EA045916B1 - ANTIBODY CONTAINING GLUTAMINE CONTAINING C-TERMINAL ELONGATION OF LIGHT CHAIN, ITS CONJUGATES, METHODS AND ROUTES OF APPLICATION - Google Patents

Description

Cross reference to related applications

This application claims priority under 35 USC §119(e) to US Provisional Application Serial No. 62/773708, filed November 30, 2018; the disclosure of which is incorporated herein by reference.

List of sequences

Incorporated herein by reference in its entirety is the Sequence Listing entitled 191017_SEQT_13142WOPCT_YC.txt containing SEQ ID NO: 1 through SEQ ID NO: 40, which includes the nucleic acid sequences and/or amino acid sequences disclosed herein. The sequence listing has been provided herein in ASCII text format via EFS-Web and is thus presented in both paper and machine-readable form. The sequence listing was first generated using PatentIn 3.5 on October 26, 2018 and is approximately 14 kb in size.

BACKGROUND OF THE INVENTION

The present disclosure relates to modified transglutaminase enzyme-conjugated antibodies and conjugates derived from such antibodies.

Currently, there is great interest in the type of biological product in which the antibody is covalently linked to a partner molecule (conjugate or immunoconjugate). Thus, the conjugate consists of three components: (1) the antibody, (2) the partner molecule, and (3) a linker that covalently connects the first two components.

The partner molecule may be a therapeutic agent, such as an anticancer drug, an adjuvant, another protein, or a radioisotope. An antibody is an antibody whose antigen is expressed by a target cell or tissue. The antibody, binding to the antigen, serves to deliver the conjugate to the target. Once there, cleavage of the covalent bond or degradation of the antibody releases the therapeutic agent at the target location. Conversely, while the conjugate circulates in the blood system, the therapeutic agent remains inactive due to its covalent bond to the antibody, reducing the risk of side effects. For a review of conjugates in anticancer treatment, see Gerber et al. 2013.

As an alternative to a therapeutic agent, the partner molecule may be an analytical agent for diagnosis, disease detection, or monitoring of a medical condition. In such a case, the analytical agent may be, for example, biotin, a fluorescent label, a radiolabel, or a deuterated polymer. Smith et al. 2019 discloses a conjugate containing a deuterated polymer for MRI imaging. In such a case, cleavage of the linker at the target site is not necessary and may in fact be undesirable. For such applications, the linker may be of a non-cleavable type.

The main step in the preparation of a conjugate is the covalent coupling step, also called the conjugation step. Many methods for performing conjugation have been disclosed. Recently, conjugation mediated by the enzyme transglutaminase (EC 2.3.2.13) has attracted considerable interest.

There are many known variants of transglutaminases, which are either produced naturally by various organisms or created through bioengineering. In the food industry, Streptomyces mobaraensis transglutaminase, produced by fermentation or recombinant expression, is widely used to texturize proteins. As used herein, the term transglutaminase is used generically unless a specific type or source is indicated.

Transglutaminase forms an amide bond between the carboxamide side chain of glutamine (amine acceptor or reciprocal acyl donor) and the ε-amino group of lysine (amine donor or reciprocal acyl acceptor). In terms of specificity, transglutaminase is selective for the glutamine residue, requiring it to be located in the flexible part of the protein loop and flanked by certain amino acids, but is non-selective for the lysine residue, for example, readily accepting the amino group of an alkylenamino compound as a surrogate for the ε-amino lysine. See Fontana et al. 2008.

In a typical transglutaminase-mediated conjugation, the glutamine residue is located on the antibody while the amino group is located on a moiety of the linker partner molecule, as shown below:

~T~ 9 Transglutaminase

Gin—(CH2) ₂ -C-NH ₂ + H ₂ X-[Linker]-[Partner molecule] >.

Antibody “T~O

Ii

Gin—(C H 2) 2' C - N ^- [Linker] - [Partner molecule] In

Conjugate

The location of the glutamine residue in the polypeptide chain significantly affects its availability in

- 1 045916 as an amine acceptor. Typically, none of the glutamine residues in an antibody are accessible, and some modification of the antibody is required to make them accessible. Typically, the antibody is glycosylated at position 297 of asparagine (N297) of the heavy chain (N-linked glycosylation). Jeger et al. 2010 found that deglycosylation of an antibody by eliminating the glycosylation site via N297A substitution or post-translational enzymatic deglycosylation makes the nearby glutamine at position 295 (Q295) available for transamidation by S. mobaraensis transglutaminase. They also showed that the N297Q substitution not only eliminates glycosylation, but also introduces a second glutamine residue (at position 297), which is also an amine acceptor. Thus, simple deglycosylation generates two transglutaminase-active glutamine residues per antibody (one per heavy chain, at position Q295), whereas an antibody with the N297Q substitution generates four such glutamine residues (two per heavy chain, at positions Q295 and Q297). .

In addition to the N297A and N297Q substitutions disclosed by Jeger et al. 2010, there have been other disclosures regarding modification of an antibody or other protein to make it a substrate for transglutaminase.

(a) Strop et al. 2017 and Farias et al. reveal the Fc regions of antibodies constructed using glutamine-containing tags, such as LLQGG, LSLSQG, GGGLLQGG, GLLQG, etc., while the glutamine in the tag can act as an amine acceptor and be located at various places in the heavy or light chain of the antibody, including their carboxy ends.

(b) Chen et al. 2005 discloses a modification of a protein with a QSKVX tag, where X is L or I, and this protein can then be conjugated to a transglutaminase.

(c) Fischer et al. 2015 discloses the inclusion of glutamine ((9)-containing a label of the formula (Q)-NH-(C)-XL-(V-(Y-(M or Z) _z )q) _r in an antibody fragment not containing an Fc domain (d) Rao-Naik et al 2018 discloses the addition of glutamine-containing C-terminal heavy chain extensions to an antibody to render it transglutaminase responsive.

In an approach complementary to modifying an antibody to make it active against transglutaminase, Rao-Naik et al. 2017 disclose modification of transglutaminase to make it capable of conjugating to a wild-type antibody.

Substrate specificity studies of transglutaminase have also been carried out using small peptide-containing molecules: Ando et al. 1989, Kamiya et al. 2011, Ohtsuka et al. 2000.

Other disclosures regarding the conjugation of antibodies or other proteins using transglutaminase: Bregeon 2016, Bregeon et al. 2016, Bregeon et al. 2017, Dennler et al. 2014, Innate Pharma 2013, Lin et al. 2006, Mero et al. 2009, Mindt et al. 2008, Sato 2002, Sato et al. 2001, Schibli et al. 2007, and Sugimura et al. 2007.

It is also known to attach cysteine-containing terminal extensions to an antibody to achieve conjugation via a Michael addition reaction to the maleimide group. Liu et al. 2014 disclose the addition of such extensions to the C-terminus of the heavy chain. Babcook et al. 2017 disclose the addition of such extensions to the C-terminus of the light chain.

Complete references to the documents cited herein, with first author or inventor and year of issue, are listed at the end of this specification.

Brief Disclosure of the Present Invention

The location of the conjugation site can affect the stability and pharmacokinetics of the conjugate. Strop et al. 2013. Thus, it is desirable to provide alternative conjugation sites and conjugate structures to diversify the options available for biologics development.

Disclosed herein are antibodies having C-terminal glutamine-containing extensions on the light chain for conjugation with transglutaminase. In accordance with one embodiment, a full-length antibody is provided having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO:40.

In another aspect, provided herein is a conjugate of formula (IV) ⁰ s Ab-CNLD ( ^IV ) wherein the Ab is a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension comprising an amino acid sequence selected from the group , consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO : 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24 , NO: 25, NO: 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO : 39 and NO: 40;

- 2 045916

L is a linker moiety that binds Ab via an amide bond

with glutamine in glutamine-containing extension; And

D is selected from the group consisting of a protein, a radioisotope, an analytical agent and a therapeutic agent.

In another aspect, provided herein is a method of producing an antibody conjugate comprising the steps of (a) mixing a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13 , NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO : 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40 , with an amine donor compound containing a primary amine and a moiety selected from the group consisting of a protein, a radioisotope, an analyte and a therapeutic agent, in the presence of transglutaminase; and (b) allowing transglutaminase to catalyze the formation of an amide bond between the glutamine side chain carboxamide of the glutamine-containing extension and the primary amine of the amine donor compound, thereby resulting in the formation of an antibody conjugate.

In another aspect, provided herein is a method of producing an antibody conjugate comprising the steps of (a) mixing a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13 , NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO : 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40 , with a first compound, wherein the first compound is an amine donor compound having a primary amine and a first reactive functional group, in the presence of transglutaminase;

(b) allowing transglutaminase to catalyze the formation of an amide bond between the glutamine side chain carboxamide of the glutamine-containing extension and the primary amine of the first compound, forming an adduct of the antibody and the first compound;

(c) contacting the adduct with a second compound having a second reactive functional group and a moiety selected from the group consisting of a protein, a radioisotope, an analyte, and a therapeutic agent; wherein the second reactive functional group is capable of reacting with the first reactive functional group to form a covalent bond therebetween; and (d) allowing the first and second reactive functional groups to react and form a covalent bond between them, thereby resulting in the formation of an antibody conjugate.

If the fragment (in the first connection or second connection, as appropriate) is a protein, the resulting conjugate is a fusion protein. If the fragment is a radioisotope, the resulting conjugate can be used for radiation therapy or radioimaging. The moiety may be an analytical agent such as a fluorescent tag, a deuterated polymer, or a ligand-like biotin, in which case the conjugate may be used for medical condition diagnosis, treatment monitoring, or analytical applications. Preferably, the fragment is a therapeutic agent (in which case the product is also called an antibody-drug conjugate or ADC) that can be used in various treatments, especially in the treatment of cancer.

Brief description of drawings

In fig. 1 is a schematic diagram of an antibody light chain having a C-terminal extension (SEQ ID NO: 1) as disclosed herein.

In fig. 2 presents a comparison of one- and two-step methods for producing conjugates using transglutaminase (BTG).

Detailed Disclosure of the Present Invention Definitions

The term antibody means whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single-chain variants thereof. A complete antibody is a protein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region containing three domains, _CH1 , _CH2 and _CH3 . Each light chain contains

- 3 045916 has a light chain variable region (V _L or V _k ) and a light chain constant region containing one single domain, C _L . The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), with insertions of more conserved framework regions (FRs). Each VH and VL contains three CDRs and four FRs, located from the amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Variable regions contain a binding domain that interacts with the antigen. Constant regions can mediate binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. An antibody is said to specifically bind to antigen X if the antibody binds to antigen X with a KD of 5 x 10 -8 M or less, more preferably 1 x 10 ^-8 M or less, more preferably 6 x 10 ^-9 M or less, more preferably 3 x 10 ^-9 M or less, even more preferably 2x10 ^-9 M or less. The antibody may be chimeric, humanized, or preferably human. The heavy chain constant region can be designed to affect the type or extent of glycosylation, increase the half-life of the antibody, increase or decrease interactions with effector cells or the complement system, or modulate certain other properties. Design can be accomplished by replacing, adding or deleting one or more amino acids, or replacing a domain with a domain from another type of immunoglobulin or a combination of the above.

Antigen-binding fragment and antigen-binding portion of an antibody (or simply antibody portion or antibody fragment) mean one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody, such as (i) the Fab fragment, a monovalent fragment consisting of the _VL , VH, CL and _CH1 domains; (ii) fragment F(ab') ₂ , a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (see, for example, Abbas et al., Cellular and Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) Fd fragment consisting of VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of one arm of the antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a _VH domain; (vii) dedicated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing one variable domain and two constant domains. Preferred antigen binding fragments are Fab, F(ab') ₂ , Fab', Fv and Fd fragments. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be joined using recombinant methods using a synthetic linker that allows them to exist as a single protein chain, in which the V _L and V _H regions combine in pairs to form monovalent molecules (known as single-chain Fv or scFv); see, for example, Bird et al. (1988) Science 242:423426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883/ Such single chain antibodies are also encompassed by the term antigen binding portion of an antibody.

Unless otherwise indicated, for example by reference to linear numbering in SEQ ID NO:, references to the numbering of amino acid positions in the variable region of the heavy or light chain of an antibody (VH or VL) correspond to the Kabat system (Kabat et al., Sequences of proteins immunological interest, 5th ed., Pub. No. 91-3242, US Dept. Health & Human Services, NIH, Bethesda, Md., 1991, hereinafter Kabat) and references to the numbering of amino acid positions in the constant region of the heavy or light chain of the antibody ( C _H1 , C _H2 , C _H3 or C _L ) correspond to the EU index as set out according to Kabat. See Lazar et al., US 2008/0248028 A1, the disclosure of which is incorporated herein by reference. In addition, the ImMunoGeneTics Information System (IMGT) provides a table on its website called IMGT Scientific Chart: Correspondence between C Numberings, which shows the correspondence between its numbering system, EU numbering, and Kabat numbering for the heavy chain constant region.

The term isolated antibody means an antibody that is substantially free of other antibodies having a different antigen specificity (eg, an isolated antibody that specifically binds antigen X is substantially free of antibodies that specifically bind antigens other than antigen X). However, an isolated antibody that specifically binds antigen X may be cross-reactive with other antigens, such as antigen X molecules from other species. In certain embodiments, the isolated antibody specifically binds to human X antigen and does not cross-react with other (non-human) X antigens. Moreover, the isolated antibody may essentially be free of other cellular material and/or chemicals.

Monoclonal antibody or monoclonal antibody composition means a preparation of antibody molecules with a single molecular composition that exhibits a single binding specificity and affinity for a particular epitope.

Human antibody means an antibody having variable regions in which both

- 4 045916 the casing region and CDR regions (and constant region, if present) are derived from human germline immunoglobulin sequences. Human antibodies may include later modifications, including natural or synthetic modifications. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced randomly or by site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term human antibody does not include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, are grafted onto human framework sequences.

Human monoclonal antibody means an antibody exhibiting a single binding specificity that has variable regions in which both the framework region and the CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that contains a B cell derived from a transgenic non-human animal, for example, a transgenic mouse, the genome of which contains a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

Embodiments

Typically, transglutaminase-mediated preparation of an antibody conjugate can be one-step or two-step, as schematically shown in FIG. 2. In a one-step process, transglutaminase binds an extended glutamine carboxamide, acting as an amine acceptor, and an amine donor compound H ₂ N-LD, where L is a linker moiety and the partner molecule D is a protein, radioisotope, analyte, or therapeutic agent , with direct formation of the conjugate. In a two-step process, transglutaminase catalyzes the formation of an initial transamidation adduct between the glutamine carboxamide in the extension, which acts as an amine receptor, and the first compound (H ₂ N-L'-R'), which is the amine donor compound, where L' represents the first linker moiety and R' represents the first reactive functional group. The adduct is then reacted with a second compound (RLD), wherein R is a second reactive functional group capable of reacting with R, L is a second linker moiety, and D is as defined above. Sometimes a one-step process is called an enzymatic process and a two-step process a chemoenzymatic process because it includes both a chemical and an enzymatic step. Each of L, L' and L may represent an alkyl chain -(CH ₂ ) _Ш -, where m represents equals an integer from 2 to 10 inclusive, or may represent, especially in the case of L and L, a more complex structure such as described below.

The amine donor, H2N-LD or H ₂ N-L'-R', is often used in large excess to suppress unwanted transamidation between the glutamine carboxamide and the ε-amino group of the antibody lysine. If fragment D is expensive or difficult to obtain, using a large excess may not be practical. In such cases, a two-step process may be preferred, even if it requires an additional step.

As a demonstration, an anti-mesothelin antibody having the same heavy and light chain CDRs as antibody 6A4 from Terrett et al, US 8268970 B2 (2012) was conjugated. Its heavy and light chain sequences are shown as SEQ ID NO: 27 and SEQ ID NO: 28, respectively. C-terminal light chain extensions having the sequences described below were attached, and the thus modified antibodies were then conjugated to either compound (A) or compound (B) as an amine donor using a one-step process.

[THK7 agonist]-[Self-cleaving group] - N

Me*^CO ₂ H

Me Me

In compound (A), the therapeutic agent is a Toll-like receptor 7 (TLR7) agonist, which can be used as an adjuvant for vaccines and immunotherapies in the treatment of various conditions. Examples of TLR7 agonists are disclosed in US series applications

- 5 045916 No. 16/103210, 16/103511, 16/103581, 16/013601 and 16/103619; each filed August 14, 2018; the disclosures of which are incorporated herein by reference. In compound (B), the therapeutic agent is a tubulisin analogue, a cytotoxin that can be used in the treatment of cancer. The preparation of compound (B) is described in US provisional application Serial No. 62/688737, filed June 22, 2018; the disclosure of which is incorporated herein by reference.

In accordance with one embodiment, the C-terminal light chain extensions disclosed herein comprise one glutamine. Examples are listed in table. And below along with the drug-antibody ratio (DAR) of the conjugates obtained from them. Since there are two light chains per antibody, each of which is extended, the theoretical maximum DAR is 2.0. In fig. 1 is a schematic representation of an antibody light chain having a C-terminal extension of SEQ ID NO: 1.

Table A - Extensions with Glutamine Alone SEQ Ш NO: Subsequence elongation DAR (connection A) DAR (connection B) 1 GGVLQRAS 1.99 2.0 2 GGVLQGAS 1.84 1.6 3 GGVLQRPS 1.96 2.0 4 GGVLQGPS 1.97 2.0 5 GGVLQSPS 1.94 1.77 6 GGVLQYAS 1.87 1.7 7 GGGGVLQRAS 1.99 2.0 8 GGGGVLQGAS 1.95 2.0 9 GGGGVLQRPS 1.99 2.0 10 GGGGVLQGPS 1.94 2.0 AND GGGGVLQSPS 1.99 2.0 12 GGGGVLQYAS 2 2.0 13 VLQYAS 0.95 0.97

In another embodiment, the C-terminal light chain extension contains two glutamines. Examples are listed in table. Below along with the drug-antibody ratio (DAR) of the conjugates derived from them.

Since there are two light chains per antibody, each extended with two glutamines, the theoretical maximum DAR is 4.0.

- 6 045916

Table B - Extensions with two glutamines SEQ Ш NO: Subsequence elongation DAR (connection A) DAR (connection B) 14 GGVLQRQS 2.37 2.05 15 GGVLQGQS 2.04 2.04 16 GGVLQRQRPS 2.23 2.01 17 GGVLQGQGPS 3.27 3.19 18 GGVLQSQSPS 1.86 2.01 19 GGVLQYQS 2.90 2.60 20 GGGGVLQRQS 2.95 2.88 21 GGGGVLQGQS 2.60 2.89 22 GGGGVLQRQRPS 2.96 2.88 23 GGGGVLQGQGPS 3.72 3.71 24 GGGGVLQSQSPS 2.37 2.84 25 GGGGVLQYQS 3.32 3.58 26 VLQYQS 1.95 2.67 29 GGVLQVLQS h,o thirty GGVLQGVLQS 3.8 31 GGVLQGGVLQS 3.4 32 GGVLQVLQGPS 2.5 33 GGVLQGGQGPS h,o 34 GGVLQGGGQGPS 3.1 35 GGGGVLQVLQS 3.2 36 GGGGVLQGVLQS 4.0 37 GGGGVLQGGVLQS 4.0 38 GGGGVLQVLQGPS 3.2 39 GGGGVLQGGQGPS 3.5 40 GGGGVLQGGGQGPS z, z

It is possible that in C-terminal extensions containing two glutamines, it is useful to separate the glutamines from each other further than as they are presented in Table 1. B, inserting between two and four amino acids.

Since the C-terminus of the light chain is somewhat hidden, it is desirable to provide spacers so that the glutamine in the extension is more protruding and accessible to transglutaminase. This effect can be achieved by using multiple glycines (two to four) at the N-terminus of the extension. Referring to the table. And, it can be seen that extensions without glycines (SEQ ID NO: 13) result in a lower DAR than those with two or four glycines.

In accordance with one embodiment, the antibody has a C-terminal light chain extension containing two glycine spacers and one glutamine. Examples of such extensions are provided under SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5 and NO: 6.

In another embodiment, the antibody has a C-terminal light chain extension containing four glycine spacers and one glutamine. Examples of such extensions are provided under SEQ ID NO: 7, NO: 8, NO: 9, NO: 10, NO: 11 and NO: 12.

In another embodiment, the antibody has a C-terminal light chain extension containing two glycine and two glutamine spacers. Examples of such extensions are provided under SEQ ID NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33 and NO: 34.

In another embodiment, the antibody has a C-terminal light chain extension containing four glycine and two glutamine spacers. Examples of such extensions are provided under SEQ ID NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40.

Preferably, the C-terminal light chain extensions contain a valine-leucine (VL) dipeptide on the N-terminal side of glutamine.

Antibodies that can be modified and conjugated by the methods described in this disclosure include antibodies that recognize the following antigens: mesothelin, prostate specific membrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H3, B7H4 (also known as O8E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucoside-GMi, CTLA4 and CD44. The antibody may be animal (eg, murine), chimeric, humanized, or preferably human. The antibody is preferably a monoclonal antibody, especially a human monoclonal antibody

- 7 045916 scrap. The production of human monoclonal antibodies to some of the above antigens is disclosed in Korman et al., US 8609816 B2 (2013; B7H4, also known as 08E; in particular, antibodies 2A7, 1G11 and 2F9); Rao-Naik et al, 8097703 B2 (2012; CD19; specifically antibodies 5G7, 13F1, 46E8, 21D4, 21D4a, 47G4, 27F3 and 3C10); King et al., US 8481683 B2 (2013; CD22; in particular antibodies 12C5, 19A3, 16F7 and 23C6); Keler et al., US 7387776 B2 (2008; CD30; in particular antibodies 5F11, 2H9 and 17G1); Terrett et al., US 8124738 B2 (2012; CD70; in particular antibodies 2H5, 10B4, 8B5, 18E7 and 69A7); Korman et al., US 6984720 B1 (2006; CTLA-4; specifically antibodies 10D1, 4B6 and 1E2); Vistica et al., US 8,383,118 B2 (2013, fucosyl-GM1, in particular antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5) Korman et al., US 8008449 B2 (2011; PD-1; in particular antibodies 17D8, 2D3, 4Н1, 5С4, 4А11, 7D3 and 5F4); Huang et al., US 2009/0297438 A1 (2009; PSMA, in particular antibodies 1C3, 2A10, 2F5, 2C6); Cardarelli et al., US 7875278 B2 (2011; PSMA; in particular antibodies 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3); Terrett et al., US 8,222,375 B2 (2012; PTK7; specifically antibodies 3G8, 4D5, 12C6, 12C6a and 7C8); Terrett et al., US 8680247 B2 (2014; glypican-3; specifically antibodies 4A6, 11E7 and 16D10); Harkins et al., US 7335748 B2 (2008; RG1; specifically antibodies A, B, C and D); Terrett et al., US 8268970 B2 (2012; mesothelin; in particular antibodies 3C10, 6A4 and 7B1); Xu et al., US 2010/0092484 A1 (2010; CD44; in particular antibodies 14G9.B8.B4, 2D1.A3.D12 and 1A9.A6.B9); Deshpande et al., US 8258266 B2 (2012; IP10; in particular, antibodies 1D4, 1E1, 2G1, 3C4, 6A5, 6A8, 7C10, 8F6, 10A12, 10A12S and 13C4); Kuhne et al., US 8450464 B2 (2013; CXCR4; in particular antibodies F7, F9, D1 and E2); and Korman et al., US 7,943,743 B2 (2011; PD-L1; specifically, antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4); the disclosures of which are incorporated herein by reference.

With respect to conjugates of formula (IV) ⁰ and Ab-CNLD ( ^IV ) (a) D is preferably, in accordance with one embodiment, a cytotoxic drug.

(b) In another preferred embodiment, D is a TLR7, STING, NRLP3 or RIG-1 agonist.

(c) In a preferred embodiment, L is co6oy-( _CH2 ) _2-6 .

(d) According to another preferred embodiment, L is o < ^|a '>

where T represents a self-cleaving group;

t is 0 or 1;

AA ^a and each AA ^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine , lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;

p is 1, 2, 3 or 4;

q equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and r equals 1, 2, 3, 4 or 5.

One- and two-step conjugation processes are discussed in more detail below.

According to a preferred embodiment, the amine donor compound in the one-step process is represented by formula (I):

H ₂ N-(CH ₂ ) ₂ . ₆ D (I) where D represents a protein, radioisotope, analytical agent or therapeutic agent.

According to another preferred embodiment, the amine donor compound for the one-step process has the structure represented by formula (Ia):

o < ^|a >

where D is a protein, radioisotope, analytical agent or therapeutic agent;

T represents a self-cleaving group;

t is 0 or 1;

AA ^a and each AA ^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine , lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, trip

- 8 045916 tophan, tyrosine and valine;

p is 1, 2, 3 or 4;

q equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and r equals 1, 2, 3, 4 or 5.

In formulas (Ia), (Ia') and (III) (below), -AA ^a -[AA ^b ] _p - is a polypeptide whose length is determined by the value of p (dipeptide if p is 1, tetrapeptide if p is 3 etc.). AA ^a is at the carboxyl terminus of the polypeptide, and its carboxyl group forms a peptide (amide) bond with the amine nitrogen D (or T, if present). In contrast, the last AA ^b is at the amino terminus of the polypeptide, and its α-amino group forms a peptide bond with

Preferred -AA ^a -[LL ^b ]p- polypeptides are ValCit, Val-Lys, Lys-Val-Ala, Asp-ValAla, Val-Ala, Lys-Val-Cit, Ala-Val-Cit, ValGly, ValGln and Asp -Val-Cit, written in the normal N-to-C direction, as in H2NValCitCO2H. More preferably, the polypeptide is Val-Cit, Val-Lys or Val-Ala. Preferably, the AA polypeptide ^a [AA ^b ]p is cleaved by an enzyme found within the target cell, for example cathepsin and especially cathepsin B, or by an enzyme in the environment of the target organ or tissue.

If the q index is different from 0, compound (Ia) contains a poly(ethylene glycol) (PEG) group, which can advantageously improve the solubility of compound (Ia) by facilitating conjugation with the antibody, a step that is carried out in an aqueous environment. In addition, the PEG group can serve as a spacer between the antibody and the peptide -AA ^a -[AA ^b ] _p - such that the bulk of the antibody does not sterically interfere with the action of the enzyme that cleaves the peptide.

As indicated by the subscript, if t is 0 or 1, the self-cleaving group T is not necessarily present. A self-cleaving group is a group whose cleavage from AA ^a or AA ^b , as the case may be, initiates a sequence of reactions whereby the self-cleaving group separates from D and frees the latter to perform its therapeutic function. If present, a self-cleaving T group is preferably a paminobenzyloxycarbonyl (PABC) group, the structure of which is shown below with an asterisk (*) indicating the end of PABC bound to the amine nitrogen of drug D and a wavy line indicating the end bound to the polypeptide -AA ^a -[AA ^b ] _p -. The RABC group may be substituted as disclosed in US Provisional Application Serial No. 62/677307, filed May 29, 2018.

Another self-cleaving group that can be used is a substituted thiazole, as described in Feng, US 7375078 B2 (2008).

Compounds (A) and (B) illustrate the construction of amine donor compounds according to formula (Ia) with the arrangement of their various elements as shown. Compound (A), but not compound (B), contains a self-cleaving group.

[THK7 agonist]-[Self-cleaving group] -N ν n °oA

Me^

H • N^NH ₂

ABOUT

N g (A) nh ₂ 'Me

H

Therapeutic Self-cleaving agent group

Cleavable Alkylamine Donor Peptide

nh ₂

Therapeutic agent

Me Me

Cleavable Alkylamine Donor Peptide (B)

In two-step conjugation, many combinations of R' and R groups can be used. Suitable combinations of R' and R (or, alternatively, R and R') include:

- 9 045916 (a) a maleimide group and a sulfhydryl group to form a Michael addition adduct, as in

(b) a dibenzocyclooctyne group and an azide group to form a cycloaddition product via click chemistry as in

(c) N-hydroxysuccinimide ester and amine to form an amide as in

(d) an aldehyde or ketone (where alkyl is preferably Ozalkyl) and hydroxylamine to form an oxime, as in

Thus, R' can be selected from while reciprocally R can be selected from

A suitable first amine donor compound for the two-step process is depicted in formula (II)

H ₂ N-(CH2) ₂ -8-R' (II) where R' is defined above and is preferably

A corresponding suitable RLD compound is shown in formula (III) o _R ' - _(CH2)r 4o^ ^L[AAb]p “ ^AAa “ ^{(T)rD (111)} where R' is defined above and is preferably

and r, q, AA ^b , p, AA ^a , T, t and D are defined above in accordance with formula (Ia).

In the case where the conjugate is intended for use in the treatment of cancer, the therapeutic

- 10 045916 the agent may be a cytotoxic drug that causes the death of the target cancer cell. Cytotoxic drugs that can be used in conjugates include the following types of compounds, their analogs and derivatives:

(a) enediynes such as calicheamicin (see, for example, Lee et al, J. Am. Chem. Soc. 1987, 109, 3464 and

3466) and uncialamycin (see, for example, Davies et al., WO 2007/038868 A2 (2007); Chowdari et al., US 8709431 B2 (2012); and Nicolaou et al., WO 2015/023879 A1 (2015) );

(b) tubulisins (see, for example, Domling et al., US 7778814 B2 (2010); Cheng et al., US 8394922 B2 (2013); and Cong et al., US 8980824 B2 (2015));

(c) DNA alkylators such as CC-1065 analogues and duocarmycin (see, for example, Yang et al., US 2018/0051031 A1 (2018); Boger, US 65458530 B1 (2003); Sufi et al., US 8461117 B2 (2013); and Zhang et al., US 8852599 B2 (2014));

(d) epothilones (see, for example, Vite et al., US 2007/0275904 Al (2007) and US RE42930 E (2011));

(e) auristatins (see, for example, Senter et al., US 6844869 B2 (2005), and Doronina et al., US 7498298 B2 (2009));

(f) benzodiazepine dimers (see, for example, Zhang et al., US 9527871 B2 (2016); Zhang et al., US 9688694 B2 (2017); McDonald et al., US 9526801 B2 (2016); Howard et al., US 2013/0059800 A1 (2013); US 2013/0028919 A1 (2013); and WO 2013/041606 A1 (2013)); and (g) maytansinoids such as DM1 and DM4 (see, for example, Chari et al., US 5208020 (1993), and Amphlett et al., US 7374762 B2 (2008)).

In one embodiment, the cytotoxic drug is a DNA alkylator, tubulisin, auristatin, benzodiazepine dimer, enediyne, or maytansinoid. Specific examples are provided by way of illustration and not limitation.

The immune system has receptors whose natural ligands are pathogen-associated molecular patterns (PAMs). Binding of PAMP to its cognate receptor

- 11 045916 activates the immune system to protect against infection caused by an associated pathogen. In addition, these receptors can also be activated by synthetic agonists, which have an adjuvant effect on the action of vaccines and immunotherapies in the treatment of a variety of conditions other than the actual pathogenic infection. Immuno-oncology agents such as ipilimumab, nivolumab and pembrolizumab in particular may benefit from this adjuvant effect. Receptors that can be activated by synthetic agonists include TLR3, TLR7, TLR9 (Toll-like receptor-3, -7, and -9, respectively), STING (gene stimulator; also known as MPYS, TMEM173, MITA, or ERIS), NLRP3 ( NOD-like receptor protein 3) and RIG-I (retinoic acid-inducible gene I). Thus, according to an alternative embodiment, the therapeutic agent is a TLR3, TLR7, TLR9, STING, NLRP3, or RIG-I agonist. In particular, the therapeutic agent may be a TLR7 agonist, as disclosed in Poudel et al., US 2019/0055243 A1 (2019); Young et al., US 2019/0055244 A1 (2019); Poudel et al., US 2019/0055245 A1 (2019); He et al., US 2019/0055246 A1 (2019); He et al., US 2019/0055247 A1 (2019); and Purandare et al., PCT application PCT/US2019/028697, filed April 23, 2019.

The above-mentioned therapeutic agents can be used in conjugates obtained using either a one-step or two-step method.

Examples

The practical application of the present invention can be further understood by reference to the following examples, which are provided by way of illustration and not limitation.

Example 1 - Preparation of modified antibodies

ExpiCHO cells were used for antibody expression. ExpiCHO cells were grown in ExpiCHO medium at 37°C, 8% CO2 atmosphere in plastic flasks. The heavy chain and light chain expression vector were mixed in OptiMEM, the PEI solution in OptiMEM was added, and the resulting PEI/DNA complex was incubated at room temperature for 30 minutes before adding to the cells. 24 hours after transfection, Feed B and VPA were added to the cells and the cells were stored at 37°C with agitation. After 7 days, the culture supernatant was collected by centrifugation and filtration. The supernatant was purified with MabSelect SuRe LX resin. Briefly, cell supernatant was incubated with the resin overnight at 4°C, washed with 10 CV PBS followed by elution with 4 CV 0.1 M citrate pH 3.5, immediately neutralized with 1/10 volume of 1 M Tris pH 8.

Example 2 - Conjugation of Modified Antibodies

Conjugation of an antibody modified as described herein to an amine donor using transglutaminase was carried out according to the protocol specified below. We used a BTGase-activating dispase with point mutations V65I and Y75F. It was dialyzed in 50 mM sodium acetate pH 5.5 from the formulation (buffer 20 mM acetate, 10% glycerol pH 4) before use.

Antibody at a concentration of ~2 mg/ml in 50 mM Tris-HCl, pH 8.0, or 20 mM histidine, 50 mM imidizaol, 10% sucrose, pH ~ 7.8 reacted with a 10-fold molar excess at the donor site amine in the presence of a 0.2 molar excess of transglutaminase per antibody. The reaction proceeded overnight at 37°C with continuous gentle stirring.

The antibody-drug conjugate was filtered through 0.2 μm and purified using a mAb Select SuRe™ column (GE Healthcare). The conjugate was loaded onto a column pre-equilibrated with 50 mM Tris-HCl, pH 8.0, and washed with 10 CV (column volume) of equilibration buffer, followed by 10 CV of 50 mM Tris-HCl, 17% acetonitrile, pH 8.0, to remove unreacted amine donor. The column was re-equilibrated with 50 mM Tris-HCl, pH 8.0, before eluting with 0.1 M citrate, pH 3.5, in 1 ml fractions and neutralized with 1/10 of the elution volume with 1 M Tris, pH 8.0. The desired fractions were dialyzed into 20 mM histidine, 10% sucrose, pH 6.0 preparation buffer and analyzed by LC-MS (ESI-QTOF), RP-HPLC and SDS-PAGE to determine purity and drug-antibody ratio (DAR) ).

Example 3 - Conjugated Modified Antibody Assay

Antibody at a concentration of 5 mg/ml in 50 mM imidazole, 10% sucrose, pH 8, reacted with a 10-fold molar amount at the amine donor site in the presence of a 0.2 molar excess of recombinant bacterial transglutaminase per antibody. After overnight incubation at 37°C with continuous gentle stirring, the reaction mixture was analyzed by LC-MS (ESIQTOF) to evaluate DAR.

The foregoing detailed description of the present invention includes portions that primarily or exclusively relate to specific parts or aspects of the present invention. It should be understood that this is for clarity and convenience, that a particular feature may relate to more than just the portion in which it is disclosed, and that the present disclosure herein includes all relevant combinations of information found in the various portions. Likewise, while the various figures and descriptions herein refer to specific embodiments of the present invention, it is to be understood that where a particular feature is disclosed in the context of a particular drawing or embodiment, such feature

- 12 045916 may also be used to an appropriate extent in the context of another drawing or embodiment in combination with another feature or in the present invention as a whole.

Moreover, although the present invention has been specifically described in terms of certain preferred embodiments, the present invention is not limited to such preferred embodiments. Preferably, the scope of the present invention is defined by the appended claims.

Literary sources

The complete references to the following references, cited in abbreviated form by the first author (or inventor) and dated earlier in this specification, are provided below. Each of these references is incorporated herein by reference for all purposes.

Ando et al., Agri. Biol. Chem. 1989, 53, 2613, Purification and Characteristics of a Novel Transglutaminase Derived from Microorganisms.

Babcook et al., US 2017/0008970 (2017).

Bregeon, US 2016/0114056 A1 (2016).

Bregeon et al., US 9,427,478 B2 (2016).

Bregeon et al, US 9,717,803 B2 (2017).

Chen et al., US 2005/0136491 A1 (2005).

Dennler et al., Bioconjug. Chem. 2014,25, 569, Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates.

Farias et al., US 2016/0193356 A1 (2016).

Fischer et al., US 2015/0284713 A1 (2015).

Fontana et al., Adv. Drug Deliv. Rev. 2008, 60, 13, Site-Specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase.

Gerber et al., Nat. Prod. Rep. 2013, 30, 625, The antibody-drug conjugate: an enabling modality for natural product-based cancer therapies.

Innate Pharma, A New Site Specific Antibody Conjugation Using Bacterial Transglutaminase, presentation at ADC Summit, San Francisco, California, Oct. 15, 2013.

Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995, Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase.

Kamiya et al., US 2011/0184147 A1 (2011).

Lin et al., J. Am. Chem. Soc. 2006, 128, 4542, Transglutaminase-Catalyzed Site-Specific Conjugation of Small-Molecule Probes to Proteins in Vitro and on the Surface of Living Cells.

Liu et al., US 8,865,875 B2 (2014).

Mero et al., Bioconjug. Chem. 2009, 20, 384, Transglutaminase-Mediated PEGylation of Proteins: Direct Identification of the Sites of Protein Modification by Mass Spectrometry Using a Novel Monodisperse PEG.

Mindt et al, Bioconjug. Chem. 2008, 19, 271, Modification of Different IgG1 Antibodies via Glutamine and Lysine using Bacterial and Human Tissue Transglutaminase.

Ohtsuka et al., Biosci. Biotechnol Biochem. 2000, 64, 2608, Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl Donors.

Rao-Naik et al., WO 2017/059158 A1 (2017).

Rao-Naik et al., US 2018/0037921 A1 (2018).

Sato, Adv. Drug Deliv. Rev. 2002, 54, 487, Enzymatic procedure for site-specific pegylation of proteins.

Sato et al., US 6,322,996 B1 (2001).

Schibli et al., US 2007/0184537 A1 (2007).

Smith et al., US 2019/0099505 A1 (2019).

Strop et al., Chemistry & Biology 2013, 20, 161, Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates.

Strop et al., US 9,676,871 B2 (2017).

Sugimura et al., J. Biotechnol. 2007, 131, 121, Novel site-specific immobilization of a functional protein using a preferred substrate sequence for transglutaminase 2.

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Claims (23)

CLAIM 1. A full-length antibody having at the C-terminus of its light chain a glutamine-containing extension containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40. 2. The full-length antibody of claim 1, wherein the extension has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5 and NO: 6. 3. The full-length antibody of claim 1, wherein the extension has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, NO: 8, NO: 9, NO: 10, NO: 11 and NO: 12. 4. The full-length antibody of claim 1, wherein the extension has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, NO: 15, NO: 16, NO: 17, NO: 18 and NO: 19. 5. The full-length antibody of claim 1, wherein the extension has an amino acid sequence selected from the group consisting of SEQ ID NO: 20, NO: 21, NO: 22, NO: 23, NO: 24 and NO: 25. 6. The full-length antibody of claim 1, wherein the glutamine-containing extension has a valine-leucine (VL) on the N-terminal side of the glutamine. 7. Conjugate of formula (IV) 9 n Ab-CNLD ( ^IV ) wherein Ab is a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3 , NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO : 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 26, NO: 29, NO: 30 , NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40; L is a linker moiety linked to Ab via an amide bond with glutamine in glutamine-containing extension; And D is selected from the group consisting of a protein, a radioisotope, an analytical agent and a therapeutic agent. 8. The conjugate according to claim 7, wherein D is a cytotoxic drug. 9. The conjugate of claim 7, wherein D is a TLR3, TLR7, TLR9, STING, NLRP3 or RIGI agonist. 10. Conjugate according to claim 7, where L represents -(CH ₂ )2-6-· 11. The conjugate according to claim 7, where L represents O —gene 1 ( ^|a ') (OH ₂ ) _G [ U where T is a self-cleaving group; t is 0 or 1; AA ^a and each AA ^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine , lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; p is 1, 2, 3 or 4; q equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and r equals 1, 2, 3, 4 or 5. 12. A method for producing an antibody conjugate, comprising the steps of (a) mixing a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40, with an amine donor compound containing a primary amine and a moiety selected from the group consisting of protein, radioisotope, analyte and therapeutic agent, in the presence of transglutaminase; and (b) allowing transglutaminase to catalyze the formation of an amide bond between the glutamine side chain carboxamide of the glutamine-containing extension and the primary amine - 14 045916 amine donor compound, thereby leading to the formation of an antibody conjugate. 13. The method according to claim 12, where the amine donor compound has the structure H ₂ NLD where L is a linker moiety and D is a protein, radioisotope, assay or therapeutic agent. 14. The method according to claim 13, where the amine donor compound has the structure H ₂ N-(CH ₂ ) ₂ . ₆ D (I) 15. The method according to claim 12, wherein the donor-amine compound has the structure represented by formula (Ia) ABOUT HN-<CHlAoM ^JL[AAbl ''^{_AA8_<T)rD (|E)} H ₂ N (CH ₂ ) _g [ and ] where D is a protein, radioisotope, analytical agent or therapeutic agent; T represents a self-cleaving group; t is 0 or 1; AAa and each AA ^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; p is 1, 2, 3 or 4; q equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and r equals 1, 2, 3, 4 or 5. 16. The method of claim 12, wherein the fragment is a therapeutic fragment. 17. The method of claim 16, wherein the therapeutic agent is a cytotoxic drug. 18. The conjugate of claim 16, wherein the therapeutic agent is a TLR3, TLR7, TLR9, STING, NLRP3 or RIG-I agonist. 19. A method for producing an antibody conjugate, comprising the steps of (a) mixing a full-length antibody having at the C-terminus (carboxy-terminus) of its light chain a glutamine-containing extension containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11, NO: 12, NO: 13, NO: 14, NO: 15, NO: 16, NO: 17, NO: 18, NO: 19, NO: 20, NO: 21, NO: 22, NO: 23, NO: 24, NO: 25, NO: 26, NO: 29, NO: 30, NO: 31, NO: 32, NO: 33, NO: 34, NO: 35, NO: 36, NO: 37, NO: 38, NO: 39 and NO: 40, with the first connection, while the first compound is an amine donor compound having a primary amine and a first reactive functional group, in the presence of transglutaminase; (b) allowing transglutaminase to catalyze the formation of an amide bond between the glutamine side chain carboxamide of the glutamine-containing extension and the primary amine of the first compound, forming an adduct of the antibody and the first compound; (c) contacting the adduct with a second compound having a second reactive functional group and a moiety selected from the group consisting of a protein, a radioisotope, an analyte, and a therapeutic agent; wherein the second reactive functional group is capable of reacting with the first reactive functional group to form a covalent bond therebetween; and (d) allowing the first and second reactive functional groups to react and form a covalent bond between them, thereby resulting in the formation of an antibody conjugate. 20. The method according to claim 19, where the first compound has the structure H ₂ N-L'-R' where L' represents the first linker moiety and R' represents the first reactive functional group and the second compound has the structure R”-L”-D where R represents a second reactive functional group capable of reacting with R', L represents a second linker moiety, and D represents a protein, radioisotope, assay, or therapeutic agent. 21. The method according to claim 20, where R' is selected from - 15 045916 and reciprocally R is chosen from 22. The method according to claim 20, where the first compound has the structure represented by formula (II) H ₂ N-(CH2) ₂ _ ₈ -R' (II) and the second compound has the structure represented by formula (III) ABOUT R-(snaAo'CHU' ¹ --m.- ^D (I) where R' represents the first reactive group; R represents a second reactive group capable of reacting with R'; D is a protein, radioisotope, analytical agent or therapeutic agent; T is a self-cleaving group; t is 0 or 1; AA ^a and each AA ^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine , lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; p is 1, 2, 3 or 4; q equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and r equals 1, 2, 3, 4 or 5. 23. The method according to claim 22, where R' is selected from and reciprocally R is chosen from -