Novel engineered cysteine sites for selective conjugation

Abstract

The present disclosure relates to methods of developing, designing, and producing therapeutic antibodies to improve developability of an antibody or antibody drug conjugate. In particular, the disclosure relates to the introduction of engineered cysteine sites for selective conjugation of linker-payloads to antibodies.

Description

NOVEL ENGINEERED CYSTEINE SITES FOR SELECTIVE CONJUGATIONSEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30466_WO” created 2 April 2026 and is 5.4 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.BACKGROUND

[0002] The present invention is in the field of medicine. More particularly, the present invention provides methods of developing, designing, and producing therapeutic antibodies and conjugates thereof. In particular, the invention relates to the introduction of engineered cysteine sites for selective conjugation of linker-payloads to antibodies to produce antibody -drug conjugates.

[0003] For an antibody drug conjugate (ADC) antibody to be effective, it must bind specifically to a target antigen to deliver a payload to the target cell, avoiding significant binding to nontarget cells. Therefore, it is essential that engineered antibodies maintain their specific binding ability even when conjugated to a pay load. Additionally, the ADC must be capable of delivering the payload to the target cell, being internalized by the cell, and releasing the payload within the appropriate compartment inside the cell.

[0004] Payload drugs are typically conjugated to the antibodies through lysine side chains or by reducing interchain disulfide bonds to create activated cysteine sulfhydryl groups. Using nonspecific conjugation methods have several disadvantages. It can disrupt protein folding by breaking cystine bonds and results in a heterogeneous mixture of antibodies with varying drug-to-antibody ratios (DAR) and a complex mix of antibodies conjugated at different positions. Even if it were possible to purify enough antibodies with a desired drug-to-antibody ratio, the fraction would still consist of a complex mix of antibodies conjugated at various positions. Each species could have distinct therapeutic properties, and maintaining batch-to-batch consistency would be challenging, posing obstacles to the success of ADCs in therapy.

[0005] Antibodies and their derivatives have become integral tools in therapeutic, diagnostic, and imaging applications due to their high specificity and affinity for target antigens. In particular, in the context of antibody drug conjugates, antibody fragments may offer someadvantages over full-length antibodies, such as improved tissue penetration, reduced immunogenicity, and ease of recombinant production.

[0006] Fragments or portions of antibodies can be chemically or biologically linked to functional payloads to create antibody drag conjugates. These payloads may include cytotoxic drags, imaging agents, radiolabels, enzymes, or other molecules intended to confer or enhance diagnostic or therapeutic functionality.SUMMARY OF INVENTION

[0007] The present invention provides site-specific conjugation mutations to improve the developability and stability of antibody drag conjugates. Antibody drag conjugates comprising the specific amino acid residues provided by the present invention demonstrate several beneficial properties compared to antibody drag conjugates not comprising the specific amino acid residues. The present invention provides antibody drag conjugates wherein the lipophilic pay loads can be shielded by the antibody surface, thereby reducing the overall hydrophobicity of the antibody drag conjugates. The present invention also provides antibody drag conjugates with improved biophysical stability. Furthermore, the pharmacokinetics of antibody drag conjugates comprising the amino acid residues provided by the invention are improved compared to antibody drag conjugates not comprising the specific amino acid residues. The antibody drag conjugates provided by the present invention also provide improved drag:antibody ratio (DAR) stability with reduced retro-Michael release of payload in circulation.

[0008] The present invention provides an antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering); and / or a cysteine at amino acid residue 398 (EU numbering).

[0009] In some embodiments, the antibody the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 378 (EU numbering); and a cysteine at amino acid residue 398 (EU numbering).

[0010] In some embodiments, the human IgG heavy chain constant region comprises a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises; a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; and / or a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

[0011] In some embodiments, the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; and a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

[0012] The present invention also provides an antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering); and / or a cysteine at amino acid residue 378 (EU numbering).

[0013] In some embodiments, the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering); and a cysteine at amino acid residue 378 (EU numbering).

[0014] In some embodiments, the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and / or a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain.

[0015] In some embodiments, the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain.

[0016] The present invention also provides an antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering); and / or a cysteine at amino acid residue 398 (EU numbering).

[0017] In some embodiments, the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering); and a cysteine at amino acid residue 398 (EU numbering).

[0018] In some embodiments, the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and / or a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

[0019] In some embodiments, the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

[0020] In some embodiments, the antibody further comprises a human IgG light chain constant region and wherein the human IgG light chain constant region comprises: a cysteine at amino acid residue 206 (EU numbering).

[0021] The present invention also provides an antibody comprising a human IgG heavy chain constant region and a human IgG light chain constant region, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 378 (EU numbering); a cysteine at amino acid residue 398 (EU numbering); and wherein the human IgG light chain constant region comprises a cysteine at amino acid residue 206 (EU numbering).

[0022] In some embodiments, the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain; and wherein the human IgG light chain constant region comprises a cysteine at amino acid residue 206 (EU numbering).

[0023] The present invention also provides an antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering); a cysteine at amino acid residue 378 (EU numbering); and a cysteine at amino acid residue 398 (EU numbering).

[0024] In some embodiments, the human IgG heavy chain constant region comprises a CHI, a CH2, and a CH3 domain, wherein the human IgG heavy chain constant region comprises: a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; and a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

[0025] In some embodiments, the human IgG heavy chain constant region comprises a CH2 domain and wherein the human IgG heavy chain constant region further comprises: an alanine at amino acid residue 234 (EU numbering) of the CH2 domain; an alanine at amino acid residue 235 (EU numbering) of the CH2 domain; and / or a serine at amino acid residue 265 (EU numbering) of the CH2 domain.

[0026] In some embodiments, the human IgG heavy chain constant region comprises a CH2 domain and wherein the human IgG heavy chain constant region further comprises: an alanine at amino acid residue 234 (EU numbering) of the CH2 domain; an alanine at amino acid residue 235 (EU numbering) of the CH2 domain; and a serine at amino acid residue 265 (EU numbering) of the CH2 domain.

[0027] In some embodiments, the antibody further comprises: a proline at amino acid residue 228 (EU numbering).

[0028] In some embodiments, the human IgG heavy chain constant region comprises a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises: a lysine at amino acid residue 274 (EU numbering) of the CH2 domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; and / or a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.

[0029] In some embodiments, the human IgG heavy chain constant region comprises a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises: a lysine at amino acid residue 274 (EU numbering) of the CH2 domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.

[0030] In some embodiments, the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises: a lysine at amino acid residue 274 (EU numbering) of the CH2 domain: an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; a glycine at amino acid residue 137 (EU numbering) of the CHI domain; an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a glycine at amino acid residue 137 (EU numbering) ofthe CHI domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of theCH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 41 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; or a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 41 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain.

[0031] In some embodiments, the antibody is a human IgGl, human IgG2, human IgG3, or human IgG4 antibody. In some embodiments, the antibody is a human IgGl antibody. In some embodiments, the antibody is a human IgG4 antibody.

[0032] The present invention also provides an antibody comprising a human IgG heavy chain constant region comprising any one of SEQ ID NOs: 1, 2 or 3.

[0033] The present invention also provides an antibody drug conjugate comprising an antibody according to the invention.

[0034] In some embodiments, the antibody of the antibody drug conjugate is conjugated to a payload molecule selected from the list consisting of: a peptide, a small molecule, an oligonucleotide, a radioligand, a fatty acid, a therapeutic agent or a diagnostic or detection agent.

[0035] In some embodiments, the antibody drug conjugate has a drug: antibody ratio of between 1 and 8. In some embodiments, the antibody drug conjugate has a drug:antibody ratio of 4, 6 or 8. In some embodiments, the antibody drug conjugate has a drug:antibody ratio of 4.

[0036] In some embodiments, the antibody drug conjugate has reduced hydrophobicity compared to an antibody drug conjugate not comprising the modified amino acid residues. In some embodiments, the hydrophobicity is reduced by 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 95% as measured by ESI-TOF mass spectrometry or Hydrophobicity Index Chromatography (HIC) compared to an antibody drug conjugate not comprising the modified amino acid residues.

[0037] In some embodiments, the antibody drug conjugate has increased biophysical stability compared to an antibody drug conjugate not comprising the modified amino acid residues. In some embodiments, the biophysical stability is increased by 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 least100% as measured by analytical size exclusion (SEC) chromatography compared to an antibody drug conjugate not comprising the modified amino acid residues.

[0038] In some embodiments, the antibody drug conjugate has improved pharmacokinetics compared to an antibody drag conjugate not comprising the modified amino acid residues. In some embodiments, the antibody drug conjugate has reduced retro-Michael release of payload molecules compared to an antibody drag conjugate not comprising the modified amino acid residues. In some embodiments, the retro-Michael release of pay load molecules is reduced by 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 95% as measured by DAR stability using liquid chromatography / mass spectrometry (LC / MS) compared to an antibody drag conjugate not comprising the modified amino acid residues.

[0039] The present invention also provides a nucleic acid comprising a sequence encoding the heavy chain or light chain of an antibody according to the invention.

[0040] The present invention also provides a vector comprising a nucleic acid according to the invention.

[0041] The present invention also provides a cell comprising a vector according to the invention. In some embodiments, the cell is a mammalian cell.

[0042] The present invention also provides a process of producing an antibody comprising culturing a cell according to the invention, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

[0043] The present invention also provides an antibody produced by a process according to the invention.

[0044] The present invention also provides a process for producing an antibody drag conjugate comprising an antibody according to the invention and one or more payload molecules comprising:removing capping groups on engineered cysteine residues of an antibody according to the invention in an antibody mixture by adding a reducing agent;removing unbound capping groups and the reducing agent;oxidizing the antibody mixture to reform native interchain disulfide linkages; andadding one or more payload molecules to the antibody mixture after native interchain disulfide linkages have formed and allowing the one or more payload molecules to conjugate to the engineered cysteine sites.

[0045] The present invention also provides an antibody drug conjugate produced by a process according to the invention.

[0046] The present invention also provides a pharmaceutical composition comprising the antibody or antibody drug conjugate according to the invention, and a pharmaceutically acceptable excipient, diluent or carrier.

[0047] The present invention also provides a method of administering the antibody or antibody drug conjugate according to the invention to a subject in need thereof. In some embodiments the antibody or antibody drug conjugate is administered subcutaneously to the subject. In some embodiments the antibody or antibody drug conjugate is administered intravenously to the subject.

[0048] The present invention also provides an antibody according to the invention, an antibody drug conjugate according to the invention, or a pharmaceutical composition according to the invention for use in therapy.

[0049] The present invention also provides an antibody according to the invention, an antibody drug conjugate according to the invention, or a pharmaceutical composition according to the invention for use in treating a cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

[0050] The present invention also provides a use of an antibody according to the invention, an antibody drug conjugate according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

[0051] In some embodiments, nucleic acid sequences encoding the antibodies of the present disclosure are provided. In some embodiments of the present disclosure, nucleic acids encoding a HC or LC of the antibodies are provided. In some embodiments of the present disclosure, nucleic acids encoding a VH or VL of the antibodies are provided. Some embodiments of the present disclosure, provide vectors comprising a nucleic acid sequence encoding an antibody HC or LC. Some embodiments of the present disclosure, provide vectors comprising a nucleic acid sequence encoding an antibody VH or VL.

[0052] Nucleic acids of the present disclosure may be expressed in a host cell, for example, after the nucleic acids have been operably linked to an expression control sequence. Expression control sequences capable of expression of nucleic acids to which they are operably linked are well known in the art. An expression vector may include a sequence that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectorscontaining a nucleic acid of interest (e.g., a nucleic acid encoding a heavy chain or light chain of an antibody) may be transferred into a host cell by well-known methods, e.g., stable or transient transfection, transformation, transduction or infection. Additionally, expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aide in detection of host cells transformed with the desired nucleic acid sequences.

[0053] In another aspect, provided herein are cells, e.g., host cells, comprising the nucleic acids, vectors, or nucleic acid compositions described herein. A host cell may be a cell stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of an antibody described herein. In some embodiments, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC and LC polypeptides of an antibody of the present disclosure. In some embodiments, a host cell may be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing HC polypeptides and a second vector expressing LC polypeptides of an antibody described herein. Such host cells, e.g., mammalian host cells, can express the antibodies described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NSO cells.

[0054] In some embodiments, the cell, e.g., host cell, comprises a first vector and a second vector comprising the nucleic acid sequences encoding an antibody as provided herein. In further embodiments, the host cell is a mammalian cell.

[0055] The present disclosure further provides a process for producing an antibody or antibody binding fragments thereof as described herein, by culturing the host cell described above, e.g., a mammalian host cell, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. The culture medium, into which an antibody has been secreted, may be purified by conventional techniques. Various methods of protein purification may be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice. 3rd Edition, Springer, NY (1994).

[0056] The present disclosure further provides antibodies or antibody binding fragments thereof produced by any of the processes described herein.

[0057] In another aspect, provided herein are pharmaceutical compositions comprising an antibody, antibody drag conjugate, nucleic acid, or vector described herein. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent or carrier. Pharmaceutical compositions can be prepared by methods well known in the art (e.g.,-Il- Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0058] In some embodiments, the present disclosure provides antibodies and antibody drug conjugates for use in therapy. In some embodiments, the present disclosure provides antibody and antibody drug conjugates for use in the treatment of a medical condition. In some embodiments, the medical condition is cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

[0059] In further embodiments, the present disclosure provides the use of an antibody or antibody drug conjugate in the manufacture of a medicament for the treatment of cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

[0060] In further embodiments, the present disclosure provides a method of administering a therapeutically effective amount of an antibody or antibody drug conjugate as disclosed herein, wherein the antibody or antibody drug conjugate is administered intravenously. In other embodiments, the present disclosure provides a method of administering a therapeutically effective amount of an antibody or antibody drug conjugate as disclosed herein, wherein the antibody or antibody drug conjugate is administered subcutaneously.

[0061] The term “antibody,” as used herein, refers to an immunoglobulin molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, bispecific or multispecific antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgGl, IgG2, IgG3, IgG4). An antibody as used herein, may also include antibody fragments or antigen binding fragments, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv, scFab, disulfide-linked Fvs (sdFv), a Fd fragment, which may be for example, fused to an Fc region or an IgG heavy chain constant region.

[0062] The term “conjugate”, “antibody conjugate”, “conjugated antibody” or “antibody drug conjugate,” as used interchangeably herein, refers to a complex of an antibody or a fragment thereof, which fragment can comprise an antigen-binding fragment of the antibody or a constant domain fragment of the antibody thereof, and a payload molecule. In one embodiment, the antibody and the payload molecule are connected by a linker.

[0063] The term “payload molecule” or “non-antibody molecule” as used herein, refers to a molecule that is not an immunoglobulin molecule. In one embodiment, the payload molecule is a peptide comprising 2 or more amino acid residues. In a further embodiment, the payload molecule is a small molecule drug. In a further embodiment, the payload molecule is anoligonucleotide. In some embodiments the payload molecule is a radio ligand. In a further embodiment, the payload molecule is a fatty acid (e.g., an acyl chain). In some embodiments the payload molecule is a therapeutic agent. In some embodiments the payload molecule is a diagnostic or detection agent (e.g., a radiolabelled molecule, a radioligand or a fluorescent dye). In some embodiments, the payload molecule is a lipophilic molecule (i.e. a molecule having an affinity for or the ability to dissolve in lipids, fats, oils, or non-polar solvents). In some embodiments, the payload molecule is a hydrophobic molecule (i.e. a molecule that exhibits low affinity for water and does not readily mix with or dissolve in water).

[0064] In a further embodiment, the oligonucleotide is an RNA oligonucleotide. In a further embodiment, the oligonucleotide is an RNAi oligonucleotide. In a further embodiment, the oligonucleotide is a double stranded oligonucleotide. In a further embodiment, the oligonucleotide is chemically modified.

[0065] The terms “nucleic acid” or “polynucleotide” as used interchangeably herein, refer to polymers of nucleotides, including single-stranded and I or double-stranded nucleotide-containing molecules, such as DNA, cDNA and RNA molecules, incorporating native, modified, and / or analogs of, nucleotides. Polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or a synthetic reaction.

[0066] The term “radiolabelled molecule” as used herein refers to any molecule that has been covalently or non-covalently associated with a radioactive isotope, thereby enabling detection, quantification, or therapeutic application based on the emission of radiation. The radiolabel may be incorporated into the molecule directly or via a chelator, linker, or other conjugating moiety. Suitable radioactive isotopes include, but are not limited to, alpha, beta, or gamma emitters, as well as positron-emitting isotopes, depending on the intended diagnostic or therapeutic use. Radiolabelled molecules may include, without limitation, peptides, proteins, nucleic acids and small molecules.

[0067] The term “radioligand” as used herein refers to a ligand molecule that is labeled with a radioactive isotope and is capable of selectively binding to a specific biological target, such as a receptor, enzyme, or protein. Radioligands are typically employed for diagnostic, therapeutic, or research purposes, including but not limited to imaging, receptor binding assays, and targeted radiotherapy. The radioactive isotope may be a gamma emitter, beta emitter, alpha emitter, or positron emitter, and the choice of isotope is determined by the intended application and desired radiophysical properties.

[0068] As used herein, the term drug-to-antibody ratio refers to the average number of pay load molecules conjugated to each antibody molecule in an antibody drug conjugate (ADC). It may be determined by analytical methods such as mass spectrometry, UV / Vis spectroscopy, or hydrophobic interaction chromatography (HIC), and is typically expressed as a unitless ratio (e.g., 2, 4, or 8) representing the mean number of payload molecules per antibody molecule.

[0069] An exemplary antibody is an immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The constant domain refers to a region of an antibody, which comprises the Fc region and CHI domain of the antibody heavy chain. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgGl, IgG2, IgG3, and IgG4). The numbering of the amino acid residues in the constant region is based on the EU index as in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health (1991). The term EU index numbering or EU numbering is used interchangeably herein.

[0070] As used herein the term “constant region” refers to any portion of the constant domain of an antibody that comprises the required portion of the antibody sequence containing the engineered amino acid residues of interest. In some embodiments, the constant region comprises the CH2 and CH3 domains of the constant domain. In some embodiments the constant region comprises the CHI, CH2 and CH3 domains of the constant domain.

[0071] The term “Fc region” as used herein, refers to a region of an antibody, which comprises the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of the antibody heavy chain.

[0072] The VH and VL regions can be further subdivided into regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3” and the three CDRs of the light chain are referred to as “LCDR1, LCDR2 and LCDR3”. The CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The North CDR definitions are used for the antibodies described herein.

[0073] A “wild-type” antibody as referred to herein, is an antibody comprising the natural heavy chain constant region, including a natural Fc, and a natural light chain constant region of a human IgGl, IgG2, IgG3 or IgG4 antibody.

[0074] The term "retro-Michael reaction", as used herein, refers to the chemical transformation wherein a 0-substituted carbonyl compound undergoes a cleavage reaction to yield a Michael donor (typically a nucleophile such as an enolate or carbanion equivalent) and an a,0-unsaturated compound (typically a Michael acceptor). The retro-Michael reaction is often utilized to cleave carbon-carbon bonds formed through prior conjugate additions and may degrade or uncouple the payload molecule from the antibody in an antibody drug conjugate.

[0075] The terms “bind” and “binds” as used herein, are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.

[0076] The term “subject” as used herein, refers to a mammal, including, but are not limited to, a human, chimpanzee, ape, monkey, cattle, horse, sheep, goat, swine, rabbit, dog, cat, rat, mouse, guinea pig, and the like. Preferably, the subject is a human.

[0077] The term “inhibits” as used herein, refers to for example, a reduction, lowering, slowing, decreasing, stopping, disrupting, abrogating, antagonizing, or blocking of a biological response or activity, but does not necessarily indicate a total elimination of a biological response.

[0078] The tern “treatment” or “treating” as used herein, refers to all processes wherein there may be a slowing, controlling, delaying or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.

[0079] As used herein, the term “cLogD” means the calculated logarithm of the distribution coefficient (D). This measures a compound's lipophilicity based on its tendency to partition between an organic phase and an aqueous phase. Methods of calculating cLogD are well established (Waring MJ. Lipophilicity in drug discovery. Expert Opin Drug Discov. 2010 Mar;5(3):235-48; Leo, Albert, Corwin Hansch, and David Elkins. "Partition coefficients and their uses." Chemical reviews 71.6 (1971): 525-616; Xing, Li, and Robert C. Glen. "Novel methods for the prediction of logP, p K a, and logD." Journal of chemical information and computer sciences 42.4 (2002): 796-805).

[0080] The term "about" as used herein, means within 5%.

[0081] As used herein, the term “a”, “an”, “the”, and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1 shows the mean PK profiles of ADC and parent antibody comprising 378 / 398 cysteine mutations in rats.

[0083] FIG. 2 shows the total IgG in comparative PK studies between conjugation to native cysteine residues or engineered cysteine residues (1 mg / kg in tumor free nude mice (N = 3 per time point).EXAMPLES

[0084] Example 1 : Design of IgG heavy chain and light chain constant regions containing engineered cysteine residues

[0085] Sequences containing the IgGl or IgG4 Fc with engineered cysteine mutations were cloned, expressed in Chinese hamster ovary (CHO) cells, and purified by standard Protein A followed by cation exchange chromatography. The IgGl Fc’s in some instances contained the L234A, L235A and D265S (AAS) mutations to eliminate effector function while in others wereconsistent with a native IgG sequence. The IgG4 Fc’s contained the S228P mutations to stabilize the hinge along with Q274K, Q355R and E419Q, which can help mitigate a viscosity issue that can occur with IgG4 mAbs.

[0086] The eCys mutations of interest include the L206C located in the kappa constant domain of the light chains (LC) along with the A378C and L398C in the CH3 domain of the heavy chain (HC). The combinations defined herein include the HC A378C-L398C double mutant and the LC L206C, HC A378C and HC L398C triple mutant.

[0087] The amino acid sequence for the native IgGl heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C is provided as SEQ ID NO: 1, the amino acid sequences for the IgGl AAS (L234A, L235A and D265S) heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C is provided as SEQ ID NO: 2, the amino acid sequences for the IgG4 S228P heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C is provided as SEQ ID NO: 3. The light chain kappa constant domain with the L206C is provided as SEQ ID NO: 4.

[0088] Example 2: Preparation of engineered cysteine conjugates

[0089] To prepare site-specific engineered cysteine antibody-drug conjugates (ADCs) with various DAR, the antibody was first reduced in the presence of 40-fold molar excess of dithiothreitol (DTT) for 2 hours at 37 °C or >16 hours at ~21 °C. This initial reduction step was used to remove the various capping groups, including cysteine and glutathione which are bound to the engineered cysteine during expression. Following the reduction step, the sample was purified through a desalting column to remove the unbound caps as well as the reducing agent. A subsequent 2-hour oxidation step was carried out at room temperature (~21 °C) in the presence of 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfides between the light chain and heavy chain as well as the pair of hinge disulfides. After the 2-hour oxidation step, the desired pay load of interest was added using a 10 mM stock solution solubilized in DMSO. The sample was then incubated at room temperature for 30-60 minutes to allow for efficient conjugation of the linker payload to the engineered cysteines. A subsequent polishing step, such as Size Exclusion Chromatography (SEC) or Tangential Flow Filtration (TFF) was then used to buffer exchange the samples into an appropriate formulation buffer and to remove DMSO and any excess linker-payload. Finally, the resulting ADC may be sterile filtered through a 0.2-pM filter and stored at 4 °C or -80 °C for future use.

[0090] Example 3: Preparation of conjugates using conventional stochastic conjugation

[0091] To prepare conventional antibody-drug conjugates with eight drugs per antibody (DAR 8), the IgGl antibody in a common formulation buffer, such as, 50 mM HEPES with 2 mM EDTA at pH 7.5 or PBS with 2 mM EDTA at pH 7.4 was fully reduced using a reducing reagent such TCEP at 8 to 12 molar equivalents at 37 °C for 1-2 h. The reduced antibody was then buffer exchanged using a PD-10 desalting column with 50 mM HEPES, 2 mM EDTA at pH 7.5, and the eluent was adjusted with HEPES buffer to a protein concentration between 5-10 mg / ml. An excess of linker-payloads, such as 10-12 molar equivalents, is added for 1 hour, and the conjugation reaction may be stopped by adding a substantial excess of L-cysteine, such as 6 molar equivalents. The resulting mixture of ADCs may be purified by ultracentrifugation using Amicon Ultra Centrifugal, 30kDa MWCO, with PBS pH 7.4 containing 5% isopropyl alcohol for 2 cycles at 4000x g for 20 min, followed by 3 cycles with PBS pH 7.4 at 4000 x g for 20 min. Finally, the resulting ADC may be sterile filtered through a 0.2-pM filter into appropriate formulation buffer and stored at 4 °C or -80 °C.

[0092] Example 4: Drug to Antibody Ratio (DAR) assessment

[0093] To assess the average number of linker-payloads present on the final conjugates, three analytical methods were used: 1) Hydrophobic-interaction chromatography (HIC); 2) Reverse phase (RP) HPLC and 3) Time of Flight (TOF) mass spectrometry. Methods 2 and 3 required an initial sample reduction step, which included the addition of dithiothreitol (DTT) to a final concentration of ~10mM, followed by a 5-minute incubation at 42 °C.

[0094] Reverse Phase HPLC Method:

[0095] 10 to 30 pg of the reduced antibody drug conjugate sample was injected onto BioResolve RP mAB, Polyphenyl, 450A, 2.7 pM, 2.1 x 50 mM (Water Part# 186008944). The A buffer was made up of 0.1% trifluoroacetic acid (TFA) in water while B buffer was comprised of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column temperature was set to 80°C, injection volume lOuL, flow rate- 0.2 mL / min. The column was equilibrated in 20% B buffer prior to sample injection followed by a gradient from 25% B to 33% B over 22 min. The average DAR was determined by calculating the contribution from each individual DAR species from the fractional percentage multiplied by the DAR number for each contributing species. As this value is based on a partially reduced sample and only represents half of the molecule, the number was then multiplied by 2 to account for an intact antibody drug conjugate.

[0096] Time of Flight Mass Spectrometry Method: 8 pg of the partially reduced sample was injected onto a Poroshell 300sb-C3 2.1 x 2.5 mm, 5 pM column (Agilent Part# 821075-924). Buffer A was made up of 0.1% trifluoroacetic acid (TFA) in water while buffer B comprised of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 10% B buffer prior to sample injection followed by a wash with 10% B and elution using 80% B.

[0097] The average DAR was determined by calculating the contribution from each individual DAR species from the fractional percentage multiplied by the DAR number for each contributing species. As this value is based on a partially reduced sample and only represents half of the molecule, the number was then multiplied by 2 to account for an intact antibody drug conjugate. An example DAR calculation for the IgGl 378 / 398 payload 2 conjugate are provided in Table 1DAR Ion counts DAR Contribution0 161126.73 0.001 0 0.00Total LC % 161126.73LC Avg DAR 0.00(DAR contribution from LC)0 0 0.001 4119.47 0.062 63480.39 1.873 244.24 0.014 0 0.00Total HC % 67844.1HC Avg DAR 1.94(DAR contribution from HC)(HC+LC)2 Final Avg DAR 3.89

[0098] Table 1: Quantification of the average DAR for IgGl 378 / 398 payload 2 conjugate using fractional percentages based on total ion counts from Time of Flight mass spectrometry analysis.

[0099] Example 5: Measurement of conjugation efficiency

[0100] Conjugation efficiency is monitored by standard procedures such as ESI-TOF mass spectrometry or Hydrophobicity Index Chromatography (HIC).

[0101] Aggregation propensity is measured by analytical size exclusion chromatography.LinkerDrug:antibody % Linker-payload Payload Conjugation siteratio (DAR) aggregation cLogDNone (Mab Ctrl) NA 0 0Native Cys 4 9% Native Cys 8 90% 1 3.3 eCys 378 398206 6 < 2% eCys 124, 378 3988 < 2% 206Native Cys 0.5 50% 2 11.3 eCys 378 398 3.8 < 2% eCys 378 398206 4.8 < 2% Native Cys 4 20% 3 4.1 Native Cys 8 100% eCys 378 398 8 < 2% Native Cys 4 & 8 100% 4 6.6 eCys 378 398 3.8 < 2% eCys 378 398206 5.6 < 2% Native Cys 8 < 2% eCys 378 398206 6 1% 5 2.9eCys 124378398 7.2 1%206

[0102] Table 2: Quantification of conjugation efficiency and aggregation propensity for various linker-payloads and conjugation sites

[0103] Example 6 - Measurement of high concentration stability

[0104] High concentration stability was assessed at lOOmg / mL in a common histidine formulation buffer. The ADC samples were exchanged into the formulation buffer using TFF (tangential flow filtration) and then subsequently concentrated to the target concentrations using 50KDa amicon concentrators (Millipore Cat#UFC905008). Final samples were sterile filtered and aliquoted into sterile screw cap vials. Vials were incubated at 35°C for 28 days. High molecule weight growth was measured using analytical size exclusion (SEC) chromatography. The column used for analysis was a TSKgel UP-SW3000, 4.6mm x 15cm, 2pM column (Tosoh Part# 0023449) with a flow rate of 0.35mL / min in a IX PBS buffer with 300mM NaCl.Following the 28 days 1 pl of sample was diluted into 99pl of formulation buffer and then 5ul of this dilution was injected onto the aSEC HPLC column. For high concentration assessment, DAR4 variants on both IgGl and IgG4P utilizing the heavy chain A378C-L398C were explored.Three different linker payloads with diverse structure and lipophilicity were assessed to show the translatability with different small molecules.Linker Payload Drug:antibody AHMW% Linker PayloadcLogD ratio (DAR) (35°C / 28days) None (m Ab Ctrl) N / A 0 2.76 4.13 4.4 2.9 7 3.29 3.9 1.18 4.26 4.8 1.6

[0105] Table 3: Quantification of high concentration stability for IgGl 378-398 with various linker-payloadsLinker Payload Drug:antibody AHMW% Linker PayloadcLogD ratio (DAR) (35°C / 28days) None (mAb Ctrl) N / A 0 2.66 4.13 4.2 3.2 7 3.29 3.9 2.38 4.26 4.4 2.8

[0106] Table 4: Quantification of high concentration stability for IgG4378-398 with various linker-payloads|0107| Example 7 - Measurement of viscosity

[0108] Viscosity was assessed at 125mg / mL at 125mg / mL in a common histidine formulation buffer H6S. The ADC samples were exchanged into the formulation buffer using TFF (tangential flow filtration) and then subsequently concentrated to the target concentrations using 50KDa amicon concentrators (Millipore Cat#UFC905008). Viscosity was assessed using the VROC® Initium one plus (RheoSense Cat#INI-H-1100).Linker Payload ViscosityNone (mAb Ctrl) 9.36 9.87 11.48 11.9

[0109] Table 5: Quantification of viscosity for IgGl 378-398 with various linker-payloadsLinker Payload ViscosityNone (mAb Ctrl) 9.46 7.87 9.48 11.7

[0110] Table 6: Quantification of viscosity for IgG4378-398 with various linker-payloads

[0111] Example 8 - Measurement of hydrophobicity and thermal stability

[0112] To assess relative thermal stability, the onset of aggregation (Tagg) was determined using intrinsic fluorescence and light scatter measured on a Prometheus Penta instrument (Nano Temper Technologies). Samples were prepared at 2 mg / mL and the onset of aggregation (Tagg) was determined using a thermal ramp from 20-95 °C at a rate of l°C / min. The impact of the LP on overall hydrophobicity was determined using analytical hydrophobic interaction chromatography (aHIC). A Tosoh TSKgel Butyl-NPR column with a gradient from 0-100% B in 25 minutes using the following buffers: A) 25mM Potassium phosphate, 1.5M ammonium sulfate, pH 7 and B) 25mM Potassium phosphate, 20% IPA pH 7.Linker Payload HIC Retention Time (min) Tagg (°C)None (mAb Ctrl) 8.1 78.26 8.6 79.27 11.7 78.38 9.5 78.3

[0113] Table 7: Quantification of hydrophobicity and thermal stability for IgGl 378-398 with various linker-payloadsLinker Payload HIC Retention Time (min) Tagg (°C)None (mAb Ctrl) 8.0 696 8.8 697 10.6 68.58 8.9 67

[0114] Table 8: Quantification of hydrophobicity and thermal stability for IgG4378-398 with various linker-payloads

[0115] Example 9 - Pharmacokinetics of ADC and parental antibody in rats

[0116] Plasma pharmacokinetics (PK) and linker-payload stability of ADC were evaluated in male Sprague Dawley rats following a single 5 mg / kg intravenous (IV) dose and compared to plasma PK of the parental non- ADC antibody. Whole blood samples were collected over 672 hours following dosing into tubes containing K2EDTA anticoagulant and subsequently processed to plasma. Plasma concentrations of ADC and parental antibody were determined using LC / MS methods. The mean PK profiles of ADC and parent antibody in rats is shown in Figure 1. Mean antibody CL in rats by IV dosing route is 0.196 mL / hr / kg and 0.158 mL / hr / kg, respectively (Table 9), showing that there was minimal effect on overall antibody exposure for the conjugate. Mean CL in rats for conjugated ADC by IV dosing is 0.325 mL / hr / kg, consistent with a slow release of payload over time, as expected for an ADC.Compound - Analyte Descriptor Dose (mg / kg) Route CL (mL / hr / kg) IgGl - Total IgG mAb 5 IV 0.158 IgGl 378 / 398-LP5- Total378 / 398 5 IV 0.196IgGIgGl 378 / 398-LP5- 378 / 398 5 IV 0.325Conjugate

[0117] Table 9: Mean Clearance of ADC and mab in rats|0118| Example 10 -In vivo stability assessment of ADC conjugate in rats

[0119] In vivo stability of the ADC was determined in plasma samples collected from the rat study after a single 5 mg / kg IV dose. An IP-LC / MS assay was used to determine the fully intact ADC over the time course. The percent remaining of fully intact ADC was calculated by comparing the % change in mass at the respective time point from the parental ADC dosing solution. Any loss of mass that impacts functional activity is treated as a % loss, therefore 75% remaining corresponds to 3 out of 4 linker-payloads on the ADC to be functionally active.Results are summarized in Table 10. Over the course of 168 hours (1 week) 95.7% of the ADC is a fully intact DAR4 molecule, with no loss of linker-payload up to the 72 hour time point.DoseCompound - Analyte Descriptor Route O hr 24 hr 72 hr 168 hr(mg / kg)IgGl 378 / 398-LP5 - 378 / 398 5 IV 100% 100% 100% 95.7% ADC

[0120] Table 10: In vivo stability of a IgGl 378 / 398 ADC in rats as measured by % fully intact DAR4 ADCADC# LinkerLP mol Conjugation Total AHMW% AHIC in IgGl payload wt. site DAR min 378 / 398- (Daltons) (HICADC- HICmAb) LP9 - 9 3086.77 378 / 398 4 < 2% 2.5 ADC LP10 - 10 2924.08 378 / 398 4 < 2% 0.6 ADC LP11 - 11 3005.42 378 / 398 4.1 <2% 1.4 ADC LP12 - 12 3714.99 378 / 398 4 <2% 1.5 ADC LP13 - 13 3309.56 378 / 398 3.9 <1% 2 ADC LP14- 14 3354.74 378 / 398 3.7 <1% 1.7 ADC

[0121] Table 11: Quantification of conjugation efficiency and aggregation propensity for various linker-payloads conjugated at 378 / 398 sitesPK parametersTest articleCL (mL / min / kg) TI / 2 (hr) AUC (h*nM) Vss (L / kg) mAb Ctrl 0.00351 387 15500 0.116 LP9-ADC 0.0128 108 14900 0.11 LP10-ADC 0.00674 197 19600 0.12 LP11-ADC 0.00986 140 15800 0.114

[0122] Table 12: Total IgG exposures in mouse plasma following IV dosing of ADCs at 3 mg / kg

[0123] Example 11 - Methods

[0124] LC / MS Method Summary for total antibody and conjugated payload

[0125] Concentrations of total IgG and IgG conjugated payload are determined using a beadbased anti-human IgG immuno-affinity capture. A biotinylated goat anti-human IgG (Fc specific) (Southern Biotech, Catalog number 2049-08) is used to capture ADC and parent antibody, respectively. Complexes are then removed from the plasma using streptavidin coated magnetic beads. The beads are washed to remove non-specific bindings. The captured ADC and parent antibody are eluted from the anti-human IgG capture antibody. The total IgG samples are then reduced, alkylated and treated with trypsin / lysC prior to detection of a surrogate peptide from the antibody using a Q-Exactive Plus Orbitrap mass spectrometer (Thermo). For the IgG conjugated drug assay, following immuno-affinity capture, samples are treated with Proteinase K which leads to the proteolysis of the proteinase K sensitive component of the linker and liberates dmg payload from the antibody. Following the cleavage of the linker, the pay load liberated from the antibody is quantitated using a Q-Exactive Plus Orbitrap mass spectrometer (Thermo).

[0126] LC / MS Method Summary for intact mass

[0127] Plasma samples were diluted with PBS (~3-fold for in vitro samples, dilutions varied for in vivo samples). The LC / MS assay was developed to measures fully intact ADC. Biotinylated anti-hlgG antibody (Sothern Biotech, part no.2049-08) was added to all the samples followed by an incubation of 60 min at room temperature (RT). Streptavidin magnetic beads (Promega, part no. V7820) were pre -washed with PBS / 0.1% CHAPS / PBS then added to the samples and incubated for 60 min at RT. After incubation the antibody -bead complex was washed 0.1% CHAPS followed by PBS. Bound IgG was eluted with 0.1% formic acid / 10% acetonitrile. The eluted conjugates were neutralized immediately with 0.3M Tris, pH8 containing 45mM Tris(2-carboxyethyljphosphine hydrochloride (TCEP). Samples were measured by Liquid chromatography / mass spectrometry (LC / MS) (Exploris 240 with Bio-pharma option, Thermo, San Jose, CA)

[0128] Example 12 - Native cysteine (nCys) vs engineered cysteine (eCys) DAR stability and PK results

[0129] ADCs were prepared as described in Examples 2 & 3 and injected intravenously into female Sprague Dawley rats at various concentrations. Blood draws were taken at three timepoints post dose. Total antibody concentration in plasma was measured using a standardELISA method. To determine the concentration of antibody-conjugated drug, plasma was prepared by anti-human antibody capture, purification, and enzymatic cleavage of linker, then released payload was quantified by LC / MS. The in vivo DAR was calculated by the ratio between the pM concentrations of antibody-conjugated payload and total antibody. Total IgG and average DAR loss in Pk studies (1 mg / kg in tumor free nude mice (N = 3 per time point) is shown in Figure 2.SEQUENCESSEQ ID NO: 1 (Native IgGl heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVCDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 2 (IgGl AAS - L234A, L235A and D265S - heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVCDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3 (IgG4 S228P heavy chain constant domain including the CHI, CH2 and CH3 with A378C-L398C) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVCDSDGSFFLYSRLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSLGSEQ ID NO:4 (Light chain kappa constant domain with L206C) VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCKSFNRGEC

Claims

CLAIMS:

1. An antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering); and / ora cysteine at amino acid residue 398 (EU numbering).

2. The antibody according to claim 1, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering); anda cysteine at amino acid residue 398 (EU numbering).

3. The antibody according to claim 1 or claim 2, wherein the human IgG heavy chain constant region comprises a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; and / or a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

4. The antibody according to claim 3, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; anda cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

5. An antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering); and / ora cysteine at amino acid residue 378 (EU numbering).

6. The antibody according to claim 5, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering); anda cysteine at amino acid residue 378 (EU numbering).

7. The antibody according to claim 5 or claim 6, wherein the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and / or a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain.

8. The antibody according to claim 7, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; anda cysteine at amino acid residue 378 (EU numbering) of the CH3 domain.

9. An antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering); and / ora cysteine at amino acid residue 398 (EU numbering).

10. The antibody according to claim 9, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering); anda cysteine at amino acid residue 398 (EU numbering).

11. The antibody according to claim 9 or claim 10, wherein the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; and / or a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

12. The antibody according to claim 11, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering) of the CHI domain; anda cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

13. The antibody according to any one of claims 1 to 12, wherein the antibody further comprises a human IgG light chain constant region and wherein the human IgG light chain constant region comprises:a cysteine at amino acid residue 206 (EU numbering).

14. An antibody comprising a human IgG heavy chain constant region and a human IgG light chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering);a cysteine at amino acid residue 398 (EU numbering); andwherein the human IgG light chain constant region comprises a cysteine at amino acid residue 206 (EU numbering).

15. The antibody according to claim 14, wherein the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain;a cysteine at amino acid residue 398 (EU numbering) of the CH3 domain; and wherein the human IgG light chain constant region comprises a cysteine at amino acid residue 206 (EU numbering).

16. An antibody comprising a human IgG heavy chain constant region, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering);a cysteine at amino acid residue 378 (EU numbering); anda cysteine at amino acid residue 398 (EU numbering).

17. The antibody according to claim 16, wherein the human IgG heavy chain constant region comprises a CHI, a CH2, and a CH3 domain, wherein the human IgG heavy chain constant region comprises:a cysteine at amino acid residue 124 (EU numbering) of the CHI domain;a cysteine at amino acid residue 378 (EU numbering) of the CH3 domain; anda cysteine at amino acid residue 398 (EU numbering) of the CH3 domain.

18. The antibody according to any one of claims 1 to 17, wherein the human IgG heavy chain constant region comprises a CH2 domain and wherein the human IgG heavy chain constant region further comprises:an alanine at amino acid residue 234 (EU numbering) of the CH2 domain;an alanine at amino acid residue 235 (EU numbering) of the CH2 domain; and / or a serine at amino acid residue 265 (EU numbering) of the CH2 domain.

19. The antibody according to claim 18, wherein the human IgG heavy chain constant region comprises a CH2 domain and wherein the human IgG heavy chain constant region further comprises:an alanine at amino acid residue 234 (EU numbering) of the CH2 domain;an alanine at amino acid residue 235 (EU numbering) of the CH2 domain; anda serine at amino acid residue 265 (EU numbering) of the CH2 domain.

20. The antibody according to any one of claims 1 to 19, wherein the antibody further comprises:a proline at amino acid residue 228 (EU numbering).

21. The antibody according to any one of claims 1 to 20, wherein the human IgG heavy chain constant region comprises a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises:a lysine at amino acid residue 274 (EU numbering) of the CH2 domain; an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; and / or a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.

22. The antibody according to claim 21, wherein the human IgG heavy chain constant region comprises a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises:a lysine at amino acid residue 274 (EU numbering) of the CH2 domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.

23. The antibody according to any one of claims 1 to 22, wherein the human IgG heavy chain constant region comprises a CHI, a CH2 and a CH3 domain and wherein the human IgG heavy chain constant region further comprises:a lysine at amino acid residue 274 (EU numbering) of the CH2 domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain; a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; a glycine at amino acid residue 137 (EU numbering) of the CHI domain; an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and an arginine at amino acid residue 355 (EU numbering) of the CH3 domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a glycine at amino acid residue 137 (EU numbering) of the CHI domain and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and a glycine at amino acid residue 137 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;a lysine at amino acid residue 274 (EU numbering) of the CH2 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain;an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain; ora lysine at amino acid residue 274 (EU numbering) of the CH2 domain, an arginine at amino acid residue 355 (EU numbering) of the CH3 domain, a glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, a glycine at amino acid residue 137 (EU numbering) of the CHI domain, and an asparagine at amino acid residue 203 (EU numbering) of the CHI domain.

24. The antibody according to any one of claims 1 to 23, wherein the antibody is a human IgGl, human IgG2, human IgG3, or human IgG4 antibody.

25. The antibody according to claim 24, wherein the antibody is a human IgGl antibody.

26. The antibody according to claim 24, wherein the antibody is a human IgG4 antibody.

27. An antibody comprising a human IgG heavy chain constant region comprising any one of SEQ ID NOs: 1, 2 or 3.

28. An antibody drag conjugate comprising the antibody according to any one of claims 1- 27.

29. The antibody drag conjugate of claim 28, wherein the antibody is conjugated to a payload molecule selected from the list consisting of: a peptide, a small molecule, anoligonucleotide, a radioligand, a fatty acid, a therapeutic agent or a diagnostic or detection agent.

30. The antibody drug conjugate of claim 28 or claim 29 having a drug:antibody ratio of between 1 and 8.

31. The antibody drug conjugate of claim 30 having a drug:antibody ratio of 4, 6 or 832. The antibody drug conjugate of claim 31 having a drug:antibody ratio of 4.

33. The antibody drug conjugate of any one of claims 28-32, wherein the antibody drug conjugate has reduced hydrophobicity compared to an antibody drug conjugate not comprising the modified amino acid residues.

34. The antibody drug conjugate of claim 33, wherein the hydrophobicity is reduced by 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 95% as measured by ESI-TOF mass spectrometry or Hydrophobicity Index Chromatography (HIC) compared to an antibody drug conjugate not comprising the modified amino acid residues.

35. The antibody drug conjugate of any one of claims 28-34, wherein the antibody drag conjugate has increased biophysical stability compared to an antibody drag conjugate not comprising the modified amino acid residues.

36. The antibody drag conjugate of claim 35, wherein the biophysical stability is increased by 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% as measured by analytical size exclusion (SEC) chromatography compared to an antibody drag conjugate not comprising the modified amino acid residues.

37. The antibody drug conjugate of any one of claims 28-36, wherein the antibody drug conjugate has improved pharmacokinetics compared to an antibody drug conjugate not comprising the modified amino acid residues.

38. The antibody drug conjugate of any one of claims 28-37, wherein the antibody drug conjugate has reduced retro-Michael release of payload molecules compared to an antibody drug conjugate not comprising the modified amino acid residues.

39. The antibody drug conjugate of claim 38, wherein the retro-Michael release of payload molecules is reduced by 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 95% as measured by DAR stability using liquid chromatography / mass spectrometry (LC / MS) compared to an antibody drug conjugate not comprising the modified amino acid residues.

40. A nucleic acid comprising a sequence encoding the heavy chain or light chain of the antibody according to any one of claims 1 to 27.

41. A vector comprising the nucleic acid according to claim 40.

42. A cell comprising the vector according to claim 41.

43. The cell according to claim 42, wherein the cell is a mammalian cell.

44. A process of producing an antibody comprising culturing the cell according to claim 42 or claim 43, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

45. An antibody produced by the process according to claim 44.

46. A process for producing an antibody drug conjugate comprising the antibody of any one of claims 1 to 27 or 45 and one or more pay load molecules comprising:removing capping groups on engineered cysteine residues of an antibody of any one of claims 1 to 27 or 45 in an antibody mixture by adding a reducing agent;removing unbound capping groups and the reducing agent;oxidizing the antibody mixture to reform native interchain disulfide linkages; and adding one or more payload molecules to the antibody mixture after native interchain disulfide linkages have formed and allowing the one or more payload molecules to conjugate to the engineered cysteine sites.

47. An antibody drug conjugate produced by the process according to claim 46.

48. A pharmaceutical composition comprising the antibody or antibody drug conjugate according to any one of claims 1 to 39, 45 or 47, and a pharmaceutically acceptable excipient, diluent or carrier.

49. A method of administering the antibody or antibody drug conjugate of any one of claims 1 to 39, 45 or 47 to a subject in need thereof.

50. The method of administering of claim 49, wherein the antibody or antibody drug conjugate is administered subcutaneously to the subject.

51. The method of administering of claim 49, wherein the antibody or antibody drug conjugate is administered intravenously to the subject.

52. An antibody according to any one of claims 1-27 or 45, an antibody drug conjugate according to any one of claims 28-39 or 47, or a pharmaceutical composition according to claim 48 for use in therapy.

53. An antibody according to any one of claims 1-27 or 45, an antibody drug conjugate according to any one of claims 28-39 or 47, or a pharmaceutical composition according to claim 48 for use in treating a cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

54. Use of an antibody according to any one of claims 1-27 or 45. an antibody drug conjugate according to any one of claims 28-39 or 47, or a pharmaceutical composition according to claim 48 in the manufacture of a medicament for the treatment of a cancer, a cardiovascular disease, an autoimmune disease, or a neurodegenerative disease.

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