TRANSGLUTAMINASE CONJUGATION METHOD WITH AMINO ACID-BASED LINKERS

Abstract

The present invention relates to a method for generating an antibody-payload conjugate by means of a microbial transglutaminase (MTG). The method comprises a step of conjugating a linker comprising or having the structure (shown in N—>C direction) Aax-(Sp.sub.1)-B.sub.1-(Sp.sub.2) via a primary amine in the N-terminal residue Aax to a glutamine (Gln) residue comprised in the heavy or light chain of an antibody, wherein Aax is an amino acid having the structure NH.sub.2—Y—COOH, wherein Y comprises a substituted or unsubstituted alkyl or heteroalkyl chain; (Sp.sub.1) is a chemical spacer or is absent; (Sp.sub.2) is a chemical spacer or is absent; and B.sub.1 is a linking moiety or a payload. Further the present invention relates to antibody-linker conjugates that have been generated with the method of the invention and uses thereof.

Claims

1. A method for generating an antibody-linker conjugate by means of a microbial transglutaminase (MTG), the method comprising a step of conjugating a linker comprising the structure (shown in N—>C direction)
Aax-(Sp.sub.1)-B.sub.1-(Sp.sub.2) via a primary amine in the N-terminal residue Aax to a glutamine (Gln) residue comprised in the antibody, wherein Aax is an amino acid having the structure NH.sub.2—Y—COOH, wherein Y comprises a substituted or unsubstituted alkyl or heteroalkyl chain; (Sp.sub.1) is a chemical spacer or is absent; (Sp.sub.2) is a chemical spacer or is absent; and B.sub.1 is a linking moiety or a payload.

2. The method according to claim 1, wherein Y comprises the structure —(CH.sub.2).sub.n— and wherein n is an integer from 1 to 20, optionally wherein n is an integer from 2 to 20.

3. (canceled)

4. (canceled)

5. The method according to claim 1, wherein the chemical spacers (Sp1) and (Sp2) comprise between 0 and 12 amino acid residues, respectively; and/or wherein the linker comprises not more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 amino acid residues; and/or wherein the net charge of the linker is neutral or positive; and/or wherein the linker comprises no negatively charged amino acid residues; and/or wherein the linker comprises at least one positively charged amino acid residue; and/or wherein the linker comprises a second linking moiety or payload B.sub.2, optionally wherein B.sub.2 is connected to the linker via the chemical spacer (Sp.sub.2), optionally wherein B.sub.1 and B.sub.2 are identical or differ from one another.

6-11. (canceled)

12. The method according to claim 5, wherein B.sub.1 and/or B.sub.2 are linking moieties, optionally wherein at least one of the linking moieties B.sub.1 and/or B.sub.2 comprises a bioorthogonal marker group, or a non-bio-orthogonal entity for crosslinking: optionally wherein the bioorthogonal marker group or the non-bio-orthogonal entity consists of or comprises at least one molecule or moiety selected from a group consisting of: —N—N≡N, or —N.sub.3; Lys(N.sub.3); tetrazine; an alkyne; a strained cyclooctyne; BCN; a strained alkene; a photoreactive group; —RCOH (aldehyde); acyltrifluoroborates: a protein degradation agent (‘PROTAC’): cyclopentadienes/spirolocyclopentadienes: a thio-selective electrophile; —SH; and Cysteine: optionally wherein the method comprises an additional step of linking one or more payloads to at least one of the linking moieties B.sub.1 and/or B.sub.2, optionally wherein the one or more payloads are linked to the linking moiety B.sub.1 and/or B.sub.2 via a click-reaction.

13-16. (canceled)

17. The method according to claim 5, wherein B.sub.1 and/or B.sub.2 are payloads, optionally wherein the one or more payloads comprise at least one of: a toxin; a cytokine; a growth factor; a radionuclide; a hormone; an anti-viral agent; an anti-bacterial agent; a fluorescent dye; an immunoregulatory/immunostimulatory agent; a half-life increasing moiety; a solubility increasing moiety; a polymer-toxin conjugate; a nucleic acid; a biotin or streptavidin moiety; a vitamin; a protein degradation agent (‘PROTAC’): a target binding moiety; and/or an anti-inflammatory agent: optionally wherein the toxin is at least one selected from the group consisting of pyrrolobenzodiazepines (PBD): auristatins (e.g., MMAE, MMAF); maytansinoids (maytansine, DM1, DM4, DM21): duocarmycins; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors: tubulysins: enediyenes (e.g. calicheamicin): PNUs, doxorubicins: pyrrole-based kinesin spindle protein (KSP) inhibitors: drug efflux pump inhibitors; sandramycins: cryptophycins; amanitins (e.g. α-amanitin); and a camptothecins (e.g. exatecans, deruxtecans): optionally wherein the one or more payloads further comprise a cleavable or self-immolative moiety, optionally wherein the cleavable or self-immolative moiety comprises a motif cleavable by a cathepsin and/or a p-aminobenzyl carbamoyl (PABC) moiety.

18-22. (canceled)

23. The method according to claim 1, wherein the antibody is an IgG, IgE, IgM, IgD, IgA or IgY antibody, or a fragment or recombinant variant thereof, wherein the fragment or recombinant variant thereof retains target binding properties and comprises a CH2 domain, optionally wherein the antibody is an IgG antibody, optionally wherein the antibody is glycosylated at position N297 (EU numbering) of the C.sub.H2 domain.

24-35. (canceled)

36. An antibody-linker conjugate which has been generated with a method according to claim 1.

37. An antibody-linker conjugate comprising: a) an antibody; and b) a linker comprising the structure (shown in N—>C direction)
(Aax)-(Sp.sub.1)-B.sub.1-(Sp.sub.2), wherein Aax is an amino acid having the structure NH.sub.2—Y—COOH, wherein Y comprises a substituted or unsubstituted alkyl or heteroalkyl chain; (Sp.sub.1) is a chemical spacer; (Sp.sub.2) is a chemical spacer or is absent; and B.sub.1 is a linking moiety or a payload; wherein the linker is conjugated to an amide side chain of a glutamine (Gln) residue comprised in the heavy or light chain of the antibody via a primary amine in the residue Aax.

38. The conjugate according to claim 37, wherein Y comprises the structure —(CH.sub.2).sub.n— and wherein n is an integer from 1 to 20, optionally wherein n is an integer from 2 to 20.

39. (canceled)

40. (canceled)

41. The conjugate according to claim 37, wherein the chemical spacers (Sp.sub.1) and (Sp.sub.2) comprise between 0 and 12 amino acid residues, and/or wherein the linker comprises not more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 amino acid residues; and/or wherein the net charge of the linker is neutral or positive; and/or wherein the linker comprises no negatively charged amino acid residues; and/or wherein the linker comprises at least one positively charged amino acid residue; and/or wherein the linker comprises a second linking moiety or payload B.sub.2, optionally wherein B.sub.2 is connected to the linker via the chemical spacer (Sp.sub.2), wherein B.sub.1 and B.sub.2 are identical or differ from one another.

42-47. (canceled)

48. The conjugate according to claim 41, wherein B.sub.1 and/or B.sub.2 are linking moieties, optionally wherein at least one of the linking moieties B.sub.1 and/or B.sub.2 comprises a bioorthogonal marker group, or a non-bio-orthogonal entity for crosslinking: optionally wherein the bioorthogonal marker group or the non-bio-orthogonal entity consists of or comprises at least one molecule or moiety selected from a group consisting of: —N—N≡N, or —N.sub.3; Lys(N.sub.3); tetrazine; an alkyne; a strained cyclooctyne; BCN; a strained alkene; a photoreactive group; —RCOH (aldehyde); acyltrifluoroborates; a protein degradation agent (‘PROTAC’); cyclopentadienes/spirolocyclopentadienes; a thio-selective electrophile; —SH; and Cysteine optionally wherein at least one of the linking moieties B.sub.1 and/or B.sub.2 is linked to one or more payloads, optionally wherein the one or more payloads are linked to the linking moieties B.sub.1 and/or B.sub.2 via a click-reaction.

49-52. (canceled)

53. The conjugate according to claim 41, wherein B.sub.1 and/or B.sub.2 are payloads, optionally wherein the one or more payloads comprise at least one of: a toxin; a cytokine; a growth factor; a radionuclide; a hormone; an anti-viral agent; an anti-bacterial agent; a fluorescent dye; an immunoregulatory/immunostimulatory agent; a half-life increasing moiety; a solubility increasing moiety; a polymer-toxin conjugate; a nucleic acid; a biotin or streptavidin moiety; a vitamin; a protein degradation agent (‘PROTAC’); a target binding moiety; and/or an anti-inflammatory agent; optionally wherein the toxin is at least one selected from the group consisting of pyrrolobenzodiazepines (PBD); auristatins (e.g., MMAE, MMAF); maytansinoids (maytansine, DM1, DM4, DM21); duocarmycins; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; tubulysins; enediyenes (e.g. calicheamicin); PNUs, doxorubicins; pyrrole-based kinesin spindle protein (KSP) inhibitors; cryptophycins; drug efflux pump inhibitors; sandramycins; amanitins (e.g. α-amanitin); and camptothecins (e.g. exatecans, deruxtecans) optionally wherein the one or more payloads further comprise a cleavable or self-immolative moiety, optionally wherein the cleavable or self-immolative moiety comprises the motif valine-citrulline (VC) and/or a p-aminobenzyl carbamoyl (PABC) moiety.

54-63. (canceled)

64. A pharmaceutical composition comprising the antibody-linker conjugate according to claim 37, optionally wherein the antibody-linker conjugate comprises at least one payload and, optionally, at least one further pharmaceutically acceptable ingredient.

65-68. (canceled)

69. A method for pre-, intra- or post-operative imaging, said method comprising administering to a patient in need thereof the antibody linker-conjugate according to claim 37.

70. A method for intraoperative imaging-guided cancer surgery, said method comprising administering to a patient in need thereof the antibody linker-conjugate according to claim 37.

71. (canceled)

72. A method of treating or preventing a neoplastic disease, said method comprising administering to a patient in need thereof the antibody-linker conjugate according to claim 37.

73. A method of treating or preventing a neurological disease, said method comprising administering to a patient in need thereof the antibody-linker conjugate according to claim 37.

74. A method of treating or preventing an autoimmune disease, said method comprising administering to a patient in need thereof the antibody-linker conjugate according to claim 37.

75. A method of treating or preventing an inflammatory disease, said method comprising administering to a patient in need thereof the antibody-linker conjugate according to claim 37.

76. A method of treating or preventing an infectious disease, said method comprising administering to a patient in need thereof the antibody-linker conjugate according to claim 37.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0954] FIG. 1 shows an illustration of one aspect of the present invention. MTG=microbial transglutaminase. The star symbol illustrates the payload or linking moiety B. The Aax residue, which is N-terminally in a peptide, is the substrate for MTG. Note that this process allows to maintain the glycosylation at N297. Note that in case B/star is a linking moiety, the actual payload still has to be conjugated to this moiety.

[0955] FIG. 2 shows the linkers that have been conjugated to glycosylated antibodies in Examples 2-5.

EXAMPLES

[0956] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Example 1: Conjugation Efficiency

[0957] Linkers are used as obtained and dissolved at a suitable stock concentration (e.g. 25 mM) following the manufacturers instruction, aliquots are prepared and stored at −20° C. Two antibodies of IgG-subclass (antibody 1: anti Her2 IgG1, antibody 2: anti CD38 IgG1) are modified as follows: 5 mg/mL of non-deglycosylated antibody (˜33.33 μM) is mixed with 20 molar equivalents of amino acid-based linker (i.e. ˜667 μM), 3 U/mg of antibody and a suitable buffer. The reaction mixture is incubated for 20 h at 37° C. and then subjected for LC-MS analysis under reducing conditions. Other reaction conditions have been used as indicated in the corresponding example.

Example 2: Conjugation of Peptide Linkers to Native, Non-Engineered Glycosylated Antibody (Anti Her2 IgG) Via the Primary Amine of the N-Terminal (Modified) Amino Acid

[0958] Linkers with various amino acid derivatives at the beginning of the sequence (at the N-terminus) were used for conjugation.

Methods

[0959] The antibody trastuzumab was commercially available (Herceptin®, Roche, bought from a pharmacy), as well as all peptide linkers (purchased from LifeTein LLC).

[0960] For conjugation of the peptide linkers (see FIG. 2 for structures), 5 mg/mL of native, glycosylated monoclonal antibody in 50 mM Tris pH 7.6, microbial transglutaminase (MTG, Zedira) at a concentration of 3 U/mg in 50 mM Tris pH 7.6 or water, and 20 molar equivalents of the indicated peptide linker were used and incubated for 24 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LC-MS under DTT reduced conditions. Reduction of samples was achieved by incubation of antibody-linker-conjugates (ALCs) for 15 min at 37° C. in 50 mM DTT (final) and 50 mM Tris buffer. Probes were analyzed on a Xevo G2-XS QTOF (Waters) coupled to an Acquity UPLC H-Class System (Waters) and an ACQUITY UPLC BEH C18 Column. Conjugation efficiency (CE) was calculated from deconvoluted spectra and presented in %. Intensities resulting from both glycoforms (G1F and G0F) were taken into account for the calculation, according to the formula:

[00001]$\mathrm{CE}\%=\frac{.Math.\left({\left(Int\left(G0F+G1F\right)\right)}_{\mathrm{cj}}\right)}{.Math.{\left(\mathrm{In}t\left(G0F+G1F\right)\right)}_{\mathrm{cj},\mathrm{ncj}}}$

[0961] With cC=conjugated and ncj=non-conjugated

Results

[0962] Surprisingly, all peptides having an alkyl (e.g., methyl) spacer between the primary amine and carboxyl group of the first amino acid (i.e., N-terminal amino acid) provided a significant conjugation efficiency. Of note, neither the length of the methyl groups nor the nature of the following (second) amino acid of the peptide did have a significant influence on the conjugation efficiency. It was therefore surprisingly found that any peptide having an alkyl spacer between the primary amine and the carboxy-group of the first (N-terminal) amino acid could be used to conjugate native, glycosylated antibody (Table 3). Peptides without an alkyl (e.g. methyl) spacer between the primary amine and C-alpha and/or carboxy group could not be conjugated to glycosylated antibody.

TABLE-US-00003 TABLE 3 Conjugation efficiency of peptide linkers Conjugation efficiency Peptide linker Name (%) to antibody NH.sub.2-C1-GRK(N3)-NH.sub.2 (SEQ ID NO: 40) NT24  99% NH.sub.2-C1-ARK(N3)-NH.sub.2 (SEQ ID NO: 39) NT28  89% NH.sub.2-C2-GRK(N3)-NH.sub.2 (SEQ ID NO: 42) NT37  70% NH.sub.2-C3-GRK(N3)-NH.sub.2 (SEQ ID NO: 44) NT38  53% NH.sub.2-C4-GRK(N3)-NH.sub.2 (SEQ ID NO: 46) NT41  99% NH.sub.2-C5-GRK(N3)-NH.sub.2 (SEQ ID NO: 48) NT36  84% NH.sub.2-C6-GRK(N3)-NH.sub.2 (SEQ ID NO: 50) NT42  67% NH.sub.2-C4-ValCit-NH.sub.2 (SEQ ID NO: 57) NT39  67% NH.sub.2-C4-ValArg-NH.sub.2 (SEQ ID NO: 58) NT40  96% NH.sub.2-C5-ValCit-NH.sub.2 (SEQ ID NO: 59) NT43  83% NH.sub.2-C5-ValArg-NH.sub.2 (SEQ ID NO: 60) NT44 100% NH.sub.2-C6-ThrArg-NH.sub.2 (SEQ ID NO: 83) NT64  95% NH.sub.2-C6-IleArg-NH.sub.2 (SEQ ID NO: 84) NT65  96% NH.sub.2-C6-AspArg-NH.sub.2 (SEQ ID NO: 85) NT66  64% NH.sub.2-C6-TrpArg-NH.sub.2 (SEQ ID NO: 86) NT67  82% AARK(N3)-NH.sub.2 (SEQ ID NO: 87) —   0% LGRC-NH.sub.2 (SEQ ID NO: 88) —   0% VGRC-NH.sub.2 (SEQ ID NO: 89) —   0% RGRK(N3)-NH.sub.2 (SEQ ID NO: 90) —   0% SAGRK(N3)-NH.sub.2 (SEQ ID NO: 91) —   0%

[0963] In the used nomenclature C1, C2, C3, C4, C5 or C6 the number (1 to 6) indicates the number of methylene units of the spacer between the primary amine and the carboxylic group. That is, C1 corresponds to the spacer of glycine, C2 corresponds to the spacer of β-alanin, C3 corresponds to the spacer of 4-aminobutyric acid, C4 corresponds to the spacer of 5-aminopropionic acid, C5 corresponds to the spacer of 6-aminohexanoic acid and C6 corresponds to the spacer of 7-aminoheptanoic acid.

Example 3: Conjugation of Peptide Linkers to Native, Non-Engineered Glycosylated Antibody (Anti CD38 IgG) Via the Primary Amine of the N-Terminal (Modified) Amino Acid

[0964] Linkers with various amino acid derivatives at the beginning of the sequence (at the N-terminus) were used for conjugation.

Methods

[0965] The antibody daratumumab was commercially available (Darzalex®, Janssen, bought from a pharmacy), as well as all peptide linkers (purchased from LifeTein LLC).

[0966] For conjugation of the peptide linkers (see FIG. 2 for structures), 5 mg/mL of native, glycosylated monoclonal antibody in 50 mM Tris pH 7.6, microbial transglutaminase (MTG, Zedira) at a concentration of 3 U/mg in 50 mM Tris pH 7.6 or water, and 20 molar equivalents of the indicated peptide linker were used and incubated for 24 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LC-MS under DTT reduced conditions. Reduction of samples was achieved by incubation of antibody-linker-conjugates (ALCs) for 15 min at 37° C. in 50 mM DTT (final) and 50 mM Tris buffer. Probes were analyzed on a Xevo G2-XS QTOF (Waters) coupled to an Acquity UPLC H-Class System (Waters) and an ACQUITY UPLC BEH C18 Column. Conjugation efficiency (CE) was calculated from deconvoluted spectra and presented in %. Intensities resulting from both glycoforms (G1F and G0F) were taken into account for the calculation, according to the formula:

[00002]$\mathrm{CE}\%=\frac{.Math.\left({\left(Int\left(G0F+G1F\right)\right)}_{\mathrm{cj}}\right)}{.Math.{\left(\mathrm{In}t\left(G0F+G1F\right)\right)}_{\mathrm{cj},\mathrm{ncj}}}$

[0967] With cj=conjugated and ncj=non-conjugated

Results

[0968] Surprisingly, all peptides having an alkyl (e.g., methyl) spacer between the primary amine and carboxyl group of the first amino acid (i.e., N-terminal amino acid) provided a significant conjugation efficiency. Of note, neither the length of the methyl groups nor the nature of the following (second) amino acid of the peptide did have a significant influence on the conjugation efficiency. It was therefore surprisingly found that any peptide having an alkyl spacer between the primary amine and the carboxy-group of the first (N-terminal) amino acid could be used to conjugate native, glycosylated antibody (Table 4).

TABLE-US-00004 TABLE 4 Conjugation efficiency of peptide linkers Conjugation efficiency Peptide linker Name (%) to antibody NH.sub.2-C1-GRK(N3)-NH.sub.2 (SEQ ID NO: 40) NT24 95% NH.sub.2-C4-GRK(N3)-NH.sub.2 (SEQ ID NO: 46) NT41 98% NH.sub.2-C5-GRK(N3)-NH.sub.2 (SEQ ID NO: 48) NT36 93% NH.sub.2-C4-ValArg-NH.sub.2 (SEQ ID NO: 58) NT40 89% NH.sub.2-C5-ValArg-NH.sub.2 (SEQ ID NO: 60) NT44 95% NH.sub.2-C6-ThrArg-NH.sub.2 (SEQ ID NO: 83) NT64 93% NH.sub.2-C6-IleArg-NH.sub.2 (SEQ ID NO: 84) NT65 95% NH.sub.2-C6-AspArg-NH.sub.2 (SEQ ID NO: 85) NT66 66% NH.sub.2-C6-TrpArg-NH.sub.2 (SEQ ID NO: 86) NT67 83%

[0969] In the used nomenclature C1, C2, C3, C4, C5 or C6 the number (1 to 6) indicates the number of methylene units of the spacer between the primary amine and the carboxylic group. That is, C1 corresponds to the spacer of glycine, C2 corresponds to the spacer of β-alanin, C3 corresponds to the spacer of 4-aminobutyric acid, C4 corresponds to the spacer of 5-aminopropionic acid, C5 corresponds to the spacer of 6-aminohexanoic acid and C6 corresponds to the spacer of 7-aminoheptanoic acid.

Example 4: Conjugation of Peptide Linkers Using Other Reaction Conditions (Conditions 2)

[0970] In order to demonstrate that conjugation with linkers were tolerating variation of the reaction conditions, some parameters were changed (linker equivalent, MTG amount, antibody concentration, reaction time).

Methods

[0971] The antibody trastuzumab was commercially available (Herceptin®, Roche, bought from a pharmacy), as well as all peptide linkers (purchased from LifeTein LLC).

[0972] For conjugation of the peptide linkers (see FIG. 2 for structures), 1 mg/mL of native, glycosylated monoclonal antibody in 50 mM Tris pH 7.6, microbial transglutaminase (MTG, Zedira) at a concentration of 6 U/mg in 50 mM Tris pH 7.6 or water, and 80 molar equivalents of the indicated peptide linker were used and incubated for 20 hours at 37° C. in a rotating thermomixer. These are defined as “conditions 2”. Conjugation efficiency was assessed by LC-MS under DTT reduced conditions. Reduction of samples was achieved by incubation of antibody-linker-conjugates (ALCs) for 15 min at 37° C. in 50 mM DTT (final) and 50 mM Tris buffer. Probes were analyzed on a Xevo G2-XS QTOF (Waters) coupled to an Acquity UPLC H-Class System (Waters) and a ACQUITY UPLC BEH C18 Column. Conjugation efficiency (CE) was calculated from deconvoluted spectra and presented in %. Intensities resulting from both glycoforms (G1F and G0F) were taken into account for the calculation, according to the formula:

[00003]$\mathrm{CE}\%=\frac{.Math.\left({\left(Int\left(G0F+G1F\right)\right)}_{\mathrm{cj}}\right)}{.Math.{\left(\mathrm{In}t\left(G0F+G1F\right)\right)}_{\mathrm{cj},\mathrm{ncj}}}$

[0973] With cj=conjugated and ncj=non-conjugated

Results

[0974] All peptide linkers conjugated with significant conjugation efficiency (Table 5), indicating that linkers tolerate a wide range of reaction conditions. Overall, conditions in Example 3 yielded higher conjugation efficiencies (i.e., using less than 6 U/mg of enzyme, more than lmg/ml antibody concentration and less than 80 molar equivalents of the peptide linker yields better results).

TABLE-US-00005 TABLE 5 Conjugation efficiency of peptide linkers using conditions 2 Conjugation efficiency (%) Peptide linker Name antibody with conditions 2 NH.sub.2-C1-GRK(N3)-NH.sub.2 (SEQ ID NO: 40) NT24 78% NH.sub.2-C2-GRK(N3)-NH.sub.2 (SEQ ID NO: 42) NT37 38% NH.sub.2-C3-GRK(N3)-NH.sub.2 (SEQ ID NO: 44) NT38 36% NH.sub.2-C4-GRK(N3)-NH.sub.2 (SEQ ID NO: 46) NT41 70% NH.sub.2-C5-GRK(N3)-NH.sub.2 (SEQ ID NO: 48) NT36 56% NH.sub.2-C6-GRK(N3)-NH.sub.2 (SEQ ID NO: 50) NT42 38% NH.sub.2-C4-ValCit-NH.sub.2 (SEQ ID NO: 57) NT39 63% NH.sub.2-C4-ValArg-NH.sub.2 (SEQ ID NO: 58) NT40 83% NH.sub.2-C5-ValCit-NH.sub.2 (SEQ ID NO: 59) NT43 79% NH.sub.2-C5-ValArg-NH.sub.2 (SEQ ID NO: 60) NT44 88%

[0975] In the used nomenclature C1, C2, C3, C4, C5 or C6 the number (1 to 6) indicates the number of methylene units of the spacer between the primary amine and the carboxylic group. That is, C1 corresponds to the spacer of glycine, C2 corresponds to the spacer of β-alanin, C3 corresponds to the spacer of 4-aminobutyric acid, C4 corresponds to the spacer of 5-aminopropionic acid, C5 corresponds to the spacer of 6-aminohexanoic acid and C6 corresponds to the spacer of 7-aminoheptanoic acid.

Example 5: Conjugation of Peptide Linkers Using Other Reaction Conditions (Conditions 3)

[0976] In order to demonstrate that conjugation with linkers were tolerating variation of the reaction conditions, some parameters were changed (linker equivalent, MTG amount, antibody concentration, reaction time).

Methods

[0977] The antibodies trastuzumab and daratumumab were commercially available (Herceptin®, Roche, and Darzalex®, Janssen, bought from a pharmacy), as well as all peptide linkers (purchased from LifeTein LLC).

[0978] For conjugation of the peptide linkers (see FIG. 2 for structures), 5 mg/mL of native, glycosylated monoclonal antibody in 50 mM Tris pH 7.6, microbial transglutaminase (MTG, Zedira) at a concentration of 3 U/mg in 50 mM Tris pH 7.6 or water, and 5 molar equivalents of the indicated peptide linker were used and incubated for 24 hours at 37° C. in a rotating thermomixer. These are defined as “conditions 3”. Conjugation efficiency was assessed by LC-MS under DTT reduced conditions. Reduction of samples was achieved by incubation of antibody-linker-conjugates (ALCs) for 15 min at 37° C. in 50 mM DTT (final) and 50 mM Tris buffer. Probes were analyzed on a Xevo G2-XS QTOF (Waters) coupled to an Acquity UPLC H-Class System (Waters) and a ACQUITY UPLC BEH C18 Column. Conjugation efficiency (CE) was calculated from deconvoluted spectra and presented in %. Intensities resulting from both glycoforms (G1F and G0F) were taken into account for the calculation, according to the formula:

[00004]$\mathrm{CE}\%=\frac{.Math.\left({\left(Int\left(G0F+G1F\right)\right)}_{\mathrm{cj}}\right)}{.Math.{\left(\mathrm{In}t\left(G0F+G1F\right)\right)}_{\mathrm{cj},\mathrm{ncj}}}$

[0979] With cj=conjugated and ncj=non-conjugated

Results

[0980] All peptide linkers conjugated with significant conjugation efficiency (Table 6), even when using as low as 5 eq of peptide linkers only.

TABLE-US-00006 TABLE 6 Conjugation efficiency of peptide linkers using conditions 3 Conjugation Conjugation efficiency (%) efficiency (%) Trastuzumab Daratumumab Peptide linker Name with conditions 3 with conditions 3 NH.sub.2-C1-GRK(N3)-NH.sub.2 (SEQ ID NO: 40) NT24 97% 89% NH.sub.2-C4-GRK(N3)-NH.sub.2 (SEQ ID NO: 46) NT41 88% 88% NH.sub.2-C5-GRK(N3)-NH.sub.2 (SEQ ID NO: 48) NT36 75% 72% NH.sub.2-C4-ValArg-NH.sub.2 (SEQ ID NO: 58) NT40 58% 51% NH.sub.2-C5-ValArg-NH.sub.2 (SEQ ID NO: 60) NT44 77% 73% NH.sub.2-C6-ThrArg-NH.sub.2 (SEQ ID NO: 83) NT64 92% 92% NH.sub.2-C6-IleArg-NH.sub.2 (SEQ ID NO: 84) NT65 78% 79%

[0981] In the used nomenclature C1, C2, C3, C4, C5 or C6 the number (1 to 6) indicates the number of methylene units of the spacer between the primary amine and the carboxylic group. That is, C1 corresponds to the spacer of glycine, C2 corresponds to the spacer of β-alanin, C3 corresponds to the spacer of 4-aminobutyric acid, C4 corresponds to the spacer of 5-aminopropionic acid, C5 corresponds to the spacer of 6-aminohexanoic acid and C6 corresponds to the spacer of 7-aminoheptanoic acid.