WO2014124258A2 - Sites spécifiques de modification d'anticorps pour fabriquer des immunoconjugués - Google Patents

Sites spécifiques de modification d'anticorps pour fabriquer des immunoconjugués Download PDF

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WO2014124258A2
WO2014124258A2 PCT/US2014/015302 US2014015302W WO2014124258A2 WO 2014124258 A2 WO2014124258 A2 WO 2014124258A2 US 2014015302 W US2014015302 W US 2014015302W WO 2014124258 A2 WO2014124258 A2 WO 2014124258A2
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antibody
substitution
pel
amino acid
light chain
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PCT/US2014/015302
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WO2014124258A3 (fr
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Bernhard Hubert GEIERSTANGER
Weijia Ou
Tetsuo Uno
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Irm Llc
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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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to site-specific labeling sites and processes, molecules produced thereby such as antibody drug conjugates, and their uses.
  • Heterogeneity of a pharmaceutical active ingredient is typically undesirable: It is far preferable to administer a homogeneous product, and far more difficult to fully characterize a heterogeneous one.
  • Site-specific conjugation of a cytotoxic drug to an antibody through, for example, engineered cysteine residues results in homogenous immunoconjugates that exhibit improved therapeutic index (Junutula et ah, (2008) Nat Biotechnol. 26(8):925-932).
  • ADCs Antibody drug conjugates
  • ADCs have been used for the local delivery of cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr. Opinion in Pharmacology 5:543-549, 2005).
  • ADCs allow targeted delivery of the drug moiety where maximum efficacy with minimal toxicity may be achieved.
  • site-specific conjugations that can generate homogeneous immunoconjugates with a defined drug-to-antibody ratio for use in cancer therapy.
  • site-specifically conjugated immunoconjugates exhibit improved therapeutic index, and the attachment sites described in the instant invention provide a means to prepare such site-specific and hence improved immunoconjugates.
  • the invention provides specific sites in the constant region of an antibody or antibody fragment at which a native amino acid on a parental antibody or antibody fragment can be replaced with various TAG-encoded amino acids in order to provide a modified antibody or antibody fragment.
  • the TAG sequence in a nucleic acid is normally read as a "stop" codon, but under suitable conditions it can be used to incorporate a number of different amino acids, including pyrroline- carboxy-lysine (Pel), pyrrolysine and unnatural amino acids (Noren et al, (1989) Science 14;244(4901): 182-188; Mendel et al, (1995) Annu Rev Biophys Biomol Struct.
  • TAG-encoded amino acids such as Pel
  • a payload drug moiety
  • the invention further provides engineered antibodies or fragments thereof having one or more such TAG-encoded residues in one or more specific sites (Selected TAG sites in Tables 1, 2 and 3), as well as immunoconjugates made from such engineered antibody sequences.
  • Pel is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine.
  • Pel is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. See, e.g., Ou et al, (2011) Proc Natl Acad Sci U S A. 108: 10437-10442; Cellitti et al, (2011) Nat Chem Biol. 7(8):528-30; Gaston et al, (2011) Nature 471(7340):647-50.
  • the current invention provides specific sites in the constant region of antibodies where replacing one or more native amino acids on a parental antibody or antibody fragment with Pel or other TAG-encoded amino acids provides one or more advantages as described herein, such as high expression of the corresponding TAG- encoded amino acid containing protein, good conjugation yield, and efficient conjugation loading. Because the sites identified herein are in the constant region of an antibody sequence, they can be used with various antibodies.
  • the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with Pel or another TAG-encoded amino acid on its constant region chosen from positions (Selected TAG Sites) identified herein.
  • the site for substitution is one of the Selected TAG Sites listed in Table 1, Table 2 or Table 3, or a combination of two or more of those sites.
  • the invention further provides modified antibodies or antibody fragments that are suitable for making these immunoconjugates, methods for making the
  • immunoconjugates and methods to use the immunoconjugates for treatment of disorders such as cancer or other cell proliferation disorders.
  • the invention provides an immunoconjugate of Formula
  • Ab represents an antibody or antibody fragment comprising at least one TAG-encoded amino acid residue at one of the substitution sites described herein;
  • LU is a linker unit as described herein;
  • X is a payload or drug moiety
  • n is an integer from 1 to 16.
  • LU is attached to a TAG- encoded amino acid such as Pel at one of the specific substitution sites described herein, X is a drug moiety such as an anticancer drug, and n is 2-8 when Ab is an antibody, or n can be 1-8 when Ab is an antibody fragment.
  • the invention provides a modified antibody or antibody fragment thereof comprising a substitution of a TAG-encoded amino acid for the native amino acid of a parental antibody or antibody fragment at one or more of the substitution positions (Selected TAG Sites) identified in Table 1, Table 2 and Table 3.
  • the TAG-encoded amino acid is Pel.
  • the invention provides a method to select a site where a TAG-encoded amino acid (e.g., Pel) can be substituted for a native amino acid in a parental antibody or antibody fragment.
  • a TAG-encoded amino acid e.g., Pel
  • amino acid refers to canonical, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the canonical amino acids.
  • Canonical amino acids are
  • proteinogenous amino acids encoded by the genetic code and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline serine, threonine, tryptophan, tyrosine, valine, as well as selenocysteine, pyrrolysine and pyrroline- carboxy-lysine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a canonical amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a canonical amino acid.
  • TAG-encoded amino acid refers to any amino acid that can be incorporated into a protein by expression of a TAG codon in a nucleic acid containing a TAG-codon, for example at one of the substitution sites identified herein.
  • TAG-encoded amino acids include Pyrroline- carboxy-lysine (Pel), pyrrolysine and unnatural amino acids, particularly those
  • the TAG-encoded amino acid is Pel.
  • Non-limiting examples of other TAG-encoded amino acids include reactive
  • pyrrolysine analogs such as N6-(2-(R)-propargylglycyl)-lysine (Li, Fekner, Chan, Chem Asian J. 2010, 5, 1765-9), N6-[(2-propynyloxy)carbonyl]-L-lysine (Nguyen et al, J Am Chem Soc. 2009, 131(25), 8720-1), N6-[(2-azidoethoxy)carbonyl]-L- lysine (Nguyen et al, J Am Chem Soc. 2009, 131(25), 8720-1) and others described in Fekner and Chan (Fekner and Chan, Curr Opin Chem Biol.
  • the preferred substitution sites of the invention are located in the constant region of an antibody, and are identified herein using standard numbering conventions. It is well known, however, that portions of antibodies can be used for many purposes instead of intact full-length antibodies, and also that antibodies can be modified in various ways that affect numbering of sites in the constant region even though they do not substantially affect the functioning of the constant region. For example, insertion of an S6 tag (a short peptide tag) into a loop region of an antibody has been shown to allow activity of the antibody to be retained, even though it would change the numbering of many sites in the antibody.
  • S6 tag a short peptide tag
  • substitution sites described herein are identified by a standard numbering system based on intact antibody numbering
  • the invention includes the corresponding sites in antigen binding fragments or in antibodies containing other modifications, such as S6 tag insertion.
  • the corresponding sites in those fragments or modified antibodies are thus preferred sites for substitution in fragments or modified antibodies, and references to the substitution sites by number include corresponding sites in modified antibodies or antigen binding fragments that retain the function of the relevant portion of the full-length antibody.
  • a corresponding site in a modified antibody or antibody fragment can readily be identified by aligning a segment of the antibody fragment or modified antibody with the parental antibody to identify the site in the antibody fragment or modified antibody that matches one of the preferred substitution sites of the invention. Alignment may be based on a segment long enough to ensure that the segment matches the correct portion of the full-length antibody, such as a segment of at least 20 amino acid residues, or at least 50 residues, or at least 100 residues, or at least 150 residues. Alignment may also take into account other modifications that may have been engineered into the antigen binding fragment or modified antibody, thus differences in sequence due to engineered point mutations in the segment used for alignment, particularly for conservative substitutions, would be allowed. Thus, for example, an Fc domain can be excised from an antibody, and would contain amino acid residues that correspond to the specific substitution sites described herein, despite numbering differences: sites in the Fc domain
  • substitution sites of the invention would also be expected to be advantageous sites for similar TAG-encoded substitution in the Fc domain, and are included in the scope of the invention.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a canonical amino acid.
  • the term "unnatural amino acid”, as used herein, is intended to represent amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether the same or different. In addition, such
  • unnatural amino acids typically require a modified tRNA and a modified tRNA synthetase (RS) for incorporation into a protein.
  • RS modified tRNA synthetase
  • These "selected" orthogonal tR A/RS pair are specific for the unnatural amino acid and are generated by a selection process as developed by Schultz et al. (see, e.g., Liu et al, (2010) Annu. Rev. Biochem. 79:413-444) or a similar procedure.
  • unnatural amino acid does not include the natural occurring 22 nd proteinogenic amino acid pyrrolysine (Pyl) as well as its demethylated analog pyrroline-carboxy-lysine (Pel), because incorporation of both residues into proteins is mediated by the unmodified, naturally occurring pyrrolysyl-tRNA/tRNA synthetase pair and because Pyl and Pel are generated biosynthetically (see, e.g., Ou et al, (201 1) Proc. Natl. Acad. Sci. USA. 108: 10437-10442; Cellitti et al, (201 1) Nat. Chem. Biol. 27;7(8):528-30).
  • antibody refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non- covalently, reversibly, and in a specific manner.
  • a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention).
  • the antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody.
  • the N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy -terminal domains of the heavy and light chain, respectively.
  • an antibody fragment refers to a portion of an antibody.
  • an antibody fragment can be the fragment crystallizable region (Fc region), which is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system.
  • An antibody fragment can also be an antigen binding fragment, which refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al, Nature 341 :544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.
  • scFv single-chain Fvs
  • sdFv disulfide-linked Fvs
  • Fab fragments F(ab'
  • the two domains of the Fv fragment, VL and V3 ⁇ 4 are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g., Bird et al, Science 242:423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment.”
  • antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature
  • Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703, 199, which describes fibronectin polypeptide monobodies).
  • Fn3 fibronectin type III
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al, Protein Eng. 8: 1057-1062, 1995; and U.S. Pat. No. 5,641,870).
  • the term "monoclonal antibody” or “monoclonal antibody composition” as used herein refers to polypeptides, including antibodies and antigen binding fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000).
  • the human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).
  • humanized antibody refers to an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).
  • the term “recognize” as used herein refers to an antibody or antigen binding fragment thereof that finds and interacts (e.g., binds) with its epitope, whether that epitope is linear or conformational.
  • epitope refers to a site on an antigen to which an antibody or antigen binding fragment of the invention specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
  • affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • conservatively modified variants include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative sequence modifications” are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • the term "optimized” as used herein refers to a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.
  • the optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
  • percent identical in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison algorithm typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: The cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X are used to calculate the cumulative score.
  • W word length
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 1 1-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide- nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081 ; Ohtsuka et al, (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).
  • operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to canonical amino acid polymers as well as to non-canonical amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • immunoconjugate or "antibody conjugate” as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, and the like.
  • the linkage can be covalent bonds or non-covalent interactions and can include chelation.
  • linkers known in the art, can be employed in order to form the immunoconjugate.
  • the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.
  • fusion protein refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non- human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • cytotoxin refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.
  • anti-cancer agent refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
  • drug moiety refers to a chemical moiety that is conjugated to the antibody or antigen binding fragment of the invention, and can include any moiety that is useful to attach to an antibody or antigen binding fragment.
  • a drug moiety or payload can be an anticancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent.
  • a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a
  • microtubule destabilizer an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPrV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • MetAP methionine aminopeptidase
  • an inhibitor of nuclear export of proteins CRM1 a DPPrV inhibitor
  • an inhibitor of phosphoryl transfer reactions in mitochondria a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor,
  • Suitable examples include auristatins such as MMAE and MMAF; calicheamycins such as gamma-calicheamycin; and maytansinoids such as DM1 and DM4.
  • auristatins such as MMAE and MMAF
  • calicheamycins such as gamma-calicheamycin
  • maytansinoids such as DM1 and DM4.
  • a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide, a reactive functional group such as those listed in Table 4, or a binding agent that can connect the conjugate to another moiety or surface, etc.
  • Tumor refers to a neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • anti-tumor activity means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity.
  • a possible way of showing antitumor activity is to show a decline in growth rate of abnormal cells that arises during therapy or tumor size stability or reduction.
  • Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer includes a malignancy characterized by deregulated or uncontrolled cell growth.
  • Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.
  • cancer includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from
  • metastasis the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • an optical isomer or "a stereoisomer” refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers. It is understood that a substituent may be attached at a chiral center of a carbon atom.
  • the term “chiral” refers to molecules which have the property of non- superimposability on their mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner. Therefore, the invention includes enantiomers, diastereomers or racemates of the compound.
  • Enantiomers are a pair of stereoisomers that are non-superimposable mirror images of each other.
  • a 1 : 1 mixture of a pair of enantiomers is a “racemic” mixture. The term is used to designate a racemic mixture where appropriate.
  • Diastereoisomers are stereoisomers that have at least two asymmetric atoms, but which are not mirror- images of each other.
  • the absolute stereochemistry is specified according to the Cahn-lngold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or S.
  • Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.
  • Certain compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)-.
  • the compounds can be present in the form of one of the possible isomers or as mixtures thereof, for example as pure optical isomers, or as isomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms.
  • the present invention is meant to include all such possible isomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms.
  • Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or they may be resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.
  • salt refers to an acid addition or base addition salt of a compound of the invention.
  • Salts include in particular “pharmaceutical acceptable salts”.
  • pharmaceutically acceptable salts refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable.
  • the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorotheophyllinate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/d
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table.
  • the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like.
  • Certain organic amines include isopropylamine, benzathine, cholinate,
  • diethanolamine diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, use of non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where practicable.
  • the appropriate base such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like
  • any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.
  • Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2 H, 3 H, U C, 13 C, 14 C, 15 N, 18 F 31 P, 32 P, 35 S, 36 C1, 125 I respectively.
  • the invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as 3 H and 14 C, or those into which non-radioactive isotopes, such as 2 H and 13 C are present.
  • isotopically labeled compounds are useful in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F or labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
  • substitution with heavier isotopes may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index.
  • deuterium in this context is regarded as a substituent of a compound of the formula (I).
  • concentration of such a heavier isotope, specifically deuterium may be defined by the isotopic enrichment factor.
  • isotopic enrichment factor as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope.
  • a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • a therapeutically effective amount of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
  • the term "a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease, or at least partially inhibit activity of a targeted enzyme or receptor.
  • the term “inhibit”, “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • the term “treat”, “treating” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • treat refers to preventing or delaying the onset or development or progression of the disease or disorder.
  • a subject is "in need of a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.
  • ABA compound refers to a 2- aminobenzaldehyde compound of the following formula:
  • R 30 is H
  • LU is a linker unit
  • X is a payload
  • Z is a group selected from H, halo, Ci-4 alkyl, Ci_ 4 haloalkyl, C1-4 alkoxy, and a group -LU 2 -X 2 , where LU 2 is a second linker unit and X 2 is a second payload.
  • Linker Unit refers to a covalent chemical connection between two moieties, such as an antibody and a payload.
  • Each LU can be comprised of one or more components described herein as Li, L 2 , L 3 , L 4 , L 5 and L 6 .
  • the linker unit can be selected to provide suitable spacing between the connected moieties, or to provide certain physicochemical properties, or to allow cleavage of the Linker Unit under certain conditions.
  • Cleavable linker refers to a linker or linker unit that connects two moieties by covalent connections, but breaks down to sever the covalent connection between the moieties under physiologically relevant conditions. Cleavage may be enzymatic or non-enzymatic, but generally releases a payload from an antibody without degrading the antibody. Cleavage may leave some portion of a linker or LU attached to the payload, or it may release the payload without any linker-derived residue.
  • Pel as used herein refers to pyrroline carboxy lysine, e.g.,
  • R is H, which has the following formula when incorporated into a peptide:
  • Non-cleavable linker refers to a linker or linker unit that is not susceptible to breaking down under physiological conditions, i.e., it is at least as stable as the antibody or antibody fragment portion of the
  • linker While the linker may be modified physiologically, it keeps the payload connected to the antibody until the antibody is substantial degraded, i.e., the antibody degradation precedes cleavage of the linker. Degradation of the antibody may leave some or all of the linker or LU, and even one or more amino acid groups from the antibody, attached to the payload or drug moiety that is delivered in vivo.
  • Cyclooctyne refers to an 8-membered ring containing a carbon-carbon triple bond (acetylene).
  • the ring is optionally fused to one or two phenyl rings, which may be substituted with 1-4 Ci_ 4 alkyl, C 1-4 alkoxy, halo, hydroxyl, COOH, COOLi, -C(0)NH-Li, O-Li, or similar groups, and which may contain N, O or S as a ring member.
  • cyclooctyne can be a Cs hydrocarbon ring, particularly an isolated ring that is saturated aside from the triple bond, and may be substituted with F or Hydroxy, and may be linked to a linker or LU via -0-, -C(O), C(0)NH, or C(0)0.
  • Cyclooctene refers to an 8-membered ring containing at least one double bond, especially a trans-double bond.
  • the ring is optionally fused to one or two phenyl rings, which may be substituted with 1-4 C 1-4 alkyl, Ci_ 4 alkoxy, halo, hydroxyl, COOH, COOL -C(0) H-Li, O-Li, or similar groups, and which may contain N, O or S as a ring member.
  • cyclooctene can be an isolated Cs hydrocarbon ring that is saturated aside from the trans double bond and is optionally substituted with F or Hydroxy, and may be linked to a linker or LU via -0-, -C(O), C(0)NH, or C(0)0.
  • FIG. 1 Surface accessibility plot of amino acid residues in human IgGl heavy chain (A) and kappa light chain (B). Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [A 2 ].
  • FIG. 2 Location of selected 92 TAG mutations in the structure of a human IgGl with a kappa light chain. Selected residues for TAG mutations are shown in black on only one of the two heavy chains and for one of the two kappa light chains (lHZH.pdb). Structures are shown using PyMOL, an open-source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrodinger, LLC).
  • FIG. 3 The amino acid sequence alignment of the heavy chain constant regions of trastuzumab and antibody 14090. Underlined residues in the sequences of trastuzumab antibody and antibody 14090 are the residues that have been mutated into TAG-encoded amino acids. Amino acid residues in the heavy chain are numbered by Eu numbering system (Edelman et al, 1969).
  • FIG. 4 Amino acid sequence alignment of constant regions of trastuzumab, human IgGl, IgG2, IgG3 and IgG4.
  • FIG. 5 The amino acid sequence alignment of the constant regions of human kappa and lambda light chains.
  • FIG. 6 Analysis of trastuzumab Pel antibodies by SDS-PAGE under reducing conditions after Protein A purification.
  • FIG. 7 Separation of full-length and truncated form of the trastuzumab HC-T155Pcl antibody.
  • FIG. 8 Reaction of Pel with a 2-amino-benzaldehyde (ABA) compound.
  • FIG. 9 Structure of ABA-MMAF.
  • FIG. 10 Analysis of ABA-MMAF conjugation to a trastuzumab Pel antibody by reverse phase high pressure liquid chromatography (HPLC).
  • HPLC reverse phase high pressure liquid chromatography
  • FIG. 12 Analysis of HIC-purified trastuzumab LC-T109Pcl-MMAF ADC (dashed line) and unmodified wild-type trastuzumab (solid line) by analytical size exclusion chromatography.
  • FIG. 13 Analysis of HIC-purified trastuzumab Pcl-MMAF ADCs by SDS-PAGE.
  • FIG. 14 LCMS of HIC-purified trastuzumab LC-K107Pcl-MMAF
  • FIG. 15 Thermal melting curves of unmodified wild-type trastuzumab (solid line, labeled WT anti-Her2), trastuzumab HC-E152Pcl-MMAF (dotted line) and LC-R108Pcl-MMAF (dashed line) ADCs.
  • FIG. 16 Cell proliferation assays for trastuzumab LC-K107Pcl- MMAF ADC with MDA-MB231 clone 16 and clone 40 cells.
  • FIG. 17 Cell proliferation assays for Antibody 14090 HC-E258Pcl- MMAF ADC with CMK11-5 and Jurkat cells.
  • FIG. 18 Structure of the putative toxic metabolite of a Pcl-MMAF ADC (A) and of a Pel ADC with a payload with non-cleavable linker (B).
  • FIG. 19 Pharmacokinetic studies of trastuzumab Pcl-MMAF ADCs in mice. Representative plots of plasma concentration vs. sample collection time are shown for (A) unconjugated, wild-type trastuzumab, (B) HC-E258Pcl-MMAF, (C) LC-D122Pcl-MMAF, (D) LC-R142Pcl-MMAF, (E) LC-K169Pcl-MMAF, (F) LC-S1 14Pcl-MMAF, and (G) LC-S156Pcl-MMAF ADCs. ELISA readouts with anti-hlgG antibody are shown as solid symbols while concentrations measured with anti-MMAF-ELISA are represented as open symbols. The standard deviation of measurements in three animals was used as error bars.
  • FIG. 20 In vivo efficacy studies of trastuzumab Pcl-MMAF
  • ADCs in a MDA-MB231 clone 16 xenograft mouse model were assessed for a MDA-MB231 clone 16 xenograft mouse model.
  • A Inhibition of MDA-MB231-16 tumor growth in vivo by 6 trastuzumab HC-Pcl-MMAF ADCs.
  • B Inhibition of MDA-MB231-16 tumor growth in vivo by 4 trastuzumab LC-Pcl- MMAF ADCs.
  • FIG. 21 Location of selected attachment sites in the structure of a human IgGl with a kappa light chain. Selected residues are shown in black on only one of the two heavy chains and for one of the two kappa light chains (lHZH.pdb). Three rotations of the structure are shown using PyMOL, an open- source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrodinger, LLC).
  • the present invention provides methods of site-specific labeling of antibodies or antibody fragments by replacing one or more amino acids of a parent antibody or antibody fragment at specific positions with a TAG-encoded amino acid such as pyrroline-carboxy-lysine ("Pel").
  • the engineered antibodies or antibody fragments are capable of conjugation to various agents (e.g., cytotoxic agents).
  • the present invention also provides antibody drug conjugates that are produced by using the methods described herein.
  • TAG is normally recognized as a "STOP" codon during translation of a nucleic acid into a protein, but in some systems, TAG can function as a codon for certain amino acids, including Pel, pyrrolysine, and some unnatural amino acids. It is known, for example, that the pyrrolysyl-tRNA charged with Pel (through the action of the pyrrolysyl-tRNA synthetase (RS)) naturally recognizes TAG and the ribosome inserts Pel at the TAG codon.
  • RS pyrrolysyl-tRNA synthetase
  • the release factor RF 1 competes with the pyrrolysyl-tRNA, which may result in either truncation of the nascent polypeptide chain or alternatively in Pel incorporation and continuing protein synthesis. Such competition often reduces protein yield. The extent of truncation depends on the incorporation site and is generally not predictable.
  • the present invention describes specific sites on an antibody constant region where a native amino acid of a parental antibody or antibody fragment can be replaced with a TAG-encoded amino acid such as Pel without extensive interference due to truncation.
  • a native amino acid of a parental antibody or antibody fragment can be replaced with a TAG-encoded amino acid such as Pel without extensive interference due to truncation.
  • a TAG-encoded amino acid such as Pel without extensive interference due to truncation.
  • Efficient production means the protein production yield is sufficiently high for further conjugation, and that the major product of expression is a protein that has incorporated a TAG-encoded amino acid and has thus not been truncated by the TAG codon.
  • the invention provides methods for selecting advantageous sites for such amino acid substitution in antibody sequences, and identifies advantageous sites that are useful across various antibodies.
  • the invention provides modified antibodies or antibody fragments where Pel or another TAG-encoded amino acid has been substituted for a native amino acid of a parental sequence. It has been found that different substitution sites give different efficiencies for conjugation when the TAG-encoded amino acid is Pel: the sites identified herein often provide higher conjugation yields than non-selected sites for antibodies having Pel in place of at least one native amino acid of a parental sequence.
  • the site-specific antibody labeling according to the present invention can be achieved with a variety of chemically accessible labeling reagents, such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immune potentiators, radio-imaging probes, prodrugs, and other molecules.
  • chemically accessible labeling reagents such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immune potentiators, radio-imaging probes, prodrugs, and other molecules.
  • the present invention provides methods of preparation of homogeneous immunoconjugates with a defined drug-to-antibody ratio (DAR) for use in cancer therapy, and immunoconjugates prepared thereby, as well as pharmaceutical compositions comprising these immunoconjugates.
  • DAR drug-to-antibody ratio
  • the invention provides immunoconjugates having at least one TAG-encoded amino acid (e.g., Pel) substituted for a native amino acid of a parental antibody or antibody fragment at a substitution site of the invention, e.g., the Selected TAG Sites listed in Tables 1, 2 and 3.
  • the immunoconjugate comprises an antibody or antibody fragment that comprises a sequence selected from the SEQ ID NOs listed in Table 1, Table 2 and Table 3.
  • the immunoconjugate of the invention is of
  • Ab represents an antibody or antigen binding fragment comprising at least one TAG-encoded amino acid residue such as Pel at one of the substitution sites described herein;
  • LU is a linker unit as described herein, and is preferably attached to Pel at one of the substitution sites described herein;
  • X is a payload or drug moiety; and n is a number from 1 to 16.
  • An immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375 and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.
  • An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and
  • An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain of said antibody or antibody fragment and wherein said positions are numbered according to the EU system, and wherein said light chain is a kappa light chain.
  • said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system and wherein the said light chain is a kappa light chain.
  • An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.
  • modified antibody or antibody fragment further comprises a substitution of one or more amino acids with TAG encoded amino acids on its constant region at a site selected from positions 117, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions on the heavy chain are numbered according to the EU system.
  • immunoconjugate comprises a group of the formula (IA) or (IB):
  • LU is a linker unit
  • [X] is the point of attachment for a drug moiety or payload
  • R 20 is H or methyl
  • R 30 H or methyl or phenyl.
  • said drug moiety is selected from the group consisting of a V-ATPase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a V-ATPase inhibitor an HSP90 inhibitor
  • an IAP inhibitor an mTor inhibitor
  • a microtubule stabilizer a microtubul
  • a pharmaceutical composition comprising the immunoconjugate of any of embodiments 1-22.
  • a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with TAG modified amino acid on its constant region chosen from positions 117, 119, 121, 124, 132, 134, 136, 139, 152, 153, 155, 157, 164, 165, 171, 174, 176, 177, 178, 189, 191, 195, 197, 207, 212, 246, 258, 269, 274, 282, 283, 286, 288, 290, 292, 293, 294, 320, 322, 326, 330, 333, 334, 335, 337, 344, 355, 360, 362, 375, 382, 389, 390, 392, 393, 398, 400, 413, 415, and 422 of a heavy chain, and wherein said positions are numbered according to the EU system.
  • modified antibody or antibody fragment of embodiment 24 or 25 further comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154 , 161, and 165 of a light chain, and wherein said positions are numbered according to the EU system and wherein the said light chain is a kappa light chain.
  • a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 112, 114, 122, 123, 126, 127, 129, 142, 143, 145, 152, 154, 156, 157, 159, 161, 165, 168, 169, 170, 182, 183, 188, 190, 191, 197, 199, 203, and 206 of a light chain, and wherein the positions are numbered according to the EU system. 31.
  • a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with a TAG encoded amino acid at a position selected from on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.
  • a modified antibody or antibody fragment which comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51, 55, 63, 64, 65, 73, 75, 76, 77, 81, 82, 96, 97, 98, 99, 100, 101, and 102.
  • LU is a linker unit
  • X 1 is a drug moiety or payload
  • R 20 is H or methyl
  • R 30 H or methyl or phenyl.
  • a host cell comprising the nucleic acid of claim 43.
  • a method of producing a modified antibody or antibody fragment comprising incubating the host cell of embodiment 43 under suitable conditions for expressing the antibody or antibody fragment, and isolating said antibody or antibody fragment.
  • a method to select an amino acid of an antibody that is suitable for replacement by TAG encoded amino acid to provide an advantaged site for conjugation comprising:
  • preparing a set of nucleic acids including one nucleic acid encoding a polypeptide corresponding to TAG encoded amino acid replacement of the native amino acid for each candidate site for TAG encoded amino acid substitution;
  • each nucleic acid in the set of nucleic acids expressing each nucleic acid in the set of nucleic acids, and removing from the initial set of candidate sites any site where truncation dominates over full-length polypeptide containing a TAG encoded amino acid substitution to provide a set of advantaged sites for TAG encoded amino acid substitution.
  • a method to prepare an immunoconjugate comprising providing an antibody or antibody fragment of any of embodiments 25, 26, 27, 28, 30, 31, 34, 35, or 37 comprising contacting the antibody or antibody fragment containing at least one TAG encoded amino acid residue with an ABA compound, or an ABP compound, or an AAP compound.
  • LU is a linker unit
  • X 1 is a drug moiety or payload
  • R 20 is H or methyl
  • R 30 H or methyl or phenyl.
  • LU is a linker unit
  • X 1 is a drug moiety or payload
  • R 20 is H or methyl
  • R 30 H or methyl or phenyl.
  • the Drug- Antibody ration is preferably about 2, about 4, about 6, or about 8.
  • the group LU is typically a group of formula -L1-L2-L3-L4-L5-L6-, wherein Li, L 2 , L 3 , L 4 , L 5 and L 6 are independently selected from -Ai-, -A1X 2 - and -X 2 -; wherein:
  • each X 2 is independently selected from a bond, R 8 ,
  • each R 5 is independently selected from H, Ci- 4 alkyl, phenyl or Ci- 4 alkyl substituted with 1 to 3 -OH groups;
  • R 7 is independently selected from H, C 1-4 alkyl, phenyl, pyrimidine and pyridine;
  • R 9 is independently selected from H and Ci- 6 haloalkyl
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
  • the antibodies (e.g., a parental antibody, optionally containing one or more non-canonical amino acids) of the present invention are numbered according to the EU numbering system as set forth in Edelman et ah, (1969) Proc. Natl. Acad. USA 63:78-85, except that the lambda light chain is numbered according to the Kabat numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest (1991) Fifth Edition, NIH Publication No. 91-3242.
  • Human IgGl constant region is used as a representative example throughout the application. However, the invention is not limited to human IgGl ; corresponding amino acid positions can be readily deduced by sequence alignment.
  • Figure 4 shows sequence alignment of human IgGl, IgG2, IgG3 and IgG4 heavy chain constant regions, so that an identified TAG-encoded amino acid (e.g., Pel) engineering site in the IgGl constant region can be readily identified for IgG2, IgG3, and IgG4 as shown in Figure 4.
  • TAG-encoded amino acid e.g., Pel
  • Table 1 lists the amino acid positions in the constant region of the heavy chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel.
  • Table 2 lists the amino acid positions in the constant region of the kappa light chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel.
  • Table 3 lists the amino acid positions in the constant region of the lambda light chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel.
  • TAG-encoded amino acid e.g., Pel
  • TAG sites in the heavy chain constant region of human IgGl (Sites numbered according to EU numbering system).
  • the TAG sites can be used to incorporate any other TAG-encoded amino acid.
  • TAG-encoded amino acid e.g., Pel
  • substitution sites on the kappa light chain constant region of human IgGl Sites numbered according to EU numbering system.
  • the TAG sites can be used to incorporate any other TAG-encoded amino acid.
  • TAG-encoded amino acid e.g., Pel
  • the TAG sites can be used to incorporate any other TAG-encoded amino acid.
  • findings of the invention are not limited to any specific antibodies.
  • the findings of the present invention are not limited to using Pel substitutions.
  • the positions in the antibody constant regions identified herein can be used for incorporating other amino acids, especially TAG-encoded amino acids, including non-canonical amino acids and unnatural amino acids.
  • the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids, i.e., a TAG-encoded amino acid such as Pel, on its constant region chosen from the Selected TAG Sites in Table 1, and particularly selected from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment.
  • a TAG-encoded amino acid such as Pel
  • the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof containing at least one TAG-encoded amino acid such as Pel at a site selected from the ones identified herein, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises an amino acid sequence selected from SEQ ID NOs 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51 and 55.
  • the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids on its heavy chain constant region chosen from positions identified in Table 1.
  • the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain.
  • an immunoconjugate comprising a modified antibody or an antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 1 17, 124, 136, 139, 152, 155, 171, 174
  • immunoconjugate of the invention or a modified antibody of the invention, comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 117 and 136, 117 and 139, 117 and 152, 1 17 and 155, 1 17 and 171, 117 and 174, 117 and 258, 117 and 286, 1 17 and 188, 1 17 and 191, or 117 and 375; 136 and 139, 136 and 152, 136 and 155, 136 and 171, 136 and 171, 136 and 174, 136 and 258, 136 and 186, 136 and 288, 136 and 292, 136 and 375; 139 and 152, 139 and 155, 139 and 171, 139 and 174, 139 and 258, 139 and 186, 139 and 188, 139 and 292, 139 and 375; 152 and 155, 139 and 171,
  • an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 117, 136 and 139; 1 17, 136 and 152; 1 17, 136 and 155; 1 17, 136 and 171; 1 17, 136 and 174; 1 17, 136 and 258; 1 17, 136 and 286; 117, 136 and 288; 1 17, 136 and 292; 117, 136 and 375; 117, 139 and 152; 117, 139, and 155; 117, 139 and 171 ; 1 17, 139, and 174; 1 17, 139 and 258; 1 17, 139 and 286; 117, 139 and 288; 1 17, 139 and 292; 1 17, 139 and 375; 1 17, 152, and 155; 1 17, 152, and 171; 117, 152, and 171; 117, 152, and 171; 117, 152, and 171
  • the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its constant region chosen from the Selected TAG sites in Table 2, particularly positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment, wherein the light chain is a human kappa light chain.
  • a TAG-encoded amino acid such as Pel is substituted for the native amino acid of the parental antibody sequence in at least one of the specific substitution sites identified herein.
  • the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NOs: 63, 64, 65, 73, 75, 76,77, 81 and 82.
  • an immunoconjugate of the invention or a modified antibody of the invention, comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 107 and 108; 107 and 109;
  • an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 107, 108 and 109; 107, 108 and 145; 107, 108 and 152; 107, 108 and 154; 107, 109 and 145; 107, 109 and 152; 107, 109 and 154; 107, 145 and 152; 107, 145 and 154; 107, 152 and 154; 108, 109 and 145; 108, 109 and 152; 108, 109, and 154; 109, 145 and 152; 107, 145 and 154; 107, 152 and 154; 108, 109 and 145; 108, 109 and 152; 108, 109, and 154; 109, 145 and 152; 109, 145 and 154; and 109, 152 and 154 of the human kappa light chain
  • the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its constant region chosen from the Selected TAG sites in Table 3, particularly positions 143, 145, 147, 156, 159, 163 and 168 of the light chain of said antibody or antibody fragment, wherein the light chain is a human lambda light chain, using the Kabat numbering system.
  • a TAG-encoded amino acid such as Pel is substituted for the native amino acid of the parental antibody sequence in at least one of the specific substitution sites identified herein.
  • the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NOs: 96, 97, 98, 99, 100, 101 and 102.
  • an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 143 and 145; 143 and 147; 143 and 156; 143 and 156; 143 and 159; 143 and 163; 143 and 168; 145 and 147; 145 and 156; 145 and 159; 145 and 163; 145 and 168; 147 and 156; 147 and 159; 147 and 163; 147 and 163; 147 and 168; 156 and 159; 156 and 163; 156 and 168; 159 and 163; 159 and 168; and 163; 159 and 163; 159 and 168; 156 and 159; 156 and 163; 156 and 168; 159 and 163; 159 and 168; and 163 and 168 of the human lambda light chain.
  • an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 143, 145 and 147; 143, 145 and 156; 143, 145 and 159; 143, 145 and 163; 143, 145 and 168; 144, 147 and 156; 145, 147 and 159; 145, 147 and 163; 145, 147, and 168; 147, 156 and 159; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 163; 147, 156 and 168; 156, 159 and 163; 156, 159 and 168; and 159, 163 and 168 of the human lambda light chain.
  • the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG Sites in Table 1, particularly from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment, in combination with a second substitution, which may be a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment, or a position chosen from positions 143, 145, 147, 156, 159, 163 and 168 of a lambda light chain.
  • a modified antibody or antibody fragment according to the present invention whether alone or as part of an
  • immunoconjugate may comprise a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 136
  • Positions on the human kappa light chain are described using the Eu numbering system. Additional embodiments of the invention include any of these antibodies or antibody fragments further including a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain; and a second substitution in the light chain that is different from the one in the foregoing embodiment and is selected from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the human kappa light chain.
  • a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 3
  • a modified antibody or antibody fragment according to the present invention may comprise a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pcl substitution on position 159 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a
  • Additional embodiments of the invention include any of these antibodies or antibody fragments further including a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain; and a second substitution in the light chain that is different from the one in the foregoing embodiment and is selected from positions 143, 145, 147, 156, 159, 163, and 168 of the human lambda light chain.
  • a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain; and a second substitution in the light chain that is different from the
  • the amino acid substitution described herein is Pel, though other TAG-encoded amino acid can be used as well. Where Pel is introduced, it is typically used as the site of conjugation to which a drug moiety is attached. Conjugates of Formula IA or IB are embodiments of this aspect of the invention.
  • the immunoconjugates of the invention comprise a drug moiety selected from the group consisting of a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a drug moiety selected from the group consisting of a V-ATPase inhibitor, a HSP90 inhibitor
  • the immunoconjugates of the invention comprise a drug moiety that is an anti-cancer agent.
  • the modified antibody or antibody fragments of the present invention can be any formats known in the art, such as a monoclonal, chimeric, humanized, fully human, bispecific, multispecific antibody or antibody fragment thereof.
  • the modified antibody heavy chain and/or light chain may contain 1, 2, 3, 4, 5, 6, 7, 8, or more Pel substitutions in its constant regions.
  • the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more Pel substitutions in its constant regions.
  • the parental antibody (antibody without Pel substitution) is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgGl antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • the present invention also provides modified antibodies or antibody fragments thereof comprising a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG sites in Table 1, particularly selected from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment.
  • the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51 and 55.
  • the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of at least one TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG Sites in Table 2, especially positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment.
  • the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 73, 75, 76, 77, 81, and 82.
  • the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of at least one TAG-encoded amino acid such as Pel for one or more amino acids on the constant region of a lambda light chain chosen from the Selected TAG sites in Table 3, especially positions 143, 145, 147, 156, 159, 163, and 168 of the lambda light chain of said antibody or antibody fragment, using the Kabat numbering system.
  • the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 98, 99, 100, 101, and 102.
  • the modified antibodies provided herein are labeled using the methods of the invention in combination with other conjugation methods known in the art including, but not limited to, chemos elective conjugation through lysine, cysteine, histidine, tyrosine, formyl-glycine, and protein tags for enzyme-mediated conjugation (e.g., S6 tags).
  • Pyrrolysine is the 22nd natural, genetically encoded amino acid found in certain methanogenic Archaea of the family
  • Methanosarcinaceae and two unrelated bacterial species are unrelated bacterial species. Specifically, pyrrolysine is found in MtmBl, the monomethylamine (MMA) methyltransferase which initiates methane formation in such Archaea bacteria, (see Srinivasan et ah, (2002), Science, 296, 1459-62; Soares, et al., (2005) Journal of Biological Chemistry, 280, 36962-9; Hao et al, (2002) Science, 296, 1462-6; Krzycki (2005) Current Opinion in Microbiology, 8, 706-12; Krzycki (2004) Current Opinion in Chemical Biology, 8, 484-91, and Ambrogelly et ah, (2007) Nature Chemical Biology 3, 29-35).
  • MtmBl the monomethylamine (MMA) methyltransferase which initiates methane formation in such Archaea bacteria
  • Pyrrolysine is considered a dipeptide wherein the ⁇ - amine of lysine is linked to the D-isomer of 4-methyl-pyrroline-5-carboxylate via an amide bond (see, Polycarpo et ah, (2006) FEBS Letters, 580, 6695-700).
  • the structure of pyrrolysine was deduced from the crystal structure of MtmBl and from the residue's mass (see, J. Biol. Chem. 2005, 44, 36962-36969; PNAS, 2007, 104, 1021-1026).
  • the mtmBl gene encoding MtmBl possesses an in-frame amber (TAG) codon, which is normally a canonical stop codon.
  • TAG in-frame amber
  • the UAG codon encoded as TAG on the DNA level, does not terminate translation during production of the MtmB 1 protein, but instead the UAG codon encodes pyrrolysine which is incorporated into the protein. Pyrrolysine is endogenously synthesized and is co-translationally incorporated at such in-frame UAG codons as the free amino acid.
  • pyrrolysine The biosynthesis and incorporation of pyrrolysine are facilitated by the natural genes pylT, pylS, pylB, pylC and pylD.
  • pylT encodes pyrrolysyl-tRNA
  • pylS encodes pyrrolysyl-tRNA synthetase
  • pylB, pylC and pylD encode proteins required for the biosynthesis of pyrrolysine.
  • pylT and pylS genes along with pylB, pylC and pylD genes form a pylTSBCD gene cluster which is a natural genetic code expansion cassette whose transfer allows the UAG codon to be translated as pyrrolysine, which is incorporated into a protein at the UAG site.
  • D-glutamate D-glutamate
  • D-isoleucine D-proline
  • D-ornithine D- ornithine was stated to be the most effective precursor for pyrrolysine biosynthesis in Escherichia coli transformed with a plasmid carrying the natural genes pylT, pylS, pylB, pylC and pylD.
  • the modified Pel-containing antibodies of the invention can be prepared by using a pyrrolysine analogue, pyrroline-carboxy-lysine (Pel) that is naturally encoded, biosynthetically generated and incorporated into antibodies or antibody fragments using the natural genes, pylT, pylS, pylB, pylC and pylD, and D-ornithine as a precursor.
  • a pyrrolysine analogue pyrroline-carboxy-lysine (Pel) that is naturally encoded, biosynthetically generated and incorporated into antibodies or antibody fragments using the natural genes, pylT, pylS, pylB, pylC and pylD, and D-ornithine as a precursor.
  • Pel pyrroline-carboxy-lysine
  • D-arginine is a precursor to D-ornithine
  • the modified Pel-containing antibodies of the invention can be prepared by using a pyrrolysine analogue, Pel, that is naturally encoded, biosynthetically generated and incorporated into proteins using the natural genes, pylT, pylS, pylB, pylC and pylD, and D-arginine as a precursor.
  • Pel or certain synthetic Pel analogs
  • PylD is involved in the activation of (2S)-2-amino-6-((R)- 2,5- diaminopentanamido)hexanoic acid into the semialdehyde ((S)-2-amino-6-((R)- 2-amino-5- oxopentanamido)hexanoic acid) that spontaneously cyclizes to Pcl-A as suggested by in vitro NMR measurements with PylD, L-lysine-N-e-D-ornithine dipeptide, ATP and NAD + (Cellitti et al. (201 1) Nat Chem Biol. 7(8):528-30).
  • the present invention provides conjugation methods encompassing site specific incorporation of biosynthetically generated pyrrolysine and/or pyrroline-carboxy-lysine ((S)-2-amino-6-(3,4-dihydro-2H-pyrrole- 2-carboxamido) hexanoic acid (Pcl-A) or (S)-2-amino-6-(3,4-dihydro-2H-pyrrole-5- carboxamido)hexanoic acid (Pcl-B)), where the Pcl-A or Pcl-B is incorporated at one of the substitution sites identified herein, e.g., at one of the sites listed in Table 1, Table 2 or Table 3.
  • the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell.
  • the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney (293 FreestyleTM) cells, human epitheloid carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells.
  • the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells.
  • the insect cells used in the methods provided herein include, but are not limited to, Spodoptera frugiperda (sf9 and sf21) cells, Trichoplusia ni (BTI TN-5B 1-4 or High-Five(TM)) cells and Mammestra brassicae cells.
  • the prokaryotic cell is a bacterium
  • the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis.
  • such methods for the site specific incorporation of biosynthetically generated pyrrolysine and Pel involves introducing the genes pylT, pylS, pylB, pylC and pylD, and the gene for the desired protein (e.g., antibody), into prokaryotic cells and/or eukaryotic cells, and optionally adding a precursor for pyrrolysine or Pel to the growth media of the transfected cells.
  • the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L- ornithine.
  • the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5- diaminopentanamido)hexanoic acid.
  • the precursor is (2S)-2-amino-6-((R)-
  • the precursor is 2,5-diamino-3- methylpentanoic acid.
  • the precursor is (2R,3R)-2,5-diamino-3- methylpentanoic acid.
  • the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell.
  • the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney 293 FreestyleTM cells, human epitheloid carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK- 21 cells and Chinese hamster ovary CHO cells.
  • the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells.
  • the insect cells used in the methods provided herein include, but are not limited to, Spodoptera frugiperda sf9 and sf21 cells, Trichoplusia ni (BTI TN-5B 1-4 or High-Five(TM)) cells and Mammestra brassicae cells.
  • the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis.
  • PylC has sequence homology with D-alanyl-D-alanine ligases and in the biosynthesis of Pel or pyrro lysine, could catalyze the attachment of D-omithine to the epsilon-amino group of lysine to give (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.
  • PylB is the iron-sulfur SAM enzyme believed to be required to generate 3 -methyl-D -ornithine from L-lysine in the biosynthesis of pyrrolysine (Quitterer et al. (2012) Angew Chem Int Ed Engl. 51(6): 1339-42; Gaston et al. (2011) Nature 471(7340):647-50)). Even in the presence of the pylB gene, the relative amounts of Pel and pyrrolysine containing proteins varied from fermentation to fermentation with Pel protein typically being more prominent. These observations suggest that PylB's activity or the required co-factors are limiting for efficient pyrrolysine biosynthesis in Escherichia coli and mammalian cells.
  • modified pylB genes are used in the biosynthesis of pyrrolysine or other pyrrolysine analogues.
  • pyrrolysine Pel and other pyrrolysine analogues in Escherichia coli, mammalian and other host cells, one or more of the pylB, pylC and pylD genes may be modified.
  • modifications may include using homologous genes from other organisms, including but not limited to other species of Methanosarcinae, or mutated genes.
  • site-directed mutagenesis is used, while in other embodiments random mutagenesis combined with selection is used.
  • Such methods also include the addition of the DNA of the desired protein and the inclusion of the pylT and pylS genes to incorporate the pyrrolysine, Pel or pyrrolysine analogues into the protein.
  • the formation of intermediates in the biosynthesis of pyrrolysine, Pel and/or other pyrrolysine analogues from D-ornithine or the biosynthesis of pyrrolysine may be limited by the function of host enzymes and proteins.
  • low activity or concentration of one or more host enzymes may be limiting the formation of intermediates required in the biosynthesis of pyrrolysine, Pel or other pyrrolysine analogues.
  • the activity of host enzymes may divert the intermediates from the pathway leading to pyrrolysine, Pel or other pyrrolysine analogues to other metabolic pathways, or may be inhibiting the formation of such intermediates.
  • one or more host enzyme may be modified.
  • Such modification include, but are not limited to, the overexpression, activation, suppression or inhibition of such host enzyme by genetic or chemical means, the addition of the DNA encoding such host enzymes, the addition of silencing RNA (siRNA) to suppress mRNA translation, and the addition of cofactors required for the formation of said intermediates from D-ornithine.
  • siRNA silencing RNA
  • the modified antibody or antibody fragment thereof provided herein is site- specifically labeled by post-translational modification of a pyrrolysine and/or desmethyl pyrrolysine (Pel) residue that has been incorporated into the antibody or antibody fragment thereof.
  • Methods for this modification are known in the art, see, e.g., WO2010/048582.
  • the modified antibody or antibody fragments can be converted into conjugates by known methods, also, providing a conjugate having the general formula:
  • IA IB where LU is a linker unit and X 1 is a drug moiety or payload; and R 20 and R 30 are as defined herein. Note that IA can be reduced as described herein to form IB.
  • R 20 is H or Me
  • R 30 is H or Me or phenyl.
  • LU can be a group of the formula -L2-L3-L4-L5-L6 where these linker components are as defined herein.
  • Immunoconjugates comprising one or more, e.g., 1-8, modified Pel residues of Formula IA or IB at one or more of the Pel substitution sites identified herein are embodiments of the invention. In some embodiments, of these immunoconjugates, R 20 is H and R 30 is H or Methyl.
  • the modified antibodies of the invention typically contain 1-12, frequently 2-8, and preferably 2, 4 or 6 -LU-X 1 (Linker Unit- Payload) moieties.
  • an antibody light or heavy chain is modified to incorporate two Pel residues at two of the specific sites identified herein for substitutions (or alternatively one Pel is incorporated in the light chain and one in the heavy chain), so the tetrameric antibody ultimately contains four conjugation sites.
  • the antibody can be modified by replacement of 3 or 4 of its native amino acids with Pel at the specific sites identified herein, in light chain or heavy chain or a combination thereof, resulting in 6 or 8 conjugation sites in the tetrameric antibody.
  • X 1 in these conjugates represents a payload, which can be any chemical moiety that is useful to attach to an antibody.
  • X 1 is a drug moiety selected from a cytotoxin, an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, an immune potentiator, and an anesthetic agent or any other therapeutic, or biologically active moiety or drug moiety.
  • X 1 is a label such as a biophysical probe, a fluorophore, an affinity probe, a spectroscopic probe, a radioactive probe, a spin label, or a quantum dot.
  • X 1 is a chemical moiety that modifies the antibody's physicochemical properties such as a lipid molecule, a polyethylene glycol, a polymer, a polysaccharide, a liposome, or a chelator.
  • X 1 is a functional or detectable biomolecule such as a nucleic acid, a ribonucleic acid, a protein, a peptide (e.g., an enzyme or receptor), a sugar or polysaccharide, an antibody, or an antibody fragment.
  • X 1 is an anchoring moiety such as a nanoparticle, a PLGA particle, or a surface, or any binding moiety for specifically binding the conjugate to another moiety, such as a histidine tag, poly-G, biotin, avidin, streptavidin, and the like.
  • X 1 is a reactive functional group that can be used to attach the antibody conjugate to another chemical moiety, such as a drug moiety, a label, another antibody, another chemical moiety, or a surface.
  • the Linker Unit can be any suitable chemical moiety that covalently attaches the Pcl-derived cyclic group to a payload.
  • Many suitable LUs are known in the art.
  • LU can be comprised of one, two, three, four, five, six, or more than six linker components referred to herein as L 1; L 2 , L 3 , L 4 , L 5 and L 6 .
  • LU comprises a non-enzymatically cleavable linker, a non- cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo- cleavable linker or any combination thereof, and the LU optionally contains a self- immolative spacer.
  • one of the components of LU is a bond
  • that component represents a direct bond between the components flanking it, so the groups on either side of that component are directly bonded to each other.
  • LU is a group of the formula -Li-L-L 3 -
  • L 3 , L 4 , L 5 and L 6 can be selected from:
  • each X 2 is independently selected from a bond, R 8 ,
  • each R 5 is independently selected from H, Ci- 4 alkyl, phenyl or Ci- 4 alkyl substituted with 1 to 3 -OH groups;
  • R 7 is independently selected from H, phenyl, pyrimidine and
  • R 8 is independently selected
  • R 9 is independently selected from H and
  • each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8 and 9.
  • L6 is a stable, or non-cleavable, linker.
  • at least one of Li, L2, L3, L 4 , L5 and L6 is a cleavable linker, which may be chemically cleavable (hydrazones, disulfides) or enzymatically cleavable.
  • the enzymatically cleavable linker is one readily cleaved by a peptidase: the Val-Cit linker (valine-citrulline) , a dipeptide of two known amino acids, is one such linker.
  • the enzymatically cleavable linker is one that is triggered by activity of a glucuronidase.
  • a linker which comprises a self-immolative spacer that falls apart spontaneously under physiological conditions once glucuronidase cleaves the glycosidic linkage:
  • the modified antibodies or antibody fragment thereof provided herein are labeled by a one-step method wherein the post-trans lational modification occurs by reacting a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) with either an aminobenzaldehyde (ABA) analogue linked to an X 1 group, an amino-acetophenone (AAP) analogue linked to an X 1 group or an amino-benzophenone (ABP) analogue linked to an X 1 group.
  • ABA aminobenzaldehyde
  • AAP amino-acetophenone
  • ABS amino-benzophenone
  • the modified antibodies or antibody fragment thereof are labeled by a two- step method wherein the post-translational modification involves first reacting a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) with either an aminobenzaldehyde (ABA) analogue linked to an X a group, an amino- acetophenone (AAP) analogue linked to an X a group or an amino-benzophenone (ABP) analogue linked to an X a group, followed by coupling the X a group with an X b group that is directly attached or linked to an X 1 .
  • ABA aminobenzaldehyde
  • AAP amino- acetophenone
  • ABS amino-benzophenone
  • the X a and X b groups are complementary reactive or coupling groups such as those illustrated herein that combine covalently so that X a and X b together form a linker selected from the options described herein for linkers Li, L 2 , L 3 , L 4 , L 5 and L 6 : exemplary two-step methods are shown in Schemes (Ila)-(IId) below.
  • the modified antibodies or antibody fragment thereof are labeled by a two-step method wherein the post-translational modification involves reducing the labeled antibodies or antibody fragment thereof obtained by the one-step method.
  • two-step methods are shown in Schemes (Ile)-(IIf) below.
  • the modified antibodies or antibody fragment thereof are labeled by a three- step method wherein the post-translational modification involves reducing the labeled antibodies or antibody fragment thereof obtained by the two-step method.
  • a three-step method wherein the post-translational modification involves reducing the labeled antibodies or antibody fragment thereof obtained by the two-step method.
  • R 20 is H or CH 3 ;
  • R 30 is H, CH 3 or phenyl
  • LU is a Linker Unit (LU)
  • X 1 is a drug moiety selected from an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, and an anesthetic agent,
  • X 1 is a biophysical probe, a fluorophore, an affinity probe, a chelator, a
  • spectroscopic probe a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a polysaccharide, or a surface.
  • the Linker Unit comprises a linker selected from a non- enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker, a photo-cleavable linker or any combination thereof, and the Linker Unit (LU) optionally contains a linker that comprises a self- immolative spacer.
  • the Linker Unit is -L 1 -L 2 -L3-L4-, wherein
  • Li is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L 2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker, and
  • L 4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • the Linker Unit is -L1-L2-L 3 -L4-, wherein
  • Li is a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker;
  • L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L 3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L 4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • the Linker Unit is -L1-L2-L 3 -L4-, wherein
  • Li is a bond, -Ai-, -A1X 2 - or -X 2 -; where Ai and X 2 are as defined above;
  • L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L 3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • the Linker Unit is -L1-L2-L 3 -L4-, wherein
  • Li is a bond, -Ai-, -A1X 2 - or -X 2 -; where Ai and X 2 are as defined above;
  • L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker
  • L 4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • the Linker Unit is -Lx-I ⁇ -Ls-I ⁇ -, wherein
  • Li is a bond, -Ai-, -A 1 X 2 - or -X 2 -;
  • each X 2 is independently selected from a bond, R 8 ,
  • each R 5 is independently selected from H, Ci- 4 alkyl, phenyl or
  • each R 6 is independently selected from H, fluoro, benzyloxy substituted with -
  • Ci_ 4 alkyl substituted with -C( 0)OH;
  • R 7 is independently selected from H, phenyl and pyridine
  • R 8 is independently selected
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9;
  • L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • L 4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an
  • the Linker Unit is -L1-L2-L3-L4-, wherein
  • Li is a bond, -Ai-, -A1X 2 - or -X 2 -;
  • each X 2 is independently selected from a bond, R 8 ,
  • each R 5 is independently selected from H, Ci- 4 alkyl, phenyl or
  • each R 6 is independently selected from H, fluoro, benzyloxy substituted with -
  • Ci_ 4 alkyl substituted with -C( 0)OH;
  • R 7 is independently selected from H, phenyl, pyrimidine, and pyridine;
  • R 8 is independently selected
  • R 9 is independently selected from H and
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9;
  • L 2 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker
  • L3 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker
  • L 4 is a bond, an enzymatically cleavable linker or a linker that comprises a linker that comprises a self-immolative spacer.
  • the Linker Unit is -L 1 -L 2 -L3-L 4 -, wherein
  • Li is a bond, -Ai-, -A 1 X 2 - or -X 2 -;
  • L 2 is a bond, -A 2 -, or -A 2 X 2 -;
  • L 3 is a bond, -A3-, or -A 3 X 2 -;
  • L 4 is a bond, -A 4 -, -A 4 X 2 -,
  • each R 5 is independently selected from H, Ci_ 4 alkyl, phenyl or Ci_ 4 alkyl substituted with 1 to 3 -OH groups;
  • each R 6 is independently selected from H, fluoro, benzyloxy substituted with -
  • Ci_ 4 alkyl substituted with -C( 0)OH;
  • R 7 is independently selected from H, phenyl and pyridine
  • R 9 is independently selected from H and Ci- 6 haloalkyl
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
  • the Linker Unit is -L1-L2-L3-L4-, wherein
  • Li is a bond, -Ai-, -A1X 2 - or -X 2 -;
  • L 2 is a bond, -A 2 -, or -A 2 X 2 -;
  • L 3 is a bond, -A3-, or -A 3 X 2 -;
  • each X 2 is independently selected from a bond
  • each R 5 is independently selected from H, Ci_ 4 alkyl, phenyl or Ci_ 4 alkyl substituted with 1 to 3 -OH groups;
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
  • the Linker Unit is -L1-L2-L3-L4-, wherein Li is a bond, -Ai-, -A1X 2 - or -X 2 -;
  • L 2 is a bond, -A 2 -, or -A 2 X 2 -;
  • L 3 is a bond, -A3-, or -A 3 X 2 -;
  • L 4 is a bond
  • each R 5 is independently selected from H, Ci- 4 alkyl, phenyl or substituted with 1 to 3 -OH groups;
  • R 7 is independently selected from H, phenyl and pyridine
  • R 9 is independently selected from H and Ci- 6 haloalkyl
  • each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and
  • each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
  • L 4 is a bond or a val-cit linker of this formula:
  • [X] indicates the point of attachment to a payload.
  • R 20 is H and R 30 is H or Methyl.
  • the X 1 group is a maytansinoid such as
  • DM1 or DM4 or a dolostatin 10 compound, e.g. auristatins MMAF or MMAE, or a calicheamicin such as N-acetyl-y-calicheamicin, or a label or dye such as rhodamine or tetramethylrhodamine.
  • auristatins MMAF or MMAE or a calicheamicin such as N-acetyl-y-calicheamicin
  • a label or dye such as rhodamine or tetramethylrhodamine.
  • a "linker” is any chemical moiety that is capable of linking an antibody or a fragment thereof to an X 1 group.
  • Linkers can be susceptible to cleavage, such as, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • linkers can be substantially resistant to cleavage.
  • a linker may or may not include a self-immolative spacer.
  • Non-limiting examples of the non-enzymatically cleavable linkers as used herein to conjugate an X 1 group to the modified antibodies or antibody fragment thereof provided herein include, acid-labile linkers, linkers containing a disulfide moiety, linkers containing a triazole moiety, linkers containing a hydrazine moiety, linkers containing a thioether moiety, linkers containing a diazo moiety, linkers containing an oxime moiety, linkers containing an amide moiety and linkers containing an acetamide moiety.
  • Non-limiting examples of the enzymatically cleavable linkers as used herein to conjugate an X 1 group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers that are cleaved by a protease, linkers that are cleaved by an amidase, and linkers that are cleaved by -glucuronidase.
  • such enzyme cleavable linkers are linkers which are cleaved by cathepsin, including cathepsin Z, cathepsin B, cathepsin H and cathepsin C.
  • the enzymatically cleavable linker is a dipeptide cleaved by cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin B, cathepsin H or cathepsin C.
  • the enzymatically cleavable linker is a cathepsin B-cleavable peptide linker.
  • the enzymatically cleavable linker is a cathepsin B-cleavable dipeptide linker. In certain embodiments the enzymatically cleavable linker is valine-citrulline or phenylalanine- lysine.
  • Other non-limiting examples of the enzymatically cleavable linkers as used herein conjugate an X 1 group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers which are cleaved by ⁇ -
  • glucuronidase e.g.,
  • Self-immolative spacers are bifunctional chemical moieties covalently linked at one terminus to a first chemical moiety and at the other terminus to a second chemical moiety, thereby forming a stable tripartate molecule.
  • a linker can comprise a self-immolative spacer bonded to a third chemical moiety that is cleavable from the spacer either chemically or enzymatically. Upon cleavage of a bond between the self-immolative spacer and the first or third chemical moiety, self- immolative spacers undergo rapid and spontaneous intramolecular reactions and thereby separate from the second chemical moiety.
  • the first or third moiety is an enzyme cleavable group and this cleavage results from an enzymatic reaction, while in other embodiments the first or third moiety is an acid labile linker and this cleavage occurs due to a change in local pH.
  • the second moiety is the "Payload" group as defined herein.
  • cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a proteolytic enzyme, while in other embodiments it results from cleaved by a hydrolase or glucosidase. In certain embodiments, cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a cathepsin enzyme or a
  • the enzyme cleavable linker is a peptide linker and the self-immolative spacer is covalently linked at one of its ends to the peptide linker and covalently linked at its other end to a drug moiety.
  • This tripartite molecule is stable and pharmacologically inactive in the absence of an enzyme, but which is enzymatically cleavable by enzyme at the bond covalently linking the spacer moiety and the peptide moiety.
  • the peptide moiety is cleaved from the tripartate molecule which initiates the self-immolating character of the spacer moiety, resulting in spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.
  • a linker comprises a self-immolative spacer that connects to the peptide, either directly or indirectly at one end, and to a payload at the other end; and the spacer is attached to a third moiety that can be cleaved from the spacer enzymatically, such as by a glucuronidase. Upon cleavage of the third moiety, the spacer degrades or rearranges in a way that causes the payload to be released.
  • a linker with this type of self-immolative spacer is this glucuronidase-cleavable linker:
  • Non-limiting examples of the self-immolative spacer optionally used in the conjugation of an X 1 group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, moieties which include a benzyl carbonyl moiety, a benzyl ether moiety, a 4-aminobutyrate moiety, a hemithioaminal moiety or a N-acylhemithioaminal moiety.
  • self-immolative spacers include, but are not limited to, p-aminobenzyloxycarbonyl groups, aromatic compounds that are electronically similar to the p-aminobenzyloxycarbonyl group, such as 2- aminoimidazol-5-methanol derivatives and ortho or para-aminobenzylacetals.
  • self-immolative spacers used herein which undergo cyclization upon amide bond hydrolysis include substituted and unsubstituted 4-aminobutyric acid amides and 2-aminophenylpropionic acid amides.
  • the self-immolative spacer is
  • n 1 or 2.
  • the self-immolative spacer is
  • the self-immolative spacer is , where n is 1 or
  • R is H.
  • R 30 is H or Me.
  • the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled at one (or more) of the substitution sites identified herein by a one-step method as shown in Scheme (la) or Scheme (lb), wherein an X 1 group linked to either an aminobenzaldehyde (ABA) analogue, an amino-acetophenone (AAP) analogue or an amino-benzophenone (ABP) analogue reacts with a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) engineered into the antibody or antibody fragment thereof.
  • ABA aminobenzaldehyde
  • AAP amino-acetophenone
  • ABSP amino-benzophenone
  • the one step method includes the steps of:
  • R 30 Linker Unit (LU) and X 1 are as described herein.
  • the one step method includes the steps of:
  • R 30 , Li, L 2 , L 3 , L 4 and X 1 are as defined herein.
  • R 20 is H and R 30 is H or Methyl.
  • the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled by a two-step method, wherein, in the first step a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) which has been incorporated into the antibody or antibody fragment thereof at one (or more) of the substitution sites identified herein is reacted with either an aminobenzaldehyde (ABA) analogue linked to an X a group, an amino-acetophenone (AAP) analogue linked to an X a group or an amino-benzophenone (ABP) analogue linked to an X a group.
  • ABA aminobenzaldehyde
  • AAP amino-acetophenone
  • ABS amino-benzophenone
  • an X b group which is directly attached or linked to an X 1 , is reacted with the X a group on the modified pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue), thereby directly attaching the X 1 group to the modified antibody or antibody fragment thereof or attaching the X 1 group to the modified antibody or antibody fragment thereof via a Linker Unit (LU).
  • LU Linker Unit
  • alkene, alkyne, triaryl phosphine, cyclooctyne, oxanobornadiene, diaryl tetrazine, monoaryl tetrazine and norbornene of X a and X b are optionally substituted.
  • the Two-Step Method of Scheme (Ha) includes the steps of:
  • R 30 , X a , X b , A h L 2 , L 3 , and X 1 are as defined herein.
  • the Two-Step Method of Scheme (lib) includes the steps of: (a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues and/or one or more desmethyl pyrrolysine (Pel) residues at one (or more) of the substitution sites identified herein;
  • R 30 , X a , X b , , A 2 , , and X 1 are as defined herein.
  • the Two-Step Method of Scheme (lie) includes the steps of: (a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues and/or one or more desmethyl pyrrolysine (Pel) residues at one (or more) of the substitution sites identified herein;
  • R 30 , X a , X b , , , A 3 , and X 1 are as defined herein.
  • the Two-Step Method of Scheme (lid) includes the steps of:
  • R 30 , X a , X b , , , , A 4 and X 1 are as defined herein.
  • Suitable reactive functional groups for Xa, and complementary reactive functional groups for Xb, include those in Table 4.
  • R 20 , R 30 , LU and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • R 20 , R 30 , , , , and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled by a three-step method, wherein the modified antibodies or antibody fragment thereof obtained using the two-step methods shown in Schemes (Ila)-(IId) are reduced. Certain embodiments of such three-step methods are shown in Schemes (Ilia)- (Hid) below.
  • X a and a corresponding X b are as given in Table 4, and where R 20 , R 30 , A ⁇ , X 2 , L 2 , L3, L 4 and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • X a and a corresponding X b are as given in Table 4, and where R 20 , R 30 , Ai, X 2 , L 2 , L3, L 4 and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • X a and a corresponding X b are as given in Table 4, and where R 20 , R 30 , Ai, X 2 , L 2 , L3, L 4 and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • X a and a corresponding X b are as given in Table 4, and where R 20 , R 30 , A ⁇ , X 2 , L 2 , L 3 , L 4 and X 1 are as defined herein.
  • the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.
  • R 20 , R 30 , X a , X b and X 1 are as defined herein, and Y 1 is
  • Table 7 shows certain embodiments of compounds of Formula (II-c) which are used in the Two-step methods or the Three-step methods described herein to react with an R a group coupled to at least one pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue incorporated into a modified antibody or antibody fragment thereof.
  • the resulting modified pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue located in the modified antibody or antibody fragment thereof are also shown.
  • L l5 A 2 , L 3 , L 4 , R 20 , R 30 , X a , X b and X 1 are as defined herein,
  • Table 8 shows certain embodiments of compounds of Formula
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (F):
  • R 20 , R 30 , LU and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (G):
  • R 20 , R 30 , LU and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (H):
  • R 20 , R 30 , , , , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (J):
  • R 20 , R 30 , , , , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (K):
  • R 20 , R 30 , A h X 2 , L 2 , L 3 , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (L):
  • R 20 , R 30 , , A 2 , X 2 , , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof
  • IT , R , h , A 3 , X , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**)
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (N):
  • IT , R , h , , A 4 , X and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**)
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (O):
  • R 20 , R 30 , A h X 2 , L 2 , L 3 , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (P):
  • R , R , U, A 2 , X , , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (Q):
  • IT , R , h , A 3 , X , and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the X 1 group is linked to the antibody or antibody fragment thereof by a linking group of Formula (R):
  • R , R , U, , , A 4 , X and X 1 are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X 1 group.
  • the modified antibody or antibody fragment thereof provided herein are labeled with an "X 1 group-to-antibody” ratio of 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibody or antibody fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 pyrrolysine and/or or desmethyl pyrrolysine (Pel) residues.
  • a "X 1 group-to-antibody” ratio of 4 is achieved by incorporating a Pel residue into the heavy chains and a Pel residue into the light chains of an antibody resulting in 4 conjugation sites, two in the two heavy chains and two in the two light chains.
  • a ratio greater than about 8 is typically best achieved by combining the Pel substitution methods of the
  • the methods of the invention can be combined with methods such as reactions at cysteine sulfur, acylations at lysine, or conjugation via S6 tags.
  • the payload to antibody ratio has an exact value for a specific conjugate molecule, it is understood that the value will often be an average value when used to describe a sample containing many molecules, due to some degree of inhomogeneity, typically in the conjugation step.
  • the average loading for a sample of an immunoconjugate is referred to herein as the drug to antibody ratio, or DAR.
  • the DAR is between about 1 and about 16, and typically is about 1, 2, 3, 4, 5, 6, 7, or 8.
  • at least 50% of a sample by weight is compound having the average ratio plus or minus 2, and preferably at least 50% of the sample is a conjugate that contains the average ratio plus or minus 1.
  • Preferred embodiments include immunoconjugates wherein the DAR is about 2 or about 8, e.g., about 2, about 4, about 6 or about 8.
  • a DAR of 'about n' means the measured value for DAR is within 10% of n.
  • the present invention provides site-specific labeled immunoconjugates.
  • the immunoconjugates of the invention may comprise modified antibodies or antibody fragments thereof that further comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody.
  • framework modifications are made to decrease the immunogenicity of the antibody.
  • one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence.
  • an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • the somatic mutations can be "back-mutated” to the germline sequence by, for example, site- directed mutagenesis.
  • Such "back-mutated” antibodies are also intended to be encompassed by the invention.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.
  • antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • modifications within the Fc region typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al.
  • one or more amino acids of an antibody or antibody fragment thereof of the present invention are replaced by one or more allotypic amino acid residues, such as those shown in Figure 4 for the IgGl subclass and the kappa isotype.
  • Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et a/., MAbs. 1 :332-338 (2009).
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta.
  • the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al, J. Biol. Chem. 276:6591- 6604, 2001).
  • the glycosylation of an antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen.”
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Such an approach is described in, e.g., U.S. Patent Nos.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered
  • EP 1, 176, 195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al, (2002) J. Biol. Chem. 277:26733-26740).
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • the antibody is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, and T256F, as described in U.S. Patent No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121,022 by Presta et al.
  • the present invention provides site-specific labeling methods for incorporating a TAG-encoded amino acid such as Pel at one (or more) of the substitution sites identified herein, modified antibodies and antibody fragments thereof, and immunoconjugates prepared accordingly, comprising a TAG-encoded amino acid such as Pel at one (or more) of the substitution sites identified herein.
  • a modified antibody or antibody fragments thereof can be conjugated to any moiety that is useful to connect to an antibody.
  • Some of the payloads to which the antibody can be conjugated include a label, a biophysical probe, immunopotentiator, enzyme, RNA, DNA, saccharide or polysaccharide, reactive functional group, or a drug moiety, e.g., an anti-cancer agent, an autoimmune treatment agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent, or an imaging reagent.
  • An antibody or antibody fragments can also be conjugated using several identical or different labeling moieties combining the methods of the invention with other conjugation methods.
  • the immunoconjugates of the present invention comprise a drug moiety selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPrV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a drug moiety selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an m
  • the modified antibodies or antibody fragments of the present invention may be conjugated to a payload such as a drug moiety that modifies a given biological response.
  • Drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be an immune potentiator, such as an immune potentiator, a small molecule immune potentiator, a TLR agonist, a CpG oligomer, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope peptide or a like.
  • the drug moiety may also be an oligonucleotide, a siRNA, a shRNA, a cDNA or a like.
  • the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response
  • the modified antibodies or antibody fragments of the present invention are conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • a drug moiety such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • cytotoxin include but not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos.
  • DNA-alkylating agents e.g., CC-1065 analogs
  • anthracyclines e.g., tubulysin analogs
  • duocarmycin analogs e.g., auristatin E
  • auristatin F e.g., maytansinoids
  • cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et ah, Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et ah, J. Antibiot.
  • colchicin colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine
  • ablating agents e.g., mechlorethamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU)
  • cyclothosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine
  • An example of a calicheamicin antibody conjugate is commercially available (Mylotarg Tm ; Wyeth-Ayerst).
  • modified antibodies or antibody fragments thereof can be conjugated to a payload comprising a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as
  • radioimmunoconjugates examples include, but are not limited to, iodine 131 , indium 111 , yttrium 90 , and lutetium 177 . Methods for preparing radioimmunoconjugates are established in the art. Examples of
  • radioimmunoconjugates are commercially available, including ZevalinTM (DEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.
  • the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et al, (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al, (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al, (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.
  • the present invention further provides modified antibodies or fragments thereof that specifically bind to an antigen.
  • the modified antibodies or fragments may be conjugated or fused to a payload such as a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the invention provides fusion proteins comprising an antibody fragment described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide.
  • modified antibody fragments without antibody specificity such as but not limited to, modified Fc domains with engineered Pcl(s) according to the present invention, are used to generate fusion proteins comprising such an antibody fragment (e.g., engineered Fc) and a heterologous protein, polypeptide, or peptide.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson et al, (1999) J. Mol. Biol.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the modified antibodies or antibody fragments thereof of the present invention can be conjugated to payloads such as marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, (1984) Cell 37:767), and the "FLAG” tag (A. Einhauer et al, J. Biochem. Biophys. Methods 49: 455-465, 2001).
  • HA hemagglutinin
  • FLAG A. Einhauer et al, J. Biochem. Biophys. Methods 49: 455-465, 2001.
  • antibodies or antibody fragments can also be conjugated to tumor-penetrating peptides in order to enhance their efficacy.
  • modified antibodies or antibody fragments of the present invention are conjugated to a diagnostic or detectable agent as a payload.
  • a diagnostic or detectable agent as a payload.
  • Such immunoconjugates can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to,
  • fluorescent materials such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
  • fluorescent materials such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610,
  • Modified antibodies or antibody fragments of the invention may also be attached to solid supports, which are particularly useful for
  • Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • compositions including immunoconjugates are mixed with a pharmaceutically acceptable carrier or excipient.
  • the compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynacomastica, and
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001 ; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al.
  • an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects.
  • the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.
  • compositions comprising antibodies or fragments thereof of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided
  • the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.
  • the dosage is between 0.1 and 10 mg/kg, or between 1 and 10 mg/kg.
  • the dosage of the antibodies or fragments thereof of the invention may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • Doses of the immunoconjugates the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In a specific embodiment, does of the immunoconjugates of the invention are repeated every 3 weeks.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard et ah, A Handbook of SOPs for Good Clinical Practice,
  • the route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et ah, Biopolymers 22:547-556, 1983; Langer et ah, J. Biomed. Mater. Res. 15: 167-277, 1981 ; Langer, Chem. Tech. 12:98-105, 1982; Epstein et ah, Proc. Natl. Acad. Sci.
  • composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
  • composition of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • Selected routes of administration for the immunoconjugates of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the immunoconjugates of the invention is administered by infusion.
  • the immunoconjugates of the invention is administered by infusion.
  • immunoconjugates of the invention is administered subcutaneously.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al, Surgery 88:507, 1980; Saudek et al, N. Engl. J. Med. 321 :574, 1989).
  • Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23 :61, 1983; see also Levy et al, Science 228: 190, 1985; During et al, Ann. Neurol. 25:351, 1989; Howard et al, J. Neurosurg. 7 1 : 105, 1989; U.S. Pat. No.
  • polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138, 1984).
  • compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops.
  • prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.
  • Additional therapies which can be administered in combination with the immunoconjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the immunoconjugates of the invention
  • the immunoconjugates of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,81 1; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153 : 1038); antibodies (Bloeman et al, (1995) FEBS Lett. 357: 140; Owais et al, (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al, (1995) Am. J. Physiol. 1233: 134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
  • biotin
  • the invention provides protocols for the administration of pharmaceutical composition comprising immunoconjugates of the invention alone or in combination with other therapies to a subject in need thereof.
  • the therapies e.g., prophylactic or therapeutic agents
  • the therapy e.g., prophylactic or therapeutic agents
  • the combination therapies of the present invention can also be cyclically administered.
  • Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.
  • a first therapy e.g., a first prophylactic or therapeutic agent
  • a second therapy e.g., a second prophylactic or therapeutic agent
  • the therapies e.g., prophylactic or therapeutic agents
  • the combination therapies of the invention can be administered to a subject concurrently.
  • each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • each therapy can be administered to a subject separately, in any appropriate form and by any suitable route.
  • the therapies e.g., prophylactic or therapeutic agents
  • two or more therapies are administered to a within the same patient visit.
  • prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical
  • the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions.
  • the prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.
  • Example 1 Selection of 92 sites for Pel mutation in human IgGl heavy chain and kappa light chain.
  • IgGl heavy and human kappa light chains were identified in a crystal structure of an hlgGl/kappa antibody (Protein Databank structure entry lHZH.pdb, Table 9, Table 10, Figure 1) using the computer program Surface Racer 5.0, as described by Tsodikov et al (2002) J. Comput. Chem. 23 :600-609.
  • 92 residues were selected for a single TAG substitution; 60 residues in the hlgG heavy chain and 32 in the human kappa light chain, based on the following criteria: 1) select residues in CHI, CH2 and CH3 domains of the constant regions of heavy chain and the constant regions of light chain; 2) select surface exposed residues; 3) focus on polar or charged residues such as Ser, Thr, Lys, Arg, Glu, and Asp; and 4) exclude residues in FcRn binding domain, Protein A binding domain and heavy chain hinge region. [00248] Criterion 1), namely the selection of Pel substitution sites in the constant region of the antibody, assures transferability of the conjugation sites to many different antibodies.
  • Criterion 2 is based on observation of inter-antibody dimer formation for Pel substitutions of prominently exposed residues (residues excluded based on this criteria are listed in Table 8). Based on the IgG crystal structure, the putative orientation of the Pel side chain was taking into consideration: Residues for which the Pel side chain may be partially shielded from interactions with another antibody but may still react with a small molecular payload, were favored over residues with larger surface accessibility but with an orientation that may enable interactions with a large macromolecule, such as dimer formation.
  • Criterion 3 was implemented to favor conservative mutations in order to minimize destabilizing effects of the mutations on the antibody.
  • criterion 4 was used to avoid functional changes to the antibody such as effects on FcRn and Protein A binding, which may affect the antibody's pharmacokinetic properties or may result in the loss of a purification handle, respectively. Residues excluded based on criterion 4 are listed in Table 9.
  • TAG-encoded amino acids include but are not limited to unnatural amino acids such as ara-acetyl-phenylalanine, para-azido- phenylalanine and reactive pyrrolysine analogs (Liu and Schultz, 2010; Neumann, 2012), but also include the natural amino acids pyrrolysine (O) and pyrroline- carboxy-lysine (Pel; Z). It is hence important to realize that the incorporation sites of the invention can be used for the site-specific incorporation of many different TAG-encoded amino acids. For all proteins prepared for this study, however, Pel was used as the TAG-encoded amino acid.
  • Table 9 Surface accessibility of amino acid residues in human IgGl heavy chain. Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [A 2 ]. Reasons for exclusion" indicate the sites that are excluded from selection due to the reasons mentioned in the table. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid. Surface Reason for

Abstract

La présente invention concerne des sites spécifiques de modification d'anticorps ou de fragments d'anticorps par le remplacement d'au moins un acide aminé dans la région constante d'un anticorps ou d'un fragment d'anticorps parental par un acide aminé codé par TAG tel que Pcl, qui peut être utilisé au niveau d'un site de fixation d'une charge ou d'une combinaison lieur-charge. L'invention comprend des anticorps ou des fragments d'anticorps modifiés, divers conjugués formés à partir de ceux-ci et des utilisations des anticorps ou des fragments d'anticorps modifiés et de leurs conjugués.
PCT/US2014/015302 2013-02-08 2014-02-07 Sites spécifiques de modification d'anticorps pour fabriquer des immunoconjugués WO2014124258A2 (fr)

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