WO2019092148A1

WO2019092148A1 - Antibodies with functionalized glutamine residues

Info

Publication number: WO2019092148A1
Application number: PCT/EP2018/080686
Authority: WO
Inventors: Delphine Bregeon; Laurent Gauthier
Original assignee: Innate Pharma
Priority date: 2017-11-10
Filing date: 2018-11-08
Publication date: 2019-05-16

Abstract

The present application relates to methods for the functionalization of immunoglobulins, in particular with drugs. Also disclosed herein are linking reagents, functionalized antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.

Description

ANTIBODIES WITH FUNCTION ALIZED GLUTAMINE RESIDUES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/584,137, filed 10 November 2017; which isare incorporated herein by reference in its entirety; including any drawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled "TGase12_ST25", created 1 1 November 2018, which is 12 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for the functionalization of immunoglobulins.

BACKGROUND

Immunoglobulins conjugated to a drug of interest, generally known as antibody drug conjugates (ADCs), are a promising area of therapeutic research. Recent developments in ADC technology have focused on linker technology that provides for intracellular cleavage or more recently, non-cleavable linkers that provide greater in vivo stability and reduced toxicity. The feasibility of a non-cleavable linker-based approach, however, may be more dependent on the cellular target than in the case of cleavable linkers. ADCs with noncleavable linkers must be internalized and degraded within the cell, whereas compounds with cleavable linkers may be active against targets that are poorly internalized through extracellular drug release and drug entry into cells. In the case of cytotoxic ADCs, immunostimulatory ADCs and immunosuppressive ADCs, the respective killing of bystander antigen-negative cells through targeting of antigen-positive cells (collateral toxicity), immune stimulation at the disease site or anti-inflammatory action at the disease site is presumably only possible with cleavable linkers. As a consequence, it is generally believed that no general linker design exists for all ADCs and that each antibody must be examined separately. Additionally, the efficacy of a drug linked to a toxin may vary, e.g. depending on the cell type or particular tumor cell, such that it may also be necessary to test a variety of drugs against a given target and further in combination with a particular linker system. Development of ADCs therefore remains an expensive and time-consuming process and there is a need in the field for improved linker systems.

Transglutaminases (TGases) have been exploited for some time in the food industry for their ability to cross-link proteins. TGases have been shown to be capable of conjugating glutamine and lysine residues, including antibodies (see, e.g., Josten et al. (2000) J. Immunol. Methods 240, 47-54; Mindt et al (2008) Bioconjug. Chem. 19, 271 -278; Jeger et al (2010) Angew. Chem. Int. Ed. 49: 9995-9997); Kamiya et al (2003) Enzyme. Microb. Technol. 33, 492-496 and U.S. patent publication no. 201 1/0184147. More recently, TGases have been found to be able to site-specifically conjugate glutamines naturally present in or inserted into the Fc domain of antibodies, as well as in peptide tags fused to heavy or light chain constant regions or to antibody variable region fragments (see, e.g., PCT publication WO2014/072482). Such conjugation takes place without conjugation to the glutamines present in the variable regions of the antibodies, including many human-derived variable region sequences (see PCT publication WO2013/092998). TGases have been proposed for the conjugation of therapeutic agents to glutamines including, inter alia, the glutamine naturally present at Kabat position 295 within the CH2 domain of the Fc portion, including in antibodies that retain N-linked glycosylation at nearby Kabat residue N297 and in deglycosylated or aglycosylated antibodies (see, e.g., PCT publications WO2013/092983, WO2013/092998, WO2014/202773, WO2014/202775, WO2016/102632, WO2017/059158). TGase technology has been found to present many advantages, including for potential therapeutic and diagnostic use, but also to permit rapid screening of candidate antibody-drug conjugates (see, e.g., WO2014/009426). Furthermore, antibody-drug conjugates having a functionalized glutamine at Kabat position 295 can be bound by affinity media such as Protein A or Protein G, enabling them to be manufactured using well-known processes (see, e.g., WO2014/140300). Finally, while TGase can have decreased conjugation efficiency with some large or hydrophobic substrates, a two-step process for conjugating antibodies has been developed that enables TGase-mediated conjugation of large, charged and/or hydrophobic drugs in a highly complete manner, e.g. reaching DARs of more than 1.8 or 1.9 in an antibody having two available acceptor glutamines (see, e.g. WO2013/092983, WO2014/202775, WO2016/102632).

Conjugation of therapeutic agents, molecular markers, and other moieties of interest to antibodies are of considerable commercial interest. However, the rules which govern selection by TGases of glutamine residues for modification are still largely unknown. There is therefore a need in the art for improved methods to conjugate drus with antibodies using TGases. SUMMARY OF THE INVENTION

Past efforts to develop ADCs have largely focused on targeting tumor cells through the use of highly-potent cytotoxic agents, and consequently generally have sought to retain effector function of the Fc domain in order to have maximum tumor-killing functions. However, as shown herein, anti-tumor antibodies conjugated with highly-potent cytotoxic agents, notably pyrrolobenzodiazepine- (PBD) based agents) display maximal efficacy at concentrations well below that at which the ADCC mechanism has any significant contribution. Notably, anti-tumor antigen antibody-PBD conjugates showed an EC₅₀ for in vitro tumor cell killing of less than 0.01 μg ml assays as well as strong anti-tumoral activity in mice tumor models at a dose of only 0.05 mg/kg once weekly, leading to substantially complete elimination of all tumors. By comparison, antibodies that mediate ADCC generally show acivity in such mice models in the range of 10 mg/kg. As a result, it is believed that the potential interactions of Fc-competent antibodies with human FcyRI (and possibly other human Fey receptors) can cause significant unwanted and off-target toxicity that results in an overall decrease of the therapeutic window for the ADC.

The present invention arises, inter alia, from the development of an approach to make use of enzyme-mediated conjugation onto Fc domains that bind to the human neonatal Fc receptor (e.g. human FcRn) without substantial binding to human FcyRI , and/or more generally to all FcyR human receptors (e.g., CD16A, CD16B, CD32A, CD32b, CD64) Through the use of enzymatic-mediated conjugation, notably TGase-mediated conjugation, immunoconjugates with highly homogenous drug:antibody ratios (high % of antibodies conjugated at the desired DAR) were achieved for mutated heavy chain constant regions. It is shown herein that TGase is capable of conjugation with high efficiency onto acceptor glutamines in the human CH2 domain, including at Kabat residue 295, in antibodies whose constant domains comprise amino acid modifications that abolish binding to human CD64 (and further also CD16A, CD16B, CD32A and CD32b). Conjugation at residue 295 is particularly advantageous because the residue is naturally present in human IgG antibodies, furthermore no non-immunoglobulin amino acid sequences are introduced that could give rise to immunogenicity or that could alter the physicochemical or other biological properties of the antibody. The antibodies and Fc regions of the disclosure that bind to human FcRn proteins with a binding affinity that is similar (or not less than, not substantially less than, a KD within 1 -log of) that of native human lgG1 antibodies or Fc domains. Affinity can be characterized as KD as determined by SPR. For example the antibodies can be characterized as having a KD for binding to recombinant FcRn protein in a SPR assay (e.g. Biacore, according to the methods of Example 4)) that is within 0.5-log or 1-log of the KD observed for binding of a native wild type human lgG1 antibody to recombinant FcRn protein, or optionally wherein the KD is lower than that observed with the wild type human lgG1 antibody. In one embodiment, the modifications are amino acid substitutions that decrease binding to human CD16A, CD16B, CD32A, CD32b and CD64 are amino acid substitutions in the hinge and/or CH2 domain. In one embodiment, a constant domain is a human lgG1 domain comprising a substitution at 234, 235 and/or 237 (e.g. comprising one, two or three of the substitutions), and a substitution at residues 330 and/or 331.

Despite the inability to induce Fey receptor-mediated crosslinking of a target antigen at the surface, the antibodies are capable of intracellular internalization when bound to an antigen of interest on a mammalian cell expressing such antigen. When an immunoconjugate comprises an antibody or other Fc-containing protein conjugated to highly potent cytotoxic agents, the resulting immunoconjuates have advantageous pharmaceutical characteristics, including advantageous therapeutic window, reduced off-target toxicity and side effects. The methods and compositions that lack binding to human CD16A, CD16B, CD32A, CD32b, CD64 can also advantageously be used prepare immunoconjuates that are designed to be non-depleting toward target cells expressing the antigen target of the antibody. Such immunoconjuates may also benefit from advantageous therapeutic window, and reduced off-target effects. For example, the immunoconjugate may comprise an antibody or other Fc-containing protein coupled to an anti-inflammatory agent. In another embodiment, the immunoconjugate may comprise an antibody or other Fc-containing protein coupled to an immunostimulatory agent. Finally, the antibodies of the description have good stability (low propensity to aggregate in solution).

In one embodiment of any aspect herein, an immunoconjugate comprises an agent of interest (e.g. a therapeutic agent, a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent) covalently bound to an acceptor glutamine residue (Q)) within an immunoglublin heavy chain constant region (e.g. within an Fc domain, within an Fc domain of an antibody or antibody fragment). In one embodiment, the acceptor glutamine is in the CH2 and/or CH3 domain, optionally in the C'E loop of a CH2 domain, optionally at Kabat position 295 and/or 297 (Kabat EU numbering) of a heavy chain. In one embodiment, the antibody comprises an Fc region that lacks natural N-linked glycosylation at Kabat residue 297, e.g., the glycan naturally present at residue N297 is absent. In another embodiment, the antibody comprises an Fc region that bears N-linked glycosylation at Kabat residue 297; in one embodiment the glycans naturally present at residue N297 are present; in another embodiment, a modified (e.g. truncated) glycan is present at residue N297. In one embodiment, the immunoconjugate (or the Fc domain, antibody or anibody fragment) binds to human FcRn protein (e.g. comparably to a human lgG1 antibody or dimeric Fc domain) but does not substantially bind to a human CD64 protein.

In one aspect, an immunoconjugate (or Fc-domain containing protein, antibody or antibody fragment), comprises an Fc domain comprising an amino acid substitution (e.g. compared to a reference Fc domain, e.g. a human lgG1 Fc domain) in a heavy chain constant region at Kabat positions 234, 235 and 331 , optionally at Kabat positions 234, 235, 237 and 331 , or optionally at Kabat positions 234, 235, 237, 330 and 331 , and wherein the protein (e.g., the Fc domain) comprises a functionalized glutamine residue (Q) comprising the structure:

a functionalized amino acid residue (Q) comprising the structure:

(Q)-L"-Y-Z

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

Q is a glutamine residue present in or appended to (e.g., naturally present in the primary sequence of, inserted into, substituted into, fused to) a constant domain of the Fc-domain containing protein;

L" is a linker covalently bonded to the glutamine Q;

Y is a spacer system; and

Z comprises a moiety of interest, a moiety that improves the pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety.

In one embodiment, the structure of the functionalized glutamine residue (Q) further comprises a conditionally-cleavable moiety (V) placed between L" and Z (e.g., between L" and Y or between Y and Z). In one embodiment, V is a moiety conditionally-cleavable moiety, optionally following prior conditional transformation, following intracellular internalization (e.g. intracellular cleavage of V ultimately leading to intracellular release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety".

In one embodiment, the structure of the functionalized glutamine residue (Q) further comprises an addition product (RR') placed between L" and Z, preferably between L" and Y, wherein RR' is an addition product between a reactive moiety R and a complementary reactive moiety R'.

Optionally Q is present within the Fc domain, optionally within in a human CH2 domain (or a C'E loop thereof). Optionally Q appended to the Fc domain, for example the Q is present within a peptide tag, e.g., a TGase recognition tag, appended to the Fc domain. Optionally Q is present in or appended to the constant domain is a human CH3 domain. In one embodiment Q is present in (e.g., naturally present in the primary sequence, inserted into, substituted into, fused to) a human Fc domain, optionally a CH2 constant domain or a C'E loop thereof, optionally at Kabat residues 295 and/or 297, or a CH3 constant domain.

In one aspect, provided is an antibody or antibody fragment comprising an Fc domain of human γ isotype, wherein the antibody or antibody fragment: (a) binds to a human FcRn protein, (b) substantially lacks binding to human Fey receptor CD64, and (c) comprises a functionalized glutamine residue (Q) at Kabat residue 295. In one embodiment, the functionalized glutamine residue (Q) comprises the structure:

(Q)-L"-Z

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present at Kabat residue 295;

L" is a lysine-based linker in which the nitrogen atom is covalently bonded to the γ carbon of Q as a secondary amine; and

In any embodiment herein, where residue Kabat 297 is not an acceptor glutamine, the residue at Kabat 297 can be asparagine having or lacking N-linked glycosylation, or a residue other than asparagine or glutamine. Optionally, when an acceptor glutamine is present at Kabat 295, the residue at Kabat 297 is a residue other than aspartic acid.

In one embodiment, the Fc domain comprises a CH2 and/or CH3 domain of human lgG1 isotype. In one embodiment, the Fc domain comprises a hinge domain of human lgG1 isotype. In one embodiment, the Fc domain comprises a CH1 domain of human lgG1 isotype. In one embodiment, the Fc domain or constant domain has an amino acid substitution in a heavy chain constant region at any three, four, five or more of residues selected from the group consisting of: 234, 235, 237, 322, 330 and 331 (Kabat numbering). Optionally, a phenylalanine or an alanine is present at Kabat position 234. Optionally, a glutamic acid is present at position 235. Optionally, an alanine is present at position 237. Optionally, a serine is present at position 330. Optionally, a serine is present at position 331.

In one aspect, provided is an Fc-domain containing protein (e.g. an antibody or antibody fragment of the disclosure), wherein the Fc domain comprises an amino acid substitution (e.g. compared to a reference Fc domain, e.g. a human lgG1 Fc domain) in a heavy chain constant region at Kabat positions 234, 235 and 331 , optionally at Kabat positions 234, 235, 237 and 331 , or optionally at Kabat positions 234, 235, 237, 330 and 331 , wherein the protein comprises a functionalized amino acid residue (B) comprising the structure:

(Q)-L"-RR'-Y-Z

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

Q is a glutamine residue (Q) present in or appended to (e.g., naturally present in the primary sequence of, inserted into, substituted into, fused to) a constant domain of the Fc-domain containing protein;

L" is a linker covalently bonded to the glutamine Q;

(RR') is an addition product between a reactive moiety R and a complementary reactive moiety R';

Y is a spacer system; and

Z comprises a moiety of interest, optionally a moiety that improves the pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety, optionally, wherein (Z) is a hydrophobic compound, a charged organic compound and/or organic compound having a molecular weight of at least 500, 700 or 800 g/mol.

In one embodiment, the structure of the functionalized glutamine residue (Q) further comprises a conditionally-cleavable moiety (V) placed between RR' and Z (e.g., between RR' and Y or between Y and Z). In one embodiment, V is a moiety conditionally-cleavable moiety, optionally following prior conditional transformation, following intracellular internalization (e.g. intracellular cleavage of V ultimately leading to intracellular release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety).

In one aspect, provided is a composition comprising a plurality of Fc-domain containing proteins (e.g. an antibody or antibody fragment), wherein the Fc domain comprises an amino acid substitution (e.g. compared to a reference Fc domain, e.g. a human lgG1 Fc domain) in a heavy chain constant region at Kabat positions 234, 235 and 331 , optionally at Kabat positions 234, 235, 237 and 331 , or optionally at Kabat positions 234, 235, 237, 330 and 331 , wherein at least 90% of the Fc-domain containing proteins in said composition have (m) functionalized acceptor glutamine residues (Q) per antibody or fragment, wherein m is an integer selected from 2 or 4, wherein each of the functionalized acceptor glutamine residues has the structure:

(Q)-L"-Y-Z

or a pharmaceutically acceptable salt or solvate thereof,

wherein: Q is a glutamine residue present in or appended to (e.g., naturally present in the primary sequence, inserted into, substituted into, fused to) a constant domain of the Fc-domain containing protein;

L" is a lysine-based linker in which the nitrogen atom is covalently bonded to the γ carbon of Q as a secondary amine;

Y is a spacer system; and

In one embodiment, the plurality of Fc-domain containing proteins in the composition share the same amino acid sequence.

(Q)-L"-RR'-Y-Z

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in or appended to (e.g., naturally present in the primary sequence, inserted into, substituted into, fused to) a constant domain of the Fc-domain containing protein;

Y is a spacer system; and

In one embodiment, the plurality of Fc-domain containing proteins in the composition share the same amino acid sequence. In one aspect, provided is a method for preparing an Fc-domain containing protein comprising a moiety of interest bound thereto, comprising the steps of:

a) providing an Fc-domain containing protein (e.g. an antibody or antibody fragment) comprising at least one acceptor glutamine residue, wherein the Fc domain comprises an amino acid substitution (e.g. compared to a reference Fc domain, e.g. a human lgG1 Fc domain) in a heavy chain constant region at Kabat positions 234, 235 and 331 , optionally at Kabat positions 234, 235, 237 and 331 , or optionally at Kabat positions 234, 235, 237, 330 and 331 ; and

b) reacting said Fc-domain containing protein with a linker comprising a primary amine and a moiety of interest (Z), in the presence of a TGase, under conditions sufficient to obtain an Fc-domain containing protein comprising an acceptor glutamine linked to moiety of interest (Z) via the linker (a functionalized glutamine residue). In one embodiment, the glutamine residue is present in or appended to (e.g., naturally present in the primary sequence, inserted into, substituted into, fused to) a constant domain of the Fc-domain containing protein.

Optionally, in any of the composition or methods, the linker is a compound of formula la:

G-NH-C-X-L-(V-(Y-(Z)_z)_q)_r Formula la; or a pharmaceutically acceptable salt or solvate thereof

wherein:

G is an H, amine protecting group, or Fc-domain containing protein (e.g., attached via an amide bond);

C is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally where the carbon adjacent to the nitrogen is unsubstituted, optionally wherein any carbon of the chain is substituted alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide (e.g. with a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide); wherein C has a chain length from among the range of 2 to 20 atoms, preferably 3 to 6 atoms;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework comprises a linear framework of 3 to 30 carbon atoms optionally substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), an amino acid, a di-, tri-, tetra-, or oligopeptide, other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1 , 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4; and

z is an integer selected from among 1 , 2, 3 or 4;

V is independently absent, a bond or a continuation of a bond if L is a bond, a non- cleavable moiety or a conditionally-cleavable moiety (e.g. following intracellular internalization), optionally following prior conditional transformation, which can be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process (e.g. cleavage of V ultimately leading to release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety";

Y is independently absent, a bond or a continuation of a bond if V is a bond or continuation of a bond, or a spacer system (e.g., a self-eliminating spacer system or a non- self-elimination spacer system) which is comprised of 1 or more spacers; and

Z is a moiety that improves the pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety.

In one aspect, provided is a method for preparing an Fc-domain containing protein comprising a moiety of interest bound thereto, comprising the steps of:

b) reacting said Fc-domain containing protein with a linker comprising a primary amine and a reactive group (R), in the presence of a TGase, under conditions sufficient to obtain an Fc-domain containing protein comprising an acceptor glutamine linked to a reactive group (R) via said linker; and

(c) reacting, an Fc-domain containing protein of step (b) comprising an acceptor glutamine linked to a reactive group (R) with a compound comprising a moiety of interest (Z) and a reactive group (R') capable of reacting with reactive group R, under conditions sufficient to obtain an Fc-domain containing protein comprising an acceptor glutamine linked to moiety of interest (Z) via the linker (a functionalized glutamine residue). In one embodiment, the glutamine residue is present in or appended to (e.g., naturally present in the primary sequence, inserted into, substituted into, fused to) a constant domain of the Fc- domain containing protein.

Optionally, the reaction mixture in step (b) is free of organic solvent or contains less than 10%, optionally less than 5%, (v/v) organic solvent. Optionally, the reaction in step (c) is carried out in in the presence of at least 5%, optionally at least 10%, (v/v) organic solvent.

Optionally, in any of the methods the linker is a compound of formula lb:

G-NH-C -X-L-(V-(Y-(R)_z)_q)_r Formula lb or a pharmaceutically acceptable salt or solvate thereof

wherein:

G is an H, amine protecting group, or an Fc-domain containing protein (e.g. attached via an amide bond);

C is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally where the carbon adjacent to the nitrogen is unsubstituted, optionally wherein any carbon of the chain is substituted alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide (e.g. with a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide); wherein C has a chain length of from 2 to 20 atoms, preferably 3 to 6 atoms;

X is NH, O, S, or absent;

r is an integer selected from among 1 , 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4; and

z is an integer selected from among 1 , 2, 3 or 4;

R is a reactive moiety.

Optionally, in any of the methods or compositions, the functionalized glutamine residue has a structure of formula IVa:

(Q)-NH-C-X-L- (V-(Y-(Z)_z)_q)_{r Formu}|_a ,_Va or a pharmaceutically acceptable salt thereof;

wherein:

Q is glutamine residue present in an Fc-domain containing protein;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein any carbon of the chain is optionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide (e.g. a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide); wherein C has a chain length of from 2 to 20 atoms;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework, preferably of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework comprises a linear framework of 3 to 30 carbon atoms optionally substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligopeptide (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected among 1 , 2, 3 or 4;

q is an integer selected among 1 , 2, 3 or 4;

z is an integer selected among 1 , 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or a conditionally-cleavable moiety (e.g. following intracellular internalization) that can optionally be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process (e.g. cleavage of V ultimately leading to release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety";

Y is independently absent or a spacer (e.g., a self-eliminating spacer system or a non-self-elimination spacer system) which is comprised of 1 or more spacers; and

Z is a moiety-of-interest, optionally a moiety that improves the pharmacokinetic properties, or a therapeutic moiety or a diagnostic moiety.

Optionally, in any of the methods or compositions, a functionalized acceptor glutamine residue has a structure of Formula II (the result of reacting an antibody having an acceptor glutamine with a linking reagent of formula lb):

(Q)-NH-C-X-L- (V-(Y-(R)_z)_q)_{r Formu}|_a \\ or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an Fc-domain containing protein;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is optionally substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide; ; wherein C has a chain length of from 2 to 20 atoms;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework comprises a linear framework of 3 to 30 carbon atoms optionally substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligopeptide (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1 , 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1 , 2, 3 or 4; and

Y is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety (e.g. following intracellular internalization);

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and R is a reactive moiety.

Optionally, in any of the methods or compositions, a functionalized acceptor glutamine residue has a structure of formula IVb (the result of reacting an antibody having an acceptor glutamine of formula II with a reagent of formula III):

(Q)-NH-C-X-L- (V-(Y-(M)_z)_q)_{r Formu}|_a ,_Vb or a pharmaceutically acceptable salt or solvate thereof;

wherein:

Q is glutamine residue present in an Fc-domain containing protein;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein any carbon of the chain is optionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide; wherein C has a chain length of from 2 to 20 atoms;

X is NH, O, S, or absent;

r is an integer selected among 1 , 2, 3 or 4;

q is an integer selected among 1 , 2, 3 or 4;

z is an integer selected among 1 , 2, 3 or 4; and

Y is independently absent or a spacer (e.g., a self-eliminating spacer system or a non-self-elimination spacer system) which is comprised of 1 or more spacers; and M is independently: R or (RR') - L' - (V'-(Y'-(Z)_Z')_q )_{r ,} wherein each of L', V, Υ', ζ', q', and r' are as defined in Formula III (or are defined as L, V, Y, z, q and r, respectively,

Z is a moiety-of-interest, optionally a moiety that improves the pharmacokinetic properties, or a therapeutic moiety or a diagnostic moiety, R is a reactive moiety and wherein each (RR') is an addition product between an R and a complementary reactive group R'.

In one aspect of any of the methods or compositions herein, elements NH-C can be a lysine residue.

In one aspect of any of the methods or compositions herein, the acceptor glutamine (Q) is within or appended to a heavy and/or light chain constant region of the Fc-domain containing protein (e.g. antibody). In one aspect of any of the methods or compositions, the acceptor glutamine (Q) is in a peptide tag (e.g. TGase recognition tag), e.g. fused to or inserted into a constant region. In one aspect of any of the methods or compositions, the acceptor glutamine (Q) is in a CH2 domain and/or a CH3 domain. In one aspect of any of the methods or compositions, the protein is an antibody and comprises one functionalized acceptor glutamine (Q) in a CH2 domain and another functionalized (Q) in a peptide tag (e.g. TGase recognition tag) fused to the C-terminus of a heavy or light chain. In one aspect of any of the methods or compositions, the acceptor glutamine is at residue 295 (Kabat EU numbering). In one aspect of any of the methods or compositions, the acceptor glutamine is at residue 297 (Kabat EU numbering). In one aspect of any of the methods, the Fc-domain containing protein (e.g. antibody) comprises an acceptor glutamine at each of residues 295 and 297 (Kabat EU numbering). In one aspect of any of the methods or compositions, the antibody is a full-length antibody. In one aspect of any of the methods or compositions, the antibody is an antibody fragment or derivative. In one aspect of any of the methods or compositions, the fragment or derivative comprises a TGase recognition tag comprising an acceptor glutamine residue.

In one embodiment, provided is a composition comprising a plurality of Fc-domain containing proteins of the disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the Fc-domain containing proteins in an antibody sample obtained by the method have the same number of functionalized acceptor glutamine residues (Q) per antibody, optionally wherein the number is 2 or 4.

In one embodiment, provided is a composition comprising a plurality of Fc-domain containing proteins of the disclosure, wherein at least 80%, 90%, 95%, 98% or 99% of the Fc-domain containing proteins in an antibody sample obtained by the method have no more or no less than (m) functionalized acceptor glutamine residues (Q) per Fc-domain containing protein, wherein m is an integer, e.g. m= 2 or 4. In one embodiment, provided is a composition comprising a plurality of Fc-domain containing proteins of the disclosure comprising a functionalized acceptor glutamine residue having a structure of Formula II or IV, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the Fc-domain containing proteins in the composition have the same number of functionalized acceptor glutamine residues (Q) per Fc-domain containing protein and at least 70%, 80%, 80%, 90%, 95%, 98% or 99% of the Fc-domain containing proteins in the composition have the same q, r and z values.

In one embodiment of any aspect herein, a composition of Fc-containing proteins having two functionalized acceptor glutamines per antibody is characterized by a mean Z (or R):antibody ratio (e.g. mean DAR) of between 1 .4 and 2.0, or between 1.7 and 2.0, between 1 .8 and 2.0, or between 1 .9 and 2.0). In one embodiment of any aspect herein, a composition of Fc-containing proteins having four functionalized acceptor glutamines per antibody is characterized by a mean Z (or R):antibody ratio (e.g. mean DAR) of between 3.0 and 4.0, or between 3.5 and 4.0, or between 3.6 and 4.0).

In one aspect of any of the methods or compositions, a linker or linking moiety (e.g. lysine based linker, the L" moiety) for linking a glutamine residue to Z is a linker comprising a NH-C- moiety, where C is a substituted or unsubstituted carbon chain, wherein any carbon of the chain is optionally substituted with a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide. Optionally, the NH-C moiety can be a lysine residue. Optionally, said linker is a linker of formula la or lb. In one embodiment, V is a conditionally-cleavable moiety following intracellular internalization, which can be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process, such as a di-, tri-, tetra-, or oligopeptide.

In one aspect of any of the methods or compositions, the antibodies are tetrameric antibodies. In one aspect of any of the methods, the antibodies are full length antibodies.

In one aspect of any of the methods or compositions, the moiety of interest (or Z) is a cytotoxic agent (e.g. an anti-cancer agent), a pro-inflammatory agent, an anti-inflammatory agent, optionally a steroid or corticosteroid agent, optionally a glucocorticoid agent. Optionally, Z is a cytotoxic agent selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysins, dolastatins and auristatins, enediynes, pyrrolobenzodiazepines, and ethylenimines. Optionally, Z is a hydrophobic compound. Optionally, Z is an organic compound having a molecular weight of at least 400 g/mol. Optionally, Z comprises a cyclic group, optionally a plurality of cyclic groups, optionally a polycyclic group. Optionally, Z is a negatively charged compound. Any of the methods or compositions herein can further be characterized as comprising any step or embodiment described in the application, including notably in the "Detailed Description of the Invention"). Further provided is an Fc-domain containing protein obtainable by any of present methods. Further provided are pharmaceutical or diagnostic formulations of the Fc-domain containing proteins. Further provided are methods of using an Fc-domain containing protein in a method of treatment or diagnosis.

These and additional advantageous aspects and features of the invention may be further described elsewhere herein. Brief Description of the Drawings

Figure 1 shows reaction schemes for thio-maleimide additions, Staudinger ligations, and Diels-Alder cycloadditions, where reactive groups of linking reagents having a single reactive functionality combine with complementary reactive group attached to a therapeutic or diagnostic moiety.

Figure 2 shows reaction schemes for Diels-Alder cycloadditions and click reactions where the reactive groups of linking reagents combine with complementary reactive group attached to an agent including a therapeutic, diagnostic, or other moiety.

Figure 3 shows the preparation of an exemplary linking reagent, and its conjugation with a protein, where: V and Y are absent, R is a thiol (sulfhydryl) reactive group that is ultimately generated from the S-acetyl protected thiol, SC(0)CH_3; r is 0; q is 0; z is 1 ; L is the two carbon comprising framework C(0)CH₂; X is NH; (C)_n is (CH₂)₅; and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein.

Figure 4 illustrates the preparation of various exemplary linking reagents, with a single S-acetyl protected thiol reactive group that can be prepared from an N-succinimidyl-S- acetylthioester reagent.

Figure 5 illustrates the preparation of an exemplary linking reagent, and its conjugation with a protein, where: V and Y are absent, R is an azide reactive group_; r is 0; q is 0; z is 1 ; L is the two carbon comprising framework C(0)CH₂; X is NH; (C)_n is (CH₂)5; and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein.

Figure 6 illustrates the preparation of various exemplary linking reagents, with a single azide reactive group that can be prepared from an N-succinimidyl-azide reagent.

Figure 7 depicts the preparation of an exemplary linking reagent, and its conjugation with a protein, where: V and Y are absent, R is an alkyne reactive group_; r is 0; q is 0; z is 1 ; L is a one carbon comprising framework CH₂; X is NH; (C)_n is (CH₂)₄CH(C0₂H); and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein.

Figure 8 shows the preparation of an exemplary linking reagent, and its conjugation with a protein, where: R is a norbornene reactive group_; r is 0; q is 0; z is 1 ; L is the one carbon comprising framework C(O); X is NH; (C)_n is(CH₂)4CH(C0₂H); and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein.

Figure 9 shows various examples of linking reagents.

Figures 10A and 10B show a general scheme for preparing conjugated antibodies. Figure 1 1 shows a scheme for preparing an antibody conjugate from a S-acetyl- cadaverine linker of Figure 3, where "R" in the figure is a moiety-of-interest Z.

Figure 12 shows a scheme for preparing an antibody conjugate from an azide- cadaverine linker of Figure 5, where "R" in the figure is a moiety-of-interest Z.

Figure 13 shows a scheme for preparing an antibody conjugate from a norbornyl- cadaverine linker of Figure 8, where "R" in the figure is a moiety-of-interest Z.

Figure 14 shows a scheme for preparing an antibody conjugate from a glycan-lysine derivative linker of Figure 7, where "R" in the figure is a moiety-of-interest Z.

Figures 15, 16, 17 and 18, respectively show that BTG was able to couple the acceptor glutamine at residue 295 substantially completely, obtaining a drug:antibody ratio of 2.0 for each of antibodies humADC2-1 , humADC2-2, humADC-3 and humADC2-4. LC/MS analysis of the glycosylated starting antibody humADC2 (top panel), the deglycosylated antibody humADC2 (middle panel), and the antibody humADC2 coupled to NH₂-PEG-N3 (one NH₂-PEG-N3 on each acceptor glutamine per heavy chain). DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

Where "comprising" is used, this can optionally be replaced by "consisting essentially of", or optionally by "consisting of".

The term "transglutaminase", used interchangeably with "TGase" or "TG", refers to an enzyme capable of cross-linking proteins through an acyl-transfer reaction between the v- carboxamide group of peptide-bound glutamine and the ε-amino group of a lysine or a structurally related primary amine such as amino pentyl group, e.g. a peptide-bound lysine, resulting in a e-(v-glutamyl)lysine isopeptide bond. TGases include, inter alia, bacterial transglutaminase (BTG) such as the enzyme having EC reference EC 2.3.2.13 (protein- glutamine-v-glutamyltransferase).

The term "acceptor glutamine", when referring to an amino acid residue of an antibody, means a glutamine residue that, under suitable conditions, is recognized by a TGase and can be cross-linked by a TGase through a reaction between the glutamine and a lysine or a structurally related primary amine such as amino pentyl group. Optionally, the acceptor glutamine is a surface-exposed glutamine.

The term "TGase recognition tag", refers to a sequence of amino acids that when incorporated into (e.g. appended to) a polypeptide sequence, under suitable conditions, is recognized by a TGase and leads to cross-linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reaction partner. The TGase recognition tag is a sequence that is not naturally present in the polypeptide comprising the TGase recognition tag. Cross-linking by the TGase may be through a reaction between a glutamine residue (an acceptor glutamine) within the TGase recognition tag and a lysine or a structurally related primary amine such as amino pentyl group.

The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

The term "antibody derivative", as used herein, comprises a full-length antibody or a fragment of an antibody, preferably comprising at least antigen-binding or variable regions thereof, wherein one or more of the amino acids are chemically modified, e.g., by alkylation, PEGylation, acylation, ester formation or amide formation or the like. This includes, but is not limited to, PEGylated antibodies, cysteine-PEGylated antibodies, and variants thereof.

By "constant region" of an antibody as defined herein is meant the region of the antibody that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of Ckappa, or Clambda, wherein numbering is according to the EU index (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 1 18-447, wherein numbering is according to the EU index.

By "Fab" or "Fab region" as used herein is meant the polypeptide that comprises the

VH, CH1 , VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein, or any other antibody embodiments as outlined herein.

By "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.

By "Fc" or "Fc region", as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cv2 and Cv3 and the hinge between Cy1 and Cv2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides include antibodies, Fc fusions, isolated Fes, and Fc fragments.

By "full length antibody" as used herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full length antibody of the IgG isotype is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, Cy1 , Cv2, and Cv3. In some mammals, for example in camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region.

By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including Vkappa and Vlambda) and/or VH genes that make up the light chain (including kappa and lambda) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL and VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia. The framework regions of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.

By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. The preferred amino acid modification herein is a substitution. By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.

"Conservative" amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

An "isolated" molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of an antibody molecule will exhibit 98%, 98%, or 99% homogeneity for antibody molecules in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use. The term "reactive moiety" herein refers to a moiety that can be coupled with another moiety without prior activation or transformation.

The term "protecting group" refers to a group that temporarily protects or blocks, i e., intended to prevent from reacting, a functional group, e.g. , an amino group, a hydroxyl group, or a carboxyl group, during the transformation of a first molecule to a second molecule.

The phrase "moiety that improves the pharmacokinetic properties", when referring to a compound (e.g. an antibody) refers to a moiety that changes the pharmacokinetic properties of the one or more moieties Z in such a way that a better therapeutic or diagnostic effect can be obtained. The moiety can for example increase the water solubility, increase the circulation time, or reduce immunogenicity.

The phrase "linking group" refers to a structural element of a compound that links one structural element of said compound to one or more other structural elements of said same compound.

The phrase "a number representing degree of branching" is used to denote that the subscript number next to a closing bracket represents how many units of the moiety within the brackets are attached to the moiety directly to the left of the corresponding opening bracket For example, A-(B)_b with b being a number representing a degree of branching means that b units B are all directly attached to A This means that when b is 2, the formula reduces to B-A-B.

The phrase "a number representing degree of polymerization" is used to denote that the subscript number next to a closing bracket represents how many units of the moiety within the brackets are connected to each other. For example, Α-(Β)·ι, with b being a number representing a degree of polymerization means that when b is 2, the formula reduces to A-B- B.

As used herein, "alkyi" refers to a straight or branched hydrocarbon chain that comprises a fully saturated (no double or triple bonds) hydrocarbon group. The alkyi group may have, for example, 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer in the given range; e.g., "1 to 20 carbon atoms" means that the alkyi group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, eic, up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term "alkyi" where no numerical range is designated). The alkyi group of the compounds may be designated as "Ci-C₄ alkyi" or similar designations. By way of example only, "Ci-C₄ alkyi" indicates that there are one to four carbon atoms in the alkyi chain, i.e., the alkyi chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyi groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl. The alkyl group may be substituted or unsubstituted.

As used herein, the term "heteroalkyl" refers to a straight or branched alkyl group that contains one or more heteroatoms, that is, an element other than carbon (including but not limited to oxygen, sulfur, nitrogen, phosphorus) in place of one or more carbon atoms.

Whenever a group is described as being "substituted" that group substituted with one or more of the indicated substituents. If no substituents are indicated, it is meant that the indicated "substituted" group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamyl, thiocarbamyl, amido, sulfonamido, sulfonamido, carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, a mono-substituted amino group and a di-substituted amino group, and protected derivatives thereof.

Where the number of substituents is not specified (e.g. haloalkyl), there may be one or more substituents present. For example "haloalkyl" may include one or more of the same or different halogens. As another example, "Ci-C₃ alkoxyphenyl" may include one or more of the same or different alkoxy groups containing one, two or three atoms.

Producing antibodies

Antibodies may be produced by a variety of techniques known in the art. Typically, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide). The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). Other protocols may also be used as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization. Lymphocytes from a non-immunized non-human mammal may also be isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out. For preferred monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non- human mammal and the subsequent fusion of those splenocytes with an immortalized cell in order to form an antibody-producing hybridoma. The hybridoma colonies are then assayed for the production of antibodies that specifically bind to the polypeptide against which antibodies are desired. The assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include radioimmunoassays or fluorescence activated cell sorting. The wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be re-cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. After sufficient growth to produce the desired monoclonal antibody, the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G-Sepharose, or an anti-mouse Ig linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Biosciences, publication No. 18-1037- 46, Edition AC, the disclosure of which is hereby incorporated by reference).

Additionally, a wide range of antibodies are available in the scientific and patent literature, including DNA and/or amino acid sequences, or from commercial suppliers.

Antibodies will typically be directed to a pre-determined antigen. Examples of antibodies include antibodies that recognize an antigen expressed by a target cell that is to be eliminated, for example a proliferating cell or a cell contributing to a pathology. Examples include antibodies that recognize tumor antigens, microbial (e.g. bacterial) antigens or viral antigens. Other examples include antigens present on immune cells or non-immune cells that are contributing to inflammatory or autoimmune disease, including rejection of transplanted tissue (e.g. antigens present on T cells, e.g. Treg cells, CD4 or CD8 T cells).

As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. As used herein, the term "viral antigen" includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic.

As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens that are differentially expressed by cancer cells or are expressed by non-tumoral cells (e.g. immune cells) in tumor or tumor-adjacent tissues that have a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Still other cancer antigens can be expressed on immune cells capable of contributing to or mediating a pro-tumoral effect, e.g. cell that contributes to immune evasion, a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.

The cancer antigens are usually normal cell surface antigens which are either over- expressed or expressed at abnormal times, or are expressed by a targeted population of cells. Ideally the target antigen is expressed only on proliferative cells (e.g., tumor cells) or pro-tumoral cells present in tumor or tumor-adjacent tissues (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice. As a result, target antigens are in many cases selected on the basis of differential expression between proliferative/disease tissue and healthy tissue. Example of cancer antigens include: Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1 ), Crypto, CD4, CD20, CD30, CD19, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22 (Siglec2) or CD33 (Siglec3), CD79, CD138, CD171 , PSCA, L1 -CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferrin, Mud 6 and TMEFF2. Examples of cancer antigens also include Immunoglobulin superfamily (IgSF) such as cytokine receptors, Killer-lg Like Receptor, CD28 family proteins, for example, Killer-lg Like Receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1 , IL-6 receptor. Examples also include MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), or optionally an antigen other than MICA and/or MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g. nectin- 4), proteins of the UL16-binding protein (ULBP) family, proteins of the retinoic acid early transcript-1 (RAET1 ) family, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1 , prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mijllerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as Receptor Tyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1 ), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, members of the human EGF-like receptor family, e.g., HER- 2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1 , CA125, integrin receptors, ανβ3 integrins, α5β1 integrins, al^3-integrins, PDGF beta receptor, SVE-cadherin, IL-8 receptor, hCG, IL- 6 receptor, CSF1 R (tumor-associated monocytes and macrophages), a-fetoprotein, E- cadherin, ocatenin, β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1 , P1A, EBV-encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-1 , SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is not intended to be exhaustive. In one aspect, the antigen of interest is an antigen (e.g. any one of the antigens listed above) capable of undergoing intracellular internalization, for example when bound by a conventional human lgG1 antibody, either in the presence of absence of Fey receptor cells.

In one embodiment, the antigen of interest is a cancer antigen, e.g. one of the cancer antigens listed above known to internalize (e.g. Immunoglobulin superfamily (IgSF) members, for example cytokine receptor a or β chains, Killer-lg Like Receptors, CD28 family proteins, B7-H3, B7-H4, B7-H6, KIR3DL2, PTK7, ROR1 , L1 -CAM, Siglec family members, EGF receptor and EGF-like receptor family members, EGFR, HER-2, integrins, anti- Mullerian hormone Type II receptor, CSF-1 R, NOTCH3, PTK7, EFNA4 (ephrin-A4, TROP-2 (TACSTD2) and others). In one embodiment, the antigen of interest is a polypeptide present on an immune cell capable of mediating an immunosuppressive and/or pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.

In one embodiment, the antibody binds to an antigen expressed by a proinflammatory cell, e.g. a cell contributing to an inflammatory or autoimmune disorder. An example of an antigen is PRLR (prolactin receptor).

Wild-type full-length IgG antibodies of human isotype will possess a conserved acceptor glutamine at residue 295 (Kabat) of the heavy chain which will be accessible to a TGase and therefore reactive with a compound of Formula I in the presence of a TGase, under suitable conditions, to form a conjugate from the antibody and the compound of Formula II or IV. Such an antibody will bear natural glycosylation at the asparagine at residue 297 (Kabat) of the heavy chain. Wild-type full-length IgG antibodies of human isotype can also be modified so as to eliminate or truncate the natural glycosylation (e.g., lacking glycosylation) at the asparagine at residue 297, for example by enzymatic deglycosylation or through the substitution of the asparagine at residue 297 by another residue.

In one embodiment, the constant regions and/or Fc regions of the proteins of the disclosure are of human origin, optionally comprising amino acid sequences partly or fully derived from a human lgG1 isotype, optionally constant regions and/or Fc regions. In one embodiment, a heavy chain is a chimeric heavy chain comprising amino acid sequences derived from two or more human isotypes (e.g. a heavy chain of lgG1 isotype comprising amino acid sequences derived from a human lgG2, lgG3, or lgG4 isotype). In one embodiment, a heavy and/or light chain of an Fc domain containing protein (e.g. antibody) comprises a TGase recognition tag, i.e. a sequence of 2, 3, 4, 5 or more residues specifically recognized by an enzyme (an enzyme that conjugates a moiety of interest to an antibody), e.g. a TGase, etc. In another embodiment, a heavy and/or light chain is free of a Tgase recognition tag. In one embodiment, a constant domain or Fc region comprises an acceptor amino acid residue naturally present in a wild-type human constant region. In one embodiment, an Fc domain comprises a TGase recognition tag fused to the C-terminus of the (e.g., each CH3 domain. In one embodiment, an antibody comprises a TGase recognition tag fused to the C-terminus of the (e.g. each) antibody light chain. In one embodiment, an antibody comprises a TGase recognition tag fused to the C-terminus of the (e.g. each) antibody heavy chain.

It should be noted that a single site mutation that provides a glutamine that is accessible to a TGase will yield more than one engineered glutamine residue that can be conjugated when the antibody comprises more than one engineered chain. For example, a single site mutation will yield two engineered glutamine residues in a tetrameric IgG due to the dimeric nature of the IgG antibody. The engineered glutamine residues will be in addition to any acceptor glutamine already present in an antibody, if any. In one embodiment, the Fc domain lacks N-linked glycosylation at Kabat residue 297. In one embodiment, an asparagine at amino acid position 297 (EU Index of Kabat (1991 )) is substituted, optionally with a glutamine residue, optionally with a non-aspartic acid residue. Such an antibody will have a constant region with a N297Q substitution (a N297Q variant antibody). An antibody having a N297Q substitution and the native glutamine present at residue 295 (EU Index of Kabat (1991 ) will therefore have two acceptor glutamines and thus two conjugation sites per heavy chain. In tetravalent form, the antibody will therefore have four conjugates per antibody. Such antibody will lack N297-linked glycosylation. In one embodiment, the glutamine naturally present in a human Fc domain at amino acid position 295 is substituted with a non-glutamine residue. An antibody having a Q295X substitution will be understood to have an introduced glutamine at a different position, e.g., the antibody may have a N297Q substitution, or comprise a TGase recognition tag within the CH2 domain, CH3 domain or fused to the C-terminus terminus of a heavy and/or light chain.

In one embodiment, the asparagine naturally present at amino acid position 297 is substituted with a non-asparagine, non-glutamine residue. The antibody can have a constant region with a N297X substitution (e.g., Q295X/N297X), or a S298X and/or T299X substitution (a N297X, S298X and/or T299X variant antibody), wherein X is any amino acid (other than a glutamine or the residue Q, N, S or T naturally present at the respective 295, 297, 298 or 299 residue), optionally wherein the substitution is a conservative substitution.

In one embodiment, an antibody comprises a TGase recognition tag, e.g. inserted into a heavy chain constant region (e.g. inserted into a CH2 domain), and/or optionally a TGase recognition tag fused to the C-terminus of a heavy chain CH3 domain or to the C- terminus of a light chain CK or CA domain. In one embodiment, the TGase recognition tag comprises an amino acid sequence -J1 -Q-X1-X2-, wherein Q is an acceptor glutamine, J1 is an amino acid residue having a negative electrical charge, optionally a glutamic acid (E) or an aspartic acid residue (D), X1 is any amino acid (e.g. Y (tyrosine) or F (phenylalanine), and X2 is an amino acid other than D (aspartic acid). Optionally, X2 is a non-negatively charged amino acid, any conservative substitution, an amino acid with a positively charged side chain (e.g. an arginine), an amino acid with a polar uncharged side chain, an amino acid with a hydrophobic side chain, e.g. a serine. In another embodiment, the TGase recognition tag comprises an amino acid sequence LOG, LLQ, LLQG, for example GGLLQGPP.

An antibody or Fc domain that does not have substantial specific binding to human

FcyRI (CD64), and optionally further one or more other human Fey receptors, e.g., any one or more of CD16A, CD16B, CD32A and CD32B can be prepared by modification of a human antibody IgG isotype by introducing 1 , 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to said Fey receptors. Assays to assess Fc receptor binding are described herein.

In one embodiment, an Fc domain is of human origin (e.g. of lgG1 or lgG2 isotype) and comprises one, two or three substitutions at residues 233-236, optionally 233-238 (Kabat EU numbering), and further one or two substitutions at residues 330 and/or 331 . Examples of mutations at these residues Fc lgG1 antibodies are the LALA mutant comprising L234A and L235A mutation in the lgG1 Fc amino acid sequence. In one embodiment, an Fc domain can comprise an amino acid modification (e.g. substitution) at one or more of Kabat residue(s) 233-236, optionally one or more of residues 233-237, or at one, two or three of residues 234, 235 and/or 237, and an amino acid modification (e.g. substitution) at Kabat residue(s) 330 and/or 331. One example of such an Fc domain comprises substitutions at Kabat residues L234, L235 and P331 (e.g., L234A/L235E/P331 S or (L234F/L235E/P331 S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331 S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g., L234A/L235E/G237A/A330S/P331 S). In one embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X₂ substitution, and a P331X₃ substitution, wherein X-i is any amino acid residue other than leucine, X₂ is any amino acid residue other than leucine, and X₃ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X₂ is glutamic acid or a conservative substitution thereof; optionally wherein X₃ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X₂ substitution, a G237X₄ substitution and a P331X₄ substitution, wherein X-i is any amino acid residue other than leucine, X₂ is any amino acid residue other than leucine, X₃ is any amino acid residue other than glycine, and X₄ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X₂ is glutamic acid or a conservative substitution thereof; optionally, X₃ is alanine or a conservative substitution thereof; optionally X₄ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X₂ substitution, a G237X₄ substitution, G330X₄ substitution, and a P331X₅ substitution, wherein X-i is any amino acid residue other than leucine, X₂ is any amino acid residue other than leucine, X₃ is any amino acid residue other than glycine, X₄ is any amino acid residue other than alanine, and X₅ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X₂ is glutamic acid or a conservative substitution thereof; optionally, X₃ is alanine or a conservative substitution thereof; optionally, X₄ is serine or a conservative substitution thereof; optionally X₅ is a serine or a conservative substitution thereof. In the shorthand notation used here, the format is: Wild type residue: Position in polypeptide: Mutant residue, wherein residue positions are indicated according to EU numbering according to Kabat. In one embodiment, an antibody comprises an human lgG1 Fc domain comprising a L234A/L235E/N297X/P331 S substitutions, L234F/L235E/N297X/P331 S substitutions, L234A/L235E/G237A/N297X/P331 S substitutions, or L234A/L235E/G237A/ N297X/A330S/P331 S substitutions, wherein X can be any amino acid other than an asparagine. In one embodiment, X is a glutamine; in another embodiment, X is a residue other than a glutamine (e.g. a serine).

In one embodiment, an antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E A E G G P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 1 )

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E F E G G P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 2)

In one embodiment, an antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331 (underlined): A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E A E G A P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P S S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 3)

In one embodiment, an antibody comprises a heavy chain constant region comprising the amino acid sequence below, or a sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237 and 331 (underlined):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E A E G A P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 4)

Optionally, in any of the above heavy chain constant region sequences, the N at Kabat 297 can be substituted by a residue other than an asparagine (e.g. a glutamine, a residue other than glutamine, for example a serine).

In one embodiment, an Fc domain containing protein (e.g. antibody) comprises a substitution in the Fc domain at Kabat residues 234, 235 and 322. In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235 and 331 . In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237 and 331. In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237, 330 and 331 . In one embodiment, the Fc domain is of human lgG1 subtype. Amino acid residues are indicated according to EU numbering according to Kabat.

Amino acid modifications for Fc-engineered and glutamine engineered antibodies can be prepared by a variety of methods which include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), preparation by site-directed (or oligonucleotide-mediated) mutagenesis (Carter (1985) et al Nucleic Acids Res. 13:4431 -4443; Kunkel et al (1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al (1998) J. Biol. Chem. 273:20252-20260), PCR mutagenesis (Higuchi, ( 1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991 ) Gene 102:67-70; Bernhard et al (1994) Bioconjugate Chem. 5: 126- 132; and Vallette et al ( 1989) Nuc. Acids Res. 17:723- 733) and cassette mutagenesis (Wells et al (1985) Gene 34:315-323) of an earlier prepared DNA encoding the polypeptide. Mutagenesis protocols, kits, and reagents are commercially available, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations are also generated by oligonucleotide directed mutagenesis using double stranded plasmid DNA as template by PCR based mutagenesis (Sambrook and Russel, (2001 ) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500; ZoDer, MJ. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500). Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacement(s). Standard mutagenesis techniques can be employed to generate DNA encoding such mutant cysteine engineered antibodies (Sambrook et al Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York. N.Y., 1993).

Once a desired Fc domain-containing protein (e.g. antibody) is obtained, it can first be reacted, in the presence of TGase and a linker comprising a moiety of interest, e.g. a reactive moiety (R) or a moiety Z such as a drug, under conditions suitable for the TGase to catalyze a covalent bond between the lysine-based linker and an acceptor glutamine in the primary sequence of the Fc domain-containing protein.

Alternatively, once a desired Fc domain-containing protein (e.g. antibody) is obtained, it can optionally be applied to a solid support such that the antibody is immobilized, for subsequent reaction of the immobilized antibody with TGase and a linker comprising a moiety of interest, e.g. a reactive moiety (R) or a moiety Z such as a drug. In order to generate antibodies bound to a moiety of interest Z via a linking reagent by this process, antibody bound to a solid support can be brought into contact with linking reagents (e.g. lysine based linkers of formulae la or lb) and TGase enzyme under conditions suitable for the TGase to catalyze a covalent bond between the lysine-based linker and an acceptor glutamine in the primary sequence of the antibody or antibody fragment. Lysine based linker and TGase enzyme can thus be applied (introduced) to the solid support under suitable conditions. In another embodiment, the Fc domain containing protein can first be reacted in liquid phase (solution), in the presence of TGase and a linker comprising a reactive moiety (R) to generate an Fc domain containing protein (e.g. of Formula II) having a reactive linker conjugated thereto. The resulting Fc domain containing protein (e.g. of Formula II) can then be introduced to a solid phase so as to immobilize the Fc domain containing protein having a reactive linker conjugated thereto. The immobilized Fc domain containing protein can then subsequently be reacted with a compound having a complementary reactive group (R') and a moiety of interest Z.

Generally, the solid support may be any suitable insoluble, functionalized material to which the Fc domain containing protein can be reversibly attached, either directly or indirectly, allowing them to be separated from unwanted materials, for example, excess reagents, contaminants, and solvents. Examples of solid supports include, for example, functionalized polymeric materials, e.g., agarose, or its bead form Sepharose®, dextran, polystyrene and polypropylene, or mixtures thereof; compact discs comprising microfluidic channel structures; protein array chips; pipet tips; membranes, e.g., nitrocellulose or PVDF membranes; and microparticles, e.g., paramagnetic or non-paramagnetic beads. In some embodiments, an affinity medium will be bound to the solid support and the Fc domain containing protein will be indirectly attached to solid support via the affinity medium. In one aspect, the solid support comprises a protein A affinity medium or protein G affinity medium. A "protein A affinity medium" and a "protein G affinity medium" each refer to a solid phase onto which is bound a natural or synthetic protein comprising an Fc-binding domain of protein A or protein G, respectively, or a mutated variant or fragment of an Fc-binding domain of protein A or protein G, respectively, which variant or fragment retains the affinity for an Fc-portion of an antibody. Protein A and Protein G are bacterial cell wall proteins that have binding sites for the Fc portion of mammalian IgG. The capacity of these proteins for IgG varies with the species. In general, IgGs have a higher affinity for Protein G than for Protein A, and Protein G can bind IgG from a wider variety of species. The affinity of various IgG subclasses, especially from mouse and human, for Protein A varies more than for Protein G. Protein A can, therefore, be used to prepare isotypically pure IgG from some species. Protein L has an affinity for kappa light chains from various species. It can be used to purify monoclonal or polyclonal IgG, IgA, and IgM as well as Fab, F(ab')₂, and recombinant scFv fragments that contain kappa light chains When covalently attached to a solid matrix, such as cross-linked agarose, these proteins can be used to capture and purify antigen-antibody complexes from biochemical solutions. Commercially available products include, e.g., Protein G, A or L bonded to agarose or sepharose beads, for example EZview™ Red Protein G Affinity Gel is Protein G covalently bonded to 4% Agarose beads (Sigma Aldrich Co); or POROS® A, G, and CaptureSelect® HPLC columns (Invitrogen lnc.).Affinity capture reagents are also described, for example, in the Antibody Purification Handbook, Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is hereby incorporated by reference).

After any reaction step carried out while Fc domain containing protein is immobilized on the solid support (TGase conjugation and/or reaction with complementary reactive groups R and R'), a washing step can be performed to remove any unreacted materials. Optionally, unreacted compounds are recovered; optionally, unreacted linking reagent and/or TGase are re-applied to the solid support to provide for higher completion of the reaction between antibody and substrate (linking reagent).

In order to recover Fc domain containing protein bound to a moiety of interest via a linking reagent, immobilized Fc domain containing protein conjugates can subsequently be eluted from the solid support to provide Fc domain containing protein conjugate compositions. Methods of eluting proteins from solid supports are known in the art and the skilled practitioner will be able to select an appropriate buffer for elution. For example, in embodiments, where the solid support comprises protein A or protein G resin, the Fc domain containing protein conjugates can be eluted with standard low pH buffers for elution from protein A or protein G columns.

In order to generate antibodies covalently bound to a solid support, Fc domain containing protein not covalently bound to the solid support can be eluted, leaving a solid support functionalized with Fc domain containing protein.

Lysine-based linkers

The Fc domain containing proteins can be conjugated to a moiety-of-interest via a linking reagent that can be attached, by the action of a TGase, at a glutamine residue (Q) within the sequence of the antibody. The linking reagent comprises a lysine derivative, lysine, or a functional equivalent thereof, that is connected to at least one moiety of interest (Z), optionally a reactive group (R).

Examples of lysine based linkers and their synthesis is provided in Figures 1 -15 and PCT patent application number PCT/EP2012/076606 (WO2013/092983) filed 21 December 2012, the disclosure of which is incorporated herein by reference.

The lysine derivative can be a 2 to 20 alkyl or heteroalkyl chain, or a functional equivalent thereof, with an H₂N, H₂NOCH₂, H₂NCH₂ (aminomethylene) group or a protected H₂N, H₂NOCH₂, H₂NCH₂ group positioned at one or more ends of the alkyl or heteroalkyl chain. The heteroalkyl chain can be a chain of 3 to 20 atoms where one or more nonterminal atoms can be other than carbon, for example oxygen, sulfur, nitrogen, or other atoms. The oxygen, sulfur, or nitrogen atom can be of an ether, ester, thioether, thioester, amino, alkylamino, amido or alkylamido functionality within the carbon chain.

The heteroalkyl chain can be an oligo (ethylene oxide) chain. The functionality within the alkyl or heteroalkyl chain can be included to couple the reactive group to the H₂N, H₂NOCH₂, H₂NCH₂ group or protected H₂N, H₂NOCH₂, H₂NCH₂ group. The alkyl or heteroalkyl chain can be substituted or unsubstituted. The substituents can be alkyl groups, aryl groups, alkyl aryl groups, carboxylic acid groups, amide groups, hydroxy groups, or any other groups that do not compete with the amino group for, or inhibit, conjugation with a glutamine residue of the protein. Typically, when a substituent is present, its presence is in a convenient starting material, such as the carboxylic acid group of lysine, from which the lysine derivative results. The H₂N, H₂NOCH₂, H₂NCH₂ end of an alkyl or heteroalkyl chain is necessarily included in the linking reagent.

Exemplary starting materials for the functional equivalent of lysine can be an α,ω- diaminoalkane, for example, 1 ,2-diaminoethane, 1 ,3-diaminopropane, 1 ,4-diaminobutane, 1 ,5-diaminopentane, 1 ,6-diaminohexane, 1 ,7-diaminoheptane, 1 ,8-diaminooctane, 1 ,9- diaminononane, 1 ,10-diaminodecane, 1 ,1 1 -diaminoundecane, or 1 ,12-diaminododecane. Other starting materials for the functional equivalent of a lysine derivative can be α,ω- diamino oligo (ethylene oxide), for example, H₂N(CH₂CH₂0)_xCH₂CH₂NH₂ where x is 1 to about 6. The α,ω-diamino oligo (ethylene oxide) can be a single oligomer or it can be a mixture of oligomers where x defines an average size. An exemplary protected H₂NCH₂ is the fe/t-butylcarbamate protected amine of fe/t-butyl N-(5-aminopentyl)carbamate (N-Boc- cadaverin).

The linking reagent, a pharmaceutically acceptable salt or solvate thereof, or an antibody-conjugated linking reagent may comprise the general Formula la or lb. Formulae la (having an Z group) and lb (having a R group) are shown as follows:

G-NH-C-X-L-(V-(Y-(Z)_z)_q)_r Formula la;

G-NH-C-X-L-(V-(Y-(R)_z)_q)_r Formula lb or a pharmaceutically acceptable salt or solvate thereof

wherein:

G is an H, amine protecting group, or an antibody attached via an amide bond;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally where the carbon adjacent to the nitrogen is unsubstituted, optionally wherein any carbon of the chain is substituted alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide (e.g. with a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide), optionally wherein C has a chain length of 2 to 20 atoms, preferably 3 to 6 atoms; X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), an amino acid, a di-, tri-, tetra-, or oligopeptide, other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1 , 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4; and

z is an integer selected from among 1 , 2, 3 or 4;

V is independently absent, a bond or a continuation of a bond if L is a bond, a non- cleavable moiety or a conditionally-cleavable moiety, optionally following prior conditional transformation, which can be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process (e.g. cleavage of V ultimately leading to release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety";

Y is independently absent, a bond or a continuation of a bond if V is a bond or continuation of a bond, or a spacer system (e.g., a self-eliminating spacer system or a non- self-elimination spacer system) which is comprised of 1 or more spacers;

Z is a moiety that improves the pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety; and

R is a reactive moiety, preferably a moiety comprising an unprotected or protected thiol, maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene, 1 ,2,4,5-tetrazine, norbornene, other stained or otherwise electronically activated alkene or, optionally, a protected or unprotected amine when X is absent and L, V, or Y is other than a bond or a continuation of a bond. In an alternative embodiment R is a reactive moiety, preferably a moiety comprising an unprotected or protected thiol, an unprotected or protected amine, maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene, 1 ,2,4,5-tetrazine, norbornene, other stained or otherwise electronically activated alkene, provided that R is not an amine when n= 5 and X, L, V and Y are absent. Optionally, R is not an amine when n= 4 and X, L, V and Y are absent. When more than one R group is present in a compound of the formula lb, the R groups will preferably be compatible such that no R group is a complementary reagent to any other R group.

The C group may for example be a straight, branched and/or cyclic C_2-3o alkyl, C_2-3o alkenyl, C_2-3o alkynyl, C_2-3o heteroalkyl, C_2-30 heteroalkenyl, C_2-30 heteroalkynyl, optionally wherein one or more homocyclic aromatic compound radical or heterocyclic compound radical may be inserted; notably, any straight or branched C_2-5 alkyl, C_5-10 alkyl, C_11-20 alkyl, - O- C_1-5 alkyl, -O- C_5-i₀ alkyl, -O- C_11-20 alkyl, CH₂-(CH₂-0-CH₂)_1-12-CH₂ or (CH₂- CH₂-0-)_1-12, an amino acid, an oligopeptide, glycan, sulfate, phosphate or carboxylate.

In one example the C group is a carbon comprising framework substituted with one or more O atoms. In one embodiment, the carbon adjacent to the nitrogen is substituted with an O atom. In one embodiment, the carbon adjacent to the nitrogen is unsubstituted. In one embodiment, the C group is or comprises an ethylene oxide group, e.g. a CH₂-(CH₂-0- CH₂)_n-CH₂ group or an (CH₂- CH₂-0-)_n, where n is an integer from 1 to 10.

The L group can be a carbon comprising framework, where L is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), an amino acid, a di-, tri-, tetra-, or oligopeptide, other natural oligomer, dimer, trimer, or higher oligomer (linear asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process. For example, L may comprise or be a straight, branched and/or cyclic C_2-30 alkyl, C_2-30 alkenyl, C_2-30 alkynyl, C_2-30 heteroalkyl, C_2-30 heteroalkenyl, C_2-30 heteroalkynyl, optionally wherein one or more homocyclic aromatic compound radical or heterocyclic compound radical may be inserted; notably, any straight or branched C_2-5 alkyl, C_5-10 alkyl, C_11-20 alkyl, -O- C_1-5 alkyl, -O- C_{5- 0} alkyl, -O- C_11-20 alkyl, CH₂- (CH₂-O-CH₂)_1-30-CH₂ or (CH₂- CH₂-O-)_1-30, e.g., (CH₂- CH₂-0-)₁₂, (CH₂- CH₂-0-)_1-24,an amino acid, an oligopeptide, glycan, sulfate, phosphate, carboxylate. Optionally, L is absent.

L, V and/or Y have r, q, and/or z sites of attachment for the respective V, Y, and Z or R groups, where r and q represent the degree of branching or polymerization. The sites of attachment can comprise a bond or comprise a functional group selected from an alkene, alkyne, ether, thioether, ester, thioester, amine, amide, alkylamide, or other functional group readily generated by a condensation or addition reaction.

In one example the carbon comprising framework of the L group is optionally substituted with one or more O atoms. In one embodiment, the L group comprises one or more ethylene oxide groups (CH₂-0-CH₂). Optionally, the L group comprises a carbon framework comprising a (CH₂- CH₂-0-)_n group, wherein n is an integer selected among the range of 1 to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). In Formulae la, lb, II, IVa and IVb, the linking group L links the aminopeptidyl moiety - NH-C-X to the reactive group R or Z, optionally through one or more V and/or Y moieties where present. L may be a bond connecting V, Y, R or Z directly to the aminopeptidyl moiety. In another aspect, however, L is a linking group that functionally links or spaces the one or more moieties V and/or Y reactive moiety R or moiety of interest (Z). In Formulae lb, II and IVb, spacing improves efficiency and completion of BTGase coupling, make additionally the reactive moiety R more accessible to the reaction partner, for example when the reactive moiety is present on a lysine-based linker and coupled to the antibody and then brought into contact with a reaction partner. In Formulae la and IVa, the linking group L links the aminopeptidyl moiety -NH-C-X to the moiety-of-interest (Z), optionally through one or more V and/or Y moieties where present. L may be a bond connecting V, Y or Z directly to the aminopeptidyl moiety. In another aspect, however, L is a linking group that functionally links or spaces the one or more moieties V and/or Y reactive moiety Z. In Formulae la and IVa, spacing improves efficiency and completion of BTGase coupling, providing for highly homogenous compounds. In an antibodies comprising a functionalized acceptor glutamine of Formula IVa or IVb spacing may also provide for a better accessibility of V, which in the case of enzymatic cleavage or transformation of V, may improve the rate at which V is transformed and/or cleaved.

L and C groups can be configured based on the overall structure of the linker that is to be used. Particularly when a multi-step method is used and the linker (e.g. the linker of Formula la or lb is free of or does not comprise a large, charged or hydrophobic moiety (e.g. a cyclic, polycyclic or macrocyclic moiety), the L group may be a bond or a shorter carbon framework. For example, L may represent or comprise a carbon framework of 1 , 2, 3, 4, 5, or 6 linear carbon atoms, unsubstituted or optionally substituted at one or more atoms. Preferably, where L additionally comprises other groups, the 5-20 linear carbon atoms will be adjacent to the C group, or where present, the X group.

When a linker (e.g. the linker of Formula la or lb or an antibody of Formula II, IVa or IVb) comprises a large, charged or hydrophobic moiety (e.g. a cyclic, polycyclic or macrocyclic moiety), for example, wherein V, Y and/or Z comprises a large, charged or hydrophobic moiety (e.g. a cyclic, polycyclic or macrocyclic moiety), the L group may be longer carbon framework. For example, L may represent or comprise a carbon framework of: a) 2-30 linear carbon atoms optionally substituted at one or more atoms;

b) 2-15 linear carbon atoms optionally substituted at one or more atoms;

c) 5-20 linear carbon atoms optionally substituted at one or more atoms;

d) 5-30 linear carbon atoms optionally substituted at one or more atoms;

e) 5-15 linear carbon atoms optionally substituted at one or more atoms; or f) 4, 5 or 6 linear carbon atoms optionally substituted at one or more atoms.

Preferably, the 5-20 linear carbon atoms will be adjacent to (the continuation of) the C group, or where present, the X group.

In some embodiments, L is a -(C=0)-C_1-6 alkyl group. In some embodiments, L is a Ci_-6 alkoxy-C_1-6 alkyl group. In some embodiments, L is a -(C=0)-C_1-6 alkoxy-C_1-6 alkyl group. In some embodiments, L is a

alkyl group. In some embodiments, L is a Ci_-6 alkyl group. In some embodiments, L is a C_10-2o alkyl group. In some embodiments, L is a -(C=0)-0-C_1-6 alkyl group. In some embodiments, L is a -(C=0)-0-C_2-2o alkyl group. In some embodiments, L is a -(C=0)- group. In some embodiments, L is selected from among

In some embodiments, L is or comprises an amino acid or a di-, tri- tetra- or oligopeptide. In some embodiments, L is selected from among alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

In any of the compounds (e.g. in any of Formula I, II and/or IV), linking element (L) can optionally be characterized as having a chain length of at least 2.8 Angstroms, 3, Angstroms, 4 Angstroms, 5 Angstroms, 10 Angstroms, 15 Angstroms, 18 Angstroms, 30 Angstroms, 40 Angstroms or 60 Angstroms. Optionally L has a length of no more than 100 Angstroms, optionally no more than 60 Angstroms. Optionally, L is characterized as having a length of between 2.8, 3, 4, 5, 10, 20 or 30 Angstroms and 60 Angstroms. Optionally, L is characterized as having a length of between 2.8 and 19 Angstroms, or between 4 and 19 Angstroms.

A compound may contain more than one L moiety. Any L' moiety can be defined in the same way as an L moiety. The L moieties may or may not be the same. The linking group L may be a water-soluble moiety or contain one or more water-soluble moieties, such that L contributes to the water solubility of a compound of Formula (I) - (VI). An L may also be a moiety or contain one or more moieties that reduce(s) aggregation, which may or may not be a moiety/moieties that also increase(s) the water solubility.

L may be for example a linear linker or a branched linker. In one aspect, the L moiety is branched, optionally further a dendritic structure, so that it can be connected to at least two, three, four or more V, Y or R moieties (or Z where applicable). Each V-Y moiety is however only attached once to an L moiety. Branching can occur at one or more branching atoms that may for example be carbon, nitrogen, silicon, or phosphorus. When the lysine-based linker comprises branching in L, the number of branches in L that are connected to V and/or Y will generally be prepared so as to equal the total number of branches available for reaction. That is, in preparing the lysine-based linker, chemical conversion will preferably be carried to completion, thereby maintain the controlled stoichiometry offered by the site-specific TGase-mediated conjugation approach. Thus, preferably, when L is branched, compounds will be functionalized such that each L, V or Y is connected to a R or Z moiety, such that the components of the mixture of an antibodies (or the lysine-based linker during preparation) substantially all have the same r value. For example, it can be specified that 90%, 95%, 98% of the antibodies or the lysine-based linker have the same r value. In one embodiment, L is a linear linker. In another embodiment, L is a branched linker.

Any one of the L moieties disclosed herein can be utilized in Formula la, lb, II, IVa, and IVb. Any one of the L moieties described herein can be used in combination with any of the C, X, V, Y, Z, R, M, z, q, and r groups described herein. Any one of the L' moieties disclosed herein can be utilized in Formula III. Any one of the L' moieties described herein can be used in combination with any of the R', V, Y', Z, z', q', and r' groups described herein.

Exemplary linkers of Formula la include but are not limited to:

Compound la-4

Compound la-5 o

H,N„ ,NH NH

"Val Cit PAB MMAE

o o Compound la-6

O o

NH

"Val Cit PAB MMAE o

Compound la-7 o

,0.

' Val Cit PAB MMAE Compound la-8

Compound la-9

H₂N

Compound la-10

Val Cit PAB MMAE

Compound la-1 1

Compound la-12

Compound la-13

Compound la-14 Compound la-15

Compound la-16

"MMAF

ο ο Compound la-17

Compound la-18

Compound la-19

Compound la-20

Compound la

,0. Val Cit PAB MMAE

H₂N

o o Compound la

Compound la-23

Exemplary linkers of Formula lb include but are not limited to:

Compound lb-1

Compound lb-2

Compound lb-3

Compound lb-4 Compound lb-5 Compound lb-6

Compound lb-7

Compound lb-8 Compound lb-9

Compound lb-10 Compound lb-1 1

Compound lb-12

Compound lb-13

Compound lb-14

Compound lb-15

The reactive moiety R

R is a reactive moiety, for example a moiety comprising an unprotected or protected bioorthogonal-reaction compatible reactive group, for example an unprotected or protected thiol, epoxide, maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate, sulfonate ester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde, generally any group capable of oxime and hydrazine formation, 1 ,2,4,5-tetrazine, norbornene, other stained or otherwise electronically activated alkene, a substituted or unsubstituted cycloalkyne, generally any reactive groups which form via bioorthogonal cycloaddition reaction a 1 ,3- or 1 ,5-disubstituted triazole, any diene or strained alkene dienophile that can react via inverse electron demand Diels-Alder reaction , a protected or unprotected amine, a carboxylic acid, an aldehyde, or an oxyamine.

When more than one R group is present in a compound of the formula, the R groups will preferably be compatible such that no R group is a complementary reagent to any other R group. The L, V and/or Y groups of formulae l-IV can have r, q, and/or z sites of attachment for the respective V, Y, and R groups, where r and q represent the degree of branching or polymerization. The sites of attachment can comprise a bond or comprise a functional group selected from an alkene, alkyne, ether, thioether, ester, thioester, amine, amide, alkylamide, or other functional group readily generated by a condensation or addition reaction.

The reactive group of the linking reagent can for example chosen to undergo thio- maleimide (or haloacetamide) addition, Staudinger ligation, Huisgen 1 ,3-cycloaddition (click reaction), or Diels-Alder cycloaddition with a complementary reactive group attached to an agent comprising a therapeutic moiety, a diagnostic moiety, or any other moiety for a desired function.

Optionally, two or more compatible reactive groups can be attached to the linking reagent.

In one embodiment, the reactive group is a haloacetamide, (e.g. bromo-acetamide, iodo-acetamide, cloro-acetamide). Such reactive groups will be more stable in vivo (and in serum) compared with maleimide groups.

In one embodiment, the reactive group is a reagent capable of undergoing a "click" reaction (i.e., a Click Chemistry reagent or reactive group). For example a 1 ,3-dipole- functional compound can react with an alkyne in a cyclization reaction to form a heterocyclic compound, preferably in the substantial absence of added catalyst (e.g., Cu(l)). A variety compounds having at least one 1 ,3-dipole group attached thereto (having a three-atom pi- electron system containing 4 electrons delocalized over the three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1 ,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyl diazo groups.

Examples include o-phosphenearomatic ester, an azide, a fulminate, an alkyne (including any strained cycloalkyne), a cyanide, an anthracene, a 1 ,2,4,5-tetrazine, or a norbornene (or other strained cycloalkene).

In one embodiment, R is a moiety having a terminal alkyne or azide; such moieties are described for example in U.S. patent no. 7,763,736, the disclosure of which is incorporated herein by reference. Suitable reaction conditions for use of copper (and other metal salt) as catalysts of click-reactions between terminal alkynes and azides are provided in U.S. patent no. 7,763,736.

In one embodiment, R is a substituted or unsubstituted cycloalkyne. Cycloalkynes, including heterocyclic compounds, will preferably be used in linking reagents in which an L group is present, preferably wherein L is an alkyl or heteroalkyl chain of 3-30, optionally 5-30 or 5-15 linear carbon atoms, optionally substituted at one or more atoms. Optionally, L is a (CH₂-CH2-0)_{1 -2}4 group or a (CH2)xi-(CH2-0-CH2)i-24-(CH₂)x2-, wherein x1 and x2 are independently an integer selected among the range of 0 to 20. As shown herein, presence of an L group enables high TGase-mediated coupling when cycloalkynes are used.

Cycloalkynes, including specific compounds, are described for example in U.S. Patent No. 7,807,619, the disclosure of which is incorporated herein by reference.

mbodiments, a cycloalkyne may be a compound of Formula A:

Formula A

where:

R¹ is selected from a carbonyl, an alkyl ester, an aryl ester, a substituted aryl ester, an aldehyde, an amide, an aryl amide, an alkyl halide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, a substituted aryl ketone, and a halosulfonyl;

R¹ can be at any position on the cyclooctyne group other than at the two carbons joined by the triple bond.

In some embodiments, the modified cycloalkyne is of Formula A, wherein one or more of the carbon atoms in the cyclooctyne ring, other than the two carbon atoms joined by a triple bond, is substituted with one or more electron-withdrawing groups, e.g., a halo (bromo, chloro, fluoro, iodo), a nitro group, a cyano group, a sulfone group, or a sulfonic acid group. Thus, e.g., in some embodiments, a subject modified cycloalkyne is of Formula B:

Formula B

where:

each of R² and R³ is independently: (a) H; (b) a halogen atom (e.g., bromo, chloro, fluoro, iodo); (c) -W-(CH₂)_n-Z (where: n is an integer from 1 -4 (e.g., n=1 , 2, 3, or 4); W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); (d) -(CH₂)_n-W- (CH₂)m-R⁴ (where: n and m are each independently 1 or 2; W is O, N, S, or sulfonyl; if W is O, N, or S, then R⁴ is nitro, cyano, or halogen; and if W is sulfonyl, then R⁴ is H); or (e) - CH₂)_n- R⁴ (where: n is an integer from 1 -4 (e.g., n=1 , 2, 3, or 4); and R⁴ is nitro, cyano, sulfonic acid, or a halogen); and

R¹ is selected from a carbonyl, an alkyi ester, an aryl ester, a substituted aryl ester, an aldehyde, an amide, an aryl amide, an alkyi halide, a thioester, a sulfonyl ester, an alkyi ketone, an aryl ketone, a substituted aryl ketone and a halosulfonyl. R¹ can be at any position on the cyclooctyne group other than at the two carbons linked by the triple bond.

In one embodiment, R is a substituted or unsubstituted heterocyclic strained alkyne. Cycloalkynes, including specific compounds, are described for example in U.S. Patent No. 8,133,515, the disclosure of which is incorporated herein by reference. In one embodiment, the alkyne is of the Formula C:

Formula C

wherein

each R¹ is independently selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C-I-C-IO alkyi or heteroalkyl;

each R² is independently selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C-I-C-IO organic group; X represents N-R³R⁴, N H-R⁴, CH-N-OR⁴, C-N-N R³R⁴, CHOR₄, or CH N H R₄; and each R³ represents hydrogen or an organic group and R⁴ represents linking moiety C of a linker. In one embodiment, R or R' is a DBCO (dibenzycyclooctyl) group below:

Alkynes such as those described herein above can be reacted with at least one 1 ,3- dipole-functional compound (e.g., embodied as an R' moiety in a compound of Formula III) in a cydization reaction to form a heterocyclic compound, preferably in the substantial absence of added catalyst (e.g., Cu(l)). A wide variety compounds having at least one 1 ,3-dipole group attached thereto (having a three-atom pi-electron system containing 4 electrons delocalized over the three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1 ,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyl diazo groups. The reactive moiety R is connected to L, or when present, V or Y, and is able to react with a suitable functional group (R') on a reaction partner, e.g. a complementary reagent of Formula III which undergoes a high conversion addition reaction when brought into contact with a reactive moiety R. When reactive moiety R is present in an antibody of Formula II, the reaction results in formation of an antibody of Formula IV. In this reaction, the moieties R and R' are transformed into the moiety (RR'). Any R' moiety can be defined in the same way as a R moiety, so long as R and R' are complementary when used in moieties that are to be reacted together.

A compound may contain more than one reactive moiety R. The R moieties may or may not be the same. Any one of the R moieties disclosed herein can be utilized in Formula lb and II. Any one of the R moieties described herein can be used in combination with any of the C, X, L, V, Y, z, q, and r groups described herein. Any one of the R' moieties disclosed herein can be utilized in Formula III. Any one of the R' moieties described herein can be used in combination with any of the L', V, Y', Z, z', q', and r' groups described herein.

It should be understood that, although not illustrated in Figures 1 and 2, the H₂NCH₂ group of the linking reagent may have undergone reaction with the glutamine residue of an antibody prior to the high conversion addition reaction or that the aminomethylene may be in a protected state. Alternatively, in other embodiments, the H₂NCH₂ group of the linking reagent will not have undergone reaction with the glutamine residue of an antibody prior to the high conversion addition reaction or that the aminomethylene may be in a protected state; in this case the linking reagent and reaction partner can be used to conveniently form various combinations of linkers having different V, Y, and/or Z moieties that are ready to conjugate to an antibody.

The preparation of an exemplary linking reagent and its conjugation with a protein is illustrated in Figure 3, where: V and Y are absent, R is a thiol (sulfhydryl) reactive group that is ultimately generated from the S-acetyl protected thiol, SC(0)CH_3; r is 1 ; q is 1 ; z is 1 ; L is the two carbon comprising framework C(0)CH₂; X is NH; C is (CH₂)₅; and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein. Figure 4 illustrates the preparation of various exemplary linking reagents, with a single S-acetyl protected thiol reactive group that can be prepared from an N-succinimidyl-S-acetylthioester reagent. In addition to S-acetyl, other S-protecting groups can be employed, including p-hydroxyphenylacyl, 2-quinoline, or Hqm and Hgm groups that can be deprotected by the addition of hydrazine.

Figure 5 illustrates the preparation of an exemplary linking reagent, and its conjugation with a protein, where: V and Y are absent, R is an azide reactive group_; r is 1 ; q is 1 ; z is 1 ; L is the two carbon comprising framework C(0)CH₂; X is NH; (C)_n is (CH₂)₅; and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein. Figure 6 illustrates the preparation of various exemplary linking reagents, with a single azide reactive group that can be prepared from an N-succinimidyl-azide reagent.

Figure 7 depicts the preparation of an exemplary linking reagent, and its conjugation with a protein, where: V and Y are absent, R is an alkyne reactive group_; r is 1 ; q is 1 ; z is 1 ; L is a one carbon comprising framework CH₂; X is NH; C is (CH2)4CH(C0₂H); and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein. Figure 8 shows the preparation of an exemplary linking reagent, and its conjugation with a protein, where: R is a norbornene reactive group_; r is 1 ; q is 1 ; z is 1 ; L is the one carbon comprising framework C(O); X is NH; C is(CH₂)₄CH(C0₂H); and G is transformed from the (H₃C)₃COC(0) protecting group to H and ultimately to the amide upon conjugation of a glutamine residue of a protein.

The selective and very high conversion addition reaction that can be carried out with the linking reagents can be uncatalyzed or catalyzed reactions. For example, the 2+4 Diels- Alder cycloadditions, thio-maleimide (or haloacetamide) additions, and Staudinger ligations can be carried out without a catalyst. Other very high conversion addition reactions, for example any of the click reactions, can be catalyzed with metal salts, such as Cu, Ru, Ni, Pd, and Pt salts.

The linking group (RR') in M of compounds of Formula IV represents the remainder of R when the reactive moiety R of Formula II has reacted with a reactive moiety R' in a compound of Formula III. This group (RR') then links the moiety Z (e.g. comprised in the compound of formula IV) with L, V or Y. The group that remains may be a bond.

The V moiety

The V moiety may be incorporated in the lysine-based linker (e.g. connected to L, optionally through Y). However, the V moiety may instead or in addition be incorporated in a compound comprising a moiety-of-interest Z (e.g. a compound R'-V-Y-Z of formula III) that will be reacted with an antibody conjugated with a lysine-based linker to form an antibody conjugated to the moiety-of-interest Z. Any V moiety can be defined in the same way as a V moiety.

In the compounds, the V moiety is a group that is either non-cleavable or conditionally cleavable, optionally after prior conditional transformation. In the latter case, it is designed to be transformed and/or cleaved from Y, or Z when Y is absent, by a chemical, photochemical, physical, biological, or enzymatic process, e.g. in certain conditions. This condition may for example comprise bringing a compound in an aqueous environment, which leads to hydrolysis of V, or bringing a compound in an environment that contains an enzyme that recognizes and cleaves V, or bringing a compound under reducing conditions, which leads to reduction of V, or bringing a compound in contact with radiation, e.g., UV light, which leads to transformation and/or cleavage, or bringing a compound of in contact with heat, which leads to transformation and/or cleavage, or bringing a compound under reduced pressure or bringing a compound under elevated or high pressure, which leads to transformation and/or cleavage. This condition may further be met after administrating a compound to an animal, e.g., a mammal: the condition may be met when the compound localizes to for example a specific organ, tissue, cell, subcellular target, or microbial target, for example by the presence of internal factors (e.g., target- specific enzymes or hypoxia) or application of external factors (e g., radiation, magnetic fields) or the condition may already be met directly upon administration (e.g., enzymes). In general, transformation of V will directly or indirectly lead to cleavage of V from Y, or Z when Y is absent. It may occur that two or more separate transformations and/or cleavages, requiring the same or different conditions, are required in order to cleave V completely from Y or Z. In this way, increased selectivity may be obtained. A compound may contain more than one V moiety. These V moieties may or may not be the same and may or may not require the same conditions for transformation and/or cleavage.

V may comprise for example a carbon comprising framework of 1 to 200 atoms, optionally a carbon comprising framework of at least 10 atoms, e.g. 10 to 100 atoms or 20 to 100 atoms, substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon or comprises a cyclic group, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), an amino acid, a di-, tri-, tetra-, or oligopeptide, or more generally any dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process. Generally, V may be any straight, branched and/or cyclic C2-30 alkyl, C_2-3o alkenyl, C_2- 30 alkynyl, C_2-3o heteroalkyl, C_2-30 heteroalkenyl, C_2-30 heteroalkynyl, optionally wherein one or more homocyclic aromatic compound radical or heterocyclic compound radical may be inserted; notably, any straight or branched C_2-5 alkyl, C_5-10 alkyl, C_11-20 alkyl, -O- C_1-5 alkyl, - O- C_5-10 alkyl, -O- C_11-20 alkyl, or (CH₂- CH₂-0-)_1-24 or (CH₂)_x1-(CH₂-0-CH₂)_1-24-(CH₂)_x2-group, wherein x1 and x2 are independently an integer selected among the range of 0 to 20, an amino acid, an oligopeptide, glycan, sulfate, phosphate, or carboxylate. Optionally, V may be or absent. In some embodiments, V is a C_2-6 alkyl group.

In one aspect, a compound is used to target one or more therapeutic and/or diagnostic moieties Z to target cells. In this instance, V may for example contain a substrate molecule that is cleaved by an enzyme present in the vicinity of the target cells or inside the target cells, for example tumor cells. V can for example contain a substrate that is cleaved by an enzyme present at elevated levels in the vicinity of or inside the target cells as compared to other parts of the body, or by an enzyme that is present only in the vicinity of or inside the target cells.

If target cell specificity is achieved solely based upon the selective transformation and/or cleavage of V at the target site, the condition (eventually) causing the cleavage should preferably, at least to a certain degree, be target cell-specific, whereas the presence of another target-specific moiety in the compound, for instance when the ABD recognizes an antigen present on a target cell with a degree of specificity, reduces or takes away this requirement. For example, when an antibody causes specific internalization into a target cell, an enzyme also present in other cells may transform and/or cleave V. In one embodiment, transformation and/or cleavage of V occurs intracellular^. In another embodiment, transformation and/or cleavage of V occurs extracellularly.

In one embodiment, the V moiety is a conditionally cleavable moiety.

In one embodiment, V contains a di-, tri-, tetra-, or oligopeptide which consists of an amino acid sequence recognized by a protease, for example plasmin, a cathepsin, cathepsin B, prostate-specific antigen (PSA), urokinase-type plasminogen activator (u-PA), or a member of the family of matrix metalloproteinases, present in the vicinity of or inside the target cells, for example tumor cells. In one embodiment provided is a conjugate wherein V is a dipeptide, tripeptide, tetrapeptide, or oligopeptide moiety comprised of natural L amino acids, unnatural D amino acids, or synthetic amino acids, or a peptidomimetic, or any combination thereof. In one embodiment, V is a peptide. In another embodiment, V is a dipeptide. In another embodiment, V is a tripeptide. In another embodiment, V is a tetrapeptide. In yet another embodiment, V is a peptidomimetic.

In one embodiment, V contains a substrate for an enzyme. In another embodiment, V contains a beta-glucuronide that is recognized by beta- glucuronidase present in the vicinity of or inside tumor cells.

In one embodiment, V contains a substrate for an extracellular enzyme. In another embodiment, V contains a substrate for an intracellular enzyme.

In yet another embodiment, V contains a substrate for a lysosomal enzyme.

In yet another embodiment, V contains a substrate for the serine protease plasmin.

In yet another embodiment, V contains a substrate for one or more of the cathepsins, for example cathepsin B. When V is cleaved extracellularly, the one or more Z moieties may be released extracellularly. This may provide the advantage that these Z moieties are not only able to affect or detect the cell(s) directly surrounding the site of activation, but also cells somewhat further away from the site of activation due to diffusion (bystander effect).

In one embodiment V comprises a tripeptide. The tripeptide may be linked via its C- terminus to Y. In one embodiment, the C-terminal amino acid residue of the tripeptide is selected from arginine, citrulline, and lysine, the middle amino acid residue of the tripeptide is selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, cyclohexylglycine, tryptophan and proline, and the N-terminal ammo acid residue of the tripeptide is selected from any natural or unnatural amino acid.

In another embodiment V comprises a dipeptide. The dipeptide may be linked via its C-terminus to Y. In one embodiment, the C-terminal amino acid residue of the dipeptide is selected from alanine, arginine, citrulline, and lysine, and the N-terminal amino acid residue of the dipeptide is selected from any natural or unnatural amino acid. In one embodiment, V is selected from phenylalanine-lysine and valine-citrulline.

An example of a linker comprising a lysine residue as (C)_n moiety (or NH₂-C moiety) and a valine-citrulline as the (V) moiety is shown below:

Optionally, the di-, tri-, tetra, or oligopeptide(s) comprise or consist or amino acids with non-negatively charged side chains (amino acids other than aspartic acid or glutamic acid). Optionally, the di-, tri-, tetra, or oligopeptide(s) comprise or consist or amino acids selected from: amino acids with positively charged side chains, amino acids with polar uncharged side chains, and amino acids with hydrophobic side chains. In another aspect, a compound is used to improve the pharmacokinetic properties of Z. V may in this case for example be or contain a group that is cleaved by ubiquitous enzymes, e.g., esterases that are present in the circulation, by pH-controlled intramolecular cyclization, or by acid-catalyzed, base-catalyzed, or non-catalyzed hydrolysis, or V may for example be or contain a disulfide. V may therefore, optionally together with the connecting atom of L and/or Y (or Z if Y is absent), for example form a carbonate, carbamate, urea, ester, amide, imine, hydrazone, oxime, disulfide, acetal, or ketal group. It is understood that

V can also be or contain such a moiety and/or be transformed and/or cleaved in the same or a similar way when a compound is used for other purposes than solely improving the pharmacokinetic properties of Z.

When the compounds are used for other purposes, e.g., an ex vivo diagnostic assay,

V may be or contain any of the moieties mentioned above and transformation and/or cleavage of V may occur by any one of the processes mentioned above or by any other functional transformation or cleavage process known to a person skilled in the art. For example, in a diagnostic assay, V may be cleaved or transformed by an enzyme, by reduction, or below, above, or at a certain pH.

When V is conditionally cleavable, the compounds are designed to eventually release at least one Z after cleavage and optional prior transformation of V. Release of Z from a compound via another mechanism is however not excluded.

In any embodiment, V may contain a blocking group to prevent premature transformation and/or cleavage of V before the condition is met under which V is designed to be transformed and/or cleaved.

In another aspect, V is a moiety that is non-cleavable. This means that V cannot be cleaved from Y, or Z when Y is absent, under the conditions the compound containing such a V moiety is designed to be applied, meaning that Z cannot be released in this way. Release of Z from a compound via another mechanism is however not excluded. When V is a non-cleavable moiety, Y may optionally be absent. A non-cleavable V moiety may be any moiety that cannot be cleaved, or that can be cleaved only very slowly, under the conditions the compound containing such a V moiety is designed to be applied, e.g. in vivo or in vitro. For example, when applied in vivo, V will not or only very slowly be cleaved by enzymes present in the in vivo model used or by hydrolysis or as a consequence of other biological processes that may occur in said model. Such V may therefore, optionally together with the connecting atom of L and/or Z, for example, be a carbonyl group, an amide group, an urea group, an ester group, a carbonate group, a carbamate group, or an optionally substituted methyleneoxy or methyleneamino group V may be preferred to be non-cleavable when it is not required that the one or more moieties Z are released. This may for example be the case when Z does not require to become released before it can exert its therapeutic or diagnostic properties.

In one embodiment V is connected to L via a functional group in the side chain of one of the natural or unnatural amino acids. In another embodiment, the N-terminal amino acid of V is connected via its alpha amino group to L.

Any one of the V moieties disclosed herein can be utilized in Formula la, lb, II, IVa and IVb. Any one of the V moieties described herein can be used in combination with any of the C, X, L, R, Y, Z, M, z, q, and r groups described herein. Any one of the V moieties disclosed herein can be utilized in Formula III. Any one of the V moieties described herein can be used in combination with any of the R', V, Y', Z, z', q', and r' groups described herein.

The Spacer System Y

The spacer system Y, when present, links V and optionally L to one or more moieties R, and following reaction with a compound of Formula III, a moiety-of-interest Z. In one embodiment, Y is absent. In another embodiment, Y is a self-elimination spacer system. A spacer system Y may be incorporated in a compound to for example improve the properties of Z or the compound in general, to provide suitable coupling chemistries, or to create space between V and Z. Any Y' moiety can be defined in the same way as a Y moiety.

Spacer system Y may comprise for example a carbon comprising framework of 1 to

200 atoms, optionally a carbon comprising framework of at least 10 atoms, e.g. 10 to 100 atoms or 20 to 100 atoms, substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon or comprises a cyclic group, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), an amino acid, a di-, tri-, tetra-, or oligopeptide, or more generally any dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process.

Y may be any straight, branched and/or cyclic C₂-3o alkyl, C_2-3o alkenyl, C2-30 alkynyl,

C2-30 heteroalkyl, C2-30 heteroalkenyl, C2-30 heteroalkynyl, optionally wherein one or more homocyclic aromatic compound radical or heterocyclic compound radical may be inserted; notably, any straight or branched C2-5 alkyl, C_5-io alkyl, Cn-20 alkyl, -O- Ci_-5 alkyl, -O- C_5-io alkyl, -O- Cn-20 alkyl, or (CH₂- CH₂-0-)i_-24 or (CH₂)xi-(CH2-0-CH₂)i-24 -(CH₂)_x2- group, wherein x1 and x2 are independently an integer selected among the range of 0 to 20, an amino acid, an oligopeptide, glycan, sulfate, phosphate, or carboxylate. Optionally, Y is absent. In some embodiments, Y is a C_2-6 alkyl group.

A compound may contain more than one spacer system Y. These moieties Y may or may not be the same. In some embodiments the spacer system Y is a self-elimination spacer that is connected to one or more other self-elimination spacers via a direct bond. Herein, a single self-elimination spacer may also be referred to as a spacer system. A spacer system may be branched or unbranched and contain one or more attachment sites for Z as well as V. Self-elimination spacers that are able to release only a single moiety are called 'single release spacers'. Self-elimination spacers that are able to release two or more moieties are called 'multiple release spacers'. Spacers, may be either branched or unbranched and self-eliminating through a 1 ,2+2n-elimination (n>/=1 ), referred to as "electronic cascade spacers". Spacers may eliminate through a cyclization process under formation of a cyclic urea derivative, referred to as "ω-amino aminocarbonyl cyclization spacers".

The spacer system Y may self-eliminating or non-self-eliminating. A "self-eliminating" spacer unit allows for release of the drug moiety without a separate hydrolysis step. When a self-eliminating spacer is used, after cleavage or transformation of V, the side of Y linked to V becomes unblocked, which results in eventual release of one or more moieties Z. The self- elimination spacer systems may for example be those described in WO 02/083180 and WO 2004/043493, which are incorporated herein by reference in their entirety, as well as other self-elimination spacers known to a person skilled in the art. In certain embodiments, a spacer unit of a linker comprises a p-aminobenzyl unit. In one such embodiment, a p- aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and a cytotoxic agent. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). Examples of self- eliminating spacer units further include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, e.g. US 2005/0256030 Al), such as 2- aminoimidazol-5-methanoi derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals. Spacers can be used mat undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. Chemistry Biology, 1995, 2, 223) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55. 5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacers.

A "non-self-eliminating" spacer unit is one in which part or all of the spacer unit remains bound to the moiety Z upon enzymatic (e.g., proteolytic) cleavage of the antibody - moiety-of-interest conjugate. Examples of non-self-eliminating spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Other combinations of peptidic spacers susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an antibody -moiety-of-interest conjugate containing a glycine-glycine spacer unit by a tumor- cell associated protease would result in release of a glycine-glycine-drug moiety from the remainder of the antibody -moiety-of- interest conjugate. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

In a compound, a spacer system Y may be connected to more than one V moiety. In this case, transformation and/or cleavage of one of these V moieties may trigger the release of one or more Z moieties. When V moieties that are transformed or cleaved under different conditions are connected to the same Y, release of one or more Z moieties may occur when a compound is brought under one of several different conditions.

Any one of the Y moieties disclosed herein can be utilized in Formula la, lb, II, IVa and IVb, V or VI . Any one of the Y moieties described herein can be used in combination with any of the C, X, L, V, Y, R, Z, M, z, q, and r groups described herein. Any one of the Y' moieties disclosed herein can be utilized in Formula III. Any one of the Y' moieties described herein can be used in combination with any of the R', L', V, Z, z', q', and r' groups described herein.

Conjugation of lysine-based linkers to an antibody

Enzymes of the TG-family catalyze covalent protein crosslinking by forming proteinase resistant isopeptide bonds between a lysine donor residue of one protein and an acceptor glutamine residue of another protein and is accompanied by the release of ammonia. The catalytic mechanism of transglutaminases has been proposed as follows. After the glutamine-containing first substrate (acceptor residue) binds to the enzyme, it forms a γ-glutamylthioester with the cysteine residue in the active center of TGase, known as the acylenzyme intermediate, accompanied by the release of ammonia. The second substrate (donor or K-substrate) then binds to the acylenzyme intermediate and attacks the thioester bond. The product (two proteins crosslinked by an Ne(v-glutamyl)lysine isopeptide bridge) is formed and released. This re-establishes the active-centre Cys residue of the enzyme in its original form and allows it to participate in another cycle of catalysis. The formation of the covalent acylenzyme intermediate is thought to be the rate-limiting step in these reactions. The catalytic triad of many transglutaminases is papain-like, containing Cys-His-Asp (where His is histidine and Asp is aspartic acid) and, crucially, a tryptophan (Trp) residue located 36 residues away from the active-centre Cys. In contrast, bacterial TG isolated from Streptoverticillium sp (vide supra) has an atypical catalytic triad and shows no sequence homology with the papain-like catalytic triad of other TGases.

TGases display relatively strict specificity in recognition of glutamine protein substrates. However, TGases display much broader specificity for recognition of the acyl- acceptor amine group, which can either be the ε -amino group of peptidyl lysine or a low- molecular mass primary amine (frequently a polyamine) (see, e.g. Folk, et al. (1980) J. Biol. Chem. 255, 3695-3700. For example, in addition to lysine, the small lysine-mimicking primary amine 5-pentylamine (cadaverin) and variants or fragments thereof can efficiently bind to the acylenzyme intermediate, and a pseudo-isopeptide bond with the glutamine- containing protein is formed. See, e.g., Lorand, L. et al. (1979) Biochemistry 18, 1756-1765 (1979); Murthy, S.N. et al. (1994). J. Biol. Chem. 269, 22907-2291 1 (1994); Murthy, P. et al. (2009) Biochemistry (2009).

Bacterial, archaeal and eukaryotic TGases have been characterized and differ in several ways from mammalian TGases (Lorand, L. & Graham, R.M. (2003) Nat. Rev. Mol. Cell Biol. 4, 140-156). BTG and more generally microbial TGases (EC 2.3.2.13, protein- glutamine-v-glutamyltransferase) such as Streptomyces mobaraensis are calcium- independent and have an amino acid sequence of) very different from those of mammalian TGs (Ando et al. (1989) Agric. Biol. Chem. 53, 2613-2617). BTG is furthermore much smaller (37.8 kDa versus 76.6 kDa for guinea pig liver TG). Additionally, BTG shows broader substrate specificity for the amine acceptor glutamine substrates in proteins than do mammalian TGases. BTG can optionally comprise one or more amino acid substitutions that alter performance in coupling to acceptor glutamines in the Fc domain of antibodies, including at glutamines at Kabat position 295 (see, e.g. WO2015/191883, WO2017/059158, the disclosures of which are incorporated herein by reference). These characteristics, together with a high reaction rate, low cost of production, and a decreased tendency to catalyze deamidation make BTG a preferred enzyme for use in the application herein.

When an acceptor glutamine is located within the CH2 domain, the antibodies that are to be conjugated to the lysine-based linker will optionally be free of N-linked glycosylation. Full-length wild-type IgG antibodies naturally comprise N-linked glycosylation at residue 297 of the heavy chain which may impair TGase-mediated conjugation onto glutamine residues in the CH2 domain. In other embodiments, the antibodies that are to be conjugated to the lysine-based linker will retain N-linked glycosylation. A BTG enzyme comprising amino acid substitutions can be utilized that permits improved conjugation onto Fc domain retaining N-linked glycosylation (see, e.g. WO2015/191883, WO2017/059158). Deglycosylation can be carried out as described herein or according to any suitable method. For example, antibody (1 mg) in PBS buffer (0.1 mol/L NaCI and 0.05 mol/L sodium phosphate buffer, pH 7.4) are incubated with 100 units (0.2 μΙ_) of /V-glycosidase F (PNGase F) from Flavobacterium meningosepticum (New England BioLabs, Ipswich, UK) at 37°C overnight. The enzyme is then removed by centrifugation-dialysis (Vivaspin MWCO 50 kDa, Vivascience, Winkel, Switzerland). The product can be analyzed by LC/MS.

In one embodiment, the product is analyzed for drug loading (e.g. number of conjugates per antibody. Such methods can be used to determine the mean number of conjugates per antibody (e.g., the mean DAR) as well as the distribution of number of conjugates per antibody in a composition, i.e. the percentage of total antibody with any given level of drug loading or DAR. One technique adapted to such determination and more generally drug loading is hydrophobic interaction chromatography (HIC), HIC can be carried out as described for example in Hamblett et al. (2004) Cancer Res. 10: 7063-7070; Wakankar et al. (201 1 ) mAbs 3(2): 161 -172; and Lyon et al (2012) Methods in Enzymology, Vol. 502: 123-138, the disclosure of which are incorporated herein by reference.

Examples of useful TGases include microbial transglutaminases, such as e.g. from

Streptomyces mobaraense, Streptomyces cinnamoneum and Streptomyces griseocarneum fall disclosed in US 5,156,956, which is incorporated herein by reference), and Streptomyces lavendulae (disclosed in US 5,252,469, which is incorporated herein by reference) and Streptomyces ladakanum (JP2003199569, which is incorporated herein by reference). It should be noted that members of the former genus Streptoverticillium are now included in the genus Streptomyces (Kaempfer, J Gen Microbiol, 137, 1831-1892, 1991 ). Other useful microbial transglutaminases have been isolated from Bacillus subtilis (disclosed in US 5,731 ,183, which is incorporated herein by reference) and from various Myxomycetes. Other examples of useful microbial transglutaminases are those disclosed in WO 96/06931 (e.g. transglutaminase from Bacilus lydicus) and WO 96/22366, both of which are incorporated herein by reference. Useful non-microbial transglutaminases include guinea-pig liver transglutaminase, and transglutaminases from various marine sources like the flat fish Pagrus major (disclosed in EP-0555649, which is incorporated herein by reference), and the Japanese oyster Crassostrea gigas (disclosed in US 5,736,356, which is incorporated herein by reference). A preferred TGase is bacterial transglutaminase (BTG) (see, e.g. EC 2.3.2.13, protein-glutamine-Y-glutamyltransferase). In a more preferred embodiment, the TGase is from S. mobaraense. In another embodiment, the TGase is a mutant TGase having at least 80% sequence homology with native TGase (e.g. the parental TGase, BTG, S. mobaraense TGase, etc.). A preferred example is recombinant bacterial transglutaminase derived from streptomyces mobaraensis (available from Zedira, Darmstadt, Germany). The TGase-catalyzed reaction can be carried out under mild conditions, from several hours to a day (e.g. overnight). Recombinant BTG (EC 2.3.2.13) from streptomyces mobaraensis (Zedira, Darmstadt, Germany) can be used at a concentration of between 1 and 20 U/mL, preferably between 6 U/mL and 20 U/mL. The lysine-based linker substrates are reacted with antibody (1 mg/mL) at ligand concentrations between 400 and 600 mol/L, providing a 60 to 90-fold excess of the substrates over the antibody, or optionally at lower excess of substrates, e.g. 1 - to 20-fold, or 10-20 fold. The reactions are performed in potassium-free phosphate buffered saline (PBS; pH 8) at 37 °C. After 4 h to several days (depending on the antibody and the ligand), steady-state conditions are achieved. Excess ligand and enzyme are then removed using centrifugation-dialysis (Vivaspin MWCO 50 kDa, Vivascience, Winkel, Switzerland). Reactions are monitored by LC/MS. Higher amounts of TGase can be used as a function of different lysine-derivatives and substrates.

An acceptor glutamine present on an antibody (e.g. part of the antibody's primary structure will, under suitable conditions, be recognized by a TGase and covalently bound to a lysine-based linker (e.g., compound of Formula I). The result is an antibody of Formula II or IVa (the acceptor glutamine is functionalized with the compound of Formula lb or la respectively).

Reaction partners comprising a moiety-of-interest Z and reactive group R'

Once a lysine-based linker (e.g., compound of Formula I) comprising a reactive moiety R is conjugated to an antibody (e.g., resulting in an antibody of Formula II) the antibody can be reacted with a compound comprising a moiety Z and a reactive group R', thereby forming an antibody-moiety-of-interest conjugate. In some embodiments, the conjugated antibody (e.g. the antibody of Formula II) is subjected to a deprotection step to provide an unprotected reactive group (R) and the antibody is then reacted with a compound comprising a reaction partner R'.

As discussed, this reaction step may be carried out in solution or while antibody is bound to a solid support. For example, when a lysine-based linker comprising a reactive moiety R is conjugated by a TGase while the antibody is immobilized on a solid support, the compound comprising a moiety Z and a reactive group R' can be introduced to the same solid support (e.g. following a washing step) such that the antibody is then reacted with the compound comprising a reaction partner R' while immobilized on the solid support. There is therefore no need to elute the antibody prior to the reaction with compound comprising a moiety Z and a reactive group R'.

In another variation, when a lysine-based linker comprising a reactive moiety R is conjugated by a TGase while the antibody is in solution, the reaction mixture from the TGase mediated reaction that comprises both antibody and TGase can be introduced directly to a solid support so as to immobilize the antibody but not TGase to the solid support. The compound comprising a moiety Z and a reactive group R' can then be introduced to a solid support such that the antibody is then reacted with the compound comprising a reaction partner R' while immobilized on the solid support.

In yet another variation, when a lysine-based linker comprising a reactive moiety R is conjugated by a TGase while the antibody is immobilized on a solid support, the antibody can be eluted from the solid support and the compound comprising a moiety Z and a reactive group R' can be reacted with the antibody in solution.

R' is a reactive moiety and can be defined in the same way as reactive group (R), so long as R' is complementary (reactive with) reactive group R. R' may be, for example, a moiety comprising an unprotected or protected bioorthogonal-reaction compatible reactive group, for example an unprotected or protected thiol, epoxide, maleimide, haloacetamide, o- phoshenearomatic ester, azide, fulminate, sulfonate ester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde, generally any group capable of oxime and hydrazine formation, 1 ,2,4,5- tetrazine, norbornene, other stained or otherwise electronically activated alkene, a substituted or unsubstituted cycloalkyne, generally any reactive groups which form via bioorthogonal cycloaddition reaction a 1 ,3- or 1 ,5-disubstituted triazole, any diene or strained alkene dienophile that can react via inverse electron demand Diels-Alder reaction , a protected or unprotected amine, a carboxylic acid, an aldehyde, an oxyamine, so long as such group when unprotected is reactive with R (when R' is unprotected).

When more than one R' group is present in a compound of the formula, the R' groups will preferably be compatible such that no R' group is a complementary reagent to any other R' group. The L', V and/or Y' groups of formulae l-IV can have r, q, and/or z sites of attachment for the respective V, Y', and R' groups, where r and q represent the degree of branching or polymerization. The sites of attachment can comprise a bond or comprise a functional group selected from an alkene, alkyne, ether, thioether, ester, thioester, amine, amide, alkylamide, or other functional group readily generated by a condensation or addition reaction.

In one embodiment, R' is a moiety having a terminal alkyne or azide, a substituted or unsubstituted cycloalkyne, for example a compound of Formula A (above), a modified cycloalkyne is of Formula B (above), or a substituted or unsubstituted heterocyclic strained alkyne of Formula C (above).

Any one of the R' moieties disclosed herein can be utilized in Formula III. Any one of the R' moieties described herein can be used in combination with any of the L', V, Y', Z, z', q', and r' groups described herein. The compounds of (e.g. Formula I I I) to be used in reaction with an antibody can be reacted with antibody (e.g., 1 mg/mL) at ligand concentrations between 2 and 20 (or between 4 and 20) molar equivalents to the antibody, optionally between 2 and 10 (or between 4 and 10) molar equivalents to the antibody, optionally at a less than, or about, 20, 10, 5, 4 or 2 molar equivalents to the antibody. However it will be appreciated that higher excess (equivalents of reaction partner (e.g. Formula I I I) to antibody (40 to 80 fold, 60 to 90- fold) can also be used.

The compounds of Formula I I I to be used in reaction with an antibody conjugated to a lysine-based linker (but without a moiety-of-interest), e.g., an antibody of Formula I I , as well as the resulting antibody conjugates therefore comprise one or more moieties-of-interest Z. The compounds of Formula II I may additionally comprise a moiety V and/or Y, typically depending on which elements are included in the lysine-based linker.

The compounds of Formula I I I to be used in reaction with an antibody conjugated to a lysine-based linker (e.g. an antibody of Formula II) will comprise moieties Z connected to linker L' when Y' and V are absent, connected to the spacer system Y' or, when Y' is absent, connected to V. Consequently, a compound of Formula II I may comprise a moiety Z connected to or comprising a reactive group R', optionally the moiety Z connected to a reactive group R' via a spacer system Y' or, when Y' is absent, to a reactive group R' via V, or to a reactive group R' via a V'-Y', wherein Z is preferably connected to Y' and V is connected to R' and Y'.

A compound of Formula I I I may contain one, two or more Z moieties that are the same or that differ from one another, e.g. different therapeutic moieties, and/or diagnostic moieties.

In one embodiment, the antibody of Formula I I is reacted with a compound of Formula I II comprising a moiety of interest Z comprising and a reactive group R' capable of forming a bond with reactive group R of Formula lb or II , optionally wherein the compound further comprises a V and/or Y' group. The compound comprising a moiety of interest Z comprising and a reactive group R' preferably comprises a structure of Formula I I I , below,

R' - L' - (V'-(Y'-(Z)_z.)_q.)_r. Formula I I I where:

R' is a reactive group, e.g. a reactive group complementary for forming at least one bond with reactive group R of Formula lb or II ;

L' is a bond or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

V is independently absent, a non-cleavable moiety or a conditionally-cleavable moiety that can optionally be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process, cleavage of V ultimately leading to release of one or more Z moieties. In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety",

Y' is independently absent or a spacer system (e.g., a self-eliminating spacer system or a non-self-elimination spacer system) which is comprised of 1 or more spacers,

Z is independently a reactive group (optionally protected) other than a complementary reactive group for reaction with R', a moiety that improves the pharmacokinetic properties, a therapeutic moiety, or diagnostic moiety;

q' and r' are an integer selected among 1 , 2, 3 or 4, representing degree of branching; and

z' is an integer selected among 1 , 2, 3 or 4.

Where Z is a reactive group, it can be a moiety comprising an unprotected or protected thiol, maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene, 1 ,2,4,5-tetrazine, norbornene, other stained or otherwise electronically activated alkene or, optionally, a protected or unprotected amine when X is absent and L, V, or Y is other than a bond or a continuation of a bond. In an alternative embodiment Z can be a reactive moiety, preferably a moiety comprising an unprotected or protected thiol, an unprotected or protected amine, maleimide, haloacetamide, o- phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene, 1 ,2,4,5-tetrazine, norbornene, other stained or otherwise electronically activated alkene. Preferably R is not an amine when n=5 and X, L, V and Y are absent. Preferably R is not an amine when n=4 and X, L, V and Y are absent.

The moiety R' is connected to Z, or optionally to Z via V and/or Y' and is able to react with a suitable functional group R on a reaction partner, e.g. group R on the lysine-based linker of formula lb or II. As discussed above, when the reactive moiety R' is designed to react with a reactive group R, a compound of Formula or IVb is formed.

The L' group can be a carbon comprising framework, where L' is a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, oligosaccharide, other natural oligomer, dimer, trimer, or higher oligomer resulting from any chain-growth or step-growth polymerization process, wherein L' has r', q', and/or z' sites of attachment for the respective V, Y', and R' groups, where r' and q' represent the degree of branching or polymerization. The sites of attachment can comprise a bond or comprise a functional group selected from an alkene, alkyne, ether, thioether, ester, thioester, amine, amide, alkylamide, or other functional group readily generated by a condensation or addition reaction.

The linking group (RR') in M of compounds of Formula IVb represents the R' addition product of a reactive moiety R' and a reactive moiety R. This group then links the moiety Z with L', V, and/or Y, preferably via (RR'). The group that remains may be a bond. RR' can be an addition product of a: thio-maleimide (or haloacetamide) addition, for example, a N,S- disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, for example, a Λ/,3- or Λ/,4- substitued-5-dipenylphosphinoxide-benzoic amide; Huisgen 1 ,3-cycloaddition (click reaction), for example, a /V,S-disubstituted-3-thio-pyrrolidine-2,5-dione, 1 ,4-disubstituted- 1 ,2,3-triazole, 3,5-disubstituted-isooxazole, or 3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, for example the 2,4-cycloaddition product between an O or N- substituted-5-norbornene-2-carboxylic ester or amide, /V-substituted-5-norbornene-2,3- dicarboxylic imide, O or /V-substituted-7-oxonorbornene-5-carboxylic ester or amide, or N- substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substituted anthracene or 3- substituted 1 ,2,4,5-tetrazine; or any high yield selective amidation or imidization reaction. Some reactions and the RR' reaction products are illustrated in Figures 1 and 2.

The Moiety Z

The moiety Z can be connected to Y or Y' or, when absent, to V or V, or, when absent, to L or, when absent to X, or to L' or, when absent to R', (RR'), or to C. Connections to Y, V or L may optionally be via R or RR'. Connection may be via any suitable atoms. In one embodiment, Z is coupled via oxygen (from for example a hydroxyl group or carboxyl group), carbon (from for example a carbonyl group), nitrogen (from for example a primary or secondary amino group), or sulfur (from for example a sulfhydryl group). In one embodiment, Z is coupled in the compounds via a group such that its therapeutic abilities or diagnostic characteristics are, at least partly, blocked or masked. In case a compound is to be used for treating or preventing disease in an animal, e.g., a mammal, the Z moieties are generally therapeutic moieties. In case a compound is used to make a diagnosis or used in an ex vivo or in vivo diagnostic assay, the Z moieties are generally diagnostic moieties, for example chromogenic, fluorogenic, phosphorogenic, chemiluminescent, or bio luminescent compounds.

In one embodiment, the Z moiety is compound, preferably an organic compound, having a molecular weight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol, 1000- g/mol or 2000 g/mol. In one embodiment, the Z moiety is a chemical compound displaying hydrophobic properties, optionally additionally having a molecular weight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol. 1000- g/mol or 2000 g/mol. Hydrophobic character may be determined, for example, by decreased water solubility, decreased polarity, decreased potential for hydrogen bonding, and/or an increased oil/water partition coefficient. The presently disclosed methods can be used to produce antibody conjugates where moiety of interest (Z) comprises a hydrophobic drug. As used herein, the term "hydrophobic" is a physical property of a molecule that is repelled from a mass of water. Hydrophobic compounds can be solubilized in nonpolar solvents, including but not limited to, organic solvents. Hydrophobicity can be conferred by the inclusion of apolar or nonpolar chemical groups that include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). Conversely, "hyd rophilic" molecules are capable of hydrogen bonding with a water molecule and are therefore soluble in water and other polar solvents. The terms "hydrophilic" and "polar" can be used interchangeably. Hydrophilic characteristics derive from the presence of polar or charged groups, such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.

Hydrophobic molecules are poorly water soluble, for example, having a solubility of less than about 10 mg/ml. In some embodiments, the hydrophobic compound can have a solubility of less than about 1 mg/ml in water. In other embodiments, the hydrophobic compound has a solubility in water of less than about 50, μg/ml, 10 μg/ml, and in particular embodiments, about 1 μg/ml or 2.5 μg/ml. In other embodiments, the hydrophobic compound can have a solubility of about 0.001 μg/ml to about 10 mg/ml, including but not limited to 0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, 50 μg/ml, 100 μg/ml, 500 μg/ml, 1 mg/ml, 5 mg/ml, and 10 mg/ml, and any other concentration between 0.001 μg/ml and 10 mg/ml.

Representative, non-limiting examples of hydrophobic drugs that can be formulated using the presently disclosed methods include taxanes, e.g. paclitaxel (PTX), and camptothecin (CPT), maytansanoids, duocarmycins, dolastatins and auristatins. Such drugs are poorly soluble in water, e.g. PTX has a solubility in water of less than about 1 μg/ml, CPT has a water solubility of about 2.5 μg/ml. Linkers and modified an antibodies can advantageously link hydrophobic drugs to an antibodies.

In other embodiments, in view of hydrophobic drugs being poor substrates for TGase (in the absence of improved linkers or modified an antibodies), the Z moiety may advantageously be a hydrophilic drug. Examples of hydrophilic drugs include amatoxins. Amatoxins are cyclic peptides composed of 8 amino acids as isolated from the genus Amanita. Amatoxins also include a range of chemical derivatives, semisynthetic analogs and synthetic analogs built from building blocks according to the master structure of the -5 natural compounds (cyclic, 8 amino acids), synthetic or semisynthetic analogs containing non-hydroxylated amino acids instead of the hydroxylated amino acids, synthetic or semisynthetic analogs, in which the thioether sulfoxide moiety is replaced by a sulfide, sulfone, or by atoms different from sulfur, e.g. a carbon atom as in a carbaanalog of amanitin. Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those with a functional group (e.g. a carboxylic group, an amino group, a hydroxy group, a thiol or a thiol-capturing group) that can be reacted with linker molecules or proteins. Amatoxins are described for example in European Patent publication no. 185981 1 , PCT publication nos. WO 2010/1 15630 and WO 2012/041504).

In one embodiment, the Z moiety is a large compound (e.g., molecular weight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol or 700 g/mol) comprising a polycyclic group, tricycle or one or more macrocycles. Such groups are often typical of hydrophobic and/or rigid structures. Examples of cytotoxic drugs that comprise a macrocycle (e.g. a ring of nine or more atoms) include maytansinoids, amatoxins, epothilones and taxanes. In one embodiment, the Z moiety comprises a ring of 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 atoms, or between 9 and 200 atoms. In one embodiment, the Z moiety is a chemical compound having a negative charge, optionally additionally displaying hydrophobic properties and/or having a molecular weight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol, 1000 g/mol or 2000 g/mol.

When more than one Z moiety is connected to a self-elimination spacer system Y or Y', at least one Z should be released upon self-elimination of Y or Y'. The moiety Z initially released may be a moiety that is not a fully active moiety itself. In other words, Z may be a moiety that has limited diagnostic or therapeutic abilities, e.g. a moiety that acts as a prodrug. Such a Z moiety may require further processing or metabolism, e.g., hydrolysis, enzymatic cleavage, or enzymatic modification (for example phosphorylation, reduction, or oxidation) in order to become fully active. In one embodiment, such further processing is intentionally designed for Z to for example allow Z to reach its final target or cross a biological barrier, e.g., a cell membrane or a nuclear membrane, before it is fully activated. Z may for example contain a hydrophobic moiety that enables Z to cross a cell membrane. This hydrophobic moiety may then be hydrolyzed or removed in any other way intracellularly.

In one aspect, a Z moiety may be a backbone (e.g. polymer) to which a plurality of drugs or diagnostic moieties are linked. For example, Z may be a polyacetal- or polyacetal derivative-based polymer comprising a plurality of drug molecules, see, e.g., Yurkovetskiy et al. (2004) Mol. Pharm. 1 (5): 375-382 and WO 201 1/120053, the disclosures of which are incorporated herein by reference; for example Z may be a polymer compound of Formula I of WO 201 1/120053 comprising a plurality of cytotoxic anti-cancer agents.

In one aspect, one or more moieties Z are each selected from a therapeutic or diagnostic agent. In another embodiment, one or more moieties Z are each a therapeutic agent. In another embodiment, all moieties Z are each a therapeutic agent. In yet another embodiment, the moieties Z each are the same therapeutic moiety. In yet another embodiment, the moieties Z comprise at least two different therapeutic moieties.

The moiety Z includes, for example, antineoplastic agents, drugs, toxins (such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. ricin and fragments thereof) biologically active proteins, for example enzymes, other an antibodies, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.

In one embodiment, the one or more moieties Z are each independently chosen from an antibiotic, an anti-bacterial agent, an antimicrobial agent, an anti-inflammatory agent, an immunostimulatory agent, an anti- infectious disease agent, an anti-autoimmune disease agent, an anti- viral agent, or an anticancer agent, preferably a cytotoxic anti-cancer agent.

In another embodiment, the one or more moieties Z are each an anticancer agent. In a further embodiment, the one or more moieties Z are each a hydroxyl-containing anticancer agent.

In one embodiment, Z is an alkylating agent, preferably a DNA alkylating agent. An alkylation agent is a compound that can replace a hydrogen atom with an alkyl group under physiological conditions (e.g. pH 7.4, 37 C, aqueous solution). Alkylation reactions are typically described in terms of substitution reactions by N, O and S heteroatomic nucleophiles with the electrophilic alkylating agent, although Michael addition reactions are also important. Examples of alkylating agents include nitrogen and sulfur mustards, ethylenimines, methanosulfonates, CC-1065 and duocarmycins, nitrosoureas, platinum- containing agents, agents that effectuate Topoisomerase ll-mediated site dependent alkylation of DNA (e.g. psorospermin and related bisfuranoxanthones), ecteinascidin and other or related DNA minor groove alkylation agents.

In one embodiment, Z is a DNA minor groove binding and/or alkylating agent, e.g. a pyrrolobenzodiazepine, a duocarmycin, or derivatives thereof.

In a further embodiment, the one or more moieties Z are each independently selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysins, dolastatins and auristatins, enediynes, amatoxins, pyrrolobenzodiazepines, ethylenimines, radioisotopes, therapeutic proteins and peptides, and toxins or fragments thereof.

In a further embodiment, the one or more moieties Z are each independently selected from cyclophosphamide, ifosfamide, chlorambucil, 4-(bis(2- chloroethyl)amino)phenol, 4-(bis(2-fluoroethyl)ammo)phenol, N,N-bis(2-chloroethyl)-p- phenylenediamine, N,N-bis(2-fluoro- ethyl)-p-phenylenediamine, carmustine, lomustine, treosulfan, dacarbazine, cisplatin, carboplatin, vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, inirotecan, 9-aminocamptothecin, 9- nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin, crisnatol, mitomycin C, mitomycin A, methotrexate, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, hydroxyurea, deferoxamine, 5-fluorouracil, floxuridine, doxifluridine, raltitrexed, cytarabine, cytosine arabinoside, fludarabine, 6-mercaptopurine, thioguanine, raloxifen, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, vertoporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A, interferon-alpha, interferon-gamma, tumor necrosis factor, lovastatin, staurosporine, actinomycin D, bleomycin A2, bleomycin B2, peplomycin, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, morpholino doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, thapsigargin, N⁸- acetylspermidine, tallysomycin, esperamycin, butyric acid, retinoic acid, 1, 8- dihydroxybicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine, podophyllotoxin, combretastatin A-4, pancratistatin, tubulysin A, tubulysin D, carminomycin, streptonigrin, elliptmium acetate, maytansine, maytansinol, calicheamycin, mertansine (DM1 ), N-acetyl-γ-ι'- calicheamycin, calicheamycin-γ-ι', calicheamycin-a₂', calicheamycin-a₃', duocarmycin SA, duocarmycin A, CC-1065, CBI-TMI, duocarmycin C2, duocarmycin B2, centanamycin, dolastatin, auristatin E, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), oamanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, and amanullinic acid and derivatives thereof.

One exemplary auristatin embodiment is MMAE, wherein the wavy line indicates the covalent attachment to a L, L', V, V, Y, Y', (RR'), R' or (C)_n group of a compound (e.g. a compound of Formula I, II or IV):

An exemplary auristatin embodiment is MMAF, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Cfiem. 17: 1 14-124):

Other exemplary Z embodiments include monomethylvaline compounds having phenylalanine carboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and monomethylvaline compounds having phenylalanine sidechain modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603).

An example of a linker comprising a lysine residue as (C)_n moiety, a valine-citrulline as the (V) moiety, a PAB as the (Y) moiety together with a MMAF as the (Z) moiety is shown below (corresponding to compound la-1 ):

In one embodiment, Z is or comprises a DNA minor groove binding and/or alkylating agent.

In one embodiment, the Z moiety comprises a pyrrolobenzodiazepine (PBD). In one embodiment, Z is a pyrrolobenzodiazepine monomer. In one embodiment, Z is a pyrrolobenzodiazepine dimer comprising two pyrrolobenzodiazepine units. In one embodiment, Z is a pyrrolobenzodiazepine trimer comprising three pyrrolobenzodiazepine units. In one embodiment, Z is a pyrrolobenzodiazepinemultimer comprising more than three pyrrolobenzodiazepine units. Structures of PBDs, as well as formulas and methods of producing them are described for example in PCT publications Nos: WO 2013/177481 , WO 201 1/130616, WO 2004/043880, WO 2005/085251 , WO2012/1 12687 and WO 201 1/023883, the disclosures of each of which are incorporated herein by reference.

The pyrrolo[2,1-c][1 ,4] benzodiazepines are a family of sequence-selective, minor- groove binding DNA-interactive agents that covalently attach to guanine residues. It has been reported that the (S)-chirality at the C1 1 a-position of PBDs provides them with the appropriate 3-dimensional shape to fit perfectly into the DNA minor groove. PBDs can have different effects and modes of action. PBDs can be DNA-binders or DNA-alkylators that do not cause crosslinking of DNA, or PBDs can be DNA cross-linkers.

The pyrrolobenzodiazepine unit or monomer can have a general structure as follows:

wherein the PBD can have different number, type and position of substituents, in both the aromatic A rings and pyrrolo C rings, and can vary in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N¹⁰-C¹¹ position which is the electrophilic centre responsible for alkylating DNA.

The biological activity of PBDs can be potentiated by joining two PBD monomoers or units together, typically through their C8/C8'-hydroxyl functionalities via a flexible alkylene linker.

In one aspect of the any of the embodiments herein, a pyrrolobenzodiazepine monomer or unit is a pyrrolo[2,1-c][1 ,4]benzodiazepine. In one aspect of the any of the embodiments herein, a pyrrolobenzodiazepine dimer is a C8/C8'-linked pyrrolo[2,1- c][1 ,4]benzodiazepine dimer.

A PBD can be attached to a compound comprising a reactive group R' through any suitable position, thereby yielding a compound of Formula III which comprises the PBD and a reactive group R'. For example, in the compound of Formula III, the PBD can be connected to Y' or to V, or, when absent, to L' in a compound of Formula III, via any of the positions in a PBD unit indicated below.

In one embodiment, a PBD dimer comprises the structure of the general formula below, with exemplary attachments points to other substituents or functionalities within a compound of Formula III indicated by arrows:

wherein:

R¹² and R¹² , and/or R² and R² together respectively form a double bond =CH₂ or =CH-CH₃; or

R² and R¹² are absent and R² and R¹² are independently selected from:

(iia) Ci-5 saturated aliphatic alkyl;

(iiib) C₃-6 saturated cycloalkyl;

(iic)

, wherein each of R , R and R are independently selected from H, Ci_-3 saturated alkyl, C₂-3 alkenyl, C_2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R¹² group is no more than 5;

(iid)

, wherein one of R and R is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, meth xy; pyridyl; and thiophenyl; and

iie)

, where R is selected from: H; C_1-3 saturated alkyl; C_2-3 alkenyl; C_2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR', nitro, Me₃Sn and halo; where R and R' are independently selected from optionally substituted C_1-12 alkyl, C_3-20 heterocyclyl and C_5-20 aryl groups;

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR', nitro, Me₃Sn and halo; either:

(a) R¹⁰ is H, and R¹¹ is OH, OR^A, where R^A is alkyl;

(b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or

(c) R¹⁰ is H and R¹¹ is SO_zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

R" is a C_3-i₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR^N2 (where R^N2 is H or C-M alkyl), and/or aromatic rings, e.g. benzene or pyridine;

Y and Y' are selected from O, S, or NH; and

R⁶ , R⁷ , R⁹ are selected from the same groups as R⁶, R⁷ and R⁹ respectively and R^10' and R^11' are the same as R¹⁰ and R¹¹ , wherein if R¹¹ and R¹¹ are SO_zM, M may represent a divalent pharmaceutically acceptable cation.

In another example, a PBD dimer comprises the structure of the general formula below:

wherein R⁶ , R⁷ , R⁹, R^6' , R^7' , R^9', R¹⁰ , R¹¹ , R^10' and R^11' are as defined above, and wherein the "K" ring is a substituted or unsubstituted aromatic or non-aromatic ring, optionally a 6- member ring, optionally a phenyl.

In one embodiment, the Z moiety is an epothilone or epothilone derivative. An epothilone is a cyclic molecule with a 16-membered ring and variable substituents and pharmaceutical activity as a cytostatic agent that binds to tubulin. Various epothilone derivatives are known, including variants with 14-, 15- or 18-membered rings have also been developed (e.g. WO 2011085523; WO 2009105969). Examples of epothilones or epothilone analogs or derivative include epothilone A, epothilone B, epothilone C, 13-alkyl- epothilone C derivatives, epothilone D, trans-epothilone D, epothilone E, epothilone F, an effector conjugate of epothilone, Sagopilone, or any of the epothilones referred to in the literature as ixabepilone (BMS-247550), BMS-310705, EPO-906, Patupilone, Kos-862, Kos-1584, Kos- 1803 and ABJ 879, and pharmaceutically active salts thereof. The production of epothilones, their precursors and derivatives is generally carried out according to the methods known to one skilled in the art. Suitable methods are, for example, described in DE 19907588, WO 98/25929, WO 99/58534, WO 99/2514, WO 99/67252, WO 99/67253, WO 99/7692, EP 99/4915, WO 00/485, WO 00/1333, WO 00/66589, WO 00/49019, WO 00/49020, WO 00/49021 , WO 00/71521 , WO 00/37473, WO 00/57874, WO 01/92255, WO 01/81342, WO 01/73103, WO 01/64650, WO 01/70716, US 6204388, US 6387927, US 6380394, US 02/52028, US 02/58286, US 02/62030, WO 02/32844, WO 02/30356, WO 02/32844, WO 02/14323, and WO 02/8440. Further epothilones are described in WO 93/10102, WO 98/25929, WO 99/02514, WO 99/07692, WO 99/02514, WO 99/67252, WO 00/49021 , WO 00/66589, WO 00/71521 , WO 01/027308, WO 02/080846, WO 03/074053, WO 2004/014919.

Chelated metals include chelates of di- or tripositive metals having a coordination number from 2 to 8 inclusive. Particular examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). In general the metal is preferably a radionuclide. Particular radionuclides include ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁸Co, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag, ²⁰³Pb, ²⁰⁶Bi, ²⁰⁷Bi, ¹¹¹ln, ⁶⁷Ga, ⁶⁸Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd and ⁴⁷Sc.

The chelated metal may be for example one of the above types of metal chelated with any suitable polydentate chelating agent, for example acyclic or cyclic polyamines, polyethers, (e.g. crown ethers and derivatives thereof); polyamides; porphyrins; and carbocyclic derivatives.

In general, the type of chelating agent will depend on the metal in use. One particularly useful group of chelating agents in conjugates, however, are acyclic and cyclic polyamines, especially polyaminocarboxylic acids, for example diethylenetriaminepentaacetic acid and derivatives thereof, and macrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives (for example as described in PCT publication no. WO 92/22583); and polyamides, especially desferriox-amine and derivatives thereof.

Other effector molecules may include detectable substances useful for example in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Pat. No. 4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerytbrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include ¹²⁵l, ¹³¹l, ¹¹¹ln and "Tc.

Synthetic or naturally occurring polymers for use as effector molecules include, for example optionally substituted straight or branched chain polyalkylene, polyalkenylene, or polyoxyalkylene polymers or branched or unbranched polysaccharides, e.g. a homo- or hetero-polysaccharide such as lactose, amylose, dextran or glycogen.

Particular optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups. Particular examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof. Such compounds, when used as a moiety Z can be employed as a moiety that improves the pharmacokinetic properties of the antibody.

In another embodiment, z' equals 1 , each V, Y or V-Y (including whether any V and Y is a V or Y') moiety contains a single attachment site for a functional group of Z.

In another embodiment, a one V (or V), Y, (or Y') or V-Y (or V'-Y', V-Y') moiety is attached to more than one Z moiety via multiple functional groups R on the said V, Y or V-Y moiety. Optionally, the one or more V (or V) moieties comprise a polymer, optionally an oligoethylene glycol or a polyethylene glycol or a derivative thereof.

Any one of the Z moieties disclosed herein can be utilized in Formula la, Nil, and IVa. Any one of the Z moieties described herein can be used in combination with any of the C, X, L, V, R, Y, Z, M, z, q, and r groups described herein. Any one of the Z moieties described herein can be used in combination with any of the R\ L', V, Y\ z', q', and r' groups described herein.

Antibody-Z conjugates

In one embodiment, a linking reagent (e.g. of Formula la) is directly conjugated to an antibody, without requirement for a step of reaction involving reactive groups R and R'. In one aspect, an antibody comprises a functionalized glutamine residue of Formula IVa, below,

wherein:

Q is glutamine residue present in an antibody;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein any carbon of the chain is optionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide (e.g. a O, N or S atom of an ether, ester, thioether, thioester, amine, alkylamine, amide, or alkylamide), optionally wherein C has a chain length of 2 to 20 atoms, preferably 3 to 6 atoms;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework, preferably of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected among 1 , 2, 3 or 4;

q is an integer selected among 1 , 2, 3 or 4;

z is an integer selected among 1 , 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or a conditionally-cleavable moiety that can optionally be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process (e.g. cleavage of V ultimately leading to release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety"; Y is independently absent or a spacer (e.g., a self-eliminating spacer system or a non-self-elimination spacer system) which is comprised of 1 or more spacers; and

Z is a moiety-of-interest, optionally a moiety that improves the pharmacokinetic properties, or a therapeutic moiety or a diagnostic moiety. Preferably, Z is an immunosuppressive agent, an immunostimulatory agent, or a cytotoxic anti-cancer agent, e.g. a compound selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins, enediynes, radioisotopes, therapeutic proteins and peptides, and toxins or fragments thereof.

Generally, each Z is directly coupled to either Y or V when Y is absent, or L when both Y and V are absent.

It will be appreciated that Formula IVa can for convenience also be expressed as (Ab)-NH-C-X-L- (V-(Y-(Z)z)q)_r (Formula IVa), where (Ab) is an immunoglobulin (Ab) is conjugated via a glutamine (Q) residue to an NH of the linking reagent (e.g. the compound of Formula la).

Examples of an antibodies of Formula IVa include but are not limited to an antibodies attached via an amide bond (e.g. through an acceptor glutamine residue in the primary sequence of the antibody) to a compound selected from the group consisting of compounds la-1 to la-23 (wherein the terminal NH₂- of each of said compound la-1 to la-23 is replaced by a moiety ((Q)-NH-) when attached to the antibody, wherein Q is glutamine residue present in an antibody, e.g. in the CH2 domain or in a TGase recognition tag fused to the C- terminus of the CH3 domain.

The antibody conjugates resulting from the reaction of the compounds of Formula lb or III with an antibody conjugated to a lysine-based linker will yield an antibody conjugate in which a moiety Z is connected to linker L (or L') when Y (or Y') and V (or V) are absent, to the spacer system Y (or Y') or, when Y (or Y') is absent, to V (or V). Optionally said connections are via linking group (RR') of M.

The conjugates resulting from the reaction yield an antibody which is conjugated (i.e., covalently attached) via an acceptor glutamine residue (Q) present on the antibody to a NH group of a lysine-based linker, and one or more moieties (Z) through optional linking group (RR'), optional linker (V or V) and/or optional spacer (Y or Y').

In one embodiment, the (RR') remains present in a conjugated antibody, in which case a Formula IV will comprise an (M) moiety. Such an antibody comprises a functionalized glutamine residue of Formula IVb, below, (Q)-NH-C-X-L- (V-(Y-(M)_z)_q)_{r Formu}|_a |_Vb

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

Q is glutamine residue present in an antibody;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein any carbon of the chain is optionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide, optionally wherein C has a chain length of 2 to 20 atoms, preferably 3 to 6 atoms;

X is NH, O, S, or absent;

r is an integer selected among 1 , 2, 3 or 4;

q is an integer selected among 1 , 2, 3 or 4;

z is an integer selected among 1 , 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or a conditionally-cleavable moiety that can optionally be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process (e.g. cleavage of V ultimately leading to release of one or more moieties subsequently or ultimately linked to V, for example a Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, or oligopeptide as described below in the section entitled "The V Moiety";

M is independently: R or (RR') - L' - (V'-(Y'-(Z)_z)_q )_r._, wherein each of L', V, Υ', ζ', q', and r' are as defined in Formula III (or are defined as L, V, Y, z, q and r, respectively,

Z is a moiety-of-interest, optionally a moiety that improves the pharmacokinetic properties, or a therapeutic moiety or a diagnostic moiety, R is as defined in Formula I and wherein each (RR') is an addition product between an R of Formula I and its complementary R' of Formula III (see, for example, Figure 1 and Figure 2). Optionally, V and Y are absent and V and/or Y' are present. Thus, RR' can be for example an addition product of a thio- maleimide (or haloacetamide) addition, for example, a /V,S-disubstituted-3-thio-pyrrolidine-

2.5- dione; Staudinger ligation, for example, a Λ/,3- or /V,4-substitued-5- dipenylphosphinoxide-benzoic amide; Huisgen 1 ,3-cycloaddition (click reaction), for example, a /V,S-disubstituted-3-thio-pyrrolidine-2,5-dione, 1 ,4-disubstituted-1 ,2,3-triazole, 3,5-disubstituted-isooxazole, or 3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, for example the 2,4-cycloaddition product between an O or /V-substituted-5-norbornene-2- carboxylic ester or amide, /V-substituted-5-norbornene-2,3-dicarboxylic imide, O or N- substituted-7-oxonorbornene-5-carboxylic ester or amide, or /V-substituted-7-oxonorbornene-

5.6- dicarboxylic imide and a 9-substituted anthracene or 3-substituted 1 ,2,4,5-tetrazine; or any high yield selective amidation or imidization reaction. Some reactions and the corresponding RR' reaction products are illustrated in Figures 1 and 2.

' include:

attachment of -C, X, L, L', V, V, Y, Y' or Z. RR' can be in either orientation with respect to their attachment to -C, X, L, L', V, V, Y, Y' or Z).

Optionally, the antibody conjugate comprises a group (RR') representing the remainder of a reactive moiety R when R has reacted with a reactive moiety R', wherein the group (RR') connects (a) an L to a Z, a V or a Y, (b) a V to a Z or a Y, or (c) a Y to a Z. For example, any V, Y and/or Z may be characterized as comprising a (RR') group. Any L, V, Y may be an L', V or Y', respectively.

It will be appreciated that Formula IVb can for convenience also be expressed as (mFc)-NH-C-X-L- (V-(Y-(M)_z)_q)_r , where (mFc) is an antibody conjugated via a glutamine (Q) residue to an NH of the linking reagent (e.g. the compound of Formula lb).

Examples of antibodies of Formula IVb include but are not limited to:

Compound IVb-1

Compound IVb-10

Val Cit PAB MMAE

(Q)NH

Compound IVb-1 1

Compound IVb-12

In one embodiment, the glutamine (Q) is present in the CH2 of an antibody. In one embodiment, the glutamine (Q) is at position 295 (Kabat EU numbering). In one embodiment, an acceptor glutamine (Q) is at position 297 (e.g., a N297Q substitution). In one embodiment, the antibody comprises a substitution of an asparagine at position 297 with a non-asparagine, non-aspartic acid, non-glutamine, residue.

Exemplary lysine-based linker compounds can be prepared using known synthesis methods and starting reagents. In the examples below, all chemicals are purchased from Sigma-Aldrich, Fluka or Pierce Thermo scientific unless otherwise stated. All chemicals and solvents are used without further purification. Reactions are monitored by HPLC or by thin layer chromatography (TLC) using precoated silica gel 60 F aluminum sheets (Merck), and visualized by UV absorption or stained. See, e.g., WO2016/102632, the disclosure of which is incorporated herein by reference.

Preferably, in any the methods or compositions, a composition of a plurality of antibody conjugates is obtained wherein the antibodies have a uniform ratio of functionalized acceptor glutamines:antibody. In particular, the methods permit substantially complete conjugation of antibodies, for are range of moieties Z, including large, charged and/or hydrophobic drugs. In one aspect provided is a composition wherein a high portion of antibodies in the composition (e.g. at least 80%, 85%, 90%, 95% of the antibodies) comprise at least one moiety of interest, wherein the composition is substantially free of antibodies comprising a number of moieties of interest that is greater than 2 times, optionally 1 .5 times, the mean number of conjugates per antibody (e.g., the mean DAR). In one embodiment provided is a composition comprising a plurality of antibodies of Formula II or IV, wherein at least 70%, 80%. 85%, 90%, 95%, 98% or 99% of the antibodies in the composition have the same number of functionalized acceptor glutamine residues (Q) (e.g., a functionalized acceptor glutamine of Formula II or IV) per antibody. Preferably at least 70%, 80%. 85%, 90%, 95%, 98% or 99% of the antibodies in said first antibody composition have no more or no less than (m) functionalized acceptor glutamine residues (Q) per antibody, wherein m is an integer, e.g. m=1 , 2, 3 or 4. Optionally, at least 70%, 80%, 90%, 95%, 98% or 99% of the antibodies in the composition have the same q, r and z values. It can optionally be specified that the antibodies will share the same -NH-(C)_n-X, L, V, V, Y, Y', R, RR' and/or Z moieties.

Typically, a high portion of antibodies in an antibody sample (e.g. at least 80%, 85%, 90%, 95% of the antibodies) comprise at least one moiety of interest, wherein antibody sample compositions are preferably also free of antibodies having conjugated light chains. For example, an antibody sample may comprise tetrameric antibodies covalently linked to a moiety of interest (Z), wherein the composition is characterized by a mean DAR of close to 2 (e.g., between 1 .4 and 2.0, or between 1 .5 and 2.0, or between 1 .7 and 2.0, between 1 .8 and 2.0, or between 1 .9 and 2.0), and wherein less than 10%, less than 5%, less than 2% or less than 1 % of the antibodies in the composition comprise more than two moieties of interest (Z) per antibody. Preferably, less than 25%, 20%, 15% or preferably 10% of the antibodies in the composition comprise less than two moieties of interest (Z) per antibody. Optionally antibodies in an antibody sample are covalently linked to a moiety of interest (Z), wherein the composition is characterized by a mean DAR of close to 4 (e.g., between 3.0 and 4.0, or between 3.4 and 4.0, or between 3.6 and 4.0), wherein less than 10%, less than 5%, or less than 2% of the antibodies comprise more than four functionalized acceptor glutamines per antibody. Preferably, the composition is substantially free of antibodies having more than 4 moieties of interest (Z) per antibody.

The antibody-conjugates can be used for the manufacture of a pharmaceutical preparation and/or for the treatment or diagnosis of a mammal being in need thereof. In one embodiment, provided is the use of any of the methods or any compounds defined above for the manufacture of a pharmaceutical composition and/or for the treatment of a tumor or cancer in a mammal.

Also provided are methods of manufacturing the compounds defined above, for use as a medicament or an active component or active substance in a medicament. In a further aspect provided is a method for preparing a pharmaceutical composition containing a compound as defined above, to provide a solid or a liquid formulation for administration orally, topically, or by injection. Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.

Also provided are methods of manufacturing pharmaceutical compositions comprising the compounds as defined above. A compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. The preferred form depends on the intended mode of administration and therapeutic or diagnostic application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient. Pharmaceutically acceptable carriers are well known m the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids. A pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.

EXAMPLES Example 1 : BTG coupling reaction conditions and linkers

Bacterial transglutaminase (BTG) reactions conditions and linkers were developed to permit coupling onto Fc regions following our result showing that BTG is unable to couple linkers with large, hydrophobic and/or charged payloads in quantitative fashion to antibodies.

Incomplete BTG coupling large and/or hydrophobic organic molecules to antibodies Antibody chADCI binding a tumor antigen and having a human lgG1 Fc domain was

PNGaseF-deglycosylated to expose the glutamine naturally present in the CH2 domain at

Kabat position 295 was evaluated for ability to be conjugated to a variety of substrates via BTG. However, for several types of compounds, BTG was unable to functionalize the glutamine at high levels of completion:

The chemical structure of a thiol linker coupled to maleimide-DOTA (1 ,4,7, 10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid) is shown below. The molecular weight is indicated below the structure.

MW: 702.82, 5

ChADCI antibodies and DOTA linker were reacted in the presence of BTG to modify antibodies. Quantitative enzymatic modification of chimADCI heavy chain with short DOTA thiol linker (compound 5) by BTG could not be accomplished, only unmodified chADCI heavy chain, 48945 Da, was found. Reaction conditions were explored but neither by using 1 U/ml_ (expected) nor by using 6U/ml_ BTG could significantly complete coupling be achieved. Prolonged incubation time could not influence the efficiency or completion of coupling. Compared to biotin and dansyl, DOTA has a higher molecular weight, has a more rigid structure (containing a macrocycle), and in particular is electronically negatively charged that may interfere with BTG activity.

The chemical structure of lysine-based linker (cadaverin) coupled to fluorescein is shown below.

Cadaverin-fluorescein

ChADCI antibodies and cadaverin-fluorescein linker were reacted in the presence of BTG to modify antibodies. The light chain remained unaffected. Quantitative enzymatic modification of chADCI heavy chain with short fluorescein-containing linker by BTG could not be accomplished, only unmodified chADCI heavy chain was found. Following exploration of reaction conditions, optimized conditions were tested (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 18H at 37°C) but coupling could not be achieved. Compared to biotin and dansyl, fluorescein has a higher molecular weight, has a possibly more rigid and hydrophobic structure, notably containing a polycycle, notably a tri-cycle and a further cyclic group in proximity to the site of BTG activity.

The chemical structure of the dibenzylcyclooctyne (DBCO) lysine-based linker (DBCO-amine) used is shown below.

DBCO-amine

ChADCI antibodies and the DBCO lysine-based linker were reacted in the presence of BTG to modify antibodies. The light chain remained unaffected. Quantitative enzymatic modification of chADCI heavy chain with short DBCO lysine-based linker by BTG could not be accomplished, only unmodified chADCI heavy chain was found. Following exploration of reaction conditions, optimized conditions were tested (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C) but coupling could not be achieved. Compared to biotin and dansyl linkers, the DBCO has a possibly more rigid structure, notably containing a polycycle, notably a tri-cycle group in proximity to the site of BTG activity.

The chemical structure of a TAMRA lysine-based linker is shown below.

ChADCI antibodies and TAMRA lysine-based linker were reacted in the presence of BTG to modify antibodies. The light chain remained unaffected. Quantitative enzymatic modification of chimADCI heavy chain with short TAMRA lysine-based linker by BTG could not be accomplished, only unmodified chADCI heavy chain was found. Following exploration of reaction conditions, optimized conditions were tested (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 18h at 37°C) but at best only partial coupling could be achieved, with about 50% of all heavy chains having a linker coupled thereto. Compared to biotin and dansyl, TAMRA has a higher molecular weight, has a possibly more rigid and hydrophobic structure, notably containing a polycycle, notable a tri-cycle and a cyclic group in proximity to the site of BTG activity.

A linker comprising the monomethyl auristatin F (MMAF), as well as a valine-citrulline dipeptide spacer, a 6-carbon spacer and a PAB self-elimination spacer (MW 1562, C6- MMAF linker) were reacted in the presence of BTG to modify chADCI antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). Quantitative enzymatic modification of heavy chains with MMAF linker by BTG could not be accomplished. Primarily unmodified chADCI heavy chain was found, with a major peak corresponding to unmodified heavy chain (70%) and a minor peak to heavy chain with one MMAF linker (30%) for chADCI and a major peak corresponding to unmodified heavy chain (81 %) and a minor peak to heavy chain with one MMAF linker (19%) for chCE7.

Improved lysine-based linkers for BTG-mediated direct coupling

To explore the possibility that large, charged or hydrophobic groups close to the site of BTG coupling (i.e. the primary amine) influences and inhibits BTG coupling efficiency, linkers having linear carbon-containing frameworks acting as spacers were tested.

Coupling of DOT A linkers with spacer group

The chemical structure of a spacer-containing thiol linker coupled to maleimide- DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid) and a short linker were compared. The molecular weights are indicated below the structures.

DOTA linker with spacer (MW 755.9262)

^{MW: 702}-⁸². ⁵ DOTA short linker

ChADCI antibodies and short DOTA linker or DOTA linker comprising a 6-carbon spacer (referred to as C6-DOTA) were reacted in the presence of BTG to modify antibodies. Following exploration of reaction conditions, optimized conditions were used (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 18h at 37°C).

Quantitative enzymatic modification of chADCI heavy chain with C2-DOTA linker by BTG could not be accomplished and primarily unmodified chADCI heavy chain was found, with a major peak corresponding to unmodified heavy chain (70%) and a minor peak to heavy chain with one C2-DOTA (30%). C6- DOTA linker comprising a 6-carbon spacer however achieved significantly improved coupling, with a major peak corresponding to heavy chain with one C6-DOTA (70%) and a minor peak corresponding to unmodified heavy chain (30%).

The environment of the acceptor glutamine in the heavy chain influences BTG coupling

Despite improvement with spacers, large and/or hydrophobic organic molecules representative of cytotoxic drugs could not be coupled by BTG onto acceptor glutamines of deglycosylated chADCI quantitatively (complete coupling). To explore the possibility that the environment, in terms of amino acids of the antibody, at the site of BTG-mediated coupling influences and inhibits BTG coupling efficiency, modified antibodies having amino acid substitutions were tested. Antibodies treated with PNGaseF to remove N297-linked glcoyslation will have an aspartic acid at residue 297 as a result of PNGaseF-induced deamidation at the asparagine. Modified chADCI antibodies having N297S substitution were generated which avoided N297-linked glycosylation and in turn avoided an aspartic acid or other negatively charged residue.

Unmodified (N297), PNGaseF-deglycosylated chADCI antibodies were reacted with the cadaverin-fluorescein linker in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). Quantitative enzymatic modification of chADCI heavy chain with cadaverin-fluorescein linker by BTG could not be accomplished. Only partial modification of chADCI heavy chains was found, with a substantial peak corresponding to unmodified heavy chains. However, when N297S chADCI mutant antibodies were reacted with the cadaverin-fluorescein linker in the presence of BTG, high levels of coupling was observed, with a major peak corresponding to heavy chain with one cadaverin-fluorescein linker (80%) and a minor peak to unmodified heavy chains (20%).

In another experiment, unmodified (N297), PNGaseF-deglycosylated chADCI antibodies were reacted with the cadaverin-TAMRA linker in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). Quantitative enzymatic modification of chADCI heavy chain with cadaverin-TAMRA linker by BTG could not be accomplished. Partly modified chADCI heavy chain was found, with a substantial peak corresponding to unmodified heavy chain. However, when modified N297S chADCI antibodies were reacted with the cadaverin-TAMRA linker in the presence of BTG, quantitative coupling was achieved, with a peak corresponding to heavy chains with one cadaverin-TAMRA linker and no uncoupled heavy chains.

PNGaseF treatment modifies the side chain of the asparagine at position 297 such that an aspartic acid is present at position 297 following PNGaseF treatment. It is believed that BTG activity is inhibited by negative electrical charges. One possible explanation is therefore that a negative electrical charge at the amino acid residue at the +2 position relative to the acceptor glutamine inhibits BTG's ability to couple onto the glutamine within the particular context of the Fc domain of the antibody. Antibodies can thus be modified such that aspartic acid residues are no longer present at the +2 position relative to the acceptor glutamine for the coupling of large and/or hydrophobic molecules to antibodies, or more generally to modify antibodies to avoid negative electrical charges adjacent to the acceptor glutamine, notably at the +2 position.

Combining spacers and modified antibody constant regions for direct coupling

To explore the ability of the combination of modified environment at the substrate

(implemented by use of spacer groups in the linker) and modified environment at the site of BTG coupling (implemented by use Fc domain mutants) to further improve BTG coupling, linkers comprising a different cyclic groups with and without spacers were tested using both unmodified or modified chimeric antibodies. The modified antibodies contained a substitution of the asparagine at residue 297 with either serine or glutamine to avoid formation of the negatively charged aspartic acid caused by PNGase deglysosylation. The antibodies that had a glutamine substitution at residue 297 were also modified to substituted the glutamine at residue 295 with an asparagine so as to avoid functionalization at residue 295 (functionalization occurs solely at residue 297).

DOT A (negatively charged payload)

ChADCI N297S antibodies and short DOTA linker or DOTA linker comprising a 6- carbon spacer (referred to as C6-DOTA) were reacted in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C).

While enzymatic modification of chADCI N297S heavy chain with C2-DOTA by BTG was more complete than that observed for C2-DOTA on chADCI (PNGaseF deglycosylated), quantitative coupling could not be accomplished and some unmodified chimADCI heavy chain remained. However, reacting C6-DOTA linker with chADCI N297S achieved near quantitative coupling of all heavy chains with one C6-DOTA.The combination of improved linker and protein environment therefore improved the coupling observed for C6- DOTA on chADCI (PNGaseF deglycosylated) in which only about 70% coupling was observed.

The experiments were repeated using chCE7 Q295N, N297Q antibodies and the C6-

DOTA linker using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). The reaction achieved high levels coupling of all heavy chains with one C6-DOTA, with a major peak corresponding to heavy chain modified with one C6-DOTA (greater than 80%).

DBCO (polycycle/rigid payload)

In another experiment, the chemical structure of a dibenzylcyclooctyne (DBCO) lysine-based linker comprising a "PEG" spacer and short DBCO linkers were compared (structures shown below).

DBCO linker with PEG spacer

DBCO short linker ChADCI N297S antibodies and short DBCO linker or DBCO linker comprising a 15- atom PEG spacer were reacted in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C).

Quantitative enzymatic modification of chADCI N297S heavy chain with short DBCO linker by BTG could not be accomplished and primarily unmodified chADCI heavy chain was found, with a major peak corresponding to unmodified heavy chain (70%) and a minor peak to heavy chain with one short DBCO linker (30%). However, reacting DBCO linker with spacer comprising a 15-carbon PEG spacer achieved substantially quantitative (complete) coupling of all heavy chains with one DBCO linker with spacer.

Cytotoxic agent (large, hydrophobic payload)

The linker tested comprised the monomethyl auristatin F (MMAF) as a representative large cytotoxic drug used in antibody drug conjugates, as well as a valine-citrulline dipeptide spacer, a 6-carbon spacer and a PAB self-elimination spacer. The structure is shown below. The molecular weight is indicated below the structure.

Unmodified (N297), PNGaseF deglycosylated chADCI and chCE7 antibodies were reacted with the MMAF linker in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). Quantitative enzymatic modification of chADCI heavy chain with MMAF linker by BTG could not be accomplished. Primarily unmodified chADCI or chCE7 heavy chain was found, with a major peak corresponding to unmodified heavy chain (70%) and a minor peak to heavy chain with one MMAF linker (30%) for chADCI and a major peak corresponding to unmodified heavy chain (81 %) and a minor peak to heavy chain with one MMAF linker (19%) for chCE7.

However, when modified N297S chADCI antibodies were reacted with the MMAF linker in the presence of BTG achieved, quantitative coupling was achieved, with a major peak corresponding to heavy chains with one MMAF linker (greater than 90%).

The experiments were repeated using chCE7 Q295N, N297Q antibodies and the MMAF linker using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). The reaction achieved high levels coupling of all heavy chains with one MMAF linker, with a major peak corresponding to heavy chain modified with one C6-DOTA (between 86% and 91 %) and a minor peak corresponding to 9%-14% unmodified heavy chains. Highly favorable reaction conditions were investigated to test whether direct coupling of C6-MMAF linker onto a PNGaseF-deglycosylated mAb could be pushed to completion. Conditions tested were: mAb (1 mg/ml_), 160 equivalent excess of 20mM substrate in DMSO (molar excess based on molarity of the mAb), 6U/ml_ BTGase, 200uL reaction vol., at two incubation durations, either T-i of 40 hours or T₂ of 1 10 hours, in each case at 37°C. Amount of HC + 2 x C6-MMAF could be observed compared to 16h incubation time. No difference between T-i and T₂ were observed for chADCI N297Q, and only a small difference between T-i and T₂ for PNGaseF-deglycosylated antibody. Increasing the incubation time does not push the reaction to completion for PNGaseF-deglycosylated antibodies.

Improved processes for direct coupling of auristatin

A range of processes involving different quantities of BTG and/or linkers were tested in order to develop a process involving lower amounts of cytotoxic drug substrate for direct coupling to antibodies. Briefly, antibody-linker conjugates were formed by quantitative BTG- mediated coupling of the C6-MMAF linker onto chADCI N297S and an anti-CD30 antibody referred to as chSGN35 N297S (each antibody having two acceptor glutamines per antibody) at different conditions: mAb (1 mg/ml_), 80eq., 40eq., 20eq., 10eq. excess of 20mM linker substrate in DMSO, 4U/ml_, 2U/ml_ BTG, 200uL reaction vol., 18.5h incubation time at 37°C. Equivalents (eq) are indicated as molar excess based on molarity of the mAb, thus for N297S antibodies having two acceptor glutamines, 80eq corresponds to 40 times molar excess per acceptor glutamine.

The resulting antibodies were functionalized with C6-MMAF, with only minimal unfunctionalized linker remaining, for all concentrations of BTG when 40eq C6-MMAF were used (i.e. 20 eq of C6-MMAF per acceptor glutamine), while below 40eq C6-MMAF coupling was no longer quantitative. Additionally, 80 equivalents of C6-MMAF yielded close to complete functionalization when 4U/ml or 2U/ml of BTG were used.

Improved linkers for a multi-step process

To explore the ability of a multi-step process to improve BTG coupling, various lysine-based linker comprising a reactive group were generated. The lysine-based linker can be conjugated to an antibody via BTG, followed by reaction of the conjugated antibody with a reagent comprising a reactive group capable of reacting with the reactive group on the lysine-based linker. Various lysine-based linkers were designed to be capable of quantitative coupling onto an antibody by BTG. The linkers lacked cyclic groups, notably polycyclic or macrocyclic groups proximal to the primary amine (site of BTG uptake and coupling). A first linker C2-SAc comprises a lysine based moiety and a protected thiol as reactive group, having the structure as follows.

A further linker C6-SAc comprises a lysine based moiety, a protected thiol as reactive group and an additional linear carbon-comprising framework that acts as a spacer group, and has the structure as follows.

C6-SAC

A further linker PEG-SAc comprises a lysine based moiety, a protected thiol as reactive group and an additional linear carbon-comprising PEG framework that acts as a spacer group, and has the structure as follows.

PEG-SAc

A further linker Azide-PEG4-NH2 comprises a lysine based moiety and spacer group together embodied as a linear carbon-comprising PEG framework, and an azide as reactive group, and has the structure as follows.

Ό ^{v v} O^' Azide-PEG4-NH2

A further linker Alkyne-PEG4-NH2 comprises a lysine based moiety and spacer group together embodied as a linear carbon-comprising PEG framework, and an alkyne as reactive group, and has the structure as follows.

^^O^^/°^^O^^/°^^NH_{2 A}|_kyne.p_EG4._{N H2}

A further linker DBCO-PEG4-NH2 comprises a lysine based moiety and spacer group together embodied as a linear carbon-comprising PEG framework, and as alkyne a dibenzylcyclooctyne (DBCO) as the reactive group, and has the structure as follows.

DBCO-PEG4-NH2

Unmodified chADCI and chADCI N297S antibodies and the various reactive-group- comprising linkers were reacted in the presence of BTG to modify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37°C). Quantitative enzymatic modification chADCI and chADCI N297S heavy chains with each linker by BTG could was observed. Using 6U/ml_ BTG in reaction conditions it was possible to couple the different tested thiol linkers quantitatively and stoichiometrically uniform to the heavy chain of chADCI . Various antibody-bound linkers were then reacted with reaction partners to obtain final compounds. In one series of experiments, antibody- linker conjugates were formed by quantitative BTG-mediated coupling of S-acetyl protected linker C6-SAc onto chADCI , followed by deprotection and reaction with maleimide functionalized toxin. The resulting antibodies were successfully functionalized with toxin, accompanied by a fraction of linkers that were not functionalized. In another series of experiments, antibody-linker conjugates were formed by quantitative BTG-mediated coupling of the Azide-PEG4-NH2 linker onto chADCI N297S, followed by reaction with DBCO-amine. The resulting antibodies were completely/quantitatively functionalized with DBCO-amine, with no unfunctionalized linkers remaining. Improved processes for click-chemistry functionalization

Equivalents of reaction partners for antibodies functionalized with Azide-PEG4-NH2 linker were decreased in order to develop a process involving lower amounts of cytotoxic drug substrate. Briefly, antibody-linker conjugates were formed by quantitative BTG- mediated coupling of the Azide-PEG4-NH2 linker onto chADCI N297S (two glutamines per antibody) and chADCI N297Q (four glutamines per antibody), followed by incubation with reaction partners having complementary reactive groups and a cytotoxic moiety.

DBCO-PEG4-vc-PAB-MMAE (structure shown below) 1 .5 equivalents per acceptor glutamine was reacted with antibody chADCI N297S or N297Q conjugated to the azide- PEG4-NH2 linker at room temperature for three hours, followed by purification by size exclusion chromatography. The drug antibody ratio (DAR) obtained was 2.0 on chADCI N297S and 4.0 for chADCI N297Q.

D B CO- P EG4-VC- PAB- M MAE Two-step procedure for synthesis of PBD-ADC

In order to permit coupling of a PBD at higher DAR and greater homogeneity, a method was tested in which reactive linker (NH2-PEG-N3 spacer, having the structure shown below) was coupled onto an antibody having two acceptor glutamines (one per heavy chain) without organic solvent, followed by functionalization with a compound having a complementary reactive group, cleavable linker and a PBD (DBCO-val-ala -PBD, having the structure shown below). The resulting functionalized acceptor glutamine in the antibody had the structure shown below (mAb-PEG-DBCO-PBD), wherein "mAb" indicates the acceptor glutamine-NH.

NH2-PEG-N3 spacer:

-val-ala -PBD:

-PEG-DBCO-PBD:

Briefly, 5 mg/mL mAb-N297S (mAb) was incubated with 10 equivalents of amino- PEG-azide per site of coupling and 2 U/mL BTG 5h at 37 °C in PBS. mAb-spacer was purified by affinity chromatography on protA.

Functionalized antibody (2 mg/mL in PBS/1 ,2-propane-diol 50/50 v/v) was incubated with 1.25 molar equivalent of derivatized-PBD per site of coupling (i.e. 2.5 equivalents for mAb-N297S). The mixture was incubated for 2-6 h at RT with gentle agitation. Excess of derivatized-PBD was removed by filtration on ZEBA spin desalting column (MWCO=7kDa).

The BTG-mediated coupling of the reactive PEGN3 linker was substantially completely coupled (DAR>1.9). The functionalization with DBCO-val-ala-PBD was also substantially complete, with the final DAR of greater than 1.9 for the antibody-drug conjugate. The compound exhibited purity of 90.4%, with a small amount of high molecular weight product. Molecule DAR

mAb-PEG-Na DAR > 1.9 mAb-PEG-Na-DBCO-PBD DAR > 1.9

Example 2:

Efficacy of ADCs in human cancer PDX mouse model compared to standard of care

A murine patient-derived xenograft (PDX) model of cancer was used to evaluate an antibody binding to the tumor antigen known as major histocompatibility complex class I- related chain A (MICA), conjugated to a DNA minor groove binding agent (PBD) using a two- step process (see Example 1 for two-step process). The model made use of SHO™ mice (Charles River Laboratories, Inc.), a result of intercrossing CrhHA-Prkdcscid and Crl:SKH1 - Hrhr stocks. The resulting animals are homozyogous for the Prkdcscid and the Hrhr mutations and thus exhibit the severe combined immunodeficiency phenotype characteristic of SCID mice and are also hairless.

SHO™ mice (groups of 10) were engrafted with HBCx-5 human breast cancer cells and randomized for treatment intraperitoneally (i.p.) with either bevacizumab (trade name: Avastin™), or isotype control antibody (IC) or chimeric anti-MICA antibody bearing the N297S mutation on a human lgG1 Fc domain conjugated to DNA minor groove binder (PBD) at DAR 2. 48 mice with HBCx-5 subcutaneous growing tumor (P21 .1.2/0) between 40 and 196 mm3 were allocated, according to their tumor volume to give homogenous mean and median tumor volume in each treatment arm. Bevacizumab was administered twice per week at 5 mg/kg body weight for 8 weeks. Isotype control antibody (IC) conjugated to DNA minor groove binder (PBD) at DAR 2 and chimeric anti-MICA conjugated to DNA minor groove binder (PBD) at DAR 2 were each administered at the dosage 0.05 mg/kg once per week for 8 weeks. Tumor volume was evaluated by measuring tumor diameters, with a calliper, biweekly during the whole experimental period. The formula TV (mm3) = [length (mm) x width (mm)2]/2 was used, where the length and the width are the longest and the shortest diameters of the tumor, respectively.

Results showed that the anti-MICA-PBD immunoconjugate showed strong anti- tumoral activity despite a dose of only 0.05 mg/kg once weekly, leading to substantially complete elimination of all tumors. The isotype control-PBD (IC-PBD) was not able to control tumor growth, while bevacizumab showed a partial slowing in growth of tumor volume, and this despite a dose 200 fold higher than that of anti-MICA-PBD. Interestingly, unlike what can be observed with treatment with anti-MICA antibody treatments in which an Fc-competent (e.g. human lgG1 that binds Fc yreceptors) is administered at a dose capable of mediating ADCC towards tumor cells (10 mg/kg), the anti-MICA-PBD immunoconjugates administered at a dose of 0.05 mg/g were highly effective in eliminating tumors.

Example 3: Preparation of Fc-mutated antibodies

A parental antibody chADC2 binding to an antigen expressed at the surface of a variety of solid tumor types was modified by introduction of human VH and VL acceptor frameworks to yield humADC2, verified for retention of binding affinity to the target antigen, and was produced as a human lgG1 in a variety of different variants having different mutations in the heavy chain constant regions that each caused a reduction and/or loss of binding to human Fc receptors while retaining target antigen binding. The VH and Vk sequences of each antibody were cloned into vectors containing the hulgGI CH1 constant domain and the huCk constant domain respectively. The two obtained vectors were co- transfected into the CHO cell line.

The amino acid sequence of the mutated Fc domains for each variant of the humADC2 antibody are shown below:

1. L234F/L235E/P331 S mutation (designated "humADC2-1 "):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L S G V H T F P A V L Q S S G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E F E G G P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A s I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 1 )

2. L234A/L235E/P331 S mutation (designated "humADC2-2"):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P C P A P E A E G G P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 2)

3. L234A/L235E/G237A/A330S/P331 S mutation (designated "humADC2-3"): A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S s G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T C P P c P A P E A E G A P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P S S I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 3)

4. L234A/L235E/G237A/ P331 S mutation (designated "humADC2- 1 "):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D

Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L

Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K

V D K R V E P K S C D K T H T c P P C P A P E A E G A P S V F

L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E

V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V

S V L T V L H Q D W L N G K E Y K C K V S N K A L P A s I E K

T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L

T C L V K G F Y P S D I A V E w E S N G Q P E N N Y K T T P P

V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F s C S V

M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO: 4)

Example 4: Binding to FcvR

Antibodies having the Fc domains of shown in Example 3, as well are comparator antibodies, were evaluated to assess whether they could retain binding to Fey receptors.

SPR (Surface Plasmon Resonance) measurements were performed on a Biacore

T100 apparatus (Biacore GE Healthcare) at 25°C. In all Biacore experiments HBS-EP+ (Biacore GE Healthcare) and 10 mM NaOH, 500mM NaCI served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Recombinant human FcR's (CD64, CD32a, CD32b, CD16a and CD16b) were cloned, produced and purified.

Antibodies tested included: antibodies having wild type human lgG1 domain, antibodies having a human lgG4 domain with S241 P substitution, human lgG1 antibodies having a N297S substitution, human lgG1 antibodies having L234F/L235E/P331 S substitutions, human lgG1 antibodies having L234A L235E/P331 S substitutions, human lgG1 antibodies having L234A/L235E/G237A/A330S/P331 S substitutions, and human lgG1 antibodies having L234A/L235E/G237A/P331 S substitutions. Antibodies were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC/NHS (N-ethyl-N'-(3- dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Antibodies were diluted to 10 μg ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 800 to 900 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Monovalent affinity study was assessed following a classical kinetic wizard (as recommended by the manufacturer). Serial dilutions of soluble analytes (FcRs) ranging from 0.7 to 60 nM for CD64 and from 60 to 5000 nM for all the other FcRs were injected over the immobilized bispecific antibodies and allowed to dissociate for 10 min before regeneration. The entire sensorgram sets were fitted using the 1 :1 kinetic binding model for CD64 and with the Steady State Affinity model for all the other FcRs.

The results are shown in Table 1 , below. Results showed that while full length wild type human lgG1 bound to all human Fey receptors, and human lgG4 in particular bound significantly to FcyRI (CD64) (KD shown in the Table 1 ), the L234A/L235E/G237A/A330S/P331 S substitutions and L234A/L235E/G237A/P331 S substitutions abolished binding to CD64 as well as to CD16a. Example 5: BTG-mediated coupling onto constant regions of Fc-mutated antibodies

Antibodies humADC2-1 , humADC2-2, humADC-3 and humADC2-4 produced in Example 3 were assessed for functionalization by bacterial transglutaminase. Acceptor glutamine at Kabat residue 295 naturally present in the CH2 domain were chosen for evaluation using a small lysine-based linker comprising a reactive group (an azide).

5 mg/mL of each Fc-mutated antibody of Example 3 was deglycosylated with

PNGase F overnight at 37°C. The deglycosylated mAb was then incubated with 20 equivalents of NH₂-PEG-N3 spacer per site of coupling (structure shown below) and 2 U/mL BTG overnight at 37 °C in PBS. The reaction was monitored by LC/MS (ESI-TOF). NH2-PEG-N3 spacer:

N ^^ O NH₂

Results showed that BTG was able to couple the acceptor glutamine at residue 295 substantially completely, obtaining a drug:antibody ratio of 2.0 for each of antibodies humADC2-1 , humADC2-2, humADC-3 and humADC2-4. Results are shown in Figures 15, 16, 17 and 18, respectively. Figure 15 shows LC/MS analysis of the glycosylated starting antibody humADC2-1 (top panel), the deglycosylated antibody humADC2-1 (middle panel), and the antibody humADC2-1 coupled to NH₂-PEG-N3 (one NH₂-PEG-N3 on each acceptor glutamine per heavy chain). Figure 16 shows LC/MS analysis of the glycosylated starting antibody humADC2-2 (top panel), the deglycosylated antibody humADC2-2 (middle panel), and the antibody humADC2-2 coupled to NH₂-PEG-N3 (one NH₂-PEG-N3 on each acceptor glutamine per heavy chain). Figure 17 shows LC/MS analysis of the glycosylated starting antibody humADC2-3 (top panel), the deglycosylated antibody humADC2-3 (middle panel), and the antibody humADC2-3 coupled to NH₂-PEG-N3 (one NH₂-PEG-N3 on each acceptor glutamine per heavy chain). Figure 18 shows LC/MS analysis of the glycosylated starting antibody humADC2-4 (top panel), the deglycosylated antibody humADC2-4 (middle panel), and the antibody humADC2-4 coupled to NH₂-PEG-N3 (one NH₂-PEG-N3 on each acceptor glutamine per heavy chain).

Table 1

Human Fc N297S L234F/ L234A/ L234A/ L234A/ Wild type Human lgG4 receptor KD (nM) L235E/ L235E/ L235E/ L235E/ human antibody

P331 S P331 S G237A/ G237A/ lgG1 with S241 P

KD (nM) KD (nM) A330S/ P331 S antibody KD (nM)

P331 S KD (nM) KD (nM)

KD (nM)

CD64 278 933 1553 No binding No binding 12,74 96,83

CD32a No binding 14250 19900 18190 16790 2075 3218

CD32b No binding 17410 79830 21800 16570 3914 2659

CD16a(F) Low

No binding 35580 No binding No binding No binding 961 ,9

binding

CD16a(V) No binding 8627 9924 No binding No binding 733,7 851 1

CD16b Low

No binding No binding No binding No binding No binding 15020

binding

FcRn 712 627 151 1 714 758 1272 1 176

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents. The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1 . An antibody or antibody fragment comprising an Fc domain of human lgG1 isotype that substantially lacks binding to human Fey receptors human CD16A, CD16B, CD32A, CD32B and CD64, optionally wherein the Fc domain comprises an amino acid substitution at Kabat residue(s) 234, 235, 237, 330 and/or 331 , wherein the antibody or antibody fragment, comprises a functionalized glutamine residue (Q) comprising the structure:

(Q)-L"-Z

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present within or appended to a constant region of the antibody or antibody fragment;

2. The composition of claim 1 , wherein the functionalized glutamine residue (Q) is present within the CH2 domain of the antibody or antibody fragment.

3. The composition of claims 1 or 2, wherein the functionalized glutamine residue (Q) is at heavy chain Kabat position 295 of the antibody or antibody fragment.

4. The composition of claims 1-3, wherein the Fc domain comprises a N297Q substitution and the functionalized glutamine residue (Q) is at heavy chain Kabat position 297 of the antibody or antibody fragment.

5. An antibody or antibody fragment comprising an Fc domain of human γ isotype, wherein the antibody or antibody fragment: (a) binds to a human FcRn protein, (b) substantially lacks binding to human Fey receptor CD64, and (c) comprises a functionalized glutamine residue (Q) at Kabat residue 295.

6. The composition of claim 5, wherein the functionalized glutamine residue (Q) comprises the structure:

(Q)-L"-Z or a pharmaceutically acceptable salt or solvate thereof,

wherein:

7. The composition of any one of the above claims, wherein the antibody or antibody fragments retains binding affinity for a human FcRn protein that is not substantially decreased compared to a wild-type human lgG1 antibody.

8. The composition of any one of the above claims, wherein the Fc domain comprises at least one amino acid substitution at Kabat residue(s) 234, 235 and 237, and at least one amino acid substitution Kabat residues 330 and/or 331.

9. The composition of any one of the above claims, wherein the Fc domain comprises L234A/L235E/P331 S substitutions, L234F/L235E/P331 S substitutions, L234A/L235E/G237A/P331 S substitutions, or L234A/L235E/G237A/A330S/P331 S substitutions.

10. The composition of any one of the above claims, wherein the antibody binds a pre-determined antigen, and wherein the antibody undergoes intracellular internalization when bound to a cell expressing at its surface the antigen.

1 1 . The composition of any one of the above claims, wherein the composition comprises a conditionally-cleavable moiety (V) , wherein V is placed between group L and group Z, optionally wherein V comprises a linker that is cleaved intracellularly, optionally further wherein V comprises a linker that is cleaved by an intracellular peptidase or protease enzyme, optionally, a lysosomal or endosomal protease.

12. The composition of any one of the above claims, wherein the composition further comprises a group a spacer system (Y) comprised of 1 or more spacers, wherein Y is placed between group L and group Z.

13. The composition of any of the above claims, wherein the antibody or antibody fragment comprises a functionalized glutamine residue (Q) comprising the structure comprising the structure:

(Q)-L-RR'-V-Z

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

L is a lysine-based linker in which the nitrogen atom is covalently bonded to the γ carbon of Q as a secondary amine;

V is a conditionally-cleavable moiety; and

14. The composition of any of the above claims, wherein the antibody or antibody fragment comprises a functionalized acceptor glutamine residue having formula II,

(Q)-NH-C-X-L- (V-(Y-(Z)_z)_q)_r (formula II)

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

C is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(0)S-, amine, alkylamine, amide, or alkylamide;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms, optionally, wherein the carbon comprising framework comprises a linear framework of 5 to 30 carbon atoms optionally substituted at one or more atoms,;

r is an integer selected from among 1 , 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1 , 2, 3 or 4; and

V is a conditionally-cleavable moiety;

V is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and

Z is a therapeutic agent.

15. The composition of any one of the above claims, wherein the moiety Z is a cytotoxic agent, optionally a optionally a DNA minor groove binding agent, optionally an auristain, optionally a pyrrolobenzodiazepine.

16. The composition of any one of the above claims, for use in the treatment of cancer.

17. The composition of any one of the above claims, wherein the antigen binding protein is administered in an amount between 0.01 and 0.1 mg/kg body weight, optionally between 0.01 and 0.2 mg/kg body weight, optionally between 0.05 and 0.2 mg/kg body weight.

18. A composition comprising a plurality of immunoconjugates or antibody or antibody fragments covalently bound to a moiety (Z) according to any one of the above claims, wherein the composition is characterized by a ratio of Z moieties to antigen binding protein molecules (drug:antibody ratio) of no more than about 4, optionally from 3.6 to 4.0, or no more than about 2, optionally from 1.5 to 2.5, optionally from 1.8 to 2.0.

19. A composition comprising a plurality of antibodies or antibody fragments of any of the above claims, wherein the plurality of antibodies or antibody fragments share the same amino acid sequence, and wherein at least 90% of the antibodies or antibody fragments in said composition have (m) functionalized amino acid residues (Q) per of antigen binding protein, antibody or antibody fragment, wherein m is an integer selected from 2 or 4.

20. A pharmaceutical composition comprising an antibody or antibody fragment according to any one of the above claims, and a pharmaceutically acceptable carrier.

21 . A method for the treatment or prevention of a disease in an individual, the method comprising administering to said individual an effective amount of a composition of any one of the above claims.

22. The method of claim 21 , wherein said disease is a cancer, optionally wherein said individual has a cancer having an immunosuppressive phenotype, a non-inflammatory phenotype and/or lacking or having low numbers of tumor infiltrating lymphocytes.

23. The composition of any one of claims 21-22, wherein the antibody or antibody fragment is administered in an amount between 0.01 and 0.1 mg/kg body weight, between 0.01 and 0.2 mg/kg body weight, or between 0.05 and 0.2 mg/kg body weight.

24. A method for preparing an antibody or antibody fragment comprising a moiety of interest bound thereto, comprising the steps of:

(a) immobilizing, on a solid support, an antibody comprising an Fc domain of human lgG1 isotype that comprises an acceptor glutamine and lacks binding to human Fey receptors human CD16A, CD16B, CD32A, CD32B and CD64, optionally wherein the Fc domain comprises an amino acid substitution at Kabat residue(s) 234, 235, 237, 330 and/or 331 , thereby providing an immobilized antibody, optionally comprising a step of applying an antibody-containing sample to a solid support;

(b) reacting the immobilized antibody of step (a) with a linking reagent comprising a moiety of interest, in the presence of a TGase, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a moiety-of-interest via a linker.

25. A method for conjugating a moiety of interest (Z) to an antibody, comprising the steps of:

(b) reacting the immobilized antibody of step (a) with a linking reagent comprising a reactive group, in the presence of a TGase, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a reactive group (R) via a linker; and

(c) optionally, reacting (i) the antibody obtained in step b) with (ii) a compound comprising a moiety of interest (Z) and a reactive group (R') capable of reacting with reactive group R, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a moiety of interest (Z) via a lysine-based linker.

26. The method of claims 24-25, further comprising a washing step to remove any unreacted materials.

27. The method of claims 25-25, wherein the antibody comprising an acceptor glutamine linked to a moiety-of-interest via a linker remains immobilized on a solid support and the moiety of interest is a reactive group (R), and

wherein the method further comprises a step of applying a compound comprising a moiety Z and a reactive group R' that is reactive with reactive group (R) to a solid support, to generate an antibody-moiety-of-interest (Z) conjugate.

28. A method for conjugating a moiety of interest (Z) to an antibody, comprising the steps of:

(a) reacting, in solution and in the presence of a TGase:

(i) an antibody comprising an Fc domain of human lgG1 isotype that comprises an acceptor glutamine and lacks binding to human Fey receptors human CD16A, CD16B, CD32A, CD32B and CD64, optionally wherein the Fc domain comprises an amino acid substitution at Kabat residue(s) 234, 235, 237, 330 and/or 331 , with

(ii) a linking reagent comprising a reactive group,

under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a reactive group (R) via a linker;

(b) applying an antibody-containing sample obtained in step (a) to a solid support, thereby immobilizing the antibody comprising an acceptor glutamine linked to a reactive group (R) on a solid support;

(c) reacting,

(i) the immobilized antibody obtained in step b), with

(ii) a compound comprising a moiety of interest (Z) and a reactive group (R') capable of reacting with reactive group R,

under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a moiety of interest (Z) via a lysine-based linker.

29. The method of claim 28, wherein step (b) comprises applying an antibody- containing sample of step (a) to a solid support, wherein the sample comprises TGase enzyme and antibody comprising an acceptor glutamine linked to a reactive group (R) via a linker; optionally further wherein the compound comprising a moiety of interest (Z) and a reactive group (R') is provided in an amount which is less than 10, 5, 4, 2 or 1.5 molar equivalents per acceptor glutamine.

30. The method of claims 27-28, further comprising a step of recovering unreacted compound comprising a moiety Z and a reactive group R' and re-applying said compound to the solid support to provide for higher completion of the reaction between antibody comprising reactive group (R) and compound comprising reactive group (R').

31. The method of claims 27-30, further comprising a step of eluting immobilized antibody conjugates from the solid support to provide antibody conjugate compositions.