigen recognized by the antibody.
  • Antibodies include intact immunoglobulins and the variants and portions (fragments) of antibodies well known in the art, such as single-domain antibodies (e.g. VH domain antibodies), Fab fragments, Fab' fragments, F(ab)' 2 fragments, single chain Fv proteins ("scFv”), and disulfide stabilized Fv proteins ("dsFv").
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin.
  • immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • antibody also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W. H. Freeman & Co., New York, 1997.
  • a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • H heavy chain
  • L light chain
  • ⁇ lambda
  • ⁇ kappa
  • IgM immunoglobulin heavy chain classes
  • Each heavy and light chain contains a constant region and a variable region (the regions are also known as “domains”).
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs.”
  • CDRs complementarity-determining regions
  • the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the "Kabat” numbering scheme), Chothia et al.
  • the Kabat and IMGT databases are maintained online.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are often identified by the chain in which the particular CDR is located.
  • a V H CDR3 (or HCDR3) is located in the variable domain of the heavy chain of the antibody in which it is found
  • a V L CDRl or LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • An antibody that binds 5T4, for example will have a specific V H region and the V L region sequence, and thus specific CDR sequences.
  • Antibodies with different specificities i.e.
  • V H or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
  • V L or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and/or heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • a “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds 5T4.
  • a “human” antibody also called a “fully human” antibody
  • the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence.
  • frameworks from one human antibody can be engineered to include CDRs from a different human antibody.
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor,” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).
  • ADC Antibody-drug conjugate
  • ADC A molecule that includes an antibody (or antigen- binding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent.
  • ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface.
  • exemplary drugs for use with ADCs include anti- micro tubule agents (such as maytansinoids, auristatin E and auristatin F) and interstrand crosslinking agents ⁇ e.g., pyrrolobenzodiazepines; PDBs).
  • Anti-microtubule agent A type of drug that blocks cell growth by stopping mitosis.
  • Anti-microtubule agents also referred to as “anti-mitotic agents,” are used to treat cancer.
  • Binding affinity Affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al. ⁇ Mol.
  • binding affinity is measured by an antigen/antibody dissociation rate. In another embodiment, binding affinity is measured by a competition radioimmunoassay. In another embodiment, binding affinity is measured by ELISA.
  • An antibody that "specifically binds" an antigen is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.
  • Carcinoma A cancer that arises from the epithelial tissues of the skin or the lining of the internal organs.
  • Chemotherapeutic agent Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth, such as psoriasis.
  • a chemotherapeutic agent is an agent of use in treating a 5T4- positive cancer, such as colorectal, ovarian, renal, non-small cell lung or gastric cancer.
  • a chemotherapeutic agent is a radioactive compound.
  • Combination chemotherapy is the administration of more than one agent to treat cancer.
  • One example is the administration of an antibody (or immunoconjugate or ADC) that binds 5T4 used in
  • Chimeric antigen receptor A chimeric molecule that includes an antigen- binding portion (such as a monoclonal antibody or fragment thereof) and a signaling domain, such as a signaling domain from a T cell receptor ⁇ e.g. CD3 ⁇ ).
  • CARs are comprised of a binding moiety ⁇ e.g. a scFV), a transmembrane domain and an endodomain.
  • the endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 ⁇ or FceRIy.
  • the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28 and/or CD 137.
  • Colorectal cancer A type of cancer arising from epithelium in the colon or rectum ⁇ i.e., the large intestine). Most lesions of the large bowel are adenocarcinomas. Colorectal cancer is also referred to as “colon cancer,” “rectal cancer” or “bowel cancer.”
  • Constant amino acid substitutions are those substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to 5T4.
  • a monoclonal antibody that specifically binds 5T4 can include at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and specifically bind a 5T4 polypeptide.
  • the term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody specifically binds 5T4.
  • Non-conservative substitutions are those that reduce an activity or binding to 5T4.
  • Complementarity determining region Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site.
  • the light and heavy chains of an Ig each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2 and HCDR3, respectively.
  • Placement in direct physical association includes both in solid and liquid form.
  • Degenerate variant refers to a polynucleotide encoding a 5T4 polypeptide or an antibody that binds 5T4 that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the 5T4 polypeptide or antibody that binds 5T4 encoded by the nucleotide sequence is unchanged.
  • Diagnostic Identifying the presence or nature of a pathologic condition, such as, but not limited to, cancer. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives).
  • the "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • Prognostic is the probability of development (e.g., severity) of a pathologic condition, such as cancer or metastasis.
  • Drug Any compound used to treat, ameliorate or prevent a disease or condition in a subject.
  • the drug is an anti-cancer agent, for example a cytotoxic agent, such as an anti-mitotic or anti-microtubule agent.
  • Effector molecule The portion of an antibody conjugate (or immunoconjugate) that is intended to have a desired effect on a cell to which the conjugate is targeted. Effector molecules are also known as effector moieties (EMs), therapeutic agents, diagnostic agents, or similar terms.
  • Therapeutic agents (or drugs) include such compounds as small molecules, nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids,
  • Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
  • the effector molecule can be contained within an encapsulation system, such as a liposome or micelle, which is conjugated to the antibody. Encapsulation shields the effector molecule from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; and Connor et ah, Pharm Ther 28:341-365, 1985).
  • Radioisotopes include radioisotopes and other detectable labels ⁇ e.g., fluorophores, chemiluminescent agents, and enzymes). Radioactive isotopes include 35 S, n C, 13 N, 15 0, 18 F, 19 F, 99m Tc, 131 I, 3 ⁇ 4, 14 C, 15 N, 90 Y, "Tc, m In and 125 I.
  • Epitope An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide, such as 5T4.
  • Framework region Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.
  • Gastric cancer A type of cancer that arises from the lining of the stomach (also referred to as “stomach cancer”). Gastric cancers are typically adenocarcinomas (a carcinoma of glandular origin).
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an "antigen- specific response").
  • an immune response is a T cell response, such as a CD4 + response or a CD8 + response.
  • the response is a B cell response, and results in the production of antigen- specific antibodies.
  • Isolated An "isolated" biological component, such as a nucleic acid, protein (including antibodies) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • molecule such as an antibody or a protein
  • labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • a "labeled antibody” refers to incorporation of another molecule in the antibody.
  • the label is a detectable marker, such as the
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35 S, n C, 13 N, 15 0, 18 F, 19 F, 99m Tc, 131 1, 3 H, 14 C, 15 N, 90 Y, "Tc, in In and 125 I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates.
  • labels are attached by spacer arms of various lengths to reduce
  • Linker In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. "Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a cyto toxin or a detectable label.
  • conjugating refers to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a
  • radionuclide, drug or other molecule to a polypeptide, such as an antibody or antibody fragment.
  • the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule.
  • the linkage can be either by chemical or recombinant means.
  • “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Neoplasia malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
  • NSCLC Non-small cell lung carcinoma
  • NSCLCs are adenocarcinomas, squamous cell carcinomas or large cell carcinomas.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Ovarian cancer Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells).
  • Pharmaceutical agent A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.
  • compositions and formulations suitable for pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions such as powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Preventing a disease refers to inhibiting the full development of a disease.
  • Treating refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases.
  • Treating refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • Substantial purification denotes purification from other proteins or cellular components.
  • a substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure.
  • a substantially purified protein is 90% free of other proteins or cellular components.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • Renal cancer A cancer that starts in cells of the kidney (also referred to as “kidney cancer”).
  • the majority of renal cancers are renal cell carcinomas or urothelial cell carcinomas.
  • Sample A biological specimen containing genomic DNA, RNA
  • RNA DNA
  • protein protein
  • examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.
  • a sample includes a tumor biopsy.
  • Sequence identity The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Homologs and variants of a V L or a V H of an antibody that specifically binds 5T4 or a fragment thereof are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence.
  • Small molecule A molecule, typically with a molecular weight less than about 1000 Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
  • Therapeutically effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.
  • Toxin An agent that directly or indirectly inhibits the growth of and/or kills cells.
  • Toxins include, for example, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38 and PE40), diphtheria toxin (DT), botulinum toxin, abrin, ricin, saporin, restrictocin or gelonin, or modified toxins thereof.
  • PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • 5T4 is an oncofetal antigen expressed in a number of carcinomas including colorectal, ovarian, renal, non-small cell lung and gastric carcinomas, while its expression in normal adult tissues is limited.
  • Mouse and humanized monoclonal antibodies (mAbs) have been previously used to target 5T4 as potential therapeutics against cancer.
  • mAbs monoclonal antibodies
  • a fully human therapeutic antibody would provide additional advantages in terms of reduced immunogenicity and improved tolerance. Described herein is the generation and characterization of two fully human 5T4-specific mAbs - mlOOl and ml002.
  • two naive human Fab phage display libraries were constructed and panned using biotin-labeled 5T4-Fc fusion protein.
  • the two Fab clones were converted to IgGl and tested for the ability to bind cell-surface 5T4. Both mlOOl and ml002 IgG bound cell- associated 5T4 in a concentration-dependent manner.
  • variable light (VL) and variable heavy (VH) domains of mlOOl and ml002 are provided below.
  • the CDR sequences (as determined by IMGT) are shown in bold underline.
  • the amino acid residues of the VL and VH domain CDRs are provided below each sequence.
  • One of skill in the art could readily determine the CDR boundaries using alternative numbering schemes, including the Kabat or Chothia numbering schemes.
  • DIOLTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLOPEDFATYYCOOSYSTLWTFGOGTKVEIK LCDR1 27-32
  • LCDR2 50-52
  • LCDR3 89-97 mlOOl VH (SEQ ID NO: 2)
  • HCDR3 97-110 ml002 VL (SEQ ID NO: 3)
  • LCDR3 90-95 ml002 VH (SEQ ID NO: 4)
  • HCDR3 97-112
  • isolated monoclonal antibodies or antigen-binding fragments thereof that bind (for example, specifically bind) 5T4, such as cell-surface 5T4.
  • the VL domain of the antibody or antigen-binding fragment comprises at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 1 or SEQ ID NO: 3, such as one or more (such as all three) CDR sequences from SEQ ID NO: 1 or SEQ ID NO: 3.
  • the CDR locations are determined IMGT, Kabat or Chothia.
  • the VH domain of the antibody or antigen-binding fragment comprises at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 2 or SEQ ID NO: 4, such as one or more (such as all three) CDR sequences from SEQ ID NO: 2 or SEQ ID NO: 4.
  • the CDR sequences are determined by IMGT, Kabat or Chothia.
  • the VL domain CDR sequences are at least 90% identical to the CDR sequences of SEQ ID NO: 1 and the VH domain CDR sequences are at least 90%identical to the CDR sequences of SEQ ID NO: 2; or the VL domain CDR sequences are at least 90% identical to the CDR sequences of SEQ ID NO: 3 and the VH domain CDR sequences are at least 90%identical to the CDR sequences of SEQ ID NO: 4.
  • the VL domain of the antibody or antigen-binding fragment comprises amino acid residues 27-32, 50-52 and 89-97 of SEQ ID NO: 1; and/or the VH domain of the antibody or antigen-binding fragment comprises residues 26-33, 51-58 and 97-110 of SEQ ID NO: 2.
  • the VL domain of the antibody or antigen-binding fragment comprises residues 27-33, 51-53 and 90-95 of SEQ ID NO: 3; and/or the VH domain of the antibody or antigen-binding fragment comprises residues 26-33, 51-58 and 97-112 of SEQ ID NO: 4.
  • the VL domain CDR sequences are at least 90% identical to residues 27-32, 50-52 and 89-97 of SEQ ID NO: 1 and the VH domain CDR sequences are at least 90% identical to residues 26-33, 51-58 and 97-110 of SEQ ID NO: 2; or the VL domain CDR sequences are at least 90% identical to residues 27-33, 51-53 and 90-95 of SEQ ID NO: 3 and the VH domain CDR sequences are at least 90% identical to residues 26-33, 51-58 and 97- 112 of SEQ ID NO: 4.
  • the VL domain of the antibody or antigen-binding fragment comprises SEQ ID NO: 1 and/or the VH domain of the antibody or antigen-binding fragment comprises SEQ ID NO: 2.
  • the amino acid sequence of the VL domain is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3; and/or the amino acid sequence of the VH domain is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4.
  • the VL domain of the antibody or antigen-binding fragment comprises SEQ ID NO: 3 and/or the VH domain of the antibody or antigen-binding fragment comprises SEQ ID NO: 4.
  • the monoclonal antibody or antigen-binding fragment that binds, such as specifically binds, 5T4 is a Fab fragment, a Fab' fragment, a F(ab)' 2 fragment, a single chain variable fragment (scFv), or a disulfide stabilized variable fragment (dsFv).
  • the antibody is an immunoglobulin molecule.
  • the antibody is an IgG, such as an IgGl .
  • the antibody or antigen-binding fragment is a fully human antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment is a chimeric or synthetic antibody.
  • the disclosed antibodies bind 5T4 with a dissociation constant
  • the monoclonal antibodies or antigen- binding fragments bind 5T4 with a binding affinity of about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.15 nM or about 0.1 nM.
  • ADCs antibody-drug conjugates
  • the drug is a small molecule.
  • the drug is an anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent.
  • ADCs are further discussed herein in section V below.
  • CARs chimeric antigen receptors
  • the signaling domain comprises CD3 ⁇ or FceRIy.
  • Immunoconjugates that include a 5T4- specific monoclonal antibody or antigen-binding fragment disclosed herein and an effector molecule are also provided by the present disclosure.
  • the effector molecule is a toxin, such as Pseudomonas exotoxin or a variant thereof.
  • the effector molecule is a detectable label, such as a fluorescent, radioactive or enzymatic label. Immunoconjugates are discussed in greater detail in section VII below.
  • compositions that include a therapeutically effective amount of a disclosed 5T4-speicific monoclonal antibody or antigen-binding fragment thereof, ADC, CAR or immunoconjugate and a pharmaceutically acceptable carrier.
  • ADC an antibody to a cell
  • CAR an antigen-binding fragment thereof
  • compositions and methods of their use are discussed further in section VIII below.
  • nucleic acid molecules encoding a 5T4-specific monoclonal antibody or antigen -binding fragment disclosed herein.
  • the nucleic acid molecule encodes at least a portion of the VL domain of ml 001, at least a portion of the VH domain of ml 002, at least a portion of the VL domain of ml 002, or at least a portion of the VH domain of ml002, such as a portion comprising at least one (such as all three) CDR sequences.
  • the nucleic acid molecule encodes the VL domain of SEQ ID NO: 1 or SEQ ID NO: 3, or the VH domain of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the nucleic acid molecule is operably linked to a promoter.
  • vectors that include the nucleic acid molecules disclosed herein.
  • Isolated host cells transformed with the disclosed nucleic acid molecules and vectors are further provided by the present disclosure.
  • the monoclonal antibodies disclosed herein can be of any isotype.
  • the monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGior an IgG 2 .
  • the class of an antibody that specifically binds 5T4 can be switched with another (for example, IgG can be switched to IgM), according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgGi to IgG 2 .
  • Antibody fragments are also encompassed by the present disclosure, such as single- domain antibodies ⁇ e.g., VH domain antibodies), Fab, F(ab') 2 , and Fv. These antibody fragments retain the ability to selectively bind with the antigen. These fragments include:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody such as scFv
  • scFv Single chain antibody
  • a dimer of a single chain antibody (scFV 2 ), defined as a dimer of a scFV (also known as a "miniantibody”);
  • VH single-domain antibody an antibody fragment consisting of the heavy chain variable domain.
  • antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as E. coli) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647).
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V H and the V L regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V H and/or the V L regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
  • ADCs Antibody-Drug Conjugates
  • ADCs are compounds comprised of a tumor antigen-specific antibody and a drug, typically a cytotoxic agent, such as an anti-microtubule agent. Because ADCs are capable of specifically targeting cancer cells, the cytotoxic agent can be much more potent than agents used for standard chemotherapy. The most common cytotoxic drugs currently used with ADCs have an IC50 that is 100- to 1000-fold more potent than conventional chemo therapeutic agents.
  • cytotoxic drugs include anti-microtubule agents, such as maytansinoids and auristatins (such as auristatin E and auristatin F).
  • anti-microtubule agents such as maytansinoids and auristatins (such as auristatin E and auristatin F).
  • auristatins such as auristatin E and auristatin F.
  • Other cyto toxins for use with ADCs include
  • pyrrolobenzodiazepines which covalently bind the minor groove of DNA to form interstrand crosslinks.
  • ADCs comprise a 1:2 to 1:4 ratio of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).
  • the antibody and drug can be linked by a cleavable or non-cleavable linker.
  • a linker that is stable in the circulation to prevent systemic release of the cytotoxic drug that could result in significant off-target toxicity.
  • Non- cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, digestion of the antibody by lysosomal proteases results in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).
  • Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al. , Biotechnol Prog 24:520-526, 2008).
  • Y289L-Gal-Tl U.S. Patent Application Publication Nos.
  • 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical handle for conjugation.
  • the oligosaccharide chain attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-Gl) or completely degalactosylated (IgG-GO).
  • IgG-G2 two galactose residues
  • IgG-Gl one galactose residue
  • IgG-GO completely degalactosylated
  • the mutant i,4-galactosyltransferase enzyme is capable of transferring 2-keto- galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-Gl and IgG-GO glycoforms.
  • the chemical handle on the transferred sugar enables conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al, Biotechnol Prog 24:520-526, 2008).
  • ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) 5T4.
  • a drug such as a cytotoxic agent
  • the drug is a small molecule.
  • the drug is an anti-microtubule and/or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells.
  • cytotoxic agents include, but are not limited to, a PDB, an auristatin (such as auristatin F or auristatin E), a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU- 159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.
  • auristatin such
  • the ADC comprises a pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • the natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et ah, J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al, J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs, both naturally- occurring and synthetic analogues, have been reported
  • PDB dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010/0203007).
  • Exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009/016516; US 2009/304710; US 2010/047257; US
  • the ADC comprises an antibody conjugated to one or more maytansinoid molecules.
  • Maytansinoids are derivatives of maytansine, and are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos.
  • the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588;
  • Auristatins are derivatives of the marine mollusk compound dolastatin- 10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicwb Agents and Chemother 45(12):3580-3584, 2001) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicwb Agents Chemother 42:2961-2965, 1998).
  • Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine- dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine- dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, for example, U.S. Publication No. 2013/0129753).
  • the ADC comprises an antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics, and analogues thereof, are capable of producing double- stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al, Cancer Res 58:2925-2928, 1998).
  • Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.
  • the ADC comprises an anthracycline.
  • Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can operate to kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and/or interactions of the drug molecules with the cell membrane.
  • Non- limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof.
  • PNU- 159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4): 1608-1617, 2005).
  • Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grandi et ah, Cancer Treat Rev 17: 133, 1990; Ripamonti et ah, Br J Cancer 65:703-707, 1992).
  • the ADC can further include a linker.
  • the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC.
  • ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.
  • a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond.
  • exemplary linkers with such reactive functionalities include maleimide, haloacetamides, cc-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody.
  • electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
  • a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
  • the linker is a cleavable linker, which facilitates release of the drug.
  • cleavable linkers include acid-labile linkers (for example, comprising hydrazone), protease-sensitive linkers (for example, peptidase-sensitive), photolabile linkers, and disulfide- containing linkers (Chari et al, Cancer Res 52: 127-131, 1992; U.S. Patent No. 5,208,020).
  • the ADCs disclosed herein can be used for the treatment of a 5T4-positive cancer alone or in combination with another therapeutic agent and/or in combination with any standard therapy for the treatment of cancer (such as surgical resection of the tumor, chemotherapy or radiation therapy).
  • any standard therapy for the treatment of cancer such as surgical resection of the tumor, chemotherapy or radiation therapy.
  • CARs also known as chimeric T cell receptors, artificial T cell receptors or chimeric immunoreceptors
  • CTLs cytotoxic T lymphocytes
  • CARs include a binding moiety, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs signaling functions (Cartellieri et ah, J Biomed Biotechnol 2010:956304, 2010).
  • the binding moiety is an antigen binding fragment of a monoclonal antibody, such as a scFv.
  • the endodomain can consist of a signaling chain having an IT AM, such as CD3 ⁇ or FceRIy.
  • the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28 and/or CD 137.
  • CTLs expressing CARs can be used to target a specific cell type, such as a tumor cell.
  • the monoclonal antibodies disclosed herein can be used to engineer CTLs that express a CAR containing an antigen-binding fragment of a 5T4-specific antibody, thereby targeting the engineered CTLs to 5T4-expressing tumor cells.
  • Engineered T cells have previously been used for adoptive therapy for some types of cancer (see, for example, Park et ah, Mol Ther 15(4):825- 833, 2007).
  • the use of T cells expressing CARs is more universal than standard CTL-based immunotherapy because CTLs expressing CARs are HLA unrestricted and can therefore be used for any patient having a tumor that expresses the target antigen.
  • CARs that include a 5T4-specific monoclonal antibody, or monoclonal antibody binding fragment, such as a scFv.
  • isolated nucleic acid molecules and vectors encoding the CARs, and host cells, such as CTLs, expressing the CARs.
  • CTLs expressing CARs comprised of a 5T4-specific monoclonal antibody (or antibody binding fragment) can be used for the treatment of cancers that express 5T4, such as colorectal, ovarian, renal, non-small cell lung and gastric tumors.
  • the disclosed monoclonal antibodies specific for 5T4 can be conjugated to a therapeutic agent or effector molecule.
  • Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody.
  • a therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule.
  • therapeutic agents can include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S and other labels, target moieties and ligands.
  • the choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect.
  • the therapeutic agent can be a cyto toxin that is used to bring about the death of a particular target cell (such as a tumor cell).
  • the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.
  • nucleic acids encoding antibodies and conjugates and fusion proteins thereof.
  • Effector molecules can be linked to an antibody of interest using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • the antibodies or antibody fragments disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein).
  • the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling.
  • the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or
  • An antibody that binds (for example specifically binds) 5T4 can be labeled with a detectable moiety.
  • useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, green fluorescent protein and yellow fluorescent protein.
  • An antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • enzymes that are useful for detection
  • an antibody When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label.
  • An antibody may be labeled with a magnetic agent, such as gadolinium.
  • Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese.
  • Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels.
  • An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • An antibody can also be labeled with a radiolabeled amino acid.
  • the radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect 5T4 by x-ray, emission spectra, or other diagnostic techniques.
  • Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, m In, 125 I, 131 I.
  • An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
  • PEG polyethylene glycol
  • methyl or ethyl group a methyl or ethyl group
  • carbohydrate group a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group.
  • Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins.
  • Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401).
  • the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095).
  • Pseudomonas exotoxin refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264: 14256-14261, 1989).
  • PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039;
  • the PE is PE38, comprising the following amino acid sequence: PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQ VDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANG PADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVG YHGTFLEAAQSIVFGGVRARSQDLDArWRGFYIAGDPALAYGYAQDQEPD ARGRIRNG ALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILG WPLAERTV VIPS AIPTDPRN VGGDLDPS S IPD KEQ AIS ALPD Y AS QPGKPPREDLK (SEQ ID NO: 6)
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al, Blood 113(16):3792-3800, 2009; PCT Publication No. WO
  • the PE is a variant designated PE-LR/6X (PCT Publication No. WO 2011/001100600A1100A1100A1100A1100A1100A1100A1100A1100A1100A1
  • the PE variant is PE with reducing immunogenicity, such as a PE with the following sequence:
  • the PE is a variant designated PE-LR/8M (PCT Publication No. WO 2011/032022) having the following amino acid sequence:
  • substitutions of PE are defined herein by reference to the amino acid sequence of full- length PE set forth herein as SEQ ID NO: 5. Substitutions of PE are described herein by reference to the amino acid residue present at a particular position, followed by the amino acid with which that residue has been replaced in the particular substitution. In this regard, the positions of the amino acid sequence of a particular embodiment of a PE are referred to herein as the positions of the amino acid sequence of the particular embodiment, or as the positions as defined by SEQ ID NO: 5. Thus, substitutions refer to a replacement of an amino acid residue in the amino acid sequence of a particular embodiment of a PE corresponding to the indicated position of the 613-amino acid sequence of SEQ ID NO: 5 with the understanding that the actual positions in the respective amino acid sequence may be different. In the event of multiple substitutions at two or more positions, the two or more substitutions may be the same or different - each amino acid residue of the two or more amino acid residues being substituted can be substituted with the same or different amino acid residue unless explicitly indicated otherwise.
  • Modification of PE may occur in any previously described variant, including cytotoxic fragments of PE (for example, PE38, PE-LR and PE-LR/8M).
  • Modified PEs may include any substitution(s), as described above, for one or more amino acid residues within one or more T- cell epitopes and/or B cell epitopes of PE.
  • the antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing 5T4 on their surface.
  • an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing cell-surface 5T4. This can be done for therapeutic, diagnostic or research purposes.
  • Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses.
  • Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
  • the molecule linked to an anti-5T4 antibody can be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; Connor et al, Pharm. Ther. 28:341-365, 1985).
  • Antibodies described herein can also be covalently or non-covalently linked to a detectable label.
  • Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example, 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.
  • fluorescent dyes for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels for example, 3 H, 125 1,
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • Bispecific antibodies are recombinant proteins comprised of antigen-binding fragments of two different monoclonal antibodies. Thus, bispecific antibodies bind two different antigens. Bispecific antibodies can be used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and a tumor antigen.
  • CTLs such as a CTL receptor component such as CD3
  • NK effector natural killer
  • the 5T4-specific monoclonal antibodies disclosed herein can be used to generate bispecific antibodies that target both 5T4 and CTLs, or targeting both 5T4 and NK cells, thereby providing a means to treat 5T4-expressing cancers.
  • Bi-specific T-cell engagers are a type of bispecific monoclonal antibody that are fusions of a first single-chain variable fragment (scFv) that targets a tumor antigen and a second scFv that binds T cells, such as bind CD3 on T cells.
  • Bi-specific killer cell engagers are a type of bispecific monoclonal antibody that are fusions of a first scFv that targets a tumor antigen and a second scFv that binds a NK cell activating receptor, such as CD 16.
  • bispecific monoclonal antibodies comprising a 5T4-specific monoclonal antibody, or antigen-binding fragment thereof.
  • the bispecific monoclonal antibody further comprises a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds a component of the T cell receptor, such as CD3.
  • the bispecific monoclonal antibody further comprises a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds a NK cell activating receptor, such as CD16, Ly49, or CD94.
  • the antigen-binding fragments are scFv.
  • bispecific antibodies comprising a 5T4-specific antibody, or antigen-binding fragment thereof, can be used for the treatment of cancers that express 5T4, such as colorectal, ovarian, renal, non-small cell lung or gastric cancer.
  • methods of treating a subject with cancer by selecting a subject with a cancer that expresses 5T4, and administering to the subject a therapeutically effective amount of the 5T4-targeting bispecific antibody.
  • compositions that include one or more of the disclosed antibodies that bind
  • compositions comprising ADCs, CARs (and CTLs comprising CARs), immunoconjugates, bispecific antibodies or immunotoxins are also provided.
  • the compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
  • the antibody can be formulated for systemic or local (such as intra-tumor) administration. In one example, the antibody is formulated for parenteral administration, such as intravenous administration.
  • compositions for administration can include a solution of the antibody dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a pharmaceutically acceptable carrier such as an aqueous carrier.
  • aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.
  • a typical pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of antibody per subject per day. Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).
  • Antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • Antibodies can be administered by slow infusion, rather than in an intravenous push or bolus.
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
  • Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ are generally referred to as nanoparticles, nanospheres, and
  • nanocapsules respectively.
  • Capillaries have a diameter of approximately 5 ⁇ so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 ⁇ in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219- 342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992). Polymers can be used for ion-controlled release of the antibody compositions disclosed herein.
  • polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al.,
  • ADCs, bispecific antibodies and immunoconjugates disclosed herein can be administered to slow or inhibit the growth of tumor cells or inhibit the metastasis of tumor cells, such as colorectal, ovarian, renal, non-small cell lung or gastric tumors.
  • a therapeutically effective amount of an antibody is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer.
  • Suitable subjects may include those diagnosed with a cancer that expresses 5T4, such as, but not limited to, colorectal, ovarian, renal, non-small cell lung and gastric cancer.
  • a method of treating a subject having a 5T4-positive cancer by selecting a subject with a 5T4-positive cancer and administering to the subject a therapeutically effective amount of an antibody, ADC, CAR (e.g. a CTL expressing a CAR), immunoconjugate or composition disclosed herein.
  • a method of inhibiting tumor growth or metastasis of a 5T4-positive cancer in a subject by selecting a subject with a 5T4-positive cancer and administering to the subject a therapeutically effective amount of an antibody, ADC, CAR (e.g. a CTL expressing a CAR), immunoconjugate or composition disclosed herein.
  • the 5T4-positive cancer is a colorectal, ovarian, renal, non-small cell lung or gastric cancer.
  • a therapeutically effective amount of a 5T4-specific antibody, ADC, CAR (e.g. a CTL expressing a CAR), immunoconjugate, bispecific antibody or composition will depend upon the severity of the disease and the general state of the patient's health.
  • a therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Administration of the antibodies, ADCs, CARs, immunoconjugates, bispecific antibodies and compositions disclosed herein can also be accompanied by administration of other anti- cancer agents or therapeutic treatments (such as surgical resection of a tumor).
  • Any suitable anti-cancer agent can be administered in combination with the antibodies, compositions and immunoconjugates disclosed herein.
  • Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents.
  • Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.
  • alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
  • nitrogen mustards such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil
  • alkyl sulfonates such as busulfan
  • nitrosoureas such as carmustine, lomustine, semustine, streptozocin, or dacarbazine.
  • Non-limiting examples of antimetabolites include folic acid analogs (such as
  • methotrexate methotrexate
  • pyrimidine analogs such as 5-FU or cytarabine
  • purine analogs such as mercaptopurine or thioguanine.
  • Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).
  • vinca alkaloids such as vinblastine, vincristine, or vindesine
  • epipodophyllotoxins such as etoposide or teniposide
  • antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C
  • enzymes such as L-asparaginase
  • miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II also known as cisplatin
  • substituted ureas such as hydroxyurea
  • methyl hydrazine derivatives such as procarbazine
  • adrenocrotical suppressants such as mitotane and aminoglutethimide
  • hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone).
  • adrenocorticosteroids such as prednisone
  • progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate
  • estrogens such as diethylstilbestrol and ethinyl estradiol
  • antiestrogens such as tamoxifen
  • androgens such as testerone proprionate and fluoxymesterone
  • chemotherapy drugs examples include Adriamycin, Alkeran, Ara- C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5- FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban,
  • Non-limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Jersey, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
  • Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it.
  • surgical treatment for example surgical resection of the cancer or a portion of it.
  • radiotherapy for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.
  • 5T4 expression is detected in a biological sample.
  • the sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens.
  • Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.
  • a method of determining if a subject has a 5T4-positive cancer by contacting a sample from the subject with a monoclonal antibody disclosed herein; and detecting binding of the antibody to the sample.
  • An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having a 5T4-positive cancer.
  • a method of confirming a diagnosis of a 5T4- positive cancer in a subject by contacting a sample from a subject diagnosed with a 5T4-positive cancer with a monoclonal antibody disclosed herein; and detecting binding of the antibody to the sample.
  • An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of a 5T4-positive cancer in the subject.
  • the monoclonal antibody is directly labeled.
  • the methods further include contacting a second antibody that specifically binds the monoclonal antibody with the sample; and detecting the binding of the second antibody.
  • An increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects a 5T4-positive cancer in the subject or confirms the diagnosis of a 5T4-positive cancer in the subject.
  • the cancer is a colorectal, ovarian, renal, non-small cell lung or gastric cancer, or any other type of cancer that expresses 5T4.
  • control sample is a sample from a subject without cancer.
  • sample is a blood or tissue sample.
  • the antibody that binds (for example specifically binds) 5T4 is directly labeled with a detectable label.
  • the antibody that binds (for example, specifically binds) 5T4 (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that specifically binds 5T4 is labeled.
  • a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
  • the first antibody is a human IgG
  • the secondary antibody may be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a non-limiting exemplary luminescent material is luminol; a non- limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 13 % 35 S or 3 H.
  • 5T4 can be assayed in a biological sample by a competition immunoassay utilizing 5T4 standards labeled with a detectable substance and an unlabeled antibody that specifically binds 5T4.
  • a competition immunoassay utilizing 5T4 standards labeled with a detectable substance and an unlabeled antibody that specifically binds 5T4.
  • the biological sample, the labeled 5T4 standards and the antibody that specifically bind 5T4 are combined and the amount of labeled 5T4 standard bound to the unlabeled antibody is determined.
  • the amount of 5T4 in the biological sample is inversely proportional to the amount of labeled 5T4 standard bound to the antibody that specifically binds 5T4.
  • the immunoassays and method disclosed herein can be used for a number of purposes.
  • the antibody that specifically binds 5T4 may be used to detect the production of 5T4 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of 5T4 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the 5T4 is cell-surface 5T4. In other examples, the 5T4 is soluble 5T4 (e.g. 5T4 in a cell culture supernatant or soluble 5T4 in a body fluid sample, such as a blood or serum sample).
  • kits for detecting 5T4 in a biological sample such as a blood sample or tissue sample.
  • a biological sample such as a blood sample or tissue sample.
  • a biopsy can be performed to obtain a tissue sample for histological examination.
  • a blood sample can be obtained to detect the presence of soluble 5T4 protein or fragment.
  • Kits for detecting a polypeptide will typically comprise a monoclonal antibody that specifically binds 5T4, such as any of the antibodies disclosed herein.
  • an antibody fragment such as an scFv fragment, a VH domain, or a Fab is included in the kit.
  • the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
  • kits in one embodiment, includes instructional materials disclosing means of use of an antibody that binds 5T4.
  • the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting 5T4 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to a 5T4 polypeptide.
  • the antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
  • the antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs.
  • the antibodies can also be utilized in immunoassays such as but not limited to radioimmunoassays (RIAs), ELISA, or
  • the antibodies can also be used for fluorescence activated cell sorting (FACS).
  • FACS employs a plurality of color channels, low angle and obtuse light- scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5, 061,620). Any of the monoclonal antibodies that bind 5T4, as disclosed herein, can be used in these assays.
  • the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • the 5T4 gene segment was synthesized by GenScript (Piscataway, NJ).
  • GenScript Procataway, NJ.
  • the plasmid encoding 5T4 residues 1-324 fused with IgGl Fc and Avi-tag (5T4-Fc) was transfected into 293 FREESTYLETM cells (In vitro gen) for transient expression and used for biopanning.
  • the plasmid encoding 5T4 residues 1-324 (5T4) was also transfected into 293 FREESTYLETM cells and used for ELISA. Protein purity was judged by SDS-PAGE, and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare).
  • PMBC phage display library
  • 5T4-Fc fusion protein conjugated to magnetic beads Invitrogen
  • Amplified libraries of 10 12 phage-displayed Fabs were incubated with 5, 3, 3 and 1 ⁇ g of 5T4-Fc for 2 hours at room temperature during the first, second, third and fourth rounds of biopanning, respectively.
  • Clones that bound to 5T4-Fc were identified from the third and fourth rounds of panning using monoclonal phage ELISA. The VH and VL domains of these clones were sequenced, and twelve dominant clones were identified.
  • Fabs For conversion to IgGl, the heavy and light chains of Fabs were amplified and re-cloned into the pDR12 vector. Both Fabs and IgGls were expressed using standard methods. Protein purity was estimated as >95% by SDS-PAGE and protein concentration was measured
  • the 5T4 protein was coated on a 96-well plate (Costar) at 50 ng/well in PBS overnight at 4°C.
  • phage ELISA phages from each round of panning (polyclonal phage ELISA) or clones randomly picked from the infected TGI cells (monoclonal phage ELISA) were incubated with immobilized antigen. Bound phages were detected with anti-M13-HRP polyclonal antibody (Pharmacia, Piscataway, NJ).
  • HRP-conjugated mouse anti- FLAG tag antibody Sigma- Aldrich
  • IgGl binding assay HRP-conjugated goat anti-human IgG antibody (Sigma- Aldrich) was used for detection.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • a purified recombinant antigen (in this case 5T4) is needed.
  • the 5T4 gene was fused with human IgGl Fc.
  • the 5T4-Fc purified by protein G was >95 pure as determined by polyacrylamide gel electrophoresis.
  • the purified 5T4-Fc fusion protein was used for panning of a naive human Fab phage display library derived from healthy human blood and another library derived from cord blood.
  • 5T4-Fc produced in 293 FREESTYLETM cells was labeled with biotin and used for panning of the large Fab libraries as described above. After the third and fourth rounds of panning, 200 clones were screened and 7 positive clones with different sequences were identified. Clone H4 (ml 002) and C5 (ml 001) bound with higher affinity to 5T4 than the other clones and were selected for further characterization. Fab mlOOl and ml002 were converted to the IgGl format. Fab ml 002 bound to 5T4 with an EC50 of 2 nmol/L, as measured by ELISA (FIG. 1). Specific binding of IgGl to cell surface-associated 5T4
  • IgG full-size antibodies
  • IgGl H4 ml 002
  • IgGl C5 ml 001
  • A498 cells which express 5T4 as confirmed by flow cytometry (FIG. 2A)
  • IgGl H4 and IgG C5 mlOOl
  • Example 2 5T4-specific monoclonal antibodies for detecting cancer in a subject or confirming the diagnosis of cancer in a subject
  • This example describes the use of 5T4-specific monoclonal antibodies, such as the fully human monoclonal antibodies disclosed herein, or labeled versions of these antibodies, for the detection of cancer in a subject. This example further describes the use of these antibodies to confirm the diagnosis of cancer in a subject.
  • a sample (such as a biopsy) is obtained from the patient diagnosed with, or suspected of having a 5T4-positive cancer (i.e., a cancer that overexpresses 5T4, such as colorectal, ovarian, renal, non-small cell lung or gastric cancer).
  • a sample taken from a patient that does not have cancer can be used as a control.
  • Immunohistochemistry is performed to detect the presence of 5T4-expressing cells in the sample. Fluorescence can be detected using a fluorometric plate reader according to standard methods. IHC methods are well known in the art. For example, a 5T4- specific antibody conjugated to a fluorescent marker can be used to directly detect 5T4.
  • Example 3 5T4-specific monoclonal antibodies for the treatment of cancer
  • 5T4-specific monoclonal antibodies such as the fully human monoclonal antibodies disclosed herein, for the treatment of cancers that exhibit overexpression of 5T4 (referred to herein as a "5T4-positive" cancer), including, but not limited to colorectal, ovarian, renal, non-small cell lung and gastric cancer.
  • a 5T4-positive cancer including, but not limited to colorectal, ovarian, renal, non-small cell lung and gastric cancer.
  • Patients diagnosed with a 5T4-positive cancer can be treated according to standard procedures in the art.
  • patients diagnosed with a 5T4-positive cancer are administered an ADC comprising a 5T4-specific monoclonal antibody linked to a drug (such as a small molecule, for example an anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent).
  • a drug such as a small molecule, for example an anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent.
  • the ADC is administered by intravenous infusion every three weeks.
  • the dose of ADC administered to a patient varies depending on the weight and gender of the patient, and mode and time course of administration. In some cases, the ADC is administered at a dose of about 1 to about 5 mg/kg.
  • patients are evaluated for cancer progression (including tumor growth and metastasis) and other clinical signs of illness.

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Abstract

L'invention concerne des anticorps monoclonaux entièrement humains qui se lient à 5T4. Le 5T4 est un antigène associé à des tumeurs exprimé dans une variété de tumeurs de type carcinome, y compris dans des tumeurs du colon et du rectum, de l'ovaire, du rein, du poumon non à petites cellules et de l'estomac, mais ayant une expression limitée dans les tissus adultes normaux. L'invention concerne également des conjugués anticorps-médicament (ADC), des récepteurs antigéniques chimériques (CAR), des immunoconjugués et des compositions qui contiennent les anticorps monoclonaux spécifiques de 5T4 entièrement humains. Les anticorps et les compositions selon l'invention peuvent être utilisés, par exemple, dans le diagnostic et le traitement des cancers 5T4-positifs.
PCT/US2015/044253 2014-08-08 2015-08-07 Anticorps monoclonaux humains spécifiques de 5t4 et leurs procédés d'utilisation WO2016022939A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
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WO2019175198A3 (fr) * 2018-03-12 2019-11-14 Genmab A/S Anticorps
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JP2020522474A (ja) * 2017-05-23 2020-07-30 ドラゴンフライ セラピューティクス， インコーポレイテッド Ｎｋｇ２ｄ、ｃｄ１６、および腫瘍関連抗原に結合するタンパク質
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US20190374651A1 (en) * 2017-01-08 2019-12-12 Zhejiang Zova Biotherapeutics Inc. Anti-5t4 antibody-drug conjugate and use thereof
JP2020514302A (ja) * 2017-01-08 2020-05-21 浙江昭華生物医薬有限公司 抗５ｔ４抗体−薬物複合体およびその使用
EP3599249A4 (fr) * 2017-01-08 2021-04-21 Zhejiang Zova Biotherapeutics Inc. Conjugué anticorps anti-5t4-médicament et son utilisation
US11679162B2 (en) * 2017-01-08 2023-06-20 Hangzhou Adcoris Biopharma Co., Ltd. Anti-5T4 antibody-drug conjugate and use thereof
US11459394B2 (en) 2017-02-24 2022-10-04 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
EP4389226A2 (fr) 2017-02-24 2024-06-26 MacroGenics, Inc. Molécules de liaison bispécifiques capables de se lier à cd137 et à des antigènes tumoraux, et leurs utilisations
US11942149B2 (en) 2017-02-24 2024-03-26 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
CN110678197A (zh) * 2017-03-15 2020-01-10 牛津生物医学（英国）有限公司 方法
JP2020522474A (ja) * 2017-05-23 2020-07-30 ドラゴンフライ セラピューティクス， インコーポレイテッド Ｎｋｇ２ｄ、ｃｄ１６、および腫瘍関連抗原に結合するタンパク質
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US11130819B2 (en) 2018-03-12 2021-09-28 Genmab A/S Antibodies
US11008399B2 (en) 2018-03-12 2021-05-18 Genmab A/S Antibodies
WO2019175198A3 (fr) * 2018-03-12 2019-11-14 Genmab A/S Anticorps
CN111971298A (zh) * 2018-03-12 2020-11-20 健玛保 抗体
US11883432B2 (en) 2020-12-18 2024-01-30 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity
WO2023107954A1 (fr) * 2021-12-08 2023-06-15 Dragonfly Therapeutics, Inc. Anticorps ciblant 5t4 et leurs utilisations
CN117482245A (zh) * 2022-12-30 2024-02-02 英百瑞(杭州)生物医药有限公司 抗5t4抗体-自然杀伤细胞偶联物及其用途
WO2024140904A1 (fr) * 2022-12-30 2024-07-04 英百瑞(杭州)生物医药有限公司 Conjugué anticorps anti-5t4/cellule tueuse naturelle et son utilisation
WO2024183811A1 (fr) * 2023-03-08 2024-09-12 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Anticorps anti-5t4 et leurs utilisations
WO2024208145A1 (fr) * 2023-04-03 2024-10-10 非同（成都）生物科技有限公司 Anticorps anti-5t4 et son utilisation
CN118126185A (zh) * 2023-12-25 2024-06-04 上海华核药业股份有限公司 一种放射性核素标记的抗5t4纳米抗体及其制备方法与应用

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