Patent Application
20220133904
The present invention relates to methods for generating an antibody-payload conjugate by means of a microbial transglutaminase. The invention further provides linkers, linker-payload constructs and/or antibody-payload constructs.
Attaching highly potent payloads to antibodies finds increasing interest for the targeted treatment of cancer or inflammatory diseases. The constructs this produces are called antibody-payload conjugates, or antibody-drug conjugates (ADC).
Currently, seven ADCs have gained FDA-approval (Adcetris, Kadcyla, Besponsa, Mylotarg, Polivy, Padcev, Enhertu), all of which have their payload chemically attached to the antibody in a non-site specific manner. Hence, the resulting product is highly heterogeneous, both with respect to the stoichiometric relationship between antibody and payload (payload-antibody ratio, or drug-to-antibody ratio, DAR), as well concerning the conjugation sites on the antibody. Each of the resulting species, although in the same drug product, may have distinct properties that could potentially lead to a wide range of different in vivo pharmacokinetic properties and activities.
In a previous in vivo study (Lhospice et al., 2015), it was shown that a site-specific drug attachment led to a significant higher tumor uptake (˜2×) and a decreased uptake in non-targeted tissues compared to the FDA-approved ADC, also the maximal tolerated dose was at least 3× higher. These data suggest that stoichiometrically well-defined ADCs display improved pharmacokinetics and better therapeutic indexes compared to chemically modified ADCs.
As a site-specific technology, enzymatic conjugation has gained great interest since these conjugation reactions are typically fast and can be performed under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) from the species Streptomyces mobaraensis has gained increasing interest as an attractive alternative to conventional chemical protein conjugation of functional moieties including antibodies. The MTG catalyzes under physiological conditions a transamidation reaction between a ‘reactive’ glutamine of a protein or peptide and a ‘reactive’ lysine residue of a protein or peptide, whereas the latter can also be a simple, low molecular weight primary amine such as a 5-aminopentyl group (Jeger et al., 2010, Strop et al., 2014).
The bond formed is an isopeptide bond which is an amide bond that does not form part of the peptide-bond backbone of the respective polypeptide or protein. It is formed between the γ-carboxamide of the glutamyl residue of the acyl glutamine-containing amino acid donor sequence and a primary (1°) amine of the amino donor-comprising substrate according to the invention.
From the inventor's experience as well as from others, it seems that only few glutamines are typically targeted by MTG, thus making the MTG an attractive tool for site-specific and stoichiometric protein modifications.
Previously, glutamine 295 (Q295) was identified as the only reactive glutamine on the heavy chain of different IgG types to be specifically targeted by MTG with low-molecular weight primary amine substrates (Jeger et al. 2010).
Quantitative conjugation to Q295, however, was only possible upon removal of the glycan moiety at the asparagine residue 297 (N297) with PNGase F, while glycosylated antibodies could not be conjugated efficiently (conjugation efficiency <20%). This finding is also supported by the studies of Mindt et al. (2008) and Jeger et al. (2010) and Dickgiesser et al. 2020
In order to obviate deglycosylation it is also possible to insert a point mutation at the residue N297 which results in the ablation of the glycosylation called aglycosylation.
However, both approaches come with significant disadvantages. An enzymatic deglycosylation step is undesired under GMP aspects, because it has to be made sure that both the deglycosylation enzyme (e.g., PNGase F) as well as the cleaved glycan are removed from the medium, to ensure a high purity product.
The substitution of N297 against another amino acid has unwanted effects, too, because it may affect the overall stability of the CH2 domain, and the efficacy of the entire conjugate as a consequence. Further, the glycan that is present at N297 has important immunomodulatory effects, as it triggers antibody dependent cellular cytotoxicity (ADCC) and the like. These immunomodulatory effects would get lost upon deglycosylation or substitution of N297 against another amino acid.
Furthermore, the genetic engineering of an antibody for payload attachment may have disadvantages in that the sequence insertion may increase immunogenicity and decrease the overall stability of the antibody.
It is hence one object of the present invention to provide a transglutaminase based antibody conjugation approach which does not require prior deglycosylation of the antibody, in particular of N297.
It is another object of the present invention to provide a transglutaminase based antibody conjugation approach which does not require the substitution or modification of N297 in the CH2 domain.
It is one further object of the present invention to provide an antibody conjugation technology that allows the manufacture of highly homogenous conjugation products, both as regards stoichiometry as well as site-specificity of the conjugation.
These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.
The present invention relates to methods and linker structures for generating an antibody-linker conjugate and/or an antibody-payload conjugate by means of a microbial transglutaminase (MTG). The invention and general advantages of its features will be discussed in detail below.
As discussed elsewhere herein, B/star can be or comprise a linking moiety, like e.g. a bio-orthogonal group (e.g., an azide/N3-group) that is suitable for strain-promoted alkyne-azide cycloaddition (SPAAC) click-chemistry reaction to a DBCO-containing payload (e.g. a toxin or a fluorescent dye or a metal chelator, like DOTA or NODA-GA). This click-chemistry-based “two-step chemoenzymatic”-approach to attach the functional moiety to the antibody has the major advantage that it can be clicked at low molecular excess compared to the antibody, typically e.g. at 5 eq per conjugation site or lower (Dennler et al. 2014). This allows for a cost-effective generation of ADCs. In addition, virtually any probe can be clicked with this approach ranging from fluorescent dyes to metal chelators (cf. Spycher et al. 2017, Dennler et al. 2015).
B/star can also be the actual payload, e.g., a toxin. Such embodiment allows the rapid manufacture of the resulting compound in one step, facilitating purification and production.
The peptide efficiently conjugates to native IgG1 antibodies (˜77.3% as estimated from LC-MS analysis under non-optimized conditions) at position Q295.
It is important to understand that in some linker peptides shown herein, the moiety at the C-terminus is simply designated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GAR(N3) corresponds to the peptide GlyAlaArgLys(N3) or GARK(N3). That is, 6-azido-L-lysine may be abbreviated as Lys(N3) in three letter code or as K(N3) or (N3) in single letter code. It is thus to be understood that K(N3) when part of a peptide always relates to the single amino acid residue Lys(N3) but not to the dipeptide Lys-Lys(N3). A dipeptide Lys-Lys(N3), on the other hand, would be designated KK(N3) in single letter code.
It is furthermore important to understand that in different linker peptides shown herein, the C-terminus or primary amines on side chains may or may not be protected, even if shown otherwise. Protection can be accomplished by, e.g., amidation of the former, and/or acetylation of the latter. In the context of the present invention, both the protected and unprotected linker peptides are encompassed.
For example, GARK(N3) does indeed encompass two variants, with the C-terminus protected or unprotected. The following figure shows a C-terminal Lys(N3) residue wherein the C-terminus is protected by amidation:
In the two-step process, the linker peptide is Gly-(Aax)n-Cys-linking moiety. The Gly residue is conjugated to a Gln residue in the antibody via microbial transglutaminase, and the linking moiety—in this case a Cys residue with a free sulfhydryl group—is then conjugated to the payload, in this case a MMAE toxin carrying a MC/VC/PABDC linker structure, via the maleimide.
In the one-step process, the linker peptide Gly-(Aax)m is already conjugated to the payload. The Gly residue is conjugated to a Gln residue in the antibody, and the payload consists of an MMAE toxin carrying a VC/PABC structure. The valine residue of the VC structure is conjugated to the last amino acid of the linker peptide by means of a peptide bond.
Each of the linking moieties are bio-orthogonally compatible groups that can be clicked simultaneously.
These linkers thus allow to conjugate two different payloads to the Q295 of the CH2 domain of an antibody. Using a second payload allows for the development of a completely new class of antibody-payload conjugates that go beyond current therapeutic approaches with respect to efficacy and potency. Also new application fields are envisioned, for example, dual-type imaging for imaging and therapy or intra-/postoperative surgery (cf. Azhdarinia A. et al., Molec Imaging and Biology, 2012). For example, dual-labeled antibodies encompassing a molecular imaging agent for preoperative positron emission tomography (PET) and a near-infrared fluorescent (NIRF)-dye for guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of cancer (cf. Houghton J L. et al., PNAS 2015). PET and NIRF optical imaging offer complementary clinical applications, enabling the non-invasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. However, the generation of such dual-labeled probes up to date has been difficult due to a lack of suitable site-specific methods; attaching two different probes by chemical means results in an almost impossible analysis and reproducibility due to the random conjugation of the probes. Furthermore, in a study of Levengood M. et al., (Angewandte Chemie, 2016) a dual-drug labeled antibody, having attached two different auristatin toxins (having differing physiochemical properties and exerting complementary anti-cancer activities) imparted activity in cell line and xenograft models that were refractory to ADCs comprised of the individual auristatin components. This suggests that dual-labeled ADCs enable to address cancer heterogeneity and resistance more effectively than the single, conventional ADCs alone. Since one resistance mechanism towards ADCs include the active pumping-out of the cytotoxic moiety from the cancer cell, another dual-drug application may include the additional and simultaneous delivery of a drug that specifically blocks the efflux mechanism of the cytotoxic drug. Such a dual-labeled ADC could thus help to overcome cancer resistance to the ADC more effectively than conventional ADCs.
Similar structures in which alkynes or tetrazine/trans-cyclooctenes are being used as linker are equally suitable and covered by the scope and gist of the present invention.
It is important to understand that in some linker peptides shown herein, the moiety at the C-terminus is simply designated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GAR(N3) or GARK(N3) does actually mean GARK1, with K1=Lys(N3), or GlyAlaArgLys(N3).
It is furthermore important to understand that in different linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection may be accomplished by amidation of the C-terminus. Since conjugation of the linker to an antibody is achieved via the primary amine of the N-terminal glycine residue of the linker, the N-terminus of the linker is preferably unprotected. In the context of the present invention, both the protected and unprotected linker peptides are encompassed. For example, GARK(N3) does indeed encompass two variants, with a) both termini unprotected as discussed above, or b) only the C-terminus protected as discussed above.
The question whether or not the C-terminus is amidated is a practical question, depending on the conjugation conditions (buffer, medium, reactivity of the other reaction components, etc).
It is important to understand that in some linker peptides shown herein, the moiety at the C-terminus is simply designated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GAR(N3) or GARK(N3) does actually mean GARK1, with K1=Lys(N3), or GlyAlaArgLys(N3).
Before the invention is described in detail, it is to be understood that this invention is not limited to the particular components or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.
Furthermore, the content of the documents referred to herein is incorporated by reference. This refers, particularly, for documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.
According to a first aspect, a method for generating an antibody-payload conjugate or an antibody-linker conjugate by means of a microbial transglutaminase (MTG) is provided, which method comprises the step of conjugating a linker comprising the peptide structure (shown in N->C direction)
Gly-(Aax)m-B-(Aax)n
via the N-terminal primary amine of the N-terminal glycine (Gly) residue to a glutamine (Gln) residue comprised in the heavy or light chain of an antibody,
wherein
As used herein, the term “primary amine” relates to an amine substituted with two hydrogen atoms, of the general formula R—NH2.
In certain embodiments, the peptide linker may comprise two or more linking moieties and/or payloads. That is, the linker may have the peptide structure (shown in N->C direction)
Gly-(Aax)m-B1-(Aax)n-B2-(Aax)o
wherein
In other embodiments, the peptide linker may comprise three linking moieties and/or payloads. That is, the linker may have the peptide structure (shown in N->C direction)
Gly-(Aax)m-B1-(Aax)n-B2-(Aax)o-B3-(Aax)p
wherein
It is to be understood, that the invention also encompasses linkers comprising more than three linking moieties and/or payloads, such as 4, 5 or 6 linking moieties and/or payloads. In this case, the peptide structure of the linkers follows the same pattern as described above for the linkers comprising 2 or 3 linking moieties and/or payloads.
In certain embodiments, a method for generating an antibody-payload conjugate by means of a microbial transglutaminase (MTG) is provided, which method comprises the step of conjugating a linker having the peptide structure (shown in N->C direction)
Gly-(Aax)m-B-(Aax)n
via the N-terminal primary amine of the N-terminal glycine (Gly) residue to a glutamine (Gln) residue comprised in the heavy or light chain of an antibody, wherein
In certain embodiments, the invention relates to a method for generating an antibody-payload conjugate or an antibody-linker conjugate by means of a microbial transglutaminase (MTG), which method comprises the step of conjugating a linker having the peptide structure (shown in N->C direction)
Gly-(Aax)m-B-(Aax)n
via the N-terminal primary amine of the N-terminal glycine (Gly) residue to a glutamine (GM) residue comprised in the heavy or light chain of an antibody. In this case, it is to be understood that the moiety B may comprise more than one payload and/or linking moiety. For example, B may stand for (B′-(Aax)o-B″), wherein B′ and B″ are payloads and/or linking moieties and wherein o is an integer between ≥0 and ≤12. Alternatively, B may stand for (B′-(Aax)o-B″-(Aax)p-B′″), wherein B′, B″ and B′″ are payloads and/or linking moieties and wherein o and p are integers between ≥0 and ≤12.
Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the linker comprises two or more payloads and/or linking moieties. In another embodiment, the invention relates to a method according to the invention, wherein the two or more payloads and/or linking moieties B differ from one another.
That is, the linker according to the invention may comprise a single payload or linking moiety. In certain embodiments, the linker comprises two linking moieties, wherein the two linking moieties are identical. In other embodiments, the linker comprises two linking moieties, wherein the two linking moieties are different. In yet another embodiment, the linker comprises two identical or different payloads. The invention further encompasses linkers comprising one or more payload and one or more linking moiety.
It is further to be understood that not all payloads or linking moieties can function as an intrachain payload or linking moiety, for example, because they do not have the functional groups to form peptide or amide bonds with the C-terminal carboxyl group of a first Aax moiety and the N-terminal amine group of a second Aax moiety. In this case, it is preferred that such payload or linking moieties are located at the C-terminal end of the linker, where they preferably are attached to the carboxyl group of the C-terminal Aax moiety of the linker. In cases where the payload or linking moiety are at an intrachain position of the linker, it is preferred that the payload or linking moiety is an amino acid, an amino acid derivative or attached to a molecule having the general structure —NH—CHR—CO—.
In preferred embodiments, m and/or n is ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, or ≥11. In other preferred embodiments, m and/or n is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or ≤1. In further preferred embodiments, m+n is ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, or ≥11. In still further preferred embodiments m+n is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or ≤1.
Members of both ranges can be combined with another to disclose a preferred length range with lower and upper limit.
Accordingly, in a particular embodiment, the invention relates to a method according to the invention, wherein m+n, and optionally m+n+o and m+n+o+p, is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5 or ≤4.
It is important to understand that in different linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection can be accomplished by amidation of the former. In the context of the present invention, both the protected and unprotected linker peptides are encompassed.
The inventors have shown that this process is suitable to very cost effectively and quickly produce site-specific antibody-payload conjugates (24-36 hrs, or optionally 48 hrs), and hence allows the production of large libraries of such molecules, and subsequent screening thereof in high throughput screening systems.
In contrast thereto, a Cys engineering process in which an antibody payload conjugate is produced where the payload is conjugated to the antibody via a genetically (molecularly) engineered Cys residue needs at least about 3-4 weeks.
In general, the method allows conjugation of a large number of payloads to an antibody. For each payload, a suitable peptide linker structure can be identified from a large linker pool to deliver optimal clinical and non-clinical characteristics. This is not possible in other methods where the linker structure is fixed. In addition, the method according to the invention allows to generate antibody-payload conjugates comprising two or more different payloads, wherein each payload is conjugated to the antibody in a site-specific manner. Thus, the method according to the invention may be used to generate antibodies with novel and/or superior therapeutic or diagnostic capacities.
The linker may comprise any amino acid, including, without limitation, α-, β-, γ-, δ- and ε-amino acids. In the case of α-amino acids, the linker may comprise any naturally occurring L- or D-amino acid. A naturally occurring L- or D-amino acid encompasses any L- or D-amino acid that can be found in nature. That is, the term “naturally occurring L- or D-amino” acid encompasses all canonical or proteogenic amino acids that are used as building blocks in naturally-occurring proteins. In addition, the term “naturally occurring L- or D-amino” acid encompasses all non-canonical L- or D-amino acids that can be found in nature, for example as metabolic intermediates or degradation products or as building blocks for other non-proteogenic macromolecules.
Further, the linker may comprise non-naturally occurring L- or D-amino acids. A non-naturally occurring L- or D-amino acid encompasses any molecule having the general structure H2N—CHR—COOH, which has not previously been found in nature.
The skilled person is aware of resources and databases to consult when determining whether an L- or D-amino acid is naturally or non-naturally occurring. However, in cases of doubt, it is to be understood that the term “naturally or non-naturally occurring L- or D-amino acid” encompasses the L- and D-isomer of any molecule having the general structure H2N—CHR—COOH, irrespective of the origin of said molecule.
In certain embodiments, the linker of the invention may also comprise naturally or non-naturally occurring, non-chiral amino acids having the general structure H2N—CR1R2—COOH.
Furthermore, the linker of the invention may comprise amino acid derivatives. An amino acid derivative is a compound that has been derived from a naturally or non-naturally occurring amino acid by one or more chemical reactions, such as chemical reactions of the α-amino group, the α-carboxylic acid group and/or the amino acid side chain. That is, the term amino acid derivative encompasses any molecule having the structure —NH—CHR—CO—, which has been derived from a naturally or non-naturally occurring L- or D-amino acid. Since it is envisioned that the amino acid derivative of the invention is part of a peptide-based linker, it is preferred that the amino acid derivative has been obtained by one or more chemical reactions of the amino acid side chain of a naturally or non-naturally occurring L- or D-amino acid, or, in cases where the amino acid derivative is located at the C-terminal end of the peptide, the alpha-carboxylic acid group of a naturally or non-naturally occurring L- or D-amino acid. It is to be noted that naturally and non-naturally occurring amino acids can be amino acid derivatives and vice versa.
Examples of non-canonical amino acids, non-naturally occurring amino acids and amino acid derivatives that may be comprised in the linker of the invention include, but are not limited to, α-aminobutyric acid, α-aminoisobutyric acid, ornithine, hydroxyproline, agmatine, {S)-2-amino-4-((2-amino)pyrimidinyl)butanoic acid, alpha-aminoisobutyric acid, p-benzoyl-L-phenylalanine, t-butylglycine, citruiline, cyclohexylalanine, desamino tyrosine, L-(4-guanidino)phenylalanine, homoarginine, homocysteine, homoserine, homolysine, n-formyl tryptophan, norleucine, norvaline, phenylglycine, (S)-4-piperidyl-(N-amidino)glycine, parabenzoyl-L-phenylalanine, sarcosine and 2-thienyl alanine.
Besides alpha-amino acids as described above, the linker of the invention may also comprise one or more β-, γ-, δ- or ε-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. The peptidomimetic may not exclusively contain classical peptide bonds that are formed between two α-amino acids but may additionally or instead comprise one or more amide bonds that are formed between an alpha amino acid and a β-, γ-, δ- or ε-amino acid, or between two β-, γ-, δ- or ε-amino acids. An example of a linker that is a peptidomimetic and comprises an amide bond between an α-amino acid and a β-amino acid is shown in
The term “D-amino acid” is understood to comprise the D-counterparts of both naturally occurring amino acids as well as of non-naturally occurring amino acids.
Since the peptide linkers of the present invention are peptide-based, they are likely to be hydrolyzed by a host cell peptidase once the antibody-payload conjugate has been internalized into a target cell. Accordingly, in certain embodiments, the linker does not necessarily need to comprise a cathepsin cleavage site. Thus, in one embodiment, the linker comprising or having the peptide structure is not cleavable by cathepsin. This includes, in particular, cathepsin B. In one further embodiment, the linker comprising or having the peptide structure does not comprise a valine-alanine motif or a valine-citrulline motif. However, it is to be understood that the invention also encompasses linkers that comprise a cathepsin cleavage site, such as valine-alanine or valine-citrulline. For example, linkers comprising non-canonical or D-amino acids may not be cleaved efficiently by host cell peptidases. In this case, a cathepsin cleavage site in the linker may improve the release of the payload after internalization into the host cell. The linker may further comprise other motifs or self-immolative groups that allow efficient release of the payload inside a target cell if required.
One typical dipeptide structure used in ADC linkers, yet devoid of a Lys residue, is the valine-citrulline motif, as e.g. provided in Brentuximab Vedotin, and discussed in Dubowchik and Firestone 2002. This linker can be cleaved by cathepsin B to release the toxin at the site of disease. The same applies to the valine-alanine motif, which is for example provided in SGN-CD33A.
In one further embodiment, the linker does not comprise polyethylene glycol or a polyethylene glycol derivative.
Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H—(O—CH2—CH2)n—OH. However, it is to be understood that the linkers of the invention may comprise PEG or a PEG-derivative.
It is hence important to understand that, because B can either be a payload or a linking moiety, the method according to the invention has two major embodiments, as shown in the following table 3:
That is, in certain embodiments, the payload is coupled to the linker by chemical synthesis. Accordingly, the linker may have the structure Gly-(Aax)m-Payload or Gly-(Aax)m-Payload-(Aax)n. For example, the payload may be coupled to the C-terminus of a peptide by chemical synthesis. Thus, in certain embodiments the linker may have the structure Gly-Ala-Arg-Payload, Gly-Ala-Arg-Arg-Payload, Gly-Gly-Ala-Arg-Payload, Gly-Gly-Ala-Arg-Arg-Payload or Gly-Gly-Gly-Payload.
According to one further embodiment of the invention, the antibody is at least one selected from the group consisting of
The antibody is preferably a monoclonal antibody.
The antibody can be of human origin, but likewise from mouse, rat, goat, donkey, hamster, or rabbit. In case the conjugate is for therapy, a murine or rabbit antibody can optionally be chimerized or humanized.
Fragment or recombinant variants of antibodies comprising the CH2 domain are, for example,
The antibody can also be bispecific (e.g., DVD-IgG, crossMab, appended IgG-HC fusion) or biparatopic. See Brinkmann and Kontermann (2017) for an overview.
Accordingly, in a particular embodiment, the invention relates to the method according to the invention, 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.
In a preferred embodiment, the antibody is an IgG antibody. That is, the antibody may be an IgG antibody that is glycosylated, preferably at residue N297. Alternatively, the antibody may be a deglycosylated antibody, preferably wherein the glycan at residue N297 has been cleaved off with the enzyme PNGase F. Further, the antibody may be an aglycosylated antibody, preferably wherein residue N297 has been replaced with a non-asparagine residue. Methods for deglycosylating antibodies and for generating aglycosylated antibodies are known in the art.
As discussed herein, IgG antibodies that are glycosylated at residue N297 have several advantages over non-glycosylated antibodies. In addition, it has been demonstrated that the linkers of the invention can be conjugated to antibodies that are glycosylated at residue N297 with unexpectedly high efficiency. Accordingly, in an even more preferred embodiment, the antibody is an IgG antibody that is glycosylated at residue N297 (EU numbering) of the CH2 domain.
In a particular embodiment, the invention relates to the method according to the invention, wherein (a) the linker including the payload or linking moiety B is conjugated to a Gln residue which has been introduced into the heavy or light chain of the antibody by molecular engineering or (b) the linker including the payload or linking moiety B is conjugated to a Gln residue in the Fc domain of the antibody.
According to one further embodiment of the invention, the payload or linking moiety is conjugated to a Gln residue which was introduced into the heavy or light chain of the antibody by molecular engineering.
The term “molecular engineering,” as used herein, refers to the use of molecular biology methods to manipulate nucleic acid sequences. Within the present invention, molecular engineering may be used to introduce Gln residues into the heavy or light chain of an antibody. In general, two different strategies to introduce Gln residues into the heavy or light chain of an antibody are envisioned within the present invention. First, single residues of the heavy or light chain of an antibody may be substituted with a Gln residue. Second, Gln-containing peptide tags consisting of two or more amino acid residues may be integrated into the heavy or light chain of an antibody. For that, the peptide tag may either be integrated into an internal position of the heavy or light chain, that is, between two existing amino acid residues of the heavy or light chain or by replacing them, or the peptide tag may be fused (appended) to the N- or C-terminal end of the heavy or light chain of the antibody.
In the first case, any amino residue of the heavy or light chain of an antibody may be substituted with a Gln residue, provided that the resulting antibody can be conjugated with the linkers of the invention by a microbial transglutaminase. In certain embodiments, the antibody is an antibody wherein amino acid residue N297 (EU numbering) of the CH2 domain of an IgG antibody is substituted, in particular wherein the substitution is an N297Q substitution. Antibodies comprising an N297Q mutation may be conjugated to more than one linker per heavy chain of the antibody. For example, antibodies comprising an N297Q mutation may be conjugated to four linkers, wherein a one linker is conjugated to residue Q295 of the first heavy chain of the antibody, one linker is conjugated to residue N297Q of the first heavy chain of the antibody, one linker is conjugated to residue Q295 of the second heavy chain of the antibody and one linker is conjugated to residue N297Q of the second heavy chain of the antibody. The skilled person is aware that replacement of residue N297 of an IgG antibody with a Gln residue results in an aglycosylated antibody.
In a particular embodiment, the invention relates to the method according to the invention, wherein the Gln residue that has been introduced into the heavy or light chain of the antibody by molecular engineering is comprised in a peptide that has been (a) integrated into the heavy or light chain of the antibody or (b) fused to the N- or C-terminal end of the heavy or light chain of the antibody.
Thus, instead of substituting single amino acid residues of an antibody, peptide tags comprising a Gin residue that is accessible for a transglutaminase may be introduced into the heavy or light chain of the antibody. Such peptide tags may be fused to the N- or C-terminus of the heavy or light chain of the antibody. Preferably, peptide tags comprising a transglutaminase-accessible Gln residue are based to the C-terminus of the heavy chain of the antibody. Even more preferably, the peptide tags comprising a transglutaminase-accessible Gin residue are fused to the C-terminus of the heavy chain of an IgG antibody. Several peptide tags that may be fused to the C-terminus of the heavy chain of an antibody and serve as substrate for a microbial transglutaminase are described in WO 2012/059882, WO 2016/144608, WO 2016/100735, WO 2016/096785 and by Steffen et al. (JBC, 2017) and Malesevic et al. (Chembiochem, 2015).
Exemplary peptide linkers that may be introduced into the heavy or light chain of an antibody, in particular fused to the C-terminus of the heavy chain of the antibody, are LLQGG, LLQG, LSLSQG, GGGLLQGG, GLLQG, LLQ, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, EEQYASTY, EEQYQSTY, EEQYNSTY, EEQYQS, EEQYQST, EQYQSTY, QYQS, QYQSTY, YRYRQ, DYALQ, FGLQRPY, EQKLISEEDL, LQR and YQR.
The skilled person is aware of methods to substitute amino acid residues of antibodies or to introduce peptide tags into antibodies, for example by methods of molecular cloning as described in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press.
According to one further embodiment of the invention, the payload or linking moiety is conjugated to a Gln in the Fc domain of the antibody.
That is, the linkers of the invention may be conjugated to any Gln residue in the Fc domain of the antibody that can serve as a substrate for a microbial transglutaminase.
Typically, the term Fc domain as used herein refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG (CH2 and CH3) and the last three constant region domains of IgE, IgY and IgM (CH2, CH3 and CH4). That is, the linker comprising the payload or linking moiety B may be conjugated to the CH2, CH3 and, where applicable, CH4 domains of the antibody.
According to one further embodiment of the invention, the payload or linking moiety is conjugated to the Gln residue Q295 (EU numbering) of the CH2 domain of the antibody. In a particular embodiment, the invention relates to the method according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG.
It is important to understand that Q295 is an extremely conserved amino acid residue in IgG type antibodies. It is conserved in human IgG1, 2, 3, 4, as well as in rabbit and rat antibodies amongst others. Hence, being able to use Q295 is a considerable advantage for making therapeutic antibody-payload conjugates, or diagnostic conjugates where the antibody is often of non-human origin. The method according to the invention does hence provide an extremely versatile and broadly applicable tool. Even though residue Q295 is extremely conserved among IgG type antibodies, some IgG type antibodies do not possess this residue, such as mouse IgG2a or IgG2b. Thus, it is to be understood that the antibody used in the method of the present invention is preferably an IgG type antibody comprising residue Q295 (EU numbering) of the CH2 domain.
Further, it has been shown that engineered conjugates using Q295 for payload attachment demonstrate good pharmacokinetics and efficacy (Lhospice et al. 2015), and are capable of carrying even unstable toxins prone for degradation (Dorywalska et al. 2015). It is thus expected that similar effects will be seen with this site-specific method since the same residue is modified, but of glycosylated antibodies. Glycosylation may further contribute to overall ADC stability, removal of the glycan moieties as with the mentioned approaches has been shown to result in less-stable antibodies (Zheng et al. 2011).
According to one further embodiment of the invention, the antibody to which the payload or linking moiety is conjugated is glycosylated.
Typical IgG shaped antibodies are N-glycosylated in position N297 (Asp-X-Ser/Thr-motif) of the CH2 domain.
Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody that is glycosylated at residue N297 (EU numbering) of the CH2 domain.
In the literature discussing the conjugation of linkers to a CH2 Gln residue by means of a transglutaminase, the focus has been on small, low-molecular weight substrates. However, in the prior art literature, to accomplish such conjugation, a deglycosylation step in position N297, or the use of an aglycosylated antibody, is always described as necessary (WO 2015/015448; WO 2017/025179; WO 2013/092998).
Quite surprisingly, and against all expectations, however, site-specific conjugation to Q295 of glycosylated antibodies is indeed efficiently possible by using the above discussed oligopeptide structure.
Though Q295 is very close to N297, which is, in its native state, glycosylated, the method according to the invention, using the specified linker, still allows the conjugation of the linker or payload thereto.
However, as shown, the method according to the invention does not require an upfront enzymatic deglycosylation of Q295, nor the use of an aglycosylated antibody, nor a substitution of N297 against another amino acid, nor the introduction of a T299A mutation to prevent glycosylation.
These two points provide significant advantages under manufacturing aspects. An enzymatic deglycosylation step is undesired under GMP aspects, because it has to be made sure that the both the deglycosylation enzyme (e.g., PNGase F) as well as the cleaved glycan have to be removed from the medium.
Furthermore, no genetic engineering of the antibody for payload attachment is necessary, so that sequence insertions which may increase immunogenicity and decrease the overall stability of the antibody can be avoided.
The substitution of N297 against another amino acid has unwanted effects, too, because it may affect the overall stability of the entire Fc domain (Subedi et al, 2015), and the efficacy of the entire conjugate as a consequence that can lead to increased antibody aggregation and a decreased solubility (Zheng et al. 2011) that particularly gets important for hydrophobic payloads such as PBDs. Further, the glycan that is present at N297 has important immunomodulatory effects, as it triggers antibody dependent cellular cytotoxicity (ADCC) and the like. These immunomodulatory effects would get lost upon deglycosylation or any of the other approaches discussed above to obtain an aglycosylated antibody. Further, any sequence modification of an established antibody can also lead to regulatory problems, which is problematic because often times an accepted and clinically validated antibody is used as a starting point for ADC conjugation.
Hence, the method according to the invention allows to easily and without disadvantages make stoichiometrically well-defined ADCs with site specific payload binding.
In view of the above, it is stated that the method of the present invention is preferably used for the conjugation of an IgG antibody at residue Q295 (EU numbering) of the CH2 domain of the antibody, wherein the antibody is glycosylated at residue N297 (EU numbering) of the CH2 domain. However, it is expressly stated that the method of the invention also encompasses the conjugation of deglycosylated or aglycosylated antibodies at residue Q295 or any other suitable Gln residue of the antibody, wherein the Gln residue may be an endogenous Gln residue or a Gln residue that has been introduced by molecular engineering.
The invention also encompasses the conjugation of antibodies of other isotypes than IgG antibodies, such as IgA, IgE, IgM, IgD or IgY antibodies. Conjugation of these antibodies may take place at an endogenous Gln residue, for example an endogenous Gln residue in the Fc domain of the antibody, or at a Gln residue that has been introduced into the antibody by molecular engineering.
In general, the skilled person is aware of methods to determine at which position of an antibody a linker is conjugated. For example, the conjugation site may be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS/MS analysis of the resulting fragments. For example, samples may be deglycosylated with GlycINATOR (Genovis) according to the instruction manual and subsequently digested with trypsin gold (mass spectrometry grade, Promega), respectively. Therefore, 1 μg of protein may be incubated with 50 ng trypsin at 37° C. overnight. LC-MS/MS analysis may be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). For that, 100 ng peptide solution may be loaded onto an Acquity UPLC Symmetry C18 trap column (Waters, part no. 186006527) and trapped with 5 μL/min flow rate at 1% buffer A (Water, 0.1% formic acid) and 99% buffer B (acetonitrile, 0.1% formic acid) for 3 min. Peptides may then be eluted with a linear gradient from 3% to 65% Buffer B within 25 min. Data may be acquired in resolution mode with positive polarity and in a mass range from 50 to 2000 m/z. Other instrument settings may be as follows: capillary voltage 3.2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130° C., cone gas 35 L/h, nano flow gas 0.1 bar, and purge gas 150 L/h. The mass spectrometer may be calibrated with [Glu1]-Fibrinopeptide.
Further, the skilled person is aware of methods to determine the drug-to-antibody (DAR) ration or payload-to-antibody ratio of an antibody-payload construct. For example, the DAR may be determined by hydrophobic interaction chromatography (HIC) or LC-MS.
For hydrophobic interaction chromatography (HIC), samples may be adjusted to 0.5 M ammonium sulfate and assessed via a MAB PAK HIC Butyl column (5 μm, 4.6×100 mm, Thermo Scientific) using a full gradient from A (1.5 M ammonium sulfate, 25 mM Tris HCl, pH 7.5) to B (20% isopropanol, 25 mM Tris HCl, pH 7.5) over 20 min at 1 mL/min and 30° C. Typically, 40 μg sample may be used and signals may be recorded at 280 nm. Relative HIC retention times (HIC-RRT) may be calculated by dividing the absolute retention time of the ADC DAR 2 species by the retention time of the respective unconjugated mAb.
For LC-MS DAR determination, ADCs may be diluted with NH4HCO3 to a final concentration of 0.025 mg/mL. Subsequently, 40 μL of this solution may be reduced with 1 μL TCEP (500 mM) for 5 min at room temperature and then alkylated by adding 10 μL chloroacetamide (200 mM), followed by overnight incubation at 37° C. in the dark. For reversed phase chromatography, a Dionex U3000 system in combination with the software Chromeleon may be used. The system may be equipped with a RP-1000 column (1000 Å, 5 μm, 1.0×100 mm, Sepax) heated to 70° C., and an UV-detector set to a wavelength of 214 nm. Solvent A may consist of water with 0.1% formic acid and solvent B may comprise 85% acetonitrile with 0.1% formic acid. The reduced and alkylated sample may be loaded onto the column and separated by a gradient from 30-55% solvent B over the course of 14 min. The liquid chromatography system may be coupled to a Synapt-G2 mass spectrometer for identification of the DAR species. The capillary voltage of the mass spectrometer may be set to 3 kV, the sampling cone to 30 V and the extraction cone may add up to a value of 5 V. The source temperature may be set to 150° C., the desolvation temperature to 500° C., the cone gas to 20 l/h, the desolvation gas to 600 l/h, and the acquisition may be made in positive mode in a mass range from 600-5000 Da with 1 s scan time. The instrument may be calibrated with sodium iodide. Deconvolution of the spectra may be performed with the MaxEnt1 algorithm of MassLynx until convergence. After assignment of the DAR species to the chromatographic peaks, the DAR may be calculated based on the integrated peak areas of the reversed phase chromatogram.
According to one further embodiment of the invention, the net charge of the linker is neutral or positive.
The net charge of a peptide is usually calculated at neutral pH (7.0). In the simplest approach, the net charge is determined by adding the number of positively charged amino acid residues (Arg and Lys and optionally His) and the number of negatively charged ones (Asp and Glu), and calculate the difference of the two groups. In cases where the linker comprises non-canonical amino acids, the skilled person is aware of methods to determine the charge of the non-canonical amino acid at neutral pH.
According to one further embodiment of the invention, the linker does not comprise negatively charged amino acid residues.
Preferably, the oligopeptide does not comprise the negatively charged amino acid residues Glu and Asp or negatively charged non-canonical amino acids.
According to one further embodiment of the invention, the linker comprises positively charged amino acid residues.
According to one embodiment of the invention, the linker comprises at least two amino acid residues selected from the group consisting of
In certain embodiments, the linker comprises at least one amino acid residue selected from the group consisting of
In certain embodiments, the linker comprises at least one amino acid residue selected from the group consisting of
In certain embodiments, the linker comprises at least one amino acid residue selected from the group consisting of
In certain embodiments, the linker comprises at least one arginine residue.
Table 8 shows that linkers with negative, neutral and positive net charge can be conjugated to a glycosylated antibody with the method of the invention. In particular, linkers comprising a positively charged arginine residue can be conjugated to the glycosylated antibody with high efficiency.
That is, in certain embodiments, the linker according to the invention has a neutral or positive net charge. In certain embodiments, the linker according to the invention has a neutral or positive net charge and comprises at least one arginine and/or histidine residue. In certain embodiments, the linker according to the invention has a neutral or positive net charge and comprises at least one arginine residue. In certain embodiments, the linker according to the invention does not comprise a lysine residue. In certain embodiments, the linker according to the invention has a neutral or positive net charge and does not comprise a lysine residue.
Table 8 further shows that linkers with the amino acid sequence Gly-[Gly/Ala]-Arg-B can be efficiently conjugated to a glycosylated antibody. Accordingly, in certain embodiments, the linker according to the invention has the sequence Gly-[Gly/Ala]-Arg-B or Gly-[Gly/Ala]-Arg-B-(Aax)n.
In certain embodiments, the linker comprising one or more linking moiety B is selected from a group consisting of: GDC, GRCD, GRDC, GGDC, GGCD, GGEC, GGK(N3)D, GGRCD, GGGDC, GC, GRC, GGRC, GRAC, GARC, GGHK(N3), GGK(N3)RC, GARK(N3) and GGARK(N3). In a preferred embodiment, the linker comprising one or more linking moiety B is selected from a group consisting of: GGK(N3)D, GGRCD, GC, GRC, GGRC, GARC, GGK(N3)RC, GARK(N3) and GGARK(N3). In a more preferred embodiment, the linker comprising one or more linking moiety B is selected from a group consisting of: GGRCD, GC, GGRC, GARC, GGK(N3)RC, GARK(N3) and GGARK(N3). In a most preferred embodiment, the linker comprising one or more linking moiety B is selected from a group consisting of: GC, GGRC, GARC and GGARK(N3). In certain embodiments, the linker comprising one or more linking moiety B is GGGK(N3).
According to one further embodiment of the invention, the antibody comprises the Asn residue N297 (EU numbering) in the CH2 domain of the antibody.
According to one further embodiment of the invention, the N297 residue is glycosylated.
According to one further embodiment of the invention, the linker including the payload or linking moiety B is conjugated to the amide side chain of the Gln residue. That is, the amide side chain of the Gln residue of the antibody is conjugated to the N-terminal amino group of the linker via an isopeptide bond.
According to one further embodiment of the invention, the microbial transglutaminase is derived from a Streptomyces species, in particular from Streptomyces mobaraensis, preferentially with a sequence identity of 80% to the native enzyme. Accordingly, the MTG may be a native enzyme or may be an engineered variant of a native enzyme. As shown in
One such microbial transglutaminase is commercially available from Zedira (Germany). It is recombinantly produced in E. coli. Streptomyces mobaraensis transglutaminase has an amino acid sequence as disclosed in SEQ ID NO 48. S. mobaraensis MTG variants with other amino acid sequences have been reported and are also encompassed by this invention (SEQ ID NO:28 and 49).
In another embodiment, a microbial transglutaminase Streptomyces ladakanum (formerly known as Streptoverticillium ladakanum) is being used. Streptomyces ladakanum transglutaminase (U.S. Pat. No. 6,660,510 B2) has an amino acid sequence as disclosed in SEQ ID NO 27.
Both the above transglutaminases can be sequence modified. In several embodiments, transglutaminases can be used which have 80% or more sequence identity with SEQ ID NOs 27, 28, 48 and 49.
Another suitable microbial transglutaminase is commercially from Ajinomoto, called ACTIVA TG. In comparison to the transglutaminase from Zedira, ACTIVA TG lacks 4 N terminal amino acids, but has similar activity.
Further microbial transglutaminases which can be used in the context of the present invention are disclosed in Kieliszek and Misiewicz 2014, WO 2015/191883 A1, WO 2008/102007 A1 and US 2010/0143970, the content of which is fully incorporated herein by reference.
In certain embodiments, a mutant variant of a microbial transglutaminase is used for the conjugation of a linker to an antibody. That is, the microbial transglutaminase that is used in the method of the present invention may be a variant of S. mobaraensis transgluatminase as set forth in SEQ ID NOs: 27 or 29. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutation G250D. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutations G250D and E300D. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutations D4E and G250D. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutations E120A and G250D. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutations A212D and G250D. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO:29 comprises the mutations G250D and K327T.
Microbial transglutaminase may be added to the conjugation reaction at any concentration that allows efficient conjugation of an antibody with a linker. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of less than 100 U/mL, 90 U/mL, 80 U/ml, 70 U/mL, 60 U/mL, 50 U/mL, 40 U/mL, 30 U/mL, 20 U/mL, 10 U/mL or 7 U/mL.
The method according to the invention comprises the use of a microbial transglutaminase. However, it is to be noted that an equivalent reaction may be carried out by an enzyme comprising transglutaminase activity that is of a non-microbial origin. Accordingly, also the antibody-payload conjugates according to the invention may be generated with an enzyme comprising transglutaminase activity that is of a non-microbial origin.
To obtain efficient conjugation, it is preferred that the linker is added to the antibody in molar excess. That is, in certain embodiments, the antibody is mixed with at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 molar equivalents excess of peptide linker versus the antibody.
The method according to the invention is preferably carried out at a pH ranging from 6 to 8.5. Examples 1 and 2 show that the conjugation efficiency is highest at an pH of 7.6. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein the conjugation of the linker to the antibody is achieved at a pH ranging from 6 to 8.5, more preferably at a pH ranging from 7 to 8. In a most preferred embodiment, the invention relates to a method according to the invention, wherein the conjugation of the linker to the antibody is achieved at pH 7.6.
The method of the invention may be carried out in any buffer that is suitable for the conjugation of a linker or linker-payload construct to an antibody with the method of the invention. Buffers that are suitable for the method of the invention include, without limitation, Tris, MOPS, HEPES, PBS or BisTris. Further, the buffer may comprise any salt concentration that is suitable for carrying out the method of the invention. For example, the buffer used in the method of the invention may have a salt concentration ≤150 mM, ≤140 mM, ≤130 mM, ≤120 mM, ≤110 mM, ≤100 mM, ≤90 mM, ≤80 mM, ≤70 mM, ≤60 mM, ≤50 mM, ≤40 mM, ≤30 mM, ≤20 mM, ≤10 mM or 1 mM. In certain embodiments, the buffer may be salt free.
It is to be noted that the optimal reaction conditions (e.g. pH, buffer, salt concentration) may vary between payloads and to some degree depend on the physicochemical properties of the linkers and/or payloads. However, no undue experimentation is required by the skilled person to identify reaction conditions that are suitable for carrying out the method of the invention.
According to one further embodiment of the invention, the linking moiety B is at least one selected from the group consisting of
In certain embodiments of the invention, the linking moiety B comprises
The term “bioorthogonal marker group” has been established by Sletten and Bertozzi (2011) to designate reactive groups that can lead to chemical reactions to occur inside of living systems without interfering with native biochemical processes. A “non-bio-orthogonal entity for crosslinking” may be any molecule that comprises or consists of a first functional group, wherein the first functional group can be chemically or enzymatically crosslinked to a payload comprising a compatible second functional group. Even in cases where the crosslinking reaction is a non-bio-orthogonal reaction, it is preferred that the reaction does not introduce additional modifications to the antibody other than the crosslinking of the payload to the linker. In view of the above, the linking moiety B may either consist of the “bioorthogonal marker group” or the “non-bio-orthogonal entity” or may comprise the “bioorthogonal marker group” or the “non-bio-orthogonal entity”. For example, in case of the linking moiety Lys(N3), both the entire Lys(N3) and the azide group alone may be seen as a bioorthogonal marker group within the present invention.
According to one further embodiment of the invention, the bioorthogonal marker group or the non-bio-orthogonal entity is at least one selected from the group consisting of:
These groups can for example engage in any of the binding reactions shown in table 4:
In the above table 4, the said linking moieties can either be or comprise what is called therein “binding partner 1” or “binding partner 2”.
According to one further embodiment of the invention, the linking moiety B is a Cys residue with a free sulfhydryl group.
The free sulfhydryl group of such Cys residue (or derivative) can be used to conjugate a maleimide-comprising linker toxin construct thereto. See
Toxins comprising a maleimide linker have frequently been used, and also approved by medical authorities, like Adcetris. Thus drugs comprising a MMAE toxin are conjugated to a linker comprising (i) a p-aminobenzyl spacer, (ii) a dipeptide and (iii) a maleimidocaproyl linker, which enables the conjugation of the construct to the free sulfhydryl group of a Cys residue in the antibody.
Providing a Cys-residue in the linker according to the present invention does therefore have the advantage to be able to use off-the-shelf-toxin-maleimide constructs to create antibody-payload conjugates, or, more generally, to be able to fully exploit the advantages of Cys-maleimide binding chemistry. At the same time, off-the-shelf antibodies can be used, which do not have to be deglycosylated.
In specific embodiments, the Cys residue is C-terminal, or intrachain in the peptide linker.
In another embodiment, the linking moiety B comprises an azide group. The skilled person is aware of molecules comprising an azide group which may be incorporated into a linker according to the invention, such as 6-azido-lysine (Lys(N3)) or 4-azido-homoalanine (Xaa(N3)). Linking moieties comprising an azide group may be used as substrates in various bio-orthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC) or Staudinger ligation. For example, in certain embodiments, payloads comprising a cyclooctene derivative, such as DBCO, may be coupled to a linker comprising an azide group by SPAAC (see
In yet another embodiment, the linking moiety B comprises a tetrazine. The skilled person is aware of tetrazine-comprising molecules which may be incorporated into a linker according to the invention, preferably amino acid derivatives comprising a tetrazine group (see for example
The invention further encompasses linkers comprising two different bio-orthogonal marker groups and/or non-bio-orthogonal entities. For example, a linker according to the invention may comprise an azide-comprising linking moiety, such as Lys(N3) or Xaa(N3), and a sulfhydryl-comprising linking moiety, such as cysteine. In certain embodiments, the linker according to the invention may comprise an azide-comprising linking moiety, such as Lys(N3) or Xaa(N3), and a tetrazine-comprising linking moiety, such as a tetrazine-modified amino acid. In certain embodiments, the linker according to the invention may comprise a sulfhydryl-comprising linking moiety, such as cysteine, and a tetrazine-comprising linking moiety, such as a tetrazine-modified amino acid. Linkers comprising two different bio-orthogonal marker groups and/or non-bio-orthogonal entities have the advantage that they can accept two distinct payloads and thus result in antibody-payload conjugates comprising more than one payload.
According to one further embodiment of the invention, it is provided that, in case B is a linking moiety, a further step of linking the actual payload to the linking moiety is carried out. A number of chemical ligation strategies have been developed that fulfill the requirements of bio-orthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry, Baskin et al (2007)), between nitrones and cyclooctynes (Ning et al (2010)), oxime/hydrazone formation from aldehydes and ketones (Yarema, et al (1998)), the tetrazine ligation (Blackman et al (2008)), the isonitrile-based click reaction (Stockmann et al (2011)), and most recently, the quadricyclane ligation (Sletten & Bertozzi (JACS, 2011)), Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC, Kolb & Sharpless (2003)), Strain-promoted azide-alkyne cycloaddition (SPAAC, Agard et al (2004)), or Strain-promoted alkyne-nitrone cycloaddition (SPANC, MacKenzie et al (2014)).
All these documents are incorporated by reference herein to provide sufficient enabling disclosure, and avoid lengthy repetitions.
It is to be understood that the payload is preferably coupled to the bio-orthogonal marker group or the non-bio-orthogonal entity of the linker according to the invention after said linker has been conjugated to a Gln residue of an antibody by means of a microbial transglutaminase. However, the invention also encompasses antibody-payload conjugates wherein a payload has been coupled to a linker comprising a linking moiety in a first step and wherein the resulting linker-payload construct is conjugated to the antibody by a microbial transglutaminase in a second step.
In a particular embodiment, the invention relates to the method according to the invention, wherein the payload is linked to the linking moiety B of the antibody-linker conjugate via a click-reaction, for example any one of the click reactions mentioned above. In a preferred embodiment, the click reaction is SPAAC.
According to one further embodiment of the invention, the payload B is at least one selected from the group consisting of:
Half-life increasing moieties are, for example, PEG-moieties (polyethylenglycol moieties; PEGylation), other polymer moieties, PAS moieties (oliogopeptides comprising Proline, Alanine and Serine; PASylation), or Serum albumin binders. Solubility increasing moieties are, for example PEG-moieties (PEGylation) or PAS moieties (PASylation).
Polymer-toxin conjugates are polymers that are capable of carrying many payload molecules. Such conjugates are sometimes also called fleximers, as e.g. marketed by Mersana therapeutics.
One example of a nucleic acid payload is MCT-485, which is a very small non-coding double stranded RNA which has oncolytic and immune activating properties, developed by MultiCell Technologies, Inc.
Anti-inflammatory agents are for example anti-inflammatory cytokines, which, when conjugated to a target specific antibody, can ameliorate inflammations caused, e.g., by autoimmune diseases.
The term “fluorescent dye” as used herein refers to a dye that absorbs light at a first wavelength and emits at second wavelength that is longer than the first wavelength. In certain embodiment, the fluorescent dye is a near-infrared fluorescent dye, which emits light at a wavelength between 650 and 900 nm. In this region, tissue autofluorescence is lower, and less fluorescence extinction enhances deep tissue penetration with minimal background interference. Accordingly, near-infrared fluorescent imaging may be used to make tissues that are bound by the antibody-payload conjugate of the invention visible during surgery. “Near-infrared fluorescent dyes” are known in the art and commercially available. In certain embodiments, the near-infrared fluorescent dye may be IRDye 800CW, Cy7, Cy7.5, NIR CF750/770/790, DyLight 800 or Alexa Fluor 750.
The term “radionuclide”, as used herein, relates to medically useful radionuclides, including, for example, positively charged ions of radiometals such as Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe and the like, such as 90Y, 111In, 67Cu, 77Lu, 99Tc, 161Tb, 225Ac and the like. The radionuclide may be comprised in a chelating agent. Further, the radionuclide may be a therapeutic radionuclide or a radionuclide that can be used as contrast agent in imaging techniques as discussed below. Radionuclides or molecules comprising radionuclides are known in the art and commercially available.
The term “toxin” as used herein relates to any compound that is poisonous to a cell or organism. A toxin, thus can be, e.g. small molecules, nucleic acids, peptides, or proteins. Specific examples are neurotoxins, necrotoxins, hemotoxins and cyclotoxins. According to one further embodiment of the invention, the toxin is at least one selected from the group consisting of
In certain embodiments, the payload is an auristatin. As used herein, the term “auristatin” refers to a family of anti-mitotic agents. Auristatin derivatives are also included within the definition of the term “auristatin”. Examples of auristatin include, but are not limited to, synthetic analogues of auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and dolastatin.
In certain embodiments, the payload is a maytansinoid. In the context of the present invention, the term “maytansinoid” refers to a class of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus and further maytansinol (Maytansinol) and C-3 ester of natural maytansinol (U.S. Pat. No. 4,151,042); C-3 ester analog of synthetic maytansinol (Kupchan et al., J. Med. Chem. 21: 31-37, 1978; Higashide et al., Nature 270: 721-722, 1977; Kawai et al., Chem. Farm. Bull. 32: 3441-3451; and U.S. Pat. No. 5,416,064); C-3 esters of simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); and C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; and Kawai et al., Chem. Pharm Bull. 12: 3441, 1984). Exemplary maytansinoids that may be used in the method of the invention or that may be comprised in the antibody-payload conjugate of the invention are DM1, DM3, DM4 and/or DM21.
In certain embodiments, the toxic payload molecule is duocarmycin. Suitable duocarmycins may be e.g. duocarmycin A, duocarmycin BL duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. The term “duocarmycin” should be understood as referring also to synthetic analogs of duocarmycins, such as adozelesin, bizelesin, carzelesin, KW-2189 and CBI-TMI.
The toxin, in the sense of the present invention may also be an inhibitor of a drug efflux transporter. Antibody-payload conjugates comprising a toxin and an inhibitor of a drug efflux transporter may have the advantage that, when internalized into a cell, the inhibitor of the drug efflux transporter prevents efflux of the toxin out of the cell. Within the present invention, the drug efflux transporter may be P-glycoprotein. Some common pharmacological inhibitors of P-glycoprotein include: amiodarone, clarithromycin, ciclosporin, colchicine, diltiazem, erythromycin, felodipine, ketoconazole, lansoprazole, omeprazole and other proton-pump inhibitors, nifedipine, paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil, and duloxetine. Elacridar and CP 100356 are other common P-gp inhibitors. Zosuquidar and tariquidar were also developed with this in mind. Lastly, valspodar and reversan are other examples of such agents.
The vitamin can be selected from the group consisting of folates, including folic acid, folacin, and vitamin B9.
The target binding moiety can be a protein or small molecule being capable of specifically binding to a protein or non-protein target. In one embodiment, such target binding moiety is a scFv shaped antibody, a Fab fragment, a F(ab)2 fragment, a nanobody, affibody, a diabody, a VHH shaped antibody, or an antibody mimetic, including a DARPIN.
It is to be understood that the payload can be coupled to a linking moiety of a linker by any suitable reaction, such as a click reaction, or may be attached to the linker by chemical synthesis.
According to one further embodiment of the invention, the linker has two or more linking moieties B.
In such embodiment, an antibody-payload conjugate can be created with, for example, an antibody to payload ratio of 4, with two payloads conjugated to each Q295 residue.
According to one further embodiment of the invention, the two or more linking moieties B differ from one another.
In such embodiment, a first linking moiety could for example be or comprise an azide (N3), while a second linking moiety could be or comprise a tetrazine. Such oligopeptide linker thus allows to conjugate two different payloads to two Gln residues of the antibody, i.e., the Q295 residues of the two CH2 domains of the antibody.
In such way, an antibody payload ratio of 2+2 can be obtained. Using a second payload allows for the development of a completely new class of antibody payload conjugates that go beyond current therapeutic approaches with respect to efficacy and potency.
Such embodiment allows, inter alia, to target two different structures in a cell, like, e.g., the DNA and microtubule. Because some cancers can be resistant to one drug, like e.g., a mirobutule toxin, the DNA-toxin can still kill the cancer cells.
According to another embodiment, two drugs could be used that are only fully potent when they are released at the same time and in the same tissue. This may lead to reduced off-target toxicity in case the antibody is partially degraded in healthy tissues or one drug is pre-maturely lost.
Furthermore, dual-labeled probes can be used for non-invasive imaging and therapy or intra/post-operative imaging/surgery. In such embodiment, a tumor patient can be selected by means of the non-invasive imaging. Then, the tumor can be removed surgically using the other imaging agent (e.g., a fluorescent dye), which helps the surgeon or robot to identify all cancerous tissue.
According to another aspect of the invention, an antibody-payload conjugate is provided which has been generated with a method according to any one of the aforementioned steps.
According to another aspect of the invention, a linker is provided comprising the peptide structure (shown in N->C direction)
Gly-(Aax)m-B-(Aax)n
wherein Gly comprises an N-terminal primary amine, and wherein
Said linker is suitable to be conjugated, via the N-terminal primary amine of the N-terminal glycine (Gly) residue, to a glutamine (Gln) residue comprised in the heavy or light chain of an antibody, by means of a transglutaminase enzyme.
Generally, the advantages and embodiments discussed above in accordance with the method of the present invention do also apply to this aspect. i.e., the linker as composition of matter. Hence, those embodiments shall be deemed disclosed also with the linker as composition of matter.
It is important to understand that in different linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection can be accomplished by amidation. In the context of the present invention, linker peptides that are protected and unprotected at the C-terminus are encompassed.
In a particular embodiment, the invention relates to the linker according to the invention, wherein the linker comprises two or more payloads and/or linking moieties B.
In certain embodiments, the linker may comprise two or more linking moieties and/or payloads. That is the linker may have the peptide structure (shown in N->C direction)
Gly-(Aax)m-B1-(Aax)n-B2-(Aax)o
wherein
In other embodiments, the linker may comprise three linking moieties and/or payloads. That is the linker may have the peptide structure (shown in N->C direction)
Gly-(Aax)m-B1-(Aax)n-B2-(Aax)o-B3-(Aax)p
wherein
It is to be understood, that the invention also encompasses linkers comprising more than three linking moieties and/or payloads, such as 4, 5 or 6 linking moieties and/or payloads. In this case, the peptide structure of the linkers follows the same pattern as described above for the linkers comprising 2 or 3 linking moieties and/or payloads.
In certain embodiments, the invention relates to a linker having the peptide structure (shown in N->C direction)
Gly-(Aax)m-B-(Aax)n
wherein
In this case, it is to be understood that the moiety B may comprise more than one payload and/or linking moiety. For example, B may stand for (B′-(Aax)o-B″), wherein B′ and B″ are payloads and/or linking moieties and wherein o is an integer between ≥0 and ≤12. Alternatively, B may stand for (B′-(Aax)o-B″-(Aax)p-B′″), wherein B′, B″ and B′″ are payloads and/or linking moieties and wherein o and p are integers between ≥0 and ≤12.
In preferred embodiments, m and/or n is ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, or ≥11. In other preferred embodiments, m and/or n is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or ≤1. In further preferred embodiments, m+n is ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, or ≥11. In still further preferred embodiments m+n is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or ≤1.
Members of both ranges can be combined with another to disclose a preferred length range with lower and upper limit.
Accordingly, in a particular embodiment, the invention relates to a linker according to the invention, wherein m+n is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5 or ≤4.
In further embodiments, the linker is not cleavable by cathepsin B, and/or the linker does not comprise a valine-alanine motif or a valine-citrulline motif, and/or the linker does not comprise Polyethylenglycol or a Polyethylenglycol derivative.
According to one embodiment, the linking moiety B is at least one selected from the group consisting of
In certain embodiments, at least one linking moiety B of the linker comprises or consists of
According to one embodiment, the bioorthogonal marker group or the non-bio-orthogonal entity is at least one selected from the group consisting of
In further embodiments, the net charge of the linker is neutral or positive, and/or the linker does not comprise negatively charged amino acid residues, and/or the linker comprises positively charged amino acid residues, and/or the linker comprises at least two amino acid residues selected from the group consisting of
In certain embodiments, the linker comprises at least one amino acid residue selected from the group consisting of
In certain embodiments, the linker comprises at least one amino acid residue selected from the group consisting of
That is, in certain embodiments, the linker according to the invention has a neutral or positive net charge. In certain embodiments, the linker according to the invention has a neutral or positive net charge and comprises at least one arginine and/or histidine residue. In certain embodiments, the linker according to the invention does not comprise a lysine residue. In certain embodiments, the linker has a neutral or positive net charge and does not comprises a lysine residue.
According to one embodiment, the primary amine group is suitable to serve as the substrate of a microbial transglutaminase (MTG).
According to one further embodiment, the linker is suitable for generating an antibody-payload conjugate by means of a microbial transglutaminase (MTG).
According to one further embodiment, the linker is selected from
In a particular embodiment, the invention relates to a linker according to the invention, wherein the linker is selected from the list as shown in table 5.
According to yet another aspect of the invention, a linker-payload construct is provided, comprising at least
In certain embodiments, a linker-payload construct is provided, comprising at least
In a particular embodiment, the invention relates to the linker-payload construct according to the invention, wherein in said construct, the one or more payloads have been covalently bound to the linking moiety B of the linker with a click reaction. That is, the one or more payloads may be attached to a linking moiety B by any of the click reactions discussed above, such as, without limitation, SPAAC, tetrazine ligation or thiol-maleimide conjugation.
Besides a click reaction between the linking moiety in the linker and the payload, the payload may be covalently bound to the linker by any enzymatic or non-enzymatic reaction known in the art. For that, the payload may be bound to the C-terminus of the linker or to an amino acid side chain of the linker.
In certain embodiments, the payload is coupled to a linker by chemical synthesis. The skilled person is aware of methods to couple a payload to a peptide linker by chemical synthesis. For example, an amine-comprising payload, or a thiol-comprising payload (for e.g. maytansine analogs), or an hydroxyl-containing payload (for e.g. SN-38 analogs) may be attached to the C-terminus of a peptide linker by chemical synthesis to obtain, for example, the linkers shown in
In a particular embodiment, the invention relates to the linker-payload construct according to the invention, wherein in said construct, the linker and/or the payload have optionally been chemically modified during binding to allow covalent or non-covalent binding, to form said construct.
In case two or more payloads are being used, the latter can be identical or different from one another.
In one embodiment, the payload is at least one selected from the group consisting of
In another embodiment, the toxin is at least one selected from the group consisting of
According to another aspect of the invention, an antibody-payload conjugate is provided comprising
In a particular embodiment, the invention relates to an antibody payload conjugate comprising
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the conjugation has been achieved with a microbial transglutaminase (MTG).
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the conjugation has been achieved before or after formation of the linker-payload construct. That is, the invention encompasses antibody-payload conjugates wherein the linkers have been conjugated to the antibody in a first step before the one or more payloads are coupled to the linking moieties of the linkers in a second step. However, the invention also encompasses antibody-payload conjugates, wherein the one or more payloads are coupled to the linking moieties of the linkers in a first step, and wherein the resulting linker-payload constructs are then conjugated to the antibody in a second step. Further, the one or more payloads may be attached to a peptide linker by means of chemical synthesis and the resulting linker-payload construct may then be conjugated to the antibody in a one-step reaction.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, 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.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody is an IgG antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody is a glycosylated antibody, a deglycosylated antibody or an aglycosylated antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the glycosylated antibody is an IgG antibody that is glycosylated at residue N297 (EU numbering) of the CH2 domain or wherein the glycosylated antibody is an antibody of a different isotype that is glycosylated at a residue that is homologous to residue N297 (EU numbering) of the CH2 domain of an IgG antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein (a) the linker-payload construct is conjugated to a Gln residue which has been introduced into the heavy or light chain of the antibody by molecular engineering or (b) the linker-payload construct is conjugated to a Gln residue in the Fc domain of the antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody or a homologous Gln residue of an antibody of a different isotype.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody that is glycosylated at residue N297 (EU numbering) of the CH2 domain.
The antibody of the method or the antibody-payload conjugate of the invention may be any antibody, preferably any IgG type antibody. For example, the antibody may be, without limitation Brentuximab, Trastuzumab, Gemtuzumab, Inotuzumab, Avelumab, Cetuximab, Rituximab, Daratumumab, Pertuzumab, Vedolizumab, Ocrelizumab, Tocilizumab, Ustekinumab, Golimumab, Obinutuzumab, Polatuzumab or Enfortumab.
That is, in certain embodiments, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Brentuximab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Trastuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Gemtuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Inotuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Avelumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Cetuximab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Rituximab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Daratumumbab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Pertuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Vedolizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Ocrelizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Tocilizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Ustekinumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Golimumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Obinutuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Polatuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is Enfortumab.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the Gln residue that has been introduced into the heavy or light chain of the antibody by molecular engineering is N297Q (EU numbering) of the CH2 domain of an aglycosylated IgG antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the Gln residue that has been introduced into the heavy or light chain of the antibody by molecular engineering is comprised in a peptide that has been (a) integrated into the heavy or light chain of the antibody or (b) fused to the N- or C-terminal end of the heavy or light chain of the antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the peptide comprising the Gln residue has been fused to the C-terminal end of the heavy chain of the antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the peptide comprising the Gln residue is selected from a group consisting of: LLQGG, LLQG, LSLSQG, GGGLLQGG, GLLQG, LLQ, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, EEQYASTY, EEQYQSTY, EEQYNSTY, EEQYQS, EEQYQST, EQYQSTY, QYQS, QYQSTY, YRYRQ, DYALQ, FGLQRPY, EQKLISEEDL, LQR and YQR.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises at least on toxin.
That is, the antibody-payload conjugate of the invention comprises an antibody that is conjugated to at least one linker, wherein the one linker comprises at least one toxin. In certain embodiments, the antibody-payload conjugate comprises two linkers, wherein each heavy chain of the antibody is conjugated to one linker, respectively. In certain embodiments, the antibody-payload conjugate comprises four linkers, wherein each heavy chain of the antibody is conjugated to two linkers, respectively. In such cases, each linker may contain one or more payloads, such as toxins.
In certain embodiments, the antibody-payload conjugate according to the invention comprises two linkers, wherein each linker comprises one payload, for example a toxin. In other embodiments, the antibody-payload conjugate according to the invention comprises two linkers, wherein each linker comprises two payloads, for example one toxin and one other payload or two identical or different toxins. In embodiments where the antibody-payload conjugate comprises two linkers, it is preferred that the linkers are conjugated to residue Q295 of the two heavy chains of an IgG antibody. Even more preferably, the antibody is an IgG antibody that is glycosylated at residue N297.
In certain embodiments, the antibody-payload conjugate according to the invention comprises four linkers, wherein each linker comprises one payload, for example a toxin. In other embodiments, the antibody-payload conjugate according to the invention comprises four linkers, wherein each linker comprises two payloads, for example one toxin and one other payload or two identical or different toxins. In embodiments where the antibody-payload conjugate comprises four linkers, it is preferred that the linkers are conjugated to residues Q295 and N297Q of the two heavy chains of an IgG antibody.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises two different toxins.
In certain embodiments, the antibody-payload conjugate according to the invention comprises two different toxins. That is, in certain embodiments, the antibody-payload conjugate may comprise two linkers, wherein each linker comprises two different toxins. Antibody-payload conjugates comprising two different toxins have the advantage that they may have increased cytotoxic activity. Such increased cytotoxic activity may be achieved by combining two toxins that target two different cellular mechanisms. For example, the antibody-payload conjugates according to the invention may comprise a first toxin that inhibits cell division and a second toxin is a toxin that interferes with replication and/or transcription of DNA.
Accordingly, in a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein a first toxin is a toxin that inhibits cell division and a second toxin is a toxin that interferes with replication and/or transcription of DNA.
A toxin that inhibits cell division, such as an anti-mitotic agent or a spindle poison, is an agent that has the potential to inhibit or prevent mitotic division of a cell. A spindle poison is a poison that disrupts cell division by affecting the protein threads that connect the centromere regions of chromosomes, known as spindles. Spindle poisons effectively cease the production of new cells by interrupting the mitosis phase of cell division at the spindle assembly checkpoint (SAC). The mitotic spindle is composed of microtubules (polymerized tubulin) that aid, along with regulatory proteins; each other in the activity of appropriately segregating replicated chromosomes. Certain compounds affecting the mitotic spindle have proven highly effective against solid tumors and hematological malignancies.
Two specific families of antimitotic agents—vinca alkaloids and taxanes—interrupt the cell's division by the agitation of microtubule dynamics. The vinca alkaloids work by causing the inhibition of the polymerization of tubulin into microtubules, resulting in the G2/M arrest within the cell cycle and eventually cell death. In contrast, the taxanes arrest the mitotic cell cycle by stabilizing microtubules against depolymerization. Even though numerous other spindle proteins exist that could be the target of novel chemotherapeutics, tubulin-binding agents are the only types in clinical use. Agents that affect the motor protein kinesin are beginning to enter clinical trials. Another type, paclitaxel, acts by attaching to tubulin within existing microtubules. Preferred toxins that inhibit cell division within the present invention are auristatins, such as MMAE and MMAF, and maytansinoids, such as DM1, DM3, DM4 and/or DM21.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein at least one of the toxins is an auristatin or a maytansinoid.
Several agents that prevent the correct replication and/or transcription of DNA molecules and have been shown to be suitable in cancer treatment are known to the person skilled in the art. For example, antimetabolites such as nucleotide or nucleoside analogs which are misincorporated into newly formed DNA and/or RNA molecules are known in the art and have been summarized by Tsesmetzis et al, Cancers (Basel), 2018, 10(7): 240. Other toxins that are known to interfere with the replication and/or transcription of DNA are duoromycins.
Accordingly, in certain embodiments, the antibody-payload conjugate according to the invention comprises two different toxins, wherein the first toxin is a duoromycin and wherein the second payload is an auristatin or a maytansinoid.
In certain embodiments, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises two different auristatins.
One main advantage of antibody-payload conjugates comprising two different toxins is that the antibody-payload conjugates may still act against target cells that have escaped the mechanism of action of one of the toxins and/or that the antibody-payload conjugate may have a higher efficacy against heterogenous tumors.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises a toxin and an inhibitor of a drug efflux transporter.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises a toxin and a solubility increasing moiety.
That is, the antibody-payload conjugate may comprise two payloads, wherein the first payload is a toxin and the second payload is a solubility increasing moiety. Structure 5 in
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises a toxin and an immunostimulatory agent.
As used herein and depending on context, the term “immunostimulatory agent” includes compounds that increase a subject's immune response to an antigen. Examples of immunostimulatory agents include immune stimulants and immune cell activating compounds. Antibody-payload conjugates of the present invention may contain immunostimulatory agents that help program the immune cells to recognize ligands and enhance antigen presentation. Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen associated molecular patterns (PAMPs), e.g., an infection-mimicking composition such as a bacterially-derived immunomodulator (a.k.a., danger signal) and damage associated molecular pattern (DAMPs), e.g. a composition mimicking a stressed or damaged cell. TLR agonists include nucleic acid or lipid compositions (e.g., monophosphoryl lipid A (MPLA)). In one example, the TLR agonist comprises a TLR9 agonist such as a cytosine-guanosine oligonucleotide (CpG-ODN), a poly(ethylenimine) (PEI)-condensed oligonucleotide (ODN) such as PEI-CpG-ODN, or double stranded deoxyribonucleic acid (DNA). In another example, the TLR agonist comprises a TLR3 agonist such as polyinosine-polycytidylic acid (poly (I:C)), PEI-poly (I:C), polyadenylic-polyuridylic acid (poly (A:U)), PEI-poly (A:U), or double stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines/cytokines, fungal beta-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises two different immunostimulatory agents.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the at least one immunostimulatory agent is a TLR agonist.
The term “TLR agonist”, as used herein, refers to a molecule which is capable of causing a signaling response through a TLR signaling pathway, either as a direct ligand or indirectly through generation of endogenous or exogenous. Agonistic ligands of TLR receptors are (i) natural ligands of the actual TLR receptor, or functionally equivalent variants thereof which conserve the capacity to bind to the TLR receptor and induce co-stimulation signals thereon, or (ii) an agonist antibody against the TLR receptor, or a functionally equivalent variant thereof capable of specifically binding to the TLR receptor and, more particularly, to the extracellular domain of said receptor, and inducing some of the immune signals controlled by this receptor and associated proteins. The binding specificity can be for the human TLR receptor or for a TLR receptor homologous to the human one of a different species.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the antibody-payload conjugate comprises a radionuclide and a fluorescent dye.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, wherein the radionuclide is a radionuclide that is suitable for use in tomography, in particular single-photon emission computed tomography (SPECT) or positron emission tomography (PET), and wherein the fluorescent dye is a near-infrared fluorescent dye.
The term “radionuclide” as used herein has the same meaning as radioactive nuclide, radioisotope or radioactive isotope.
The radionuclide is preferably detectable by nuclear medicine molecular imaging technique(s), such as, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid of SPECT and/or PET or their combinations. Single Photon Emission Computed Tomography (SPECT) herein includes planar scintigraphy (PS).
An hybrid of SPECT and/or PET is for example SPECT/CT, PET/CT, PET/IRM or SPECT/IRM.
SPECT and PET acquire information on the concentration (or uptake) of radionuclides introduced into a subject's body. PET generates images by detecting pairs of gamma rays emitted indirectly by a positron-emitting radionuclide. A PET analysis results in a series of thin slice images of the body over the region of interest (e.g., brain, breast, liver, . . . ). These thin slice images can be assembled into a three dimensional representation of the examined area. SPECT is similar to PET, but the radioactive substances used in SPECT have longer decay times than those used in PET and emit single instead of double gamma rays. Although SPECT images exhibit less sensitivity and are less detailed than PET images, the SPECT technique is much less expensive than PET and offers the advantage of not requiring the proximity of a particle accelerator. Actual clinical PET presents higher sensitivity and better spatial resolution than SPECT, and presents the advantage of an accurate attenuation correction due to the high energy of photons; so PET provides more accurate quantitative data than SPECT. Planar scintigraphy (PS) is similar to SPECT in that it uses the same radionuclides. However, PS only generates 2D-information.
SPECT produces computer-generated images of local radiotracer uptake, while CT produces 3-D anatomic images of X ray density of the human body. Combined SPECT/CT imaging provides sequentially functional information from SPECT and the anatomic information from CT, obtained during a single examination. CT data are also used for rapid and optimal attenuation correction of the single photon emission data. By precisely localizing areas of abnormal and/or physiological tracer uptake, SPECT/CT improves sensitivity and specificity, but can also aid in achieving accurate dosimetric estimates as well as in guiding interventional procedures or in better defining the target volume for external beam radiation therapy. Gamma camera imaging with single photon emitting radiotracers represents the majority of procedures.
The radionuclide may be selected in the group consisting of technetium-99m (99mTc), gallium-67 (67Ga), gallium-68 (68Ga) yttrium-90 (90Y), indium-111 (111In), rhenium-186 (186Re), fluorine-18 (18F), copper-64 (64Cu), terbium-149 (149Tb) or thallium-201 (201TI). The radionuclide may be comprised in a molecule or bound to a chelating agent.
According to another aspect of the invention, a pharmaceutical composition is provided, the composition comprising the linker according to the above description, the linker-payload construct according to the above description, and/or the antibody-payload conjugate according to the above description.
According to another aspect of the invention, a pharmaceutical product is provided, the product comprising the antibody-payload conjugate according to the above description, or the pharmaceutical composition according to the above description, and at least one further pharmaceutically acceptable carrier.
A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
Pharmaceutical formulations of the antibody-payload conjugates described herein are prepared by mixing such conjugates having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Flemington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed, (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes Zn protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
In one embodiment, the invention relates to the antibody-payload conjugate according to the invention, the pharmaceutical composition according to the invention or the pharmaceutical product according to the invention for use in therapy and/or diagnostics.
That is, the antibody-payload conjugates of the invention may be used in the treatment of a subject or in diagnosing a disease or condition in a subject. An individual or subject is a mammal. Mammals include, but are not limited to, domesticated animals (cows, sheep, cats, dogs, and horses), primates (e.g., humans and non human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
According to another aspect of the invention, the pharmaceutical composition according to the above description or the product according to the above description is provided (for the manufacture of a medicament) for the treatment of a patient
Preferably, the invention relates to the antibody-payload conjugate according to the invention, the pharmaceutical composition according to the invention or the pharmaceutical product according to the invention for use in treatment of a patient suffering from a neoplastic disease.
The term “neoplastic disease” as used herein refers to a condition characterized by uncontrolled, abnormal growth of cells. Neoplastic diseases include cancer. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. Preferred cancers include liver cancer, lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma.
That is, the antibody-payload conjugates of the invention are preferably used for the treatment of cancer. As such, in certain embodiments, the antibody-payload conjugates comprise an antibody that specifically binds to an antigen that is present on a tumor cell. In certain embodiments, the antigen is an antigen on the surface of a tumor cell. In certain embodiments, the antigen on the surface of the tumor cell is internalized into the cell together with the antibody-payload conjugate upon binding of the antibody-payload conjugate to the antigen.
If the antibody-payload conjugate is used in the treatment of cancer, it is preferred that the antibody-payload conjugate comprises at least one payload that has the potential to kill or inhibit the proliferation of the tumor cell to which the antibody-drug conjugate binds to. In certain embodiments, the at least one payload exhibits its cytotoxic activity after the antibody-payload conjugate has been internalized into the tumor cell. In certain embodiments, the at least one payload is a toxin.
According to another aspect of the invention, a method of treating or preventing a neoplastic disease is provided, said method comprising administering to a patient in need thereof the antibody-payload conjugate according to the above description, the pharmaceutical composition according to the above description, or the product according to the above description.
The inflammatory disease can be an autoimmune disease. The infectious disease can be a bacterial infection or a viral infection.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, the pharmaceutical composition according to the invention or the pharmaceutical product according to the invention for use in pre, intra- and/or postoperative imaging.
That is, the antibody-payload conjugate according to the invention may be used in imaging. For that, the antibody-payload conjugate may be visualized while binding to a specific target molecule, cell or tissue. Different techniques are known in the art to visualize particular payloads. For example, if the payload is a radionuclide, the molecules, cells, or tissues to which the antibody-payload conjugate binds may be visualized by PET or SPECT. If the payload is a fluorescent dye, the molecules, cells, or tissues to which the antibody-payload conjugate binds may be visualized by fluorescence imaging. In certain embodiments, the antibody-payload conjugate according to the invention comprises two different payloads, for example a radionuclide and a fluorescent dye. In this case, the molecule, cell or tissue to which the antibody-payload conjugate binds may be visualized using two different and/or complementary imaging techniques, for example PET/SPECT and fluorescence imaging.
The antibody-payload conjugate may be used for pre- intra- and/or post-operative imaging.
Pre-operative imaging encompasses all imaging techniques that may be performed before a surgery to make specific target molecules, cells or tissues visible when diagnosing a certain disease or condition and, optionally, to provide guidance for a surgery. Preoperative imaging may comprise a step of making a tumor visible by PET or SPECT before a surgery is performed by using an antibody-payload conjugate that comprises an antibody that specifically binds to an antigen on the tumor and is conjugated to a payload that comprises a radionuclide.
Intra-operative imaging encompasses all imaging techniques that may be performed during a surgery to make specific target molecules, cells or tissues visible and thus provide guidance for the surgery. In certain embodiments, an antibody-payload conjugate comprising a near-infrared fluorescent dye may be used to visualize a tumor during surgery by near-infrared fluorescent imaging. Intraoperative imaging allows the surgeon to identify specific tissues, for example tumor tissue, during surgery and thus may allow complete removal of tumor tissue.
Post-operative imaging encompasses all imaging techniques that may be performed after a surgery to make specific target molecules, cells or tissues visible and to evaluate the result of the surgery. Post-operative imaging may be performed similarly as pre-operative surgery.
In certain embodiments, the invention relates to antibody-payload conjugates comprising two or more different payloads. For example, the antibody-payload conjugate may comprise a radionuclide and a near-infrared fluorescent dye. Such an antibody-payload conjugate may be used for imaging by PET/SPECT and near-infrared fluorescent imaging. The advantage of such an antibody is that it may be used to visualize the target tissue, for example a tumor before and after a surgery by PET or SPECT. At the same time, the tumor may be visualized during the surgery by near-fluorescent infrared imaging.
In a particular embodiment, the invention relates to the antibody-payload conjugate according to the invention, the pharmaceutical composition according to the invention or the pharmaceutical product according to the invention for use in intraoperative imaging-guided cancer surgery.
As mentioned above, the antibody-payload conjugate of the invention may be used to visualize a target molecule, cell or tissue and to guide a surgeon or robot during a surgery. That is, the antibody-payload conjugate may be used to visualize tumor tissue during a surgery, for example by near-infrared imaging and to allow complete removal of the tumor tissue.
Said conjugate or product is administered to the human or animal subject in an amount or dosage that efficiently treats the disease. Alternatively, a corresponding method of treatment is provided.
An antibody-payload conjugate of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
Antibody-payload conjugates of the invention would be formulated, dosed, and administered in a fashion consistent of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody-payload conjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody-payload conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
For the prevention or treatment of disease, the appropriate dosage of an antibody-payload conjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody-payload conjugate, the severity and course of the disease, whether the antibody-payload conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody-payload conjugate, and the discretion of the attending physician. The antibody-payload conjugate is suitably administered to the patient at one time or over a series of treatments.
The following table 5 shows different linkers that can be used in the context of the present invention, and their SEQ ID Numbers. For the avoidance of doubt, if there is a discrepancy with the electronic WIPO ST 25 sequence listing, the sequences of this table are to be deemed the correct ones.
It is important to understand that in some linker peptides shown herein, the moiety at the C-terminus is simply designated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GARK(N3) or GlyAlaArgLys(N3) does actually mean GARK1 (with K1=Lys(N3).
It is furthermore important to understand that in different linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise.
Protection can be accomplished by amidation of the former. In the context of the present invention, both the protected and unprotected linker peptides are encompassed.
For example GARK(N3) does indeed encompass two variants, with the C-terminus protected or unprotected. On the other hand, GARK(N3)—COOH for example explicitly specifies a peptide which is not protected, i.e., has an unprotected C terminus.
The following table 5 shows some linkers that are encompassed, and suitable to be used, in the context of the present invention:
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.
All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′->3′.
Peptides were used as obtained and dissolved at a suitable stock concentration (e.g. 25 mM) following the manufacturers instruction, aliquots were prepared and stored at −20° C. Two antibodies of IgG-subclass (antibody 1: anti Her2 IgG1, antibody 2: anti CD38 IgG1) were modified as follows: 1 mg/mL of non-deglycosylated antibody (˜6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e. ˜533 μM), 6 U/mL MTG and buffer. The reaction mixture was incubated for 20 h at 37° C. and then subjected for LC-MS analysis under reducing conditions.
The following table 6 shows the conjugation efficiency of a linker according to the present invention (marked with a (*) vs other linkers:
It is clearly visible that the peptide comprising a N-terminal Gly residue, with no further primary amine except the N-terminal primary amine, has by far the best conjugation efficiency with the Q295 residue in the glycosylated antibody, even though the other peptides likewise comprise N-terminal primary amines, yet not comprised in a Gly residue
Peptides were used as obtained and dissolved at a suitable stock concentration (e.g. 25 mM) following the manufacturers instruction, aliquots were prepared and stored at −20° C. Two antibodies of IgG-subclass (antibody 1: anti Her2 IgG1, antibody 2: anti CD38 IgG1) were modified as follows: 1 mg/mL of non-deglycosylated antibody (˜6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e. ˜533 μM), 6 U/mL MTG and buffer. The reaction mixture was incubated for 20 h at 37° C. and then subjected for LC-MS analysis under reducing conditions.
The following table 7 shows the conjugation efficiency of a linker according to the present invention (marked with a (*) vs another linker βAGARK(N3) is shown in
It is clearly visible that the peptide comprising a N-terminal Gly residue, with no further primary amine except the N-terminal primary amine, has by far the better conjugation efficiency with the Q295 residue in the glycosylated antibody, compared to the structurally similar linker which has an N-terminal β-Ala residue.
Cell lines and culture: MDA-MB-231, and SK-BR-3 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 following standard cell-culture protocols.
SK-BR-3 is a breast cancer cell line isolated by the Memorial Sloan-Kettering Cancer Center in 1970 that is used in therapeutic research, especially in context of HER2 targeting. MDA-MB-231 cells are derived from human breast adenocarcinoma of the “basal” type, and are triple negative (ER, PR and HER2 negative). Adcetris (Brentuximab Vedotin) is a commercially available antibody drug conjugate that targets CD30 and is hence expected to not be active against cells which do not express CD30, e.g., MDA-MB-231, and SK-BR-3. Kadcyla (Trastuzumab emtansine) is a commercially available antibody drug conjugate that targets Her2 and is hence expected to be active against cells which express Her2 (e.g., SK-BR-3), and not active against cells which do not express Her2 (e.g., MDA-MB-231). p684 and p579 are antibody drug conjugates produced with the linker technology as specified herein, with linkers having an N-terminal glycine (GARK(N3) (P684) and GGARK(N3) (P579)). To generate the antibody-payload conjugate, a May (Maytansine) molecule coupled to a DBCO group (see below) has been clicked to the azide groups of the linkers. Both conjugates use a non-deglycosylated antibody, and target Her2, having a Drug to Antibody Ratio of 2, hence bearing two May (Maytansine) molecules. Herceptin is a non-deglycosylated, unconjugated antibody, targeting Her2.
Cell toxicity assay: Cells were seeded into 96 well plates (white walled, clear flat bottom plates) at densities of 10,000 cells per well and incubated overnight at 37° C. and 5% CO2. Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) were serially diluted 1:4 in media at a starting concentration of 10 μg/mL (66.7 nM). Media was removed from cells, and mAb/ADC dilutions were added. Cells treated with media only served as the reference for 100% viability. Cells were incubated with antibodies for three days at 37° C. and 5% CO2.
Cell viability was assessed by Cell Titer-Glo® (Promega) following manufacturer's instructions and as briefly outlined here. Plates were equilibrated to room temperature for 30 minutes. Cell Titer-Glo® reagent was made by addition of Cell Titer-Glo buffer to substrate. 50 μL per well of Cell Titer-Glo® reagent was added and incubated at room temperature with shaking for two minutes followed by an additional 30 minutes incubation at room temperature. Luminescence was detected on a Perkin Elmer 2030 Multilabel Reader Victor™ X3 plate reader using an integration time of 1 second.
The data were processed as follows: luminescence values of wells treated with media only were averaged and served as the reference for 100% viability. Percent viability of mAb/ADC treated wells was calculated using the following equation:
Normalized percent viability was plotted versus the logarithm of mAb/ADC concentration and the data were fit using GraphPad Prism 7.00.
As can be seen in
Peptides were used as obtained and dissolved at a suitable stock concentration (e.g. 25 mM) following the manufacturers instruction, aliquots were prepared and stored at −20° C. The anti-Her2 IgG1 antibody (Trastuzumab) was modified as follows: 1 mg/mL of non-deglycosylated antibody (˜6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e. ˜533 μM), 6 U/mL MTG and buffer. The reaction mixture was incubated for 20 h at 37° C. and then subjected for LC-MS analysis under reducing conditions.
The following table 8 shows the conjugation efficiencies (CE in %) of various linkers falling within the scope of the present invention.
It is important to understand that in some linker peptides shown herein, the moiety at the C-terminus is simply designated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GAR(N3) does actually mean GARK1, with K1=Lys(N3), or GlyAlaArgLys(N3).
It is furthermore important to understand that in different linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection can be accomplished by amidation. In the context of the present invention, both the protected and unprotected linker peptides are encompassed. For example GARK(N3) does indeed encompass two variants, with the C-terminus protected or unprotected.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19163810.5 | Mar 2019 | EP | regional |
This application is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/EP2020/057697, filed Mar. 19, 2020, which claims priority to European Application No. 19163810.5, filed Mar. 19, 2019, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/057697 | 3/19/2020 | WO | 00 |
