Antibody drug conjugates

Abstract

The present invention relates to a method of generating antibody payload conjugates in which protein asparaginyl ligases (PALs) and glutaminyl cyclase (QC) are used to conjugate payloads to antibodies. In a specific embodiment, it exemplified trastuzumab comprising a GGGSNQL at the C-terminus of the light chain that is conjugated to MMAE comprising the Gl motif.

Description

[0001] Antibody Drug Conjugates

[0002] Field of the Invention

[0003] The present invention relates to antibody drug conjugates and methods for producing the same

[0004] Background

[0005] The ADC field is currently rapidly expanding following the pioneering development of brentuximab (Adcetris)1and ado-trastuzumab emtansine (Kadcyla)2-3as powerful targeted therapeutics against lymphoma and breast cancer respectively. Today the market of antibodies conjugated to cytotoxic drugs, radioisotopes or PEG is estimated to exceed 100 billion USD4. Conceptually, ADCs allow specific targeting of tumor cells by highly cytotoxic agents, while sparing healthy tissues and hence limiting off- target toxicity5-7. This is a consequence of coupling a tumor-specific antibody -mostly in the IgG format- with a compound too toxic to be used in isolation compared to standard stand-alone chemotherapeutic agents4-8With proper linker design, the ADC can travel in the bloodstream and bind to tumor cells displaying the antigenic protein on its surface. Following cell internalization and lysosomal degradation, the toxic payload is then only released in the tumor micro-environment, leading to selective tumor killing and limiting off-target toxicity.

[0006] Despite several early successes, many ADCs are failing in advanced clinical trials4and their inherent toxicity limits their usage for many indications. Parameters to consider for ADC optimization are multiple and include the nature of the antigen targeted by the ADC, the precise epitope bound, the antibody, the payload drug, a chemically or enzymatically labile or non-labile linker, the chemistry employed to attach the drug to the antibody and the stoichiometry of the resulting conjugate defined as drug-to-antibody ratio (DAR). The therapeutic window of ADCs could be augmented by improving both linker and conjugation technology by affording selective delivery of an ADC with predefined DAR -typically 2 or 4 cytotoxic payloads per mAb carrier. In addition, to start addressing the pressing issue of tumor resistance to immunotherapeutic treatments, the capacity to selectively deliver two toxic payloads having two different biological targets would enrich current therapeutic options for various cancers Thus, the availability of a simple, universally applicable, yet efficient and flexible site-specific conjugation technology would help to adjust the doses of payloads delivered to the tumor, facilitate optimization of the therapeutic index and help streamline the manufacturing of ADCs.

[0007] Current conjugation technologies include chemical coupling to exposed cysteine residues via disulfide formation with a maleimide moiety present in the linker7-9Alternatively, activation by N- hydroxysuccinimide of exposed lysine e-amine groups can lead to the formation of a stable amide bond with the linker Both technologies -especially with exposed lysine residues- suffer from the non-specificity of site conjugation leading to significant heterogeneity of the ADC product requiring extensive rounds of purification In animal models, ADCs with higher DARs were observed to be cleared more rapidly from circulation with a lower therapeutic index Several alternative technologies have attempted to address this problem (reviewed in ref 9): engineered cysteine mutants have been designed that target selected positions in the antibody constant region. Alternative methods such as the genetic incorporation of unnatural amino acids10are also used to synthesize specific auristatin conjugates of trastuzumab, directed against HER21 1 . In parallel, several enzymatic-based conjugation technologies have begun to address the issue of specificity4 9These include the use of glycosyltransferases such as galactosyl- and sialyl-transferases to attach the payload to the conserved N-glycosylated Asn297 residue present in the CH2 domain of the Fc region12. Microbial transglutaminases have also been used, leading to conjugation via isopeptide bond formation of the primary amine of a lysine residue from the linker to a glutamine residue of the mAb13

[0008] Since their discovery in 2014, peptide asparaginyl ligases (PALs) from cyclotide-producing plants have emerged as attractive tools for polypeptide cyclization and bioconjugates synthesis14. These enzymes target Asx (Asn at neutral pH or Asp at acidic pH of about 4.5) as P1 residue and catalyze a transpeptidation reaction with an incoming peptide having a small residue such as Gly or Ala as N- terminal amine donor14. Several highly efficient PALs can now be efficiently overexpressed in either recombinant bacterial or insect cell host systems and can be used for site-specific bioconjugation15 16. The structural bases for their substrate recognition have also been reported17. However, the application of hyperactive recombinant PALs such as VyPAL2 from Viola yedoensis15or oaAEPI b-C247A from Oldenlandia affinis16to label large molecules such as a full-length lgG1 (150 kDa) has not been fully explored.

[0009] Xia et al., (2023) J. Am Chem Soc 145, 6838-6844 and WO2023 / 191726 describe a cascade enzymatic reaction scheme for irreversible transpeptidative protein ligation. Peptidyl aspariginyl ligases (PALs) are advantageous for bioconjugation due to their high catalytic efficiency and a minimal tripeptide recognition, and these documents describe a method in which the reversible PAL-mediated ligation is made irreversible by coupling it to glutaminyl cyclase (QC) catalyzed pyroglutamyl formation. However, they do not investigate this method for the generation of antibody payload conjugates

[0010] Beerli et al. (2015) PLoS ONE 10(7): e0131177 describes the use of sortase to enzymatically generate site-specific antibody drug conjugates Sortase recognises the LPXTG motif, and Beerli describes the incorporation of this sequence into the C-terminus of heavy and light chains of an antibody, which were then conjugated to a Glys-modified toxin

[0011] The present invention has been devised in light of the above considerations

[0012] Summary of the Invention

[0013] The present invention relates to a method for generating antibody payload conjugates in which protein asparaginyl ligases (PALs) are used to conjugate payloads to antibodies. The method provides rapid, high-efficiency and site-specific conjugation of antibody payload conjugates. Antibody payload conjugates prepared by this method have good properties of stability and efficacy and carry an amino acid signature resulting from their manner of conjugation (the PAL conjugation motif) In particular aspects, the payload is conjugated to the light chain of the antibody Light chain conjugation may be facilitated by a spacer on the light chain. Without wishing to be bound by theory, in these conjugates it appears that the payload is administered in stealth, with such conjugates exhibiting high stability and being highly toxic to the target cells, whilst sparing unrelated cells

[0014] Described herein is the assessment of the potential of recombinant PALs16-16for the rapid synthesis of ADCs bearing potent payloads such as monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor or (DXd), a DNA topoisomerase I inhibitor A cascade reaction scheme18mixing a PAL with a recombinant form of human glutaminyl cyclase (hQC) that cyclizes the departing amine group was utilized By preventing re-attack by the leaving group of the newly peptide bond formed by the PAL, this cascade reaction is demonstrated to result in a very high yield for the conjugation reaction of an IgG molecule in less than two hours.

[0015] Furthermore, the stability and tumor killing properties of ADCs prepared via our cascade enzymatic scheme (“PAL + hQC”) was compared with selected leading marketed ADCs prepared using chemical conjugation methods, such as Kadcyla2(“T-DM1"), disitamab (“RC48 / Aidixi”)19and Enhertu20(“T-DXd”). Well-validated tumor models consisting of HER2 positive breast cancers were employed for both in vitro cytotoxicity and in vivo CDX and PDX studies Either trastuzumab or disitamab were used as starting scaffolds and modified their amino-acid sequences to include the “PAL+hQC” C-terminal recognition motif “NQL” (Asn-GIn-Leu) for MMAE or DXd conjugation. The design and preparation of MMAE-based ADCs with a DAR value of 2, specifically labeled at the C-terminal ends of either the light (L) or (H) heavy chain, followed by testing in the breast cancer cell line, BT-474 (HER2, 3+) is described herein. The first set of experiments revealed that the ADC with L chain C-terminal conjugation displayed better stability in mouse plasma and anti-tumor activity in the BT-474 xenograft mouse model, compared to the ADC conjugated at the H chain C-terminal. Moreover, at a single dose of 1 mg / kg, the Light Chain conjugate (“LCc") completely eradicated the tumor within 11 days, while Kadcyla at the same dose, was less efficacious.

[0016] Next, a series of LCcs were generated and profiled in vitro with HER2-positive breast cancer cell lines and HER2-positive PDX models, including an ADC bearing branch-linked dual DXd payloads (SGZ-026, DAR 3.87), for comparison with Enhertu (DAR 8) Following a single intravenous injection of SGZ-026 or Enhertu in mice transplanted with T226 (dosed at 7 5 mg / kg) or HBCx-1 (dosed at 10 mg / kg), complete tumor eradication was achieved after 10 days and no tumor re-growth was observed over 5-6 weeks post-treatment. Remarkably, this was achieved with SGZ-026 having only about half the DAR value of Enhertu.

[0017] An aspect of the present disclosure is an antibody pay load conjugate comprising an antibody and a payload, the antibody payload conjugate comprising an antibody with a light chain and a heavy chain, wherein at least one of the light chain orthe heavy chain is conjugated to the payload by a tripeptide PAL conjugation motif, the tripeptide PAL conjugation motif having the sequence P1-P1”P2” wherein P1 is Asn or Asp, P1” is any amino acid and P2” is a hydrophobic amino acid or a ^-branched amino acid. In preferred aspects, the tripeptide PAL conjugation motif of the antibody payload conjugate has the sequence Asn-Gly-lle (NGI). In preferred aspects, the payload is conjugated to the light chain of the antibody. In payload is conjugated at the C terminus of the light chain, preferably at the C terminal of the light chain

[0018] There may be a spacer between the light chain and the tripeptide PAL conjugation motif The spacer may consist of at least 2 amino acid residues The spacer may consist of at least 5 amino acid residues In some cases, the spacer consists of 2 to 20 amino acid residues, for example 5 to 10 amino acid residues The spacer may comprise one or more glycine residues. For example, Gly-Ser, Gly-Gly-Gly- Gly-Ser or Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

[0019] In some antibody payload conjugates described herein, the payload comprises a linker, the linker conjugated to the antibody light chain via the tripeptide PAL conjugation motif. These conjugates are antibody linker payload conjugates In any of the antibody payload conjugates described herein, the payload may be a drug

[0020] In another aspect described herein, there is provided an antibody light chain or antibody heavy chain comprising a P1-P1 -P2’ tripeptide PAL motif, wherein P1 is Asn or Asp, P1 ' is Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid. Such a light or heavy chain may be provided in an antibody In other words, in some aspects, there is provided an antibody with an antibody light chain or antibody heavy chain comprising a P1-P1 ’-P2’ tripeptide PAL motif, wherein P1 is Asn or Asp, P1 ’ is Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid. In such antibodies, one or both of the light chains, one or both of the heavy chains, or one or both of the light chains and one or both of the heavy chains may comprise the P1-P1 ’-P2' tripeptide PAL motif Preferably, the PAL motif is included on at least one of the light chains. Preferably the PAL recognition motif comprises or consists of the amino acid sequence Asn-GIn-Leu (NQL) The antibody light chain or antibody heavy chain preferably includes the PAL recognition motif at the C-terminus of the light chain or heavy chain, such as at the C- terminal of the light chain or heavy chain. The antibody light chain or antibody heavy chain may additionally comprise a spacer. The spacer may consist of at least 2 amino acid residues The spacer may consist of at least 5 amino acid residues In some cases, the spacer consists of 2 to 20 amino acid residues, for example 5 to 10 amino acid residues. The spacer may comprise one or more glycine residues For example, Gly-Ser, Gly-Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser. In a further aspect, there is provided a nucleic acid encoding such an antibody, an antibody light chain or antibody heavy chain.

[0021] In a further aspect, there is provided a payload for conjugation to an antibody comprising a PAL recognition motif and optionally a linker, the linker arranged between the PAL recognition sequence and the payload. The payload is preferably a drug. The PAL recognition motif may be a PT-P2” motif, wherein P1" is any amino acid and P2" is a hydrophobic amino acid or a p-branched amino acid. In some preferred aspects P1” is Gly and P2” is Isoleucine.

[0022] As such, in some aspects described herein, there is disclosed an antibody payload conjugate having the following formula:

[0023] [Antibody]-[optional spacer]-[PAL conjugation motif]-[payload] In such aspects the antibody may be conjugated via the antibody light chain and / or antibody heavy chain, preferably the antibody light chain. The payload may be a linker-payload

[0024] In yet further aspects, methods for preparing an antibody payload conjugate are provided. Such methods involve the conjugation of an antibody comprising an antibody light chain and an antibody heavy chain to a payload using a peptide asparaginyl ligase (PAL) and glytaminy I cyclase (QC), wherein at least one of the light chain or the heavy chain includes a tripeptide PAL motif and the payload has a PAL recognition motif. These methods may involve providing an antibody comprising a light chain and an antibody heavy chain, wherein at least one of the light chain or the heavy chain includes a tripeptide PAL motif, contacting the antibody with a payload, wherein the payload has a PAL recognition motif, and incubating the antibody and the payload in the presence of a peptide asparaginyl ligase (PAL) and glutaminyl cyclase (QC) to allow the PAL to ligate the tripeptide PAL motif to the PAL recognition motif, thereby generating an antibody payload conjugate wherein the antibody is conjugated to the payload via a PAL conjugation motif. Such methods result in the preparation of an antibody payload conjugate wherein the antibody light chain or the antibody heavy chain is conjugated to the pay load by a tripeptide PAL conjugation motif. The tripeptide PAL conjugation motif may have the sequence P1-P1”P2” wherein P1 is Asn or Asp, P1” is any amino acid and P2” is a hydrophobic amino acid or a p-branched amino acid. In preferred aspects, the tripeptide PAL conjugation motif of the antibody payload conjugate has the sequence Asn-Gly-lle (NGI). In such methods, there may be a spacer arranged between the antibody light or heavy chain and the tripeptide PAL motif.

[0025] In yet further aspects, the present disclosure provides antibody payload conjugates prepared by the methods described herein

[0026] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0027] Detailed Description of the Invention

[0028] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments

[0029] Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples The whole content of such bibliographic references is herein incorporated by reference but their mention in the specification does not imply that they form part of the common general knowledge. Methods described herein are preferably performed in vitro. The term 'in vitro' is intended to encompass procedures performed with cells in culture whereas the term 'in vivo' is intended to encompass procedures with / on intact multi-cellular organisms. Described herein are methods for generating antibody payload conjugates and antibody payload conjugates generated by such methods.

[0030] Methods described herein involve a cascade enzymatic reaction scheme which overcomes the reversibility problem of PAL-mediated ligation. The methods are used to conjugate a payload to an antibody This method involves coupling QC with PAL and is disclosed in WO 2023 / 191726 (filed as PCT / SG2023 / 050219 and entitled “Improving the efficiency of PAL-Catalyzed protein ligation by a cascade enzymatic scheme), the entire contents of which are incorporated herein by reference

[0031] As used herein, the term “PAL” refers to peptide asparaginyl ligase, a type of legumain ligase that catalyzes site-specific peptide bond formation.

[0032] As used herein, the term ''QC refers to glutaminyl cyclase (QC) enzyme and QC-like enzymes QC and QC-like enzymes have identical or similar enzymatic activity, i.e , catalysing the intramolecular cyclization of N-Terminal glutaminyl and glutamyl residues of peptides and proteins to form pyroglutamyl residue (pGlu). In this regard, QC-like enzymes can fundamentally differ in their molecular structure from QC

[0033] Methods involve the use of PAL with QC to ligate a first component having a P1-PT-P2’ tripeptide PAL motif as an acyl donor, wherein P1 is Asn or Asp, PT is Gin or Glu and P2’ is a hydrophobic amino acid or a p-branched amino acid and a second component having a PT-P2" motif as an acyl acceptor at the N-terminus, wherein PT' is any amino acid and P2" is a hydrophobic amino acid or a p-branched amino acid PAL cleaves the first peptide / protein after P1 in the tripeptide PAL motif and ligates said first component to the P1 "-P2” motif of said second component, and QC cyclizes P1 ’ in the released P1 ’-P2' dipeptide motif to pyroglutamyl (pGlu)

[0034] In some preferred methods described herein, the first component is a recombinant antibody. In particularly preferred methods, the first component is the light chain of a recombinant antibody This may be referred to as an antibody light chain or a recombinant antibody light chain. In some methods described herein, the first component is the heavy chain of a recombinant antibody, also known as an antibody heavy chain, or a recombinant antibody heavy chain Methods for preparing a recombinant antibody, and particularly a recombinant antibody light chain or recombinant antibody heavy chain are well known in the art and may involve the expression of a nucleic acid encoding the recombinant antibody, light chain or heavy chain. Such methods can be used to include a P1-PT-P2’ tripeptide PAL motif in the sequence of the recombinant antibody, and particularly in the recombinant antibody light chain and / or recombinant antibody heavy chain In particularly preferred aspects, the P1-PT-P2’ tripeptide PAL motif is at the C-terminal of the antibody, such as the C-terminal of the antibody light chain or the antibody heavy chain. The P1-PT-P2’ tripeptide PAL motif may be at the C-terminus of the antibody light chain or the antibody heavy chain. The tripeptide PAL motif is included in-frame with the nucleic acid encoding the antibody, such that the tripeptide PAL motif is expressed contiguously with the antibody In preferred embodiments, the tripeptide PAL motif is included in the sequence of the recombinant antibody light chain. In some aspects and embodiments herein, only the recombinant light chain includes a P1-PT-P2’ tripeptide PAL motif. As such, in some aspects and embodiments, the recombinant heavy chain does not include a P1-PT-P2’ tripeptide PAL motif. In some alternative methods described herein, the first component is or comprises a payload The first component may further comprise a linker, in which case the first component may be referred to as a linker-payload. Where the first component is a linker-payload the P1-P1 ’-P2’ tripeptide PAL motif is arranged such that the linker is between the motif and the payload. Methods for modifying peptides to include a motif of interest are well known in the art and will be well known to those of skill in synthetic organic chemistry

[0035] Where the first component is a recombinant antibody, such as a recombinant antibody light chain or recombinant antibody heavy chain, the second component will be or will comprise a pay load In these cases, the second component may be a linker-payload. The second component will comprise a P1"-P2" motif. Methods for protein engineering are well known in the art and may be used to add a P1"-P2" motif onto a linker or payload sequence. Where the second component is a linker payload, the P1"-P2” motif will be arranged such that the linker is between the motif and the payload.

[0036] Where the first component is or comprises a payload, the second component will be a recombinant antibody, such as a recombinant antibody light chain or recombinant antibody heavy chain In such cases, similar considerations apply as to when the first component is a recombinant antibody, except that the motif will be a P1"-P2" motif.

[0037] In preferred aspects, the first component comprises a spacer between the tripeptide PAL motif and the first component. The spacer will be between the first component and the tripeptide PAL recognition motif before the first component is conjugated to the second component Where the first and second components have been conjugated, the spacer will be between the first component and the tripeptide PAL conjugation motif. The spacer may consist of at least 2 amino acid residues. In some cases, the spacer consists of at least 5 amino acid residues. The spacer may consist of 2 to 20 amino acid residues For example, 5 to 10 amino acid residues. The spacer may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. The spacer may consist of 2, 5 or 10 amino acid residues

[0038] The spacer preferably comprises a series of hydrophobic amino acids, such as Glycine (Gly), Alanine (Ala), Valine (Vai), Leucine (Leu), Isoleucine (lie), Proline (Pro), Phenylalanine (Phe), methionine (Met) or Tryptophan (Trp). The spacer may comprise a mixture of hydrophobic amino acids or it may comprise only as single amino acid species. Preferably, the spacer is a glycine-rich spacer, containing at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or more glycine residues. The spacer may optionally further contain other amino acids, preferably one or more polar amino acids such as Serine (Ser), Threonine (Thr), Cysteine (Cys), Asparagine (Asn), Glutamine (Gin), Tyrosine (Tyr). The spacer may comprise Gly-Gly-Gly-Gly-Ser The spacer may consist Gly-Gly-Gly-Gly-Ser The spacer may comprise [Gly-Gly-Gly-Gly-Ser]n, where n is 1 , 2, 3 or 4. The spacer may consist of Gly-Gly-Gly-Gly- Ser-Gly-Gly-Gly-Gly-Ser. Introducing a spacer between the P1-P1 ’-P2’ tripeptide PAL motif and the first component may improve accessibility for the PAL to catalyse the ligation and consequently improve yield, or may increase the DAR achieved. Additionally, the presence of a spacer may facilitate cleavage of the linker to release the payload, when the antibody payload conjugate reaches its target. In some cases, the first component is an antibody, and the spacer is introduced between the antibody and the P1-P1 -P2' tripeptide PAL motif In particular embodiments, the spacer is introduced between the antibody light chain and the P1-P1 -P2’ tripeptide PAL motif

[0039] It will be appreciated that PALs perform site-specific ligation reactions and require a minimal tripeptide recognition motif, P1-PT-P2’, for ligation, wherein P1 is typically Asn or Asp, and P1 ' and P2’ may be any ofthe naturally occurring amino acids Gly, Ala, Vai, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gin, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0040] For the purposes ofthe present invention, P1 is preferably Asn or Asp, P1 ’ is preferably Gin or Glu and P2' is preferably a hydrophobic amino acid or a -branched amino acid. In some embodiments, P1 is preferably Asn and P1 ’ is preferably Gin. It is known that Glu can act as a replacement for Gin at P1 ’ of the acyl donor (Seifert, F., et al , Biochemistry 48, 11831-11833 (2009)) and that Asp can act as a replacement for Asn at P1 ofthe acyl donor (Zhang, D , et al. , (2021) Journal ofthe American Chemical Society 143 (23): 8704-8712). Accordingly in some embodiments, P1 may be Glu. In various embodiments, P1 ’ may be Asp.

[0041] In some embodiments, P2’ and / or P2" may be a hydrophobic amino acid or a ^-branched amino acid. Examples of a hydrophobic amino acid may include Gly, Ala, Vai, Leu, lie, Pro, Phe, Met, Tyr and Trp

[0042] Examples of a -branched amino acid include Thr, Vai, and lie.

[0043] In some embodiments, P2’ may be selected from the group comprising Leu, Met, Phe, Tyr, Trp, Vai, lie and Thr. In various embodiments, P2” may be selected from the group comprising Leu, Phe, Tyr, Trp, Vai, lie and Thr

[0044] In some embodiments, the P1-P1 -P2’ tripeptide PAL motif of the acyl donor may be Asn-GIn-Leu.

[0045] It would be appreciated by a person skilled in the art that different PALs and variants thereof having the desired protein ligase activity may be suitable for the practice of the present invention Accordingly in some embodiments, the PAL may be a butelase-1 , butelase-2, VyPAL2, VyPAL3, OaAEP1 b-C247A, HeAEP3, AtLEGy, VuPALI , HaPALI , OaAEPI b or a functional fragment or variant thereof.

[0046] In certain embodiments, the PAL may be selected from the group comprising butelase-1 comprising the amino acid sequence set forth in SEQ ID NO: 7, butelase-2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, VyPAL2 comprising the amino acid sequence set forth in SEQ ID NO: 10, VyPAL3 comprising the amino acid sequence set forth in SEQ ID NO: 11 , OaAEPI b-C247A comprising the amino acid sequence set forth in SEQ ID NO: 12, HeAEP3 comprising the amino acid sequence set forth in SEQ ID NO: 13, AtLEGy comprising the amino acid sequence set forth in SEQ ID NO: 14, VuPALI comprising the amino acid sequence set forth in SEQ ID NO: 15, HaPALI comprising the amino acid sequence set forth in SEQ ID NO: 16, OaAEPI b comprising the amino acid sequence set forth in SEQ ID NO: 17 and a functional fragment or a variant thereof

[0047] It is also envisaged that various QCs having the desired QC enzymatic activity may be suitable for use in the practice of the present invention. Accordingly in some embodiments, the QC may be a Human glutaminyl cyclase, a Mouse glutaminyl cyclase, a Drosophila glutaminyl cyclase, an Arabidopsis g lutaminyl cyclase, a Conus glutaminyl cyclase, a Sistrurus glutaminyl cyclase, a Bacterial glutaminyl cyclase or a functional fragment or variant thereof.

[0048] In some embodiments, the QC may be selected from the group comprising Human glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 18, Mouse glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 19, Drosophila glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 20, Arabidopsis glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 21 , Conus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 22, Sistrurus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 23, Bacterial glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 24 and a functional fragment or a variant thereof.

[0049] As those skilled in the art would appreciate, a protein / enzyme's function is directly related to its structure and sequence, and that there is a positive relationship between sequence identity and function similarity In this regard, methods of determining a protein sequence identity are known in the art

[0050] Accordingly, the sequences ofthe enzymes of the present disclosure may be sufficiently varied so long as the enzymes maintain their functionality and can exhibit the required activity (for example, the QC variant being able to catalyse the intramolecular cyclization of N-Terminal glutaminyl and glutamyl residues of peptides and proteins to form pyroglutamyl residue (pGlu)).

[0051] In some embodiments, the PAL may be a butelase-1 comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 7, a butelase-2 comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in set forth in SEQ ID NO: 8 or 9, a VyPAL2 comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 10, a VyPAL3 comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 11 , a OaAEP1b-C247A comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 12, a HeAEP3 comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 13, a AtLEGy comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 14, a VuPALI comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 15, a HaPALI comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 16 or a OaAEPI b comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0052] In some embodiments, the QC may be a Human glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 18, a Mouse glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 19, a Drosophila glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 20, an Arabidopsis glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 21 , a Conus glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 22, a Sistrurus glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 23, or a Bacterial glutaminyl cyclase comprising the amino acid sequence with at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity to the amino acid sequence set forth SEQ ID NO: 24.

[0053] In some cases, the second component comprises a spacer of at least one amino acid between the P1"- P2" acyl acceptor and said second component. As described herein, introducing a spacer between the PT,-P2" acyl acceptor and said second component or protein may improve accessibility for the PAL to catalyse the ligation and consequently improve its yield, especially in cases where the second component is large enough to hinder accessibility of PAL. In some cases, the second component is the linkerpayload or payload, and the spacer is introduced between the linker-payload and the P1"-P2” acyl acceptor or between the payload and the P1"-P2” acyl acceptor. In some embodiments and aspects disclosed herein, the second component does not require a spacer In particular embodiments, the second component is a payload or a linker payload, and there is no spacer between the payload or the linker payload and the PT-P2". Similar considerations for spacer composition apply to the spacer of the second component as apply to the spacer between the tripeptide PAL conjugation motif or the PAL recognition motif and the first component.

[0054] The rate of reaction of the methods of the present disclosure may be controlled by varying the ratio of the enzyme to the substrate in question In this regard, the inventors have found that the more enzyme used, the faster the reaction proceeded In some embodiments, a small amount of enzyme (for example 0.005 % eq of QC to the substrate and 1 / 10 eq to PAL) is sufficient to carry out the invention. For some macromolecular protein substrates such as antibodies, a higher enzyme to substrate ratio may be required, such as 0 1 :1 : 100 or 0.1 :1 :50 (QC: PAL: first component). The ratio of enzyme to substrate to use is largely dependent on the substrate and the specific application and may be easily determined using standard techniques known to those skilled in the art or may be deduced by reference to the pertinent literature

[0055] In some embodiments, the ratio of QC: PAL: first component is in the range of 0.1 :1 :2000 to 1 :1 :50, respectively, and preferably in the range of 0.1 :1 :1000 to 0.1 :1 :50, respectively In some embodiments, the ratio of QC: PAL: first component or protein is 0 1 : 1 : 20 respectively.

[0056] In some methods, where the first component is an antibody and the second component is the payload, the ratio of antibody to payload is such that an excess of payload is incubated with the antibody. For example, the molar ratio of antibody to payload may be 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10. The ratio may be dependent on the potential DAR of the resultant conjugate In other words, the number of PAL recognition motifs in the antibody. Where there are more PAL recognition motifs, such as on both antibody light chains and both antibody heavy chains, an even greater excess may be appropriate to ensure the potential DAR is achieved

[0057] The antibody and payload are incubated in the presence of QC and PAL under conditions suitable for conjugation of the antibody to the payload by the PAL. The incubation may occur at temperature which is selected based on the optimal temperature for the functioning of a specific PAL In general the temperature will be around room temperature, or around 37°C In some methods, the temperature will be between 30 °C and 40 °C, between 32 °C and 38 °C, between 34 °C and 36 °C. The incubation may occur at a pH which is selected based on the optimal pH for the functioning of the PAL. In general, the pH will be around neutral, or slightly acidic, such as around pH7, or between pH6 and pH7, or around pH 6.5 The antibody and payload are incubated in the presence of QC and PAL for sufficient time for conjugation of the antibody to the payload. The exact time will depend on the efficiency of the conjugation and may vary depending on the antibody and payload to be conjugated, the specific PAL, or the conditions. The incubation may be occur until completion. That is, until substantially all of the antibody has been conjugated. However, normally the incubation will occur until sufficient antibody has been conjugated In general, the methods disclosed herein are rapid, resulting in sufficient conjugation within a couple of hours. As such, incubation may be done for about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours or about 1 hour Incubation is preferably for between 1 and 3 hours, between 1 5 and 2.5 hours, or about 2 hours

[0058] The amount of antibody conjugated to payload may be determined by determining the drug to antibody ratio (DAR) using any suitable method known in the art. For example, DAR may be determined by hydrophobic interaction chromatography, reversed-phase high performance liquid chromatography, liquid chromatography mass spectrometry, hydrophilic interaction chromatography or cathepsin B enzymatic cleavage. In the exemplified methods, hydrophobic interaction chromatography was used.

[0059] This disclosure provides novel antibody payload conjugates, such as antibody drug conjugates produced by the methods disclosed herein. These conjugates result from the conjugation of an antibody and a payload. A PAL is used to form a peptide bond between the antibody and the payload. PALs perform site-specific ligation between the antibody and the payload and require a minimal tripeptide recognition motif on one of the components and a dipeptide motif on the other component. The antibody component may include either the tripeptide recognition motif or the dipeptide motif and the payload component will comprise the other motif.

[0060] The present disclosure relates to antibody drug conjugates. As used herein, the term antibody and its plural antibodies encompasses both full-length antibodies and antigen binding molecules derived from such antibodies As such, we include a fragment or derivative thereof, or a synthetic antibody or synthetic antibody fragment.

[0061] Antigen binding regions of an antibody are made up from a light chain (which may be referred to as VL) and a heavy chain (which may be referred to as VH) Immunoglobulins of type G (i.e. IgG) are ~150 kDa glycoproteins comprising two heavy chains and two light chains From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chains comprise a VL followed by a constant region, CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g. lgG1 , lgG2 , lgG3, lgG4) , IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0062] In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

[0063] Antibodies useful in the methods disclosed herein may be monoclonal. Monoclonal antibodies are a homogenous population of antibodies specifically targeting a single epitope on an antigen. A variety of antibody classes are known, including IgG, IgA, IgD, IgE and IgM. Fragments of antibodies, such as Fab and Fab2 fragments may also be used as can genetically engineered antibodies and antibody fragments. In some cases, the antibody is an ScFv molecule, comprising covalently linked light and heavy chains.

[0064] Antibodies may be human, humanised or chimeric. In some cases, they are derived from another mammalian animal, such as a mouse, rabbit or camelid. Animal / human chimeric antigen-binding moieties can be prepared from animal antibodies by the process of chimerisation, e g as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), in Chapter 8 thereof, in particular section 3 of Chapter 8.

[0065] Humanised antigen-binding moieties can be prepared from animal antibodies by the process of humanisation, e.g. as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), in Chapter 7 thereof, in particular section 3.1 of Chapter 7 entitled 'Antibody Humanization'. Techniques for antibody humanisation are also described e.g in Safdari et al., Biotechnol Genet Eng Rev (2013) 29:175-

[0066] 86.

[0067] Aspects and embodiments of the present disclosure contemplate multispecific antigen-binding moieties. By 'multispecific’ it is meant that the antigen-binding moiety displays specific binding to more than one target or to more than one epitope of the same target. In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety. In some embodiments, the antigen-binding moiety comprises at least two different antigen-binding domains (i e. at least two antigen-binding domains, e g comprising non-identical VHs and VLs)

[0068] A large number of antibodies have been developed for therapeutic use. Any of these may be used to develop an antibody payload conjugate as described herein These include Trastuzumab, Disitamab, Vandortuzumab, Bentracimab, Datopotamab, Zenocutuzumab, Nemolizumab, Zanidatamab, Linvoseltamab, Axatilimab, Patritumab , Tarlatamab, Marstacimab, Garadacimab, Vilobelimab, Zolbetuximab, Odronextamab, Crovalimab, Camrelizumab, Serplulimab, Sugemalimab, Concizumab, Cosibelimab, Donanemab, Sintilimab, Narsoplimab, Pozelimab, Elranatamab, Rozanolixizumab, Talquetamab, Epcoritamab, Lebrikizumab, Glofitamab, Mirikizumab, Tislelizumab, Toripalimab, Retifanlimab, Lecanemab, Teplizumab, Ublituximab, Mirvetuximab, Nirsevimab, Tremelimumab, Spesolimab, Teclistamab, Mosunetuzumab, Tixagevimab, Cilgavimab, Relatlimab, Tebentafusp, Faricimab, Sutimlimab, Sotrovimab, Regdanvimab, Casirivimab, imdevimab, Tezepelumab, Tisotumab, Amivantamab, Anifrolumab, Loncastuximab, Bimekizumab, Tralokinumab, Evinacumab, Aducanumab, Dostarlimab, Ansuvimab, Margetuximab, Naxitamab, Atoltivimab, Maftivimab, and Odesivimab, Belantamab, Tafasitamab, Satralizumab, Inebilizumab, Sacituzumab govitecan, Teprotumumab, Isatuximab, Eptinezumab, Enfortumab , Crizanlizumab, Brolucizumab, Polatuzumab , Risankizumab, Romosozumab, Caplacizumab, Ravulizumab, Emapalumab, Cemiplimab, Fremanezumab, Moxetumomab, Galcanezumab, Lanadelumab, Mogamulizumab, Erenumab, Tildrakizumab, Ibalizumab, Burosumab, Durvalumab, Emicizumab, Benralizumab, Ocrelizumab, Guselkumab, Inotuzumab, ozogamicin, Sarilumab, Dupilumab, Avelumab, Brodalumab, Atezolizumab, Bezlotoxumab, Olaratumab, Reslizumab, Obiltoxaximab, Ixekizumab, Daratumumab, Elotuzumab, Necitumumab, Idarucizumab, Alirocumab, Mepolizumab, Evolocumab, Dinutuximab, Secukinumab, Nivolumab, Blinatumomab, Pembrolizumab, Ramucirumab, Vedolizumab, Siltuximab, Obinutuzumab, Ado-trastuzumab, Raxibacumab, Pertuzumab, Brentuximab, Belimumab, Ipilimumab, Denosumab, Tocilizumab, Ofatumumab, Canakinumab, Golimumab, Ustekinumab, Certolizumab, Catumaxomab, Eculizumab, Ranibizumab, Panitumumab, Natalizumab, Bevacizumab, Cetuximab, Efalizumab, Omalizumab, Tositumomab, Ibritumomab , Adalimumab, Alemtuzumab, Gemtuzumab, Trastuzumab, Infliximab, Palivizumab, Basiliximab, Daclizumab, Rituximab, Abciximab, Edrecolomab, Nebacumab and Muromonab-CD3. In some aspects, the antibody is Vandortuzumab. In some aspects described herein the antibody is Trastuzumab or Disitamab.

[0069] Antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et aL, Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety Such techniques may be used to engineer additional sequences such as a P1-PT-P2’ tripeptide PAL motif or a P1"-P2" dipeptide motif into the antigen binding molecule or antibody, such as into the light chain or the heavy chain As noted above, in some aspects and embodiments herein, only the recombinant light chain includes a P1-P1 ’-P2 ' tripeptide PAL motif In some aspects and embodiments, the recombinant heavy chain does not include a P1-P1 '-P2’ tripeptide PAL motif.

[0070] The antibody may not naturally include either the tripeptide PAL recognition motif or the dipeptide motif. As such, it may be necessary to engineer the antibody to include the relevant motif. Methods for engineering antibodies are well known in the art. For example, they may be produced by recombinant expression techniques. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety. Such methods may involve the preparation of one or more nucleotides encoding an antibody For example, the method may involve the generation of a nucleotide encoding the light chain and a nucleotide encoding the heavy chain The nucleotide may be engineered to include a sequence encoding the motif, such that when the antibody light chain or the antibody heavy chain is expressed from the nucleotide, the motif is included therein

[0071] The motif will be arranged on the antibody such that it is accessible when the antibody is contacted with the PAL The motif may be presented on the light chain or the heavy chain of the antibody. In some cases, a motif may be presented on both the light chain and the heavy chain of the antibody. The motif is preferably presented distal to the complementarity determining regions, such that conjugation with the payload does not impact the interaction between the antibody and its target. As such, the motif may be presented at the C-terminal of the light chain or the heavy chain, including at the C-terminus of the light chain or the heavy chain. The motif may be the P1 -P1 ’-P2’ tripeptide PAL motif or the P1 "-P2" dipeptide motif. Preferably, the motif is the P1-P1 ’-P2’ tripeptide PAL motif.

[0072] In some cases, a spacer is included between the antibody and the motif As such, the spacer may be included on the antibody light chain or the antibody heavy chain. Including the spacer may improve the accessibility for the PAL to catalyse the ligation and consequently improve its yield, especially in cases where the second protein is large enough to hinder accessibility of PAL. The spacer may increase the DAR achieved via PAL conjugation. The spacer may consist of at least 2 amino acid residues. In some cases, the spacer consists of at least 5 amino acid residues The spacer may consist of 2 to 20 amino acid residues For example, 5 to 10 amino acid residues The spacer may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. The spacer may consist of 2, 5 or 10 amino acid residues. The spacer preferably comprises a series of hydrophobic amino acids, such as Glycine (Gly), Alanine (Ala), Valine (Vai), Leucine (Leu), Isoleucine (lie), Proline (Pro), Phenylalanine (Phe), methionine (Met) or Tryptophan (Trp) The spacer may comprise a mixture of hydrophobic amino acids or it may comprise only as single amino acid species Preferably, the spacer is a glycine-rich spacer, containing at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or more glycine residues The spacer may optionally further contain other amino acids, preferably one or more polar amino acids such as Serine (Ser), Threonine (Thr), Cysteine (Cys), Asparagine (Asn), Glutamine (Gin), Tyrosine (Tyr). The spacer may comprise Gly-Gly-Gly-Gly-Ser. The spacer may comprise [Gly-Gly-Gly- Gly-Ser]n, where n is 1 , 2, 3 or 4. The spacer may consist Gly-Gly-Gly-Gly-Ser. The spacer may consist of Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser

[0073] Aspects and embodiments of the present disclosure relate to antigen-binding molecules comprising a pay load or a linker-payload. As used herein, a linker-payload refers to a moiety comprising one or more pay loads, and a linker for linking the payload(s) to the antigen-binding moiety of the antigen-binding molecule.

[0074] The payload may be a nucleic acid, a peptide or a protein, a carbohydrate, a lipid or a small molecule. The payload may be a detectable label or a therapeutic payload. The payload may be a cytotoxic payload or a non-cytotoxic therapeutic payload such as a non-cytotoxic immune modulator The payload may be a nucleic acid payload such as an oligonucleotide, a DNA payload, an RNA payload or an aptamer For example, the payload may be a nucleic acid associated with a gene editing system such as CRISPR, or a nucleic acid gene regulator such as a small interfering RNA (siRNA) or a microRNA (miRNA)

[0075] In some embodiments, a payload according to the present disclosure comprises or consists of a cytotoxic agent Payload moieties are described e.g. in Parslow et aL, Biomedicines. 2016 Sep; 4(3): 14, Goundry and Parker, Org. Process Res. Dev. (2022) 26, 8, 2121-2123, Fu et al , Signal Transduction and Targeted Therapy (2022) 7:93, Wang et aL, Acta Pharmaceutica Sinica B (2023) 13 (10): 4025-4059 and Conilh et aL, J Hematol & Oncol (2023) 16:3, all of which are hereby incorporated by reference in their entirety.

[0076] In some embodiments, a payload according to the present disclosure may comprise or consist of a microtubule-targeting agent, a DNA-targeting agent, an RNA-targeting agent, an immune systemactivating agent, an apoptosis-promoting agent, a metabolism-inhibiting agent, a proteasome inhibiting agent, and / or a protein-degrader agent.

[0077] In some embodiments, a payload according to the present disclosure comprises, or consists of, a microtubule-targeting agent. Microtubules play important roles in maintaining proper cellular morphology, signal transduction, organelle transportation, cell motility and cell division. Microtubules are formed of tubulin, and agents that disrupt the tubulin polymerization dynamics, resulting in cell cycle arrest and apoptosis. Tubulin inhibitors have a stronger toxicity to rapidly-dividing cancerous cells than slower- growing, non-cancerous cells. Microtubule-targeting agents include tubulin polymerization enhancers (e.g. auristatins, taxanes), and tubulin polymerization inhibitors (e.g. maytansinoids, colchicine).

[0078] In some embodiments, a payload according to the present disclosure comprises, or consists of, a maytansinoid, e g maytansine or a derivative thereof, e g. mytansine, DM1 (mertansine) or DM4 (ravtansine) In some embodiments, the maytansinoid is not DM1 . In some embodiments, the maytansinoid is not DM4. In some embodiments, a payload moiety comprises, or consists of colchicine or a derivative thereof

[0079] In some embodiments, a payload moiety comprises, or consists of, an auristatin, e.g. a dolastatin 10 derivative, e.g. monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin PE, auristatin PYE, PF-06380101 , auristatin F-hydroxypropylamide (AF-HPA) or azastatin In some embodiments, the auristatin is not MMAE. In some embodiments, the auristatin is not MMAF. In some embodiments, a payload moiety comprises, or consists of, a halichondrin B derivative, e g eribulin. In some embodiments, a payload comprises, or consists of, a tubulysin or a derivative thereof, e.g. tubulysin A, D, H, U or V. In some embodiments, a payload moiety comprises, or consists of, a cryptophycin or a derivative thereof, e.g. cryptophycin-1 , cryptophycin-52, cryptophycin-55 or cryptophycin-55gly. In some embodiments, a payload moiety comprises, or consists of, an EG5 (i e kinesin / KSP / KIF11) inhibitor, e.g. ispinesib (SB715992) or a derivative thereof, or filanesib (ARRY-520) or a derivative thereof In some embodiments, a payload moiety comprises, or consists of, a taxane, e.g. paclitaxel, docetaxel or cabazitaxel. In some embodiments, a payload moiety comprises, or consists of vinca alkaloid, e.g. vinblastine, vincristine, vindesine, vinorelbine or vinflunine. In some embodiments, a payload moiety comprises, or consists of hemiasterlin or a derivative thereof, e.g. hemiasterlin, hemisterlin A or HTI-286

[0080] In some embodiments, a payload according to the present disclosure comprises, or consists of, a DNA- targeting agent. DNA-targeting agents include agents that directly or indirectly destroy DNA through introducing / promoting the formation of single- and / or double-strand breaks (e g. enediynes, topoisomerase inhibitors), DNA alkylating agents (e.g. pyrrole [2, 1 -c][1 ,4] benzodiazepines (PBD), indolinobenzodiazpines, duocarmycins), and DNA crosslinking agents (e.g. mitomycin C) In some embodiments, the DNA alkylating agent is not a duocarmycin. In some embodiments, the DNA alkylating agent is not D6.5. In some embodiments, the DNA alkylating agent is not a PBD

[0081] In some embodiments, a payload according to the present disclosure comprises, or consists of, an enediyne, e.g. a Cal-like enediyne or an anthraquinone fusion enediyne, e.g. calicheamicin yl1 , calicheamicin 6 or uncialamycin. In some embodiments, the enediyne is not calicheamicin. In some embodiments, a payload moiety comprises, or consists of, a topoisomerase inhibitor, e.g. a TOP1 or TOP2 inhibitor, e.g. camptothecin or a derivative thereof, e g SN-38, exatecan, exatecan mesylate (DX- 8951 f), N-glycyl-exatecan or deruxtecan (DXd); e.g. an anthracycline, e g doxorubicin, daunorubicin, epirubicin, PNU-159682 or idarubicin . In some embodiments the topoisomerase inhibitor is not SN-38 In some embodiments the topoisomerase inhibitor is not doxorubicin.

[0082] In some embodiments, a payload comprises, or consists of, a pyrrolo[2, 1-c][1 ,4] benzodiazepine (PBD) dimer, or a derivative thereof, e g a PDB, KMR-28-39, SJG-136 SGD-1882 or SG3199 dimer In some embodiments, a payload moiety comprises, or consists of, indolinobenzodiazpine (IGN ; monoimine) or a derivative thereof. In some embodiments, a payload moiety comprises, or consists of, a pyridinobenzodiazepine (PDD) dimer, or a derivative thereof, e.g. a PDD or FGX5-67 dimer In some embodiments, a payload moiety comprises, or consists of, a duocarmycin or a derivative thereof, e g duocarmycin A, CC1065, duocarmycin SA, DUBA, seco-DIBA or seco-CBL

[0083] In some embodiments, a payload according to the present disclosure comprises, or consists of, a Retargeting agent. Small molecule inhibitors that target RNA can kill both dividing and dormant tumor cells. RNA-targeting agents include RNA splicing inhibitors (e.g. thailanstatin and derivatives thereof) and RNA polymerase II inhibitors (e.g. amatoxins, RNA polymerase ll-IN-2) In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, thailanstatin or a derivative thereof, e.g thailanstatin A, thailanstatin B, thailanstatin C or FR901464 In some embodiments, a payload moiety comprises, or consists of, an amatoxin, e.g. a-amanitin or p-amanitin. In some embodiments, a payload moiety comprises, or consists of, RNA polymerase ll-IN-2.

[0084] In some embodiments, a payload according to the present disclosure comprises, or consists of, an immune system-activating agent. Immune-stimulating antibody conjugates (ISACs) employ small molecule-based engagement of the innate and / or adaptive immune systems A variety of immune- modulating payloads are in development, including Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists and glucocorticoid receptor modulators (GRMs). In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a TLR agonist, e.g. an agonist of TLR7, TLR8 or TLR9. In some embodiments, a payload moiety comprises, or consists of, a STING agonist, e.g. a cyclic dinucleotide (CDN; e.g. 2,3 cGAMP; 3,3 cGAMP; c-di-GMP or c-di-AMP) or a benzimidazole. In some embodiments, a payload moiety comprises, or consists of, a glucocorticoid receptor modulator, e.g. dexamethasone or a derivative thereof.

[0085] In some embodiments, a payload according to the present disclosure comprises, or consists of, an apoptosis-promoting agent. Anti-apoptotic proteins such as Bcl-xL can play important roles in tumorigenesis, metastasis and drug resistance. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a Bcl-xL inhibitor. In some embodiments, a payload moiety comprises, or consists of, ABT-737.

[0086] In some embodiments, a payload according to the present disclosure comprises, or consists of, a metabolism-inhibiting agent. Metabolism-inhibiting agents such as niacinamide phosphate ribose transferase (NAMPT) inhibitors control the concentration of NAD+ within cells, inducing energy crisis and cell death, and antifolate antimetabolites such as methotrexate, that inhibit dihydrofolate reductase (DHFR) and thereby DNA synthesis. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a NAMPT inhibitor, e.g. FK-866 or A-1293201. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a DHFR inhibitor, e g methotrexate of a derivative thereof.

[0087] In some embodiments, a payload according to the present disclosure comprises, or consists of, a proteasome-inhibiting agent Proteasome-inhibiting agents include carmaphycins In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a carmaphycin or a derivative thereof, e.g. carmaphycin A or carmaphycin B.

[0088] In some embodiments, a payload according to the present disclosure comprises, or consists of, a proteindegrader agent, e.g. a targeted protein degrader In some embodiments, a payload moiety according to the present disclosure comprises or consists of a proteolysis targeting chimera (PROTAC). PROTACs may comprise two ligands joined by a linker; one ligand recruits the protein of interest (i.e. the protein to be degraded) and the other ligand recruits the E3 ubiquitin ligase Once the PROTAC molecule binds to the target protein and the E3 ubiquitin ligase, the E3 ligase may ubiquitinate the target protein and initiate the degradation process.

[0089] In some embodiments, the payload moiety is monomethylauristatin E (MMAE). In some embodiments, the payload moiety is deruxtecan (DXd) In some embodiments the payload moiety is PNU-159682. In some embodiments, the payload moiety is Monomethyl auristatin F (MMAF).

[0090] A linker (also known as a linker moiety) according to the present disclosure may be any moiety suitable for linking the payload moiety to the antigen-binding moiety of the antigen-binding molecule of the present disclosure. Accordingly, they generally comprise a payload connecting region, an antibody connecting region, and a linker core.

[0091] Linkers are described e.g. in Su etal., Acta Pharmaceutica Sinica B (2021) 11 (12): 3889-3907, Fu etal., Signal Transduction and Targeted Therapy (2022) 7:93

[0092] A linker according to the present disclosure may be a cleavable linker moiety or a non-cleavable moiety

[0093] The linker may be a branched or linear linker. The linker may enable the conjugation of multiple payloads (a multi payload linker). For example, the linker may be a branched linker, wherein each branch supports conjugation of a payload In this way, a single linker may enable conjugation of 1 , 2, 3, 4, or 5 payloads. In some embodiments disclosed herein, a linker is conjugated to one ortwo payloads.

[0094] Cleavable linkers typically utilise differences between the environment of systemic circulation and that in cancer cells / the tumor microenvironment to release the payload moiety in a targeted manner. Cleavable linkers include chemical cleavage linkers (e.g. acid-cleavable linkers, GSH-cleavable linkers, Fe(ll)- cleavable linkers) and enzyme cleavage linkers (e.g cathepsin-cleavable linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers, sulfatase-cleavable linkers)

[0095] In some embodiments, a linker moiety according to the present disclosure is a chemical cleavage linker In some embodiments, a linker moiety according to the present disclosure is an enzyme cleavage linker. In some embodiments, a linker moiety is an acid-cleavable linker, e.g. comprising a hydrazone group (e.g. a 6-maleimidocaproylhydrazone linker or a (4-(4-acetylphenoxy)butanoic acid) hydrazaone linker), a carbonate group or a silyl ether group In some embodiments, a linker moiety is a GSH-cleavable linker, e.g comprising a disulfide group. In some embodiments, a linker moiety is a Fe(ll)-cleavable linker, e.g comprising a 1 ,2,4-trioxolane group. In some embodiments, a linker moiety is a cathepsin-cleavable linker, e.g. comprising a dipeptide (e.g a valine-citrulline linker, a phenylalanine-lysine linker or a valinealanine linker), a triglycyl peptide (CX), a tetrapeptide (e.g. a glycine-glycine-phenylalanine-glycine (GGFG) linker) or a cBu-Cit group. In some embodiments, a linker moiety is a glucuronidase-cleavable linker, e.g. comprising a p-glucuronide group. In some embodiments, a linker moiety is a glycosidase- cleavable linker, e.g. comprising a ^-galactoside group. In some embodiments, a linker moiety is a phosphatase-cleavable linker, e g comprising a pyrophosphate group In some embodiments, a linker moiety is a sulfatase-cleavable linker, e.g. comprising an arylsulfate group. In some embodiments, a linker moiety is a photo-responsive linker, e.g. comprising a heptamethine cyanine fluorophore group, an O-nitrobenzyl group or a PC4AP group. In some embodiments, a linker moiety is a biorthogonal cleavable linker, e.g. comprising a dsProc group. In some embodiments, a linker moiety comprises a benzyl group In some embodiments, a linker moiety comprises a bis- functional electrophilic group, e.g a bis-sulfone group or a bis-sulfone bis-alkylating group

[0096] Non-cleavable linkers remain inert in common chemical and enzymatic environments in the body, with the payload moiety being released following processing of the ADC by cellular lysosomal proteases Non- cleavable linkers include linkers comprising thioether or maleimidocaproyl (MC) groups

[0097] In some embodiments, a linker moiety is a thioether linker. In some embodiments, a linker moiety is a maleimidocaproyl linker, e.g. comprising a 2-(maleimidomethyl)-1 ,3-dioxane (MD) group or a Mal-PAB group. In some embodiments, a linker moiety comprises a polyethylene glycol (PEG) group and an alkyne, triazole or piperazine group

[0098] In some embodiments, a linker-payload moiety has a maleimide group for linkage to the antigen-binding moiety. In some embodiments, a linker-payload moiety has a thiol group for linkage to the antigen-binding moiety. In some embodiments, a linker-payload moiety according to the present disclosure has an amino (-NH2) group for linkage to the antigen-binding moiety, for example by enzymatic conjugation In some of these embodiments, enzymatic conjugation with microbial transglutaminase may be used to conjugate the linker-payload moiety to the antigen-binding moiety.

[0099] In some embodiments, a linker moiety further comprises a spacer moiety. In such embodiments, a spacer will be present on both the antibody side of the PAL motif, and the linker moiety side of the PAL motif. Spacer moieties are sometimes required due to the bulky nature of payload moieties Commonly employed spacer moieties include para-aminobenzyl carbamate (PABC), hemiaminal groups, PEG groups, polar acyl sulfamide groups, polar carbamoyl sulfamide groups and HydraSpace (described e.g. in Verkade et al. , Antibodies (Basel) (2018) 7(1): 12 and WO 2016 / 053107 A1 , both of which are hereby incorporated by reference in their entirety) Para-aminobenzyl (PAB) or derivatives thereof (e g. PABC) are commonly employed as a spacer moiety in cathepsin-cleavable dipeptide linkers, p-glucuronidase- cleavable linkers, P-galactosidase-deavable linkers and phosphatase cleavable linkers. In some embodiments, a linker moiety comprises a para-aminobenzyl (PAB) as a spacer moiety. In some embodiments, the linker moiety does not comprise a glycine rich spacer, such as a pentaglycine (Glys) spacer.

[0100] In some embodiments, the linker moiety is MC-Val-Cit-PAB. In some embodiments, the linker moiety is MC-GGFG In some embodiments, a linker-payload moiety according to the present disclosure comprises more than one payload This allows for a higher drug to antibody ratio (DAR) than when the linker-payload moiety comprises a single payload. Where the linker-payload moieties are attached to specific / engineered sites on the antigen-binding moieties, such linker-payload moieties allow for higher specific drug antibody ratios. For example, where two conjugation sites are available, a DAR of 2, 4, 6 or 8 can be achieved

[0101] The antibody payload conjugates of the present disclosure may have a given drug to antibody ratio (DAR). As used herein, “drug to antibody ratio” or “drug antibody ratio" (DAR) refers to the average number of payload moieties conjugated to the antigen-binding moiety (e.g. a CNX-binding moiety). In some embodiments, the antigen-binding molecules have a DAR of between 0 and 20, e.g. between 0 and 15, between 1 and 15, between 0 and 10, between 1 and 10, between 0 and 8, between 1 and 8, between 2 and 8, between 4 and 8, between 6 and 8, between 0 and 6, between 1 and 6, between 2 and 6, between 4 and 6, between 0 and 4, between 1 and 4, between 2 and 4 In some embodiments, the antigen-binding molecules have a DAR of between 1 and 8. In some embodiments, the antigen-binding molecules have a DAR of e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the antigen-binding molecules have a DAR of 2 In some embodiments, the antigen-binding molecules have a DAR of 4.

[0102] Some antibody payload conjugates described herein have a DAR of 2 (i e there is an average of two payloads conjugated to each antibody). Such conjugates may have a single payload conjugated to each of the two light chains of the antibody Alternatively, such conjugates may have two payloads conjugated to a single light chain of the antibody using a multi pay load linker. By way of illustration, conjugate SGZ220 targets a final DAR value of 2 using a EVCitPABC linker to conjugate a single MMAE payload to each light chain of a Disitamab antibody scaffold

[0103] Some antibody payload conjugates described herein have a DAR of 4 (i e there is an average of four payloads conjugated to each antibody. Such conjugates may have a single payload conjugated to each of the two light chains of the antibody and to each of the two heavy chains of the antibody. However, in preferred aspects described herein, a DAR of 4 is achieved by using a multi payload linker on each of the light chains, such that two payloads are conjugated to each light chain of the antibody. By way of illustration, conjugate SGZ-230 targets a final DAR value of 4 using a branched EVCitPABC linker to conjugate two molecules of MMAE to each light chain of a Disitamab antibody scaffold DAR may be determined by any suitable method known in the art, such as MS or UV-Vis.

[0104] In some aspects and embodiments disclosed herein, a payload is only conjugated to the light chain In other words, the antibody payload conjugate does not contain a heavy chain conjugated to a payload or linker payload.

[0105] Due to the use of a PAL in conjugation of the antibody and the payload, antibody payload conjugates disclosed herein will retain the PAL conjugation motif, resulting from the PAL mediated ligation. The PAL, coupled with QC, is used to ligate a first component having a P1-P1 -P2’ tripeptide PAL motif as an acyl donor and a second component having a P1"-P2” motif as an acyl acceptor at the N-terminus. PAL cleaves the first peptide / protein after P1 in the tripeptide PAL motif and ligates said first component to the PT'-P2” motif of said second component, and QC cyclizes P1’ in the released PT-P2’ dipeptide motif to pyroglutamyl (pGlu) and prevents back-ligation of the released motif to the first component. This leads to efficient production of the resulting conjugate bearing the tripeptide PAL conjugation motif of P1-P1”-P2”.

[0106] As such, antibody payload conjugates disclosed herein will comprise the PAL conjugation motif of P1- P1"-P2", wherein P1 is Asn or Asp, P1" is any amino acid and P2" is a hydrophobic amino acid or a - branched amino acid. Examples of a hydrophobic amino acid may include Gly, Ala, Vai, Leu, lie, Pro, Phe, Met, Tyr and Trp. Examples of a ^-branched amino acid include Thr, Vai, and lie. In some embodiments, P1 is Asn. In various embodiments P2" may be selected from the group comprising Leu, Phe, Tyr, Trp, Vai, lie and Thr In preferred embodiments, the PAL conjugation motif has the sequence Asn-Gly-lle (NGI).

[0107] As used herein, the term "variant", refers to an amino acid sequence that is altered by one or more amino acids of the non-variant reference sequence, but retains the ability to recognize its target and affect its function. For example, a QC peptide variant is altered by one or more amino acids of the non-variant QC peptide reference sequence, but retains the ability to catalyse the intramolecular cyclization of N-Terminal glutaminyl and glutamyl residues of peptides and proteins to form pyroglutamyl residue (pGlu). The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g , replacement of leucine with isoleucine) More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc. Madison, Wisconsin, USA).

[0108] As used herein, the term "amino acid" may refer to natural and / or unnatural or synthetic amino acids, including both the D and L optical isomers, amino acid analogs (for example norleucine is an analog of leucine) and peptidomimetics As used in the context of the present application, the term “amino acid” typically refers to the 20 naturally occurring L-amino acids, namely Gly, Ala, Vai, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gin, Asp, Asn, His, Lys, Arg, Tyr, and Trp

[0109] As used herein, the terms “peptide”, “polypeptide" and “protein” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond Whereas peptides are considered to be short amino acid chains, polypeptides are long amino acid chains and proteins tend to have a stable structure and may comprise modifications (e g., glycosylation or phosphorylation). The term “protein” may encompass a naturally-occurring as well as artificial (e.g., engineered or variant) full-length protein as well as a functional fragment of the protein. It would be understood that, for the purpose of the invention, any combination of peptide, polypeptide or protein may be ligated in a reaction using PAL and QC providing one has a PAL acyl donor and the other has an acyl acceptor

[0110] As used herein, the term “functional fragment” refers to a portion of a protein that retains some or all of the activity or function (e g , biological activity or function, such as enzymatic activity) of the full-length protein, such as, e.g., the ability to catalyse a ligation reaction between two peptide. The functional fragment can be any size, provided that the fragment retains the activity / functionality of the full-length protein / enzyme.

[0111] As used herein, ‘sequence identity’ refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J., Bioinformatics (2005) 21 , 951-960), T-coffee (Notredame et al., J Mol Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics (2005) 6,298) and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4) 772-780) software When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. Sequences

[0112] Antibody payload conjugates may be characterised by reference to certain functional properties. In some embodiments, the antibody payload conjugates may possess one or more of the following properties:

[0113] High stability, including low shedding in vivo, even in murine serum High stability in circulation

[0114] High toxicity to cells expressing the target antigen

[0115] High efficacy of cell killing

[0116] A signature PAL conjugation motif between the antibody and the payload, such as NGI (Asn-Gly- lle) Binding to the target equivalent to the naked antibody (i.e the antibody in the absence of conjugation to the payload)

[0117] In vivo and / or in vitro cell killing and / or tumor reduction equivalent to or better than the equivalent ADC (i.e. and ADC conjugated via means otherthan PAL ligation)

[0118] The method of generating antibody payload conjugates described herein is associated with certain functional properties. For example:

[0119] Rapid conjugation of antibody and payload or linker-payload, even within 2 hours

[0120] Efficient conjugation of antibody and payload, resulting in high yield

[0121] Production of stable antibody payload conjugates The ability to achieve a DAR of 2 without conjugation of payload to both the heavy and light chains of the antibody

[0122] The ability to achieve a DAR of 4 without conjugation of payload to both the heavy and light chains of the antibody

[0123] No requirement for a spacer on the pay load to be conjugated

[0124] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0125] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention

[0126] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0127] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0128] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0129] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0130] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0131] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference Summary of the Figures

[0132] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0133] Figure 1. General scheme for enzymatic conjugation of IgG using a cascade enzymatic reaction with one Peptide Asparaginyl Ligases (PAL) and human glutaminyl cyclase (hQC). A. For conjugation at the C-terminus of each light chain, a five Gly-Gly-Gly-Gly-Ser spacer sequence is first genetically inserted afterthe C-terminal C214, to enhance enzyme accessibility, followed by the PAL+hQC « NQL » P1 PTP2’ motif The arrow indicates PAL cleavage site between the P1 and PT residues B. For conjugation at the C-terminus of each heavy chain, only the PAL+hQC « NQL » P1 PTP2’ motif is added (refer to amino-acid sequences in SI). In this scheme, the payload becomes conjugated to C-terminal ends of either L chains (scheme A) or heavy chains (scheme B), or both L and H chains by combining scheme A and B, giving conjugates with Drug-to-Antibody Ratio (DAR) values of 2p, 2 p, or 4p respectively, where p is the number of active cytotoxic compounds per payload (typically 1 for a single payload or 2 when using a dual payload as performed in this work).

[0134] Figure 2. Conjugation with FITC peptide on the Heavy chain. A. Reaction components are shown as molar equivalents FITC peptide was chemically synthetized with the formula GIGGIYRK{FITC}. B. SDS- PAGE analysis of the FITC peptide conjugation on the Heavy Chain by Coomassie staining and using Fluorescence imaging after 2 hours incubation at 37‘C. C. RP-HPLC and Mass spectrometry analysis of the antibody before and after conjugation with the FITC peptide. The antibody is reduced in presence of 20mM DTT at 60"C for 20 min before injection on a Jupiter C4 (Phenomenex) reverse phase column. The peak corresponding to the heavy chain is then analyzed by ESI-mass spectrometry.

[0135] Figure 3. In vitro imaging of HER2 positive cells using VyPAL2 conjugated trastuzumab to FITC payload. HER2+ SKBR3 cells and HER2- MCF7 cells were used as positive and negative controls respectively FITC conjugated antibody is incubated with HER2+ SKBR3 or HER2- MCF7 cells for 30 min and washed with fresh culture media before being imaged using a Zeiss LSM 710 confocal microscope.

[0136] Figure 4. Linker-payload structures for conjugation with PALs. Structures of payloads used, including single MMAE (A) and DXd (B) and branched MMAE (C) and DXd (D) payloads for increased DAR values, demonstrating the versatility of the conjugation method. (E) HIC analysis of conjugation efficiencies of Trastuzumab bearing spacers with varying lengths. All reactions were performed at 37 °C for 2 hours with 0.025 equivalents of VyPAL2-l244V and hQC enzyme per antibody and 10 equivalents of a single MMAE linker payload The chromatograms display the separation profiles of unconjugated and MMAE-conjugated antibody species obtained with Trastuzumab with LC NQL tag, GSNQL tag, GGGGSNQL tag, or GGGGSGGGGSNQL tag The Table notes the DAR obtained from HIC analysis.

[0137] Figure 5. Analysis of trastuzumab based antibody-drug conjugates (ADCs) with varying Drug-to- Antibody Ratios (DARs) and payload types by HIC. The chromatograms on the display the separation profiles of unconjugated and conjugated antibody species for (A) unconjugated antibody with LC tag, (B) unconjugated antibody with HC tag , (C) conjugated with single MMAE on the LC, (D) conjugated with single MMAE on the HC, (E) conjugated with single DXd on the LC, (F) conjugated with branched DXd on the LC, and (G) conjugated with branched MMAE linker payload on the LC.

[0138] Figure 6. Analysis of Disitamab based antibody-drug conjugates (ADCs) with varying Drug-to- Antibody Ratios (DAR) and payload types by HIC. The chromatograms on the display are the separation profiles of unconjugated and conjugated antibody species for (A) unconjugated antibody, (B) conjugated with single MMAE and (C) with branched MMAE linker payload

[0139] Figure 7. In vitro killing of HER2 positive cells using VyPAL2 conjugated trastuzumab to MMAE payload. HER2+ SKBR3 cells (A) and HER2- MCF7 cells (B) were grown in presence of serially diluted, unconjugated antibody (SGZ002), SGZ002-LC-MMAE, SGZ002-HC-MMAE and Kadcyla. Cell viability was determined using MTT Cell proliferation kit following user’s manual Each data points represents the mean of triplicate values and error bars represent SD Non-linear regression corresponding to a dose response-inhibition curve of the normalized growth function of the ADC concentration was performed using GraphPad Prism (C) to derive IC50 values for each condition

[0140] Figure 8. Stability of Heavy and Light chain conjugated SGZ002 in human or mouse serum. Heavy or light chain conjugated antibody was incubated in human (A) or mouse (B) plasma at 200pg / ml concentration. At each time point a sample is analyzed by LC-MS to detect the released MMAE payload Concentration of released payload is inferred from the LC spectra using a previously established calibration curve using released payload as a standard The proportion of intact ADC is estimated by subtracting the measured released payload from the initial conjugated ADC.

[0141] Figure 9. Tumor progression in CDX breast cancer BT474 model. BT474 cells were cultured in vitro until desired cell density is reached. 6x10A6 cells / 0 1 mL with 50 % Matrigel were injected subcutaneously into BALB / c female mice When tumor sizes reached 150mm3, a single dose of the indicated molecules at 1 mg / kg (A), 3 mg / kg (B) or 10 mg / kg (C) were injected. Mice in vehicle control group were treated with PBS Tumor sizes were measured every 4 days for28 days.

[0142] Figure 10. Tumor elimination using PAL conjugated ADCs. A. After 28 days study, mice were sacrificed, and tumors were weighted for each individual animal and the mean were plotted for each group (left panel). B. Tumors extracted from each animal were extracted and displayed in the panels with each row representing a group and each column within a row one animal.

[0143] Figure 11. Body weight change. BT474 cells were cultured in vitro until desired cell density is reached. 6x10A6 cells / 0.1 mL with 50 % Matrigel were injected subcutaneously into BALB / c female mice. When tumor sizes reached 150mm3a single dose of the indicated molecules were injected. Mice weights were weighed every 4 days for 28 days The data indicates the variation of the weight as compared with the weight before molecule injection.

[0144] Figure 12. IC50 curves derived from in vitro cytotoxicity measurements of PAL-mediated trastuzumab and disitamab ADCs conjugated with MMAE or DXd, Aidixi and Enhertu against HER2-positive human breast cancer cells. HER2 (3+) cell lines (A) BT-474 and (B) SKBR3, HER2 (2+) cell line, (C) JIM-T and HER2-positive patient derived xenograft cultures, (E) T226 (HER2, 3+) and (F) HBCx-1 (HER2, 2+) and a HER2-negative cancer cell line (D) MCF7 were grown in presence of serially diluted conjugates of trastuzumab-MMAE (SGZ-022, SGZ-024 and Kadcyla), trastuzumab-DXd (SGZ- 026, Enhertu) and disitamab-MMAE (SGZ220, SGZ-230 and Aidixi) Cell viability was determined using CellTite r-Glo® assay kit (Promega, Wl) according to the manufacturer's protocol. Each data-point represents the mean of duplicate (A-C) or triplicate (D & E) values and error bars represent SD. All experiments were performed at least twice Non-linear regression curve-fitting corresponding to ADC dose response-growth inhibition curves are plotted using GraphPad Prism to determine the IC50 values.

[0145] Figure 13. Anti-tumor activities of PAL-conjugated anti-HER2 ADCs in HER2-positive human breast cancer PDX models. . Groups of 8-9 athymic nude mice were implanted with T226 (HER2, 3+) and on day 1 post-tumor engraftment, a single dose of trastuzumab-DXd ADC, SGZ-026 or Enhertu was given intravenously at 1 mg / kg (A), 3 mg / kg (B) or 7 5 mg / kg (C). Additionally, groups of 8-9 athymic nude mice were implanted with HBCx-1 (HER2, 2+) and intravenously given a single 10 mg / kg dose of trastuzumab-MMAE ADCs, SGZ-022 and SGZ-024 (D), disitamab-MMAE ADCs, SGZ-230 and Aidixi (E) and trastuzumab-DXd ADCs, SGZ-026 and Enhertu (F) on day 1 post-tumor engraftment. Mice in vehicle control group were treated with PBS Tumor volumes (left panels) and body weights (right panels) were monitored for up to 5 (A-C) or 6 (D-F) weeks’ post ADC treatment

[0146] Figure 14. In vivo plasma stability of PAL-conjugated anti-HER2 ADCs in mouse. A single intravenous dose of 3 mg / kg of PAL-conjugated disitamab-MMAE ADC, SGZ230 (A), trastuzumab-MMAE ADCs, SGZ022 (C) and SGZ024 (D), trastuzumab-DXd, SGZ026 (E), as well as the marketed ADCs, Aidixi (B) and Enhertu (F) was administered to healthy BALB / c mice Blood was collected at various timepoints and the mouse plasma concentrations of intact ADC or Total antibodies were quantified by sandwich ELISA.

[0147] Figure 15. Schematic showing synthesis of GIGEVCit-PAB-MMAE (9)

[0148] Figure 16. Schematic showing synthesis of dual-MMAE linker payload (14)

[0149] Figure 17. Schematic showing synthesis of dual-MMAE linker payload (17)

[0150] Figure 18. Schematic showing synthesis of DXd linker payload (20).

[0151] Figure 19. Schematic showing synthesis of dual- DXd linker pay load (21).

[0152] Figure 20. Schematic showing synthesis of dual- DXd linker pay load (22).

[0153] Examples

[0154] Example 1: Materials and Methods

[0155] Expression and purification of VyPAL2-l244V

[0156] Expression and purification were conducted as previously described (15, 21). As VyPAL2-l244V has a 2- fold improved ligation activity overthe WT enzyme (21), it was used for the reactions. The sequence corresponding to VyPAL2 with the I Ie244 residue mutated to Vai was cloned into pFB plasmid and transformed into DH10 bacteria for bacmid generation. The bacmid was transfected in Sf9 insect cells and virus was collected after 5 days. After 2 cycles of virus amplification, the expression was carried out Protein purification from the insect cell media was performed in three steps with IMAC purification followed by ion-exchange and size-exclusion chromatography. The protein in its proenzyme form was then concentrated to 2 mg / ml and stored at 4 °C before use Activation of the proenzyme was performed by acidification at pH 4.5 (50 mM sodium citrate buffer, 1 mM DTT, 1 mM EDTA, 0.1 M NaCI) at 4 “C for 12-16 h with 0.5 mM N-lauroylsarcosine. Subsequently, active enzymes were purified on a sizeexclusion chromatography column (S75 16 / 60) (GE Life Sciences) preequilibrated in 1x PBS buffer

[0157] Expression and purification of human glutaminyl cyclase.

[0158] The sequence corresponding to the human glutaminyl cyclase with a C-terminal hexa-histidine tag was cloned into the pET28b using Ndel and Notl restriction sites E coli BL21 (DE3) competent cells transformed with pET28b-hQC were grown in TB media at 37°C until OD60Q of 0.8. 1 mM of isopropyl - D-1-thiogalactopyranoside (IPTG) was added to the culture for 48 h of expression. The cells were pelleted by centrifugation at 8000 g for 20 min at 4 °C and stored at -20 °C. For protein purification, the cells are resuspended in 50 mM Tris-HCI pH 8, 150 mM NaCI and 20 mM imidazole and disrupted by sonication. After centrifugation at 25000 g for 45 minutes, the filtered supernatant is injected on a nickel affinity column (HiTrap 5 ml, GE Healthcare) After a washing step of 5 % of 50 mM Tris-HCI pH 8, 150 mM NaCI and 500 mM imidazole (Buffer B), the protein is eluted with 100 % of Buffer B and injected on a gel filtration Superdex 75 26 / 60 (GE Healthcare) using 1x PBS The protein fractions are pooled, concentrated and stored at -80 °C.

[0159] Expression and purification of antibodies based on trastuzumab scaffold.

[0160] All sequences are listed in the supplementary information section. Briefly, the corresponding sequence was chemically synthetized and cloned into a pCDNA3 4 (Genscript). The trastuzumab anti-HER2 antibody was genetically modified to include the amino acid sequence GGGGSNQL at the C-terminus of the light chain (SGZ002-LC) and the sequence NQL (Asn-GIn-Leu) at the C-terminus of the heavy chain (SGZ002-HC). The corresponding sequences were chemically synthetized and cloned into a pCDNA3 4 (Genscript). Expression in CHO mammalian cells was carried accordingly to the ExpiCHO mammalian expression kit protocol (Invitrogen). Each antibody was purified from the culture media using a protein A affinity purification. Elution was carried with 50 mM Glycine buffer at pH 2 7 with neutralization with 1 M Tris pH 8 immediately after elution. The fraction containing the antibody were concentrated and injected on a S200 16 / 60 for a size exclusion chromatography equilibrated with PBS pH 7 4

[0161] Payload synthesis

[0162] A series of payloads were synthesized for conjugation to the antibody scaffolds These contained the linkers and payloads of ADCs already on the market, with single and branched monomethyl auristatin E (MMAE) with an EVCit-PABC linker (similar to vedotin), and single or branched Deruxtecan (DXd) with a GGFG linker. In each case, the PAL dipeptide motif was included in the linker, at the C-terminus, such that the linker was between the dipeptide motif and the drug payload. Synthesis of GIGEVCit-PAB-MMAE (9): Compound 9 was synthesized as shown in Figure 15.

[0163] Peptide 7 was synthesized using Solid phase peptide synthesis (SPPS). 2-Chlorotrityl chloride resin was used as solid support and the peptide chain was synthesized using Fmoc chemistry. Peptide cleavage from resin was achieved using 1 :1 HFIP-DCM to retain the O'Bu group on glutamic acid side chain. The cleavage solution was separated from the resin by filtration and the cleaved peptide was precipitated in the cold EtsO. The crude product was isolated by centrifugation and purified by RP-HPLC The peptide fractions after HPLC purification were lyophilized to afford the peptide in powder form.

[0164] HATU (25 mg, 0 066 mmol), DIEA (23 pL, 0.12 mmol) was added to a solution of compound 6 (50 mg, 0.04 mmol) and 7 (56 mg, 0.044 mmol) in anhydrous DMF (2.0 mL) and stirred at rt for 16 h, 20 % piperidine (0.5 mL) was added and stirred at rt for 1 h The reaction mixture was evaporated under reduced pressure and the crude compound 8 was washed with 20 % DCM in Et2O (2 x 10 mL). 1 :4 TFA: DCM (3.0 mL) was added to compound 8 at 0 ’ C and stirred for 4h at same temperature. Rm was evaporated and purified by reverse-phase HPLC purification (Buffer A: 0 045 % TFA in H2O, Buffer B: 0.045 % TFA in 90 % acetonitrile, 10 % H2O) The fractions containing the product were pooled and freeze dried to afford compound 9 (21 mg, 32 %) as off-white powder. MS (ESI): m / z [M+H]+ calc.

[0165] Synthesis of Dual MMAE linker payload (14): Compound 14 was synthesized as shown in Figure 16. The synthesis started with Fmoc-Gly-wang resin, the Fmoc was removed using piperidine in DMF followed by coupling with Fmoc-Lys(Mtt)-OH The synthesis was continued using standard solid phase peptide synthesis (SPPS) to give compound 11 , Mtt protection on lysine was removed using 4 % TFA in DCM. mPEGie-COOH was coupled to side chain amine group of lysine using HATU, followed by removal of allyl protection using PdfPPhjR The resin was cleaved using 95 %TFA and the crude was purified by column chromatography to afford Fmoc protected peptide 12.

[0166] Compound 12 was treated with commercially available NH2-E(Oallyl)VCit-PABC-MMAE (13), HATU, DIEA in DMF, the Fmoc on N-terminus was removed by in the same pot by treating with piperidine for 1 h. The crude rection was dried under vacuum and redissolved in anhydrous Methanol and treated with Pd(PPh3)4 and morpholine to remove allyl protection. The rude was evaporated and purified by reverse phase HPLC

[0167] HATU (25 mg, 0 066 mmol), DIEA (25 pL, 0.15 mmol) was added to a solution of compound 12 (50 mg, 0.033 mmol) and 13 (85 mg, 0 066 mmol) in anhydrous DMF (2 0 mL) and stirred at rt for 16 h, 20 % piperidine (0.2 mL) was added and stirred at rt for 1 h The reaction mixture was evaporated under reduced pressure and the crude was dissolved in anhydrous MeOH (3 0 mL) and treated with Pd(PPhj)4 (0.1 mmol), morpholine (3.0 equiv) and stirred at rt for 1 h. Rm was evaporated and purified by reversephase HPLC purification (Buffer A: 0.045 % TFA in H2O, Buffer B: 0.045 % TFA in 90 % acetonitrile, 10 % H2O). The fractions containing the product were pooled and freeze dried to afford compound 14 (28 mg, 22 %) as off-white powder.

[0168] Synthesis of Dual MMAE linker payload (17): Compound 17 was synthesized as shown in Figure 17.

[0169] The synthesis started with Fmoc-Gly-wang resin, the Fmoc was removed using piperidine in DMF followed by coupling with Fmoc-PEGa-OH. The synthesis was continued using standard solid phase peptide synthesis (SPPS) to give compound 15, mtt protection on lysine was removed using 4% TFA in DCM. mPEGi6-COOH was coupled to side chain amine group of lysine using HATU, followed by removal of allyl protection using Pd(PPha)4. The resin was cleaved using 95%TFA and the crude was purified by column chromatography to afford Fmoc protected peptide 16.

[0170] Compound 16 was treated with commercially available NH2-E(Oallyl)VCit-PABC-MI\ / IAE (13), HATU, DIEA in DMF, the Fmoc on N-terminus was removed by in the same pot by treating with piperidine (20 equiv) for 1 h. The crude rection was dried under vacuum and redissolved in anhydrous Methanol and treated with Pd(PPhs)4 and morpholine to remove allyl protection. The rude was evaporated and purified by reverse phase HPLC

[0171] HATU (25 mg, 0 066 mmol), DIEA (25 pL, 0.15 mmol) was added to a solution of compound 16 (50 mg, 0.025 mmol) and 13 (64 mg, 0 05 mmol) in anhydrous DMF (2.0 mL) and stirred at rt for 16h, 20 % piperidine (0.2 mL) was added and stirred at rt for 1 h The reaction mixture was evaporated under reduced pressure and the crude was dissolved in anhydrous MeOH (3 0 mL) and treated with Pd(PPha)4 (0.1 mmol), morpholine (3.0 equiv) and stirred at rt for 1 h. Rm was evaporated and purified by reversephase HPLC purification (Buffer A: 0.045 % TFA in H2O, Buffer B: 0.045 % TFA in 90 % acetonitrile, 10 % H2O). The fractions containing the product were pooled and freeze dried to afford compound 17 (22 mg, 21 %) as off-white powder.

[0172] Synthesis of GIGGGFG-am-DXd (20): Linker-payload 20 was synthesized according to Figure 18, commercially available GGFG-am-DXd mesylate was treated with FmocGIG-OH, HATU, DIEA in DMF. The Fmoc protection was removed in same pot using piperidine.

[0173] HATU (25 mg, 0 066 mmol), DIEA (25 pL, 0.15 mmol) was added to a solution of compound 18 (50 mg, 0.053 mmol) and 19 (30 mg, 0 064 mmol) in anhydrous DMF (2 0 mL) and stirred at rt for 16h, 20 % piperidine (0.2 mL) was added and stirred at room temperature (RT) for 1 h. The reaction mixture was evaporated under reduced pressure and the crude was purified by reverse-phase HPLC purification (Buffer A: 0 045 % TFA in H2O, Buffer B: 0 045 % TFA in 90 % acetonitrile, 10 % H2O) The fractions containing the product were pooled and freeze dried to afford compound 20 (23 mg, 41 %) as off-white powder.

[0174] Synthesis of dual- DXd linker payload (21): Linker-payload 21 was synthesized according to Figure 19. Commercially available GGFG-am-DXd mesylate was treated with compound 16, HATU, DIEA in DMF. The Fmoc protection was removed in same pot using piperidine.

[0175] HATU (28.5 mg, 0 075 mmol), DIEA (25 pL, 0.15 mmol) was added to a solution of compound 16 (50 mg, 0.025 mmol) and 18 (30 mg, 0 054 mmol) in anhydrous DMF (2 0 mL) and stirred at RT for 16h, 20 % piperidine (0.2 mL) was added and stirred at RT for 1 h. The reaction mixture was evaporated under reduced pressure and the crude was purified by reverse-phase HPLC purification (Buffer A: 0.045 % TFA in H2O, Buffer B: 0.045 % TFA in 90 % acetonitrile, 10 % H2O). The fractions containing the product were pooled and freeze dried to afford compound 21 (23 mg, 41 %) as off-white powder. Synthesis of dual- DXd linker payload (22): Linker-payload 21 was synthesized according to Figure 20.

[0176] Commercially available GGFG-am-DXd mesylate was treated with compound 16, HATU, DIEA in DMF.

[0177] The Fmoc protection was removed in same pot using piperidine.

[0178] HATU (38 mg, 0 099 mmol), DIEA (33 pL, 0.2 mmol) was added to a solution of compound 12 (50 mg, 0.033 mmol) and 18 (67 mg, 0 072 mmol) in anhydrous DMF (2 0 mL) and stirred at RT for 16h, 20 % piperidine (0.2 mL) was added and stirred at RT for 1 h. The reaction mixture was evaporated under reduced pressure and the crude was purified by reverse-phase HPLC purification (Buffer A: 0.045 % TFA in H2O, Buffer B: 0.045 % TFA in 90 % acetonitrile, 10 % H2O). The fractions containing the product were pooled and freeze dried to afford compound 22 (23 mg, 41 %) as off-white powder.

[0179] Antibody conjugation and Hydrophobic interaction chromatography

[0180] The fluorescent peptide used (NH2-GIGGIRK{fluorescein}-COOH) was chemically synthetized (Genscript).

[0181] The antibody is mixed with a 5-fold molar excess of linker-payload, and a 0.025 equivalent of PAL and 0.0025 equivalents of hQC in a Phosphate buffer at pH 6.5 and incubated at 37"C under agitation for 2 hours Protein A affinity chromatography was used to purify the conjugated antibody from the excess payload and enzyme, followed by a desalting step using a Desalting 26 / 10 column (Cytiva) to formulate the conjugate to the final buffer in 1x PBS pH 7.4. Drug to antibody ratio was determined by Hydrophobic interaction chromatography on a Protein PAK HIC 4 6x100mm column (Waters) in a TUV UPLC system. Column is pre-equilibrated in 50mM NaH2PO4 / Na2HPO4, 2M (NH4)2SC>4 at 40"C. Antibody to analyze is diluted in 1 M NH4SO4 solution and 10pL injection is performed at 0.4 mL per mL. Elution is performed by a gradient of 50mM NaH2PO4 / Na2HPC>4 for 15 minutes.

[0182] Tissue culture and cell imaging

[0183] SKBR3 (HTB-30, ATCC), JIMT-1 (ABC-TC504S, Accegen) and MCF7 (HTB-22, ATCC) cell lines were maintained in DMEM media (high glucose) supplemented with 10 % FBS. BT-474 (HTB-20; ATCC) was maintained in RPMI media supplemented with 10 % FBS. All cells were kept at 37 °C in an incubator under 5 % CO2. Cells were grown until 40-60 % confluency For fluorescence cell imaging trastuzumab- FITC antibody conjugate in DMEM medium at 100 nM was applied to the cells and incubated for 30 min at 37 °C. Washing was performed three times with PBS, and the cells were subsequently subjected to microscopy analysis

[0184] For cell viability cytotoxicity assays, cells were seeded into 96-well plate at the density of 5000 ce I Is / wel I and incubated at 37 °C, in 5 % CO, for 16-20 h (cancer cell lines) or 48 h (PDX cultures). MMAE and DXd ADCs conjugated by PAL, trastuzumab-emtansine (Kadcyla; MedChemExpress, NJ, USA), trastuzumab- (Enhertu, MedChemExpress, NJ, USA), or disitamab- (Aidixi, MedChemExpress, NJ, USA), 3-fold serially diluted in DMEM complete culture medium with top concentrations of either 50 or 300 nM, were applied to the cells and the plate incubated for 96-120 hours at 37°C and 5 % CO2. After the Incubation period, cell viability was assessed by using the MTT assay (Roche, IN, USA) or CellTiter-Glo® assay (Promega, Wl, USA). Briefly, for the MTT assay, culture media was replaced with MTT solution at 0.5mg / ml in PBS and incubated for 1 hour at 37"C before removal of the MTT solution. Formazan crystals were dissolved in 10OpL of DMSO and formazan concentration indicating cell viability was assessed spectrophotometrically using a TECAN microplate reader measuring the absorbance at wavelength of 575nm using 650nm wavelength as a reference For cell viability measurement with CellTiter-Glo® assay, an amount of CellTiter-Glo reagent equal to the cell culture media in the well was added to and the plate incubated for 20 min at RT followed by luminescence reading using a TECAN microplate reader. The resultant IC50 data for the tested ADCs were derived by nonlinear regression curve fit using Graph Pad Prism

[0185] ADC in vitro Plasma stability

[0186] PAL conjugates were incubated in mouse and human plasma at a concentration of 200 pg / ml and incubated at 37"C. Samples at 0, 48, 120, 168, 240 and 336 hours of incubation were analyzed by LC- MS / MS to quantify the released payload Measurements were done in duplicates.

[0187] ADC solution is added into blank human or mouse plasma to obtain ADC plasma solution with the concentration of 200 pg / mL. The ADC plasma solution for each species was divided into 24 samples (4 samples at each time point: 2 samples were used for small molecular toxin analysis (50 pL); the other 2 samples were stored at £ -60 °C as spare samples (100 pL reserved). ADC samples were incubated at 37 °C, for Oh, day 2 (48h), day 5 (120h), day 7 (168h), day 10 (240h) or day 14 (336h) and analyzed by LC-MS / MS.

[0188] The shedding rate is defined as:

[0189] Shedding rate: (measured released payload concentration I Initial payload concentration) *100

[0190] The remaining concentration of intact ADC is calculated as:

[0191] Remaining ADC= Initial ADC concentration * (1-shedding rate)

[0192] In vivo efficacy in human breast cancer BT-474 Xenograft model

[0193] BT-474 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) DMEM medium and FBS were purchased from GIBCO (Grand Island, NY, USA). Matrigel was purchased from CORNING (Corning, NY, USA). BALB / c nude mice were subcutaneously implanted with 17 -estradiol pellet (0.18 mg / pellet, 90-day release) on the lateral side of neck between the ear and the shoulder 3 days priorto BT-474 cells inoculation. BT-474 cells were maintained in DMEM medium with 10 % FBS. Cells were incubated at 37 °C in a humidified incubator with 5 % CO2. BT-474 xenograft model was established by injecting tumor cells (6x106cells / mouse with 50 % Matrigel) into the right fat pad by S.C. administration under sterile conditions When the mean tumor volume reached an appropriate size (around 200 mm3), mice were randomized to treatment groups (8 mice per group) according to the randomized grouping method. The tumor size and body weight were considered and balanced between groups. Variance of the mean tumor burdens in each group was not to exceed ±10 % of the overall mean The day of grouping was designated as Day 0, and dosing was started. Mice were dosed individually by the most recent measured body weight Dosing holidays should be given to the individual mouse with body weight loss (BWL) > 15 %, other mice in the same group received dosing as scheduled. The tumor sizes and animal body weights were measured twice per week. The tumors were measured using a caliper. Tumor volumes were estimated from measurements of the two diameters of the individual tumors. Clinical signs were recorded daily Animal survival was also recorded. Observations included general health, body weight, behavior, and any adverse effects related to the dose administration.

[0194] In vivo mouse patient-derived xenograft studies.

[0195] All in vivo studies were performed in accordance with the local guidelines of the Institutional Animal Care and Use Committee. Trastuzumab- and Kadcyla-resistant, human breast cancer PDX, T226 or trastuzumab- and pertuzumab-resistant, human breast cancer PDX, HBCx-1 , were passaged in donor mice and implanted subcutaneously as 20 mm3fragments into 4-6 weeks old female athymic nude mice T226 and HBCx-1 tumors were tested by IHC and flow cytometry for verification of HER2 3(+) and 2(+) status, respectively Animals with tumors 60-256 mm3in size were randomized into groups of 8-9 mice and injected intravenously with anti-HER2 ADCs at doses ranging from 1-10 mg / kg, or vehicle control (PBS) The day of grouping was designated as Day 0, and dosing was started on day 1 . All mice are observed clinically, for physical appearance, behavior and clinical changes, on each weighing / tumor measurement day. Animals were weighed, and tumors were measured by caliper, once or twice weekly Tumor volumes (TV) were calculated using the formula: TV (mm3) = [length (mm) x width (mm)2] x 0.5, in which length and width are the longest and the shortest diameters of the tumor, respectively. Statistical significance between ADC-treated and control groups as well as between the tested groups was evaluated with Mann-Whitney non-parametric comparison test.

[0196] In vivo mouse pharmacokinetics studies.

[0197] All in vivo studies were performed in accordance with the local guidelines of the Institutional Animal Care and Use Committee. Male BALB / c mice (Shanghai Lingchang Biotech) were dosed once intravenously with 3 mg / kg of each anti-HER2 ADCs constituted in PBS (n = 3 mice / group) Whole blood samples were collected from the animals at 1 , 2, 4 and 8 h, then 1 , 3, 7, 14, and 21 days, processed into serum by centrifugation at 10,000 g for 5 min at RT and immediately frozen at -80°C prior to analysis. ELISA-based methods were used for quantification of either Total Antibody or ADC. For total antibody measurement, recombinant His-tagged human ErbB2 / HER2 protein was used as capture antigen, followed by detection with biotinylated goat anti-human IgG Fc-HRP and TMB. For measurements of ADC conjugated with MMAE or DXd, anti-MMAE or anti-DXd antibody, respectively, were used for ADC capture, followed by binding of recombinant human ErbB2 / HER2 protein and detection with anti-H is tag-HRP. Pharmacokinetic parameters were calculated using a noncompartmental model in WinNonlin (Certara, PA, USA) and reported as mean ± SD. Example 2: Conjugation of IgG Molecules

[0198] 2.1 1gG conjugation scheme

[0199] The scheme underlying the enzymatic conjugation of IgG molecules performed in this work is summarized in Figure. 1. Briefly, a PAL+hQC tripeptide recognition tag “NQL” was genetically added to the IgG amino acid sequence of either the L (Fig 1 a) or H chain (Fig. 1b) C-terminal end. In the case of the L chain, a spacer sequence of five amino acids “GGGGS” was included upstream of the “NQL” tag, to facilitate access of the PAL to the tripeptide recognition motif (while the same three amino-acid recognition motif is largely exposed at the C-terminus of the H chain and hence the reaction proceeds rapidly without the need for a spacer) The trastuzumab sequence was used as a scaffold, as this mAb is routinely used in the clinic for treatment against HER2-positive breast cancer. Trastuzumab-derived antibodies bearing the PAL recognition motif at the C-terminus of either the L (SGZ-022 / SGZ002-LC) or H chain (SGZ002-HC) were expressed. The complete amino-acid sequences of the L and H chains for each modified mAb are given in SEQ ID NOs: 1-4. The modified IgG were overexpressed with a yield of 50-100 mg / mL and purified as described in Example 1.

[0200] 2.2 Heavy chain conjugation of Trastuzumab with FITC.

[0201] Trastuzumab bearing the PAL+hQC recognition motif at the H chain C-terminus (SGZ002-HC) at a concentration of 2 mg / mL (13 3 pM) was conjugated to a peptide having a FITC fluorescence probe attached to a lysine side-chain (GIGGIYRK{FITC), (Figure 2A). The fluorescent peptide was incubated at a final concentration of 66.5 pM to give a ~2 5-fold molar excess per H chain conjugation site (two sites per antibody molecule). The molar ratio between PAL (activated VyPAL2-l244V) used for conjugation, and the antibody was 0.025:1 and the same for the hQC:mAb molar ratio. The reaction was conducted for 2 hours at 37°C under mild agitation and then analyzed by SDS-PAGE and fluorescence imaging. Fluorescence imaging showed that the peptide is rapidly and efficiently linked to the H chain of the antibody only (Figure 2B). The sample was analyzed by HPLC-MS, after addition of 1 M of DTT to reduce the interchain disulfide bridges to resolve the light and heavy chains. The HPLC profiles show the presence of one additional peak corresponding to the ligation reaction product for the heavy chain (SGZ002-HC-FITC) and a strong reduction in the peak corresponding to the unconjugated heavy chain MALDI-TOF analysis shows that the additional peak contains a polypeptide with a mass corresponding to the H chain to which GIGGIYRK{FITC) was conjugated (Figure 2C). Conjugated SGZ002-HC-FITC species were further purified by affinity purification using a protein A column. The conjugated antibody was collected in the flow through, and buffer exchanged into PBS buffer using Desalting 26 / 10 column.

[0202] To check whether the SGZ002-HC-FITC fluorescent conjugate retained biological activity, binding to SKBR3 cells (a cancer cell line overexpressing the HER2 antigen) was assessed SKBR3 cells were grown to 60 % confluency before application of purified SGZ002-HC-FITC at a concentration of 100 nM. After 30 min incubation and washing with PBS buffer, cells were imaged in brightfield and in fluorescence mode (Figure 3, upper panel). MCF7 breast cancer cells that do not express HER2, were used as a negative control. It was observed that only SKBR3 cells, expressing HER2 on their surface, showed strong fluorescence signals while no signal was observed for MCF7 cells (Figure 3, lower panel)

[0203] 2.3 Conjugation of trastuzumab with different spacer lengths to peptides linked to MMAE payload

[0204] Next, conjugation activity of trastuzumab bearing the PAL+hQC recognition motif at the C-terminus of its L chain (SGZ002-LC) to the peptide, Glutamate-valine-citrulline-p-aminocarbamate (VC-PABC)-based linker (EVCit-PABC-MMAE; Fig 4A) was tested with different spacer lengths, NQL (0 spacer), GSNQL (2 spacer), GGGGSNQL (5 spacer) and GGGGSGGGGSNQL (10 spacer) The final antibody and peptide concentrations were 13 and 133 pM, respectively, to give a ~5-fold molar excess per target chain conjugation site (two L sites per antibody molecule). Reactions were conducted for 2 hours at 37°C under mild agitation and the ADC products were further purified by affinity using a protein A column (Cytiva) and analysed by by Hydrophobic Interaction Chromatography (HIC). The absence of a spacer yielded low conjugation efficiency, with DAR of 0 83 obtained (Fig 4E) Conjugation efficiency increased with spacer length up to 5 residues, achieving DAR of 1 86. Only a slight improvement in conjugation was observed beyond 5 residues in spacer length (Figure 4E).

[0205] 2.4 Conjugation of trastuzumab and disitamab with peptides linked to MMAE or DXd payloads

[0206] Next, trastuzumab bearing the PAL+hQC recognition motif at the H chain C-terminus (SGZ002-HC) and at the L chain C-terminus (SGZ002-LC) each at a concentration of 2 mg / mL (13 3 pM) were expressed for conjugation with a series of peptides (“GIG”) linked to Glutamate-valine-citrulline-p-aminocarbamate (VC- PABC)-based linkers (EVCit-PABC-MMAE) (Figure 4). The peptides were linked to either a single (Figures 4A & 4B) cytotoxic moiety or a dual one via a branched structure to which a PEG oligomer was added to increase solubility (Figures 4C & 4D) and were incubated at a final concentration of 133 pM to give a ~5-fold molar excess per target chain conjugation site (two L sites and two H sites per antibody molecule) (Figure 4). Conjugation of antibodies with branched-chain linker-payloads comprising two drug molecules was performed in order to increase their DAR values from two to four using the same scheme as outlined in (Figure 1) but using branched payloads as depicted in Figure 4C and 4D The PAL: IgG and hQC:mAb molar ratio were 0.025:1. Reactions were conducted for 2 hours at 37’C under mild agitation Two ADCs products were obtained: SGZ002-HC-MMAE and SGZ002-LC-MMAE which were further purified by affinity using a protein A column (Cytiva). A list summarizing all the ADCs produced in this study is given in Table 1. The conjugated antibodies were collected in the flow through, and wash steps and buffer exchanged into PBS buffer using Desalting 26 / 10 column (Cytiva). The samples were analyzed before and after conjugation by Hydrophobic Interaction Chromatography (HIC) (Figure 5) The HIC profiles showed strong reduction of the size of the peak corresponding to the unconjugated antibody (Figures 5A & 5B) and formation of additional peaks corresponding to MMAE ligated products: SGZ002- LC-MMAE and SGZ002-HC-MMAE (Figures 5C & 5D) The retention time of 18.3 on the HIC of the SGZ002-HC-MMAE conjugate with DAR 2 (Figure 5D) is longerthan forthe corresponding SGZ002-LC- MMAE conjugate (retention time of 12.06; Figure 5C). This indicates a significantly lower hydrophobicity for the light chain IgG conjugate with DAR value of 2, compared with the corresponding heavy chain conjugate. Likewise, new peaks were visible following conjugation with the single DXd payload with a DAR value of 2 (Figure 5E) and the DAR value of 4 conjugates using the branched DXd (Figure 5F) and branched MMAE (Figure 5G) which also elute later from the HIC due to their higher hydrophobicity

[0207] Table 1 : antibody conjugates used in this study.

[0208] Using similar protocols (Figure 1) disitamab-MMAE conjugates (Table 1) targeting final DAR values of 2 (SGZ220, Table 1) or 4 (SGZ-230) were also prepared (Figure 6) Conjugation was realized using 0.025 molar equivalent to the antibody of activated VyPAL2-l244V and 0.0025 equivalents of hQC The reactions were conducted for 1 hours at 25°C under mild agitation.

[0209] Example 3: Biological activity of ADCs

[0210] 3.1 PAL conjugated anti-HER2 ADCs specifically kill HER2 expressing cells. To confirm that SGZ002-HC-MMAE and SGZ002-LC-MMAE are biologically active, their cytotoxicity was assessed using HER2-expressing cells SKBR3 cells were grown to 60 % confluency before application of serially diluted trastuzumab (SGZ002 in Figure 7), SGZ002-LC-MMAE, SGZ002-HC-MMAE or trastuzumab-emtansine (Kadcyla; MedChemExpress, NJ, USA), a widely used ADC for breast cancer treatment covalently linked to DM 1 , a tubulin inhibitor. After 3 days of incubation, cell viability was measured using a MTT assay (Roche, CH) MCF7 cells were used as a negative control as they do not express HER2 proteins. Only SKBR3 cells, expressing HER2 on their surface, were killed by SGZ002- HC-MMAE, SGZ002-LC-MMAE or trastuzumab-emtansine with IC50 values of 88 pM, 79 pM and 62 pM respectively (Figure 7A - 7C) MCF7 cells were not affected by either PAL-conjugated antibodies or trastuzumab-emtansine (Figure 7B)

[0211] 3.2 In vitro plasma stability of MMAE conjugates based on the trastuzumab / SGZ002 scaffold.

[0212] SGZ002-LC-MMAE and SGZ002-HC-MMAE conjugates (Table 1) were incubated either in human or mouse plasma for up to 336 hours (14 days) at 37 "C. At the incubation time points of 0, 48, 120, 168, 240 and 336 hours, samples were analyzed by LC-MS / MS to quantify the free payload released from the antibody-drug conjugates and these data were used to infer the in vitro plasma stability of each ADC based on the SGZ002 / trastuzumab scaffold (Figure 8). As expected, both SGZ002-LC-MMAE and SGZ002-HC-MMAE were remarkably stable in human serum (Figure 8A) with MMAE-released rates of only 0 96 % and 0.62 % respectively after 336 hours of incubation. It has been noted previously that ADCs are generally less stable in mouse serum due to the presence of proteases such as serum Carboxylesterase 1C (Ces1) responsible for the premature cleavage of Val-Cit based linkers21. Of note, a significant difference in stability is observed between conjugation on the L or H chain: SGZ002-LC-MMAE appears intrinsically more stable in the mouse plasma with a release rate of MMAE of 15 % after 336 h and a proportion of intact ADC of 85 %, compared to SGZ002-HC-MMAE with half of the payload released after ~100 h and completely degraded after 336 h (Figure 8B).

[0213] 3.3 In vivo anti-tumor activity ofSGZ002 MMAE conjugates in a mouse CDX model

[0214] A BT-474 xenograft model with high level of HER2 expression (HER2 3+), was established by injection of tumor cells (6x106cells / mouse with 50 % Matrigel), into the right foot pad of BALB / c nude mice under sterile conditions Kadcyla was dosed via a single intravenous injection at either high dose (HD, 3 mg / kg of animal weight) or low dose (LD, 1 mg / kg). For comparison, SGZ002-LC-MMAE and SGZ002-HC- MMAE were also given i.v as single doses at either HD (10 mg / kg) (Figure 9C), MD (medium dose; 3 mg / kg) (Figure 9B), or LD (1 mg / kg) (Figure 9A). The experiment was concluded on day 28 and animals were euthanized after tumor and body weight measurements. On Day 28, the mean tumor volume of the vehicle group was 554.22 ± 72.58 mm3, and the corresponding mean tumor weight was 0 392 ± 0 068 g (Figures 9 and 10). The mean tumor volumes for Kadcyla at LD (1 mg / kg) and Kadcyla HD (3 mg / kg) groups were 431 04 + 63 28 mm3and 211 56 + 75.52 mm3, respectively Their mean tumor weights were 0.268 ± 0 038 g and 0.136 ± 0.057 g, respectively, with %TGITW values of 31.63 % (p>0 05) and 65.31 % (p<0 05), respectively (Figures 9 and 10). The mean tumor volumes of SGZ002-LC-MMAE LD, MD and HD groups were 197.32 + 27 19 mm3, 8.16 ± 8.16 mm3and 0.00 ± 0 00 mm3, respectively. Their mean tumor weights were 0.128 ± 0.018 g, 0.003 ± 0.003 g and 0 000 ± 0.000 g, respectively, with %TGIT values of 67.35 % (p<0.01), and 99 24 % (p<0.001) and 100 % (p<0.001), respectively (Figures 9 and 10). The mean tumor volumes of SGZ002-HC-MMAE HC conjugated LD, MD and HD groups were 278.72 ± 45 40 mm3, 46 02 ± 14 88 mm3and 0.00 ± 0.00 mm3, respectively Their mean tumor weights were 0.203 ± 0.052 g, 0.026 ± 0.010 g and 0.000 ± 0.000 g, respectively, with %TGIT values of 48.21 % (p<0 05), and 93.37 % (p<0.001) and 100 % (p<0.001), respectively (Figures 9 and 10).

[0215] In conclusion, a single dose of SGZ002-LC-MMAE and SGZ002-HC-MMAE, at all dosages tested (1 , 3 and 10 mg / kg), abrogated the growth of human breast cancer BT-474 cells with high levels of HER2 expression in BALB / c nude mice. Moreover, a strong dose-response relationship and dramatic tumor growth inhibition were observed with SGZ002-LC-MMAE LD. In contrast, Kadcyla at LD (1 mg / kg) led only to partial tumorgrowth inhibition compared to strong growth reduction of SGZ002-LC-MMAE LD (p=0 0067) and SGZ002-HC-MMAE LD (p=0.0109), whilst Kadcyla injected at HD (3 mg / kg) was not as effective as SGZ002-LC-MMAE MID (p=0 0025) and SGZ002-HC-MMAE MD (p=0 0029) in suppressing tumor growth.

[0216] Following dosing with ADCs, no body weight loss was observed in all groups of mice (Figure 11). The mean body weights of the mice in the various groups increased maximally by 7-15 % around days 11-18 and declined back to 0-10 % by day 28. On Day 28, the mean body weights of mice treated with SGZ002- HC-MMAE LD declined 0.2 % (0.08 g) compared to those on Day 0, the dosing start date. The mean body weights of the other treatment groups increased by 0.58 % - 8 72 % (0.05 g - 1 .59 g) on day 28 compared to day 0 (Figure 11) There was no drug related death observed in the present study (Figure 11).

[0217] 3.4 In vitro cytotoxicity of PAL-conjugated anti-HER2 MMAE ADCs against HER2 (3+) human breast cancer cells.

[0218] ADCs (listed in Table 1), derived from eitherthe trastuzumab or disitamab backbone with LC or HC sequence modifications (Figure 1), were conjugated with MMAE-containing branched or non-branched payloads (Figure 4) and tested against two breast cancer cell lines, SKBR3 and BT-474, expressing HER2 at high levels (HER2 “3+”) to assess their in vitro cytotoxicity (Table 2; Fig. 12). After five days of ADC treatment, cell viability was measured using a CellTiter-Glo® assay (Promega, Wl) Highly potent IC50 values ranging from 45 to 91 pM were observed (Table 2). Notably, in both cancer cell lines, SGZ- 022 and SGZ-220, which have lower DAR values of 1 .9 and 1 .8, displayed comparable IC50 values of 68-91 and 69-71 pM, respectively, and were similar to SGZ-024 and SGZ-230, that bear higher DAR values of 3 4 and 3 2, giving IC50 values of 46-61 and 45-58 pM, respectively In addition, these four ADCs obtained using PAL technology are as active as marketed anti-HER2 ADCs: Kadcyla (IC50 values of 119 pM and 132 pM) with a DAR value of 3.4, and Aidixi (IC50= 39-41 pM) with a DAR value of 4 (Table 2). Moreover, all four MMAE ADCs were highly inactive in the HER2-negative cell line, MCF7 (Table 2).

[0219] Table 2. In vitro cytotoxicity of PAL-mediated trastuzumab and disitamab ADCs conjugated with MMAE or DXd, Aidixi and Enhertu.

[0220] PAL-conjugated ADCs also inhibited growth of T226, a trastuzumab- and Kadcyla-resistant, HER2 (3+) human breast cancer PDX culture, with IC50s ranging from 400-610 pM (Table 2; Figure 12).

[0221] Remarkably, SGZ022 and SGZ220 (IC50= 480 pM for both), displayed comparable levels of inhibition as SGZ-024 and SGZ230 (IC50s = 610 and 400 pM, respectively), as well as Aidixi (IC50= 340 pM)

[0222] Activities of SGZ022 and SGZ024 against T226 were reduced by 5 to 13-fold, compared to the SKBR3 and BT-474 Likewise, SGZ220 and SGZ230 were 6-9 fold less active, whilst Aidixi was 8-9-fold less active in T226 as compared to inhibition of SKBR3 and BT-474

[0223] 3.5 In vitro cytotoxicity of PAL-conjugated anti-HER2 MMAE ADCs against Trastuzumab-resistant HER2 (2+) human breast cancer cells.

[0224] Next, PAL-mediated MMAE conjugated ADCs were profiled against the trastuzumab- and Kadcyla- resistant, intermediate HER2 (2+) breast cancer cell line, JIMT-122(Figure 12C) and a trastuzumab- and pertuzumab-resistant, intermediate HER2 (2+) breast cancer PDX culture, HBCx-1 (Table 2; Figure 12E). After five days of treatment, observations in the JIMT-1 contrasted with the observations in HER2 (3+) cell lines: SGZ022 and SGZ220, with lower DARs, displayed IC50 of 61.7 and 45.6 nM, respectively, similar to Kadcyla (IC50= 54 8 nM) These ADCs are 34-80 fold less active compared to SGZ024 and SGZ230, their counterparts with higher DARs, which displayed significantly better IC50 of 1 35 and 0.78 nM respectively

[0225] Moreover, when tested against HER2 (2+) PDX culture, HBCx-1 , inhibitory activities of all PAL- conjugated ADCs were weak with IC50 values ranging from 22 to 82 nM (Table 2; Figure 12), irrespective of their DAR values. Unexpectedly, in vitro inhibitory activities of Aidixi against JIMT-1 and HBCx-1 were 3-6-fold betterthan SGZ230, with observed IC50 values of 0.12 and 6 6 nM respectively.

[0226] 3.6 In vitro cytotoxicity of PAL-conjugated anti-HER2 DXd ADCs against HER2 (3+) human breast cancer cells.

[0227] Trastuzumab-DXd ADCs (SGZ025 and SGZ026) conjugated by PAL are mostly less active than their counterpart ADCs conjugated with MMAE in the high HER2 (3+) breast cancer cell lines, SKBR3 and BT- 474 (Table 2; Figure 12). SGZ025 (DAR 1.98), is inactive in SKBR3 and BT-474 (IC50 > 300 nM), unlike the picomolar IC50 values obtained with SGZ022. In comparison, inhibitory activity of SGZ026 (DAR 3.87), is highly potent in SKBR3 and not active in BT-474 (IC50= 157 pM vs >300 nM, respectively). This contrasts with its trastuzumab-MMAE ADC counterpart, SGZ024 (DAR 3.4) which was equally potent in both cell lines (IC50s= 46 & 61 pM) In vitro cytotoxicity of Enhertu, (DAR 8) was about 2-fold more active against SKBR3 than SGZ026 (DAR 3.87) and but inactive in BT-474 (IC50= 68 pM and >300 nM, respectively). All three DXd ADCs were highly inactive in the HER2-negative cell line, MCF7 (Table 2)

[0228] Rather unexpectedly, in vitro cytotoxicities of PAL-mediated trastuzumab-DXd ADCs (SGZ025 and SGZ026) against T226, the trastuzumab- and Kadcyla-resistant, HER2 (3+) human breast cancer PDX culture, were comparable to its trastuzumab-MMAE ADC counterparts (Table 1) SGZ025 (IC50= 1 ,120 pM) was about 2-fold less active than SGZ022 whilst SGZ026 (IC50= 520 pM) was as active as SGZ230. As seen in SKBR3 and BT-474 cells, Enhertu exhibited similar inhibition levels (IC50= 310 nM) as SGZ026 against T226, indicating that its higher DAR did not translate to better in vitro killing.

[0229] 3.7 In vitro cytotoxicity of PAL-conjugated anti-HER2 DXd ADCs against trastuzumab-resistant HER2 (2+) human breast cancer cells.

[0230] SGZ025 and SGZ026, that were conjugated with a DXd payload using PAL technology, were completely inactive in the HER2 (2+) breast cancer cell line, JIMT-1 and PDX culture, HBCx-1 (Table 2; Figure 12). At the top ADC concentrations tested in both cell types, 300 and 50 nM for JIMT-1 and HBCx-1 , respectively, no inhibition was observed. Moreover, Enhertu, despite having a 2-fold higher DAR than SGZ-026, also failed to inhibit the growth of JIMT-1 and HBCx-1 (Table 2). These data differ from the observations for ADC conjugated with MMAE (SGZ022 and SGZ024) Conjugation with higher MMAE DAR in SGZ024 had led to improved JIMT-1 cytotoxicity as compared to conjugation with lower MMAE DAR in SGZ022 In the case of PDX model, HBCx-1 , both SGZ022 and SGZ024 had displayed similarly weak inhibition (IC50 = 40-60 nM). Example 4: In vivo studies

[0231] 4.1 1n vivo anti-tumor activities of PAL-conjugated ADCs in trastuzumab- and Kadcyla-resistant, HER2 (3+) human breast cancer PDX model, T226.

[0232] In vitro cytotoxicity assays revealed similar inhibition of T226 by trastuzumab and disitamab ADCs conjugated with MMAE or DXd via PAL technology, comparable to the marketed ADCs Aidixi and Enhertu (Table 2 and Figure 12). To assess their ability to inhibit T226 tumor growth in vivo, tumor fragments were transplanted subcutaneously into athymic nude mice on Day 0 When the tumors reached 60-256 mm3(Day 1), groups of 8-9 mice received a single i.v dose of trastuztumab-DXd ADC, SGZ025 (DAR 1 .98), SGZ026 (DAR 3.87) conjugated via PAL technology or Enhertu (DAR 8) at 1 , 3 or 7.5 mg / kg. Control mice received PBS Mice were weighed and tumor sizes were measured by caliper once or twice weekly for 5 weeks post-ADC administration (Figure 13). Following tumor measurements on week 4, the vehicle control mice were euthanized for ethical reasons.

[0233] As expected, in vivo T226 growth inhibition correlated with the administered ADC dose, with single 7.5 mg / kg showing the strongest effects. In mice treated with SGZ026 (DAR 3 87) or Enhertu (DAR 8), tumor growth was completely suppressed two weeks post-treatment, with no regrowth observed during an additional three weeks of monitoring (Figure 13A). In contrast, T226 tumors were not fully eliminated in mice treated with 7.5 mg / kg of SGZ025 (DAR 1.98), as tumor regrowth occurred after two weeks (Figure 13A). Maximum tumor inhibition was observed on day 10, with SGZ025, SGZ026, and Enhertu achieving TGIs of 109 9% (p < 0 001), 124.4% (p < 0.001), and 116.1% (p < 0 001), respectively, compared to the vehicle control group (Table 3A).

[0234] In the 3 mg / kg dose group, T226 tumors were suppressed for two weeks post-dosing with SGZ026 and Enhertu, but recurred one week later (Figure 13B). This indicated that 3 mg / kg was insufficient for sustained tumor suppression, unlike the 7.5 mg / kg dose. Mice treated with SGZ025 showed only partial tumor inhibition (Figure 13B). Maximum inhibition occurred on day 10, with SGZ025, SGZ026, and Enhertu achieving TGIs of 69.2% (p < 0.01), 119.2% (p < 0 001), and 118 5% (p < 0.001), respectively (Table 3A)

[0235] The 1 mg / kg dose was the least effective Whilst Enhertu initially inhibited growth of T226, steady tumor regrowth occurred after week 3 (Figure 13C). Mice treated with SGZ025 or SGZ026 only showed partial tumor inhibition (Figure 13C). Maximum inhibition was again seen on day 10, with TGIs of 20.2% (p >0 05), 59.7% (p <0.01), and 100% (p <0.001) for SGZ025, SGZ026, and Enhertu, respectively (Table 3A)

[0236] Body weights of the mice treated with PAL-conjugated trastuzumab-DXd ADCs and Enhertu increased to similar levels as the control vehicle group over the 6 weeks’ study (<10 %) Overall, this study with T226, a HER2 (3+) breast cancer PDX model, indicated that a 7 5 mg / kg of Enhertu, was required to completely abolish tumor growth in vivo and that SGZ026 conjugated with half the DAR value via PAL technology, demonstrated comparable efficacy.

[0237] 4.2 In vivo anti-tumor activities of PAL-conjugated ADCs in trastuzumab- and pertuzumab-resistant, HER2 (2+) human breast cancer PDX model, HBCx-1. In vitro cytotoxicity assays showed weaker growth inhibition of HBCx-1 by trastuzumab and disitamab ADCs conjugated with MMAE via PAL technology compared to Aidixi, and no inhibition by trastuzumab- ADCs conjugated with DXd (Table 2 and Figure 12). To further evaluate these ADCs, their ability to inhibit in vivo HBCx-1 tumor growth were assessed. Tumor fragments of HBCx-1 were transplanted subcutaneously into athymic nude mice on Day 0 and groups of 8-9 mice were administrated with a single i.v dose of each ADC at 10 mg / kg or vehicle control (PBS) when the tumors reached 60-256 mm3in size (Day 1). Tumor regression was monitored by weighing mice and measuring tumors with calipers initially twice weekly, and subsequently once weekly after Day 14. Mice were monitored for a total of 6 weeks post-ADC administration (Figure 13)

[0238] All ADCs led to significant inhibition of HBCx-1 tumor growth in vivo Ten days after ADC treatment, trastuzumab ADCs conjugated with MMAE via PAL technology, with a DAR value of 1 9 (SGZ-022) or 3.4 (SGZ-024), significantly induced HBCx-1 tumor regression, achieving 119 9% TGI (p < 0.05) and 132.4% TGI (p < 0.01), respectively, compared to the vehicle control group (Figure 13C and Table 3B) Nevertheless, tumor re-growth was observed from day 21 and increased over the following weeks. Likewise, treatment with SGZ-230, the disitamab-MMAE ADC conjugated via PAL technology with a DAR value of 3.2, and Aidixi (DAR 4) strongly blocked tumor growth by day 10. Aidixi showed slightly better anti-tumor activity than SGZ-230, achieving 170.6 % TGI (p <0.001) compared to 153 2 % TGI (p <0 001), compared to the vehicle control group (Figure 13D and Table 3B). Growth of tumor also recurred at day 21 and progressed with time in mice treated with these ADCs Body weights of the mice treated with PAL-conjugated MMAE ADCs and Aidixi increased to similar levels as the control vehicle group overthe 6 weeks’ study (<10 %) In summary, the in vivo mouse anti-tumor effects of these ADCs on HBCx-1 corroborated their relative in vitro cytotoxic potencies with: Aid ixi> SGZ-230> SGZ-024> SGZ- 022. Nevertheless, the % TGI difference between SGZ-230 and Aidixi are not statistically significant (Table 3D) Thus, Aidixi’s 3-fold better in vitro IC50 than SGZ-230 did not translate into better in vivo efficacy.

[0239] Table 3A. T226 PDX tumor growth inhibition (%TGI) in mice treated with ADCs compared to vehicle control group.

[0240]

[0241] Table 3B. HBCx-1 PDX tumor growth inhibition (%TGI) in mice treated with ADCs compared to vehicle control group. done.

[0242] Table 3D. Statistical differences in HBCx-1 tumor growth inhibition in mice treated with ADCs or vehicle control, PBS (* = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001); ns= not statistically significant.

[0243] Remarkably, treatment with SGZ026 (DAR 3.87) and with Enhertu (DAR 8) completely abrogated tumor growth at 10 days post-treatment with 205 % TGI (p <0.001) and 212.4 % TGI (p <0 001) respectively, compared to the vehicle control group (Figure 13C and Table 3A) Body weights of the mice treated with SGZ026 and Enhertu increased to similar levels as the control vehicle group over the 6 weeks' study (about 10 %) No tumor re-growth was observed in mice up to 6 weeks post-treatment with either ADC Such dramatic in vivo tumor killing are at odds with their lack of in vitro cytotoxicity against HBCx-1 and demonstrate that ADCs bearing DXd payloads are more efficacious in v / vo than those conjugated with MMAE payloads. More importantly, the experiment also revealed that SGZ026, that was obtained using PAL-technology, displays the same capacity to eradicate the tumor as Enhertu, despite having only half the DAR value. 4.2 In vivo mouse pharmacokinetics studies.

[0244] To ascertain if the difference in in vivo anti-tumor efficacies noted above between ADCs directed against HER2 bearing either a MMAE or DXd payload was due to their pharmacokinetics properties, a single i.v. dose of 3 mg / kg of PAL-conjugated ADCs, Aidixi or Enhertu were injected into separate groups of male BALB / c mice (n=3). Blood was collected from the mice at 1 , 2, 4 and 8 h, then 1 , 3, 7, 14, and 21 days post-ADC administration and plasma levels oftotal Ab or ADC in the samples were determined by ELISA. For total antibody measurement, recombinant His-tagged human ErbB2 / HER2 protein was used as capture antigen, followed by binding with biotinylated goat anti-human IgG Fc-HRP. For measurements of the amount of ADC conjugated with MMAE or DXd, an antibody directed against MMAE or DXd was used for ADC capture, followed by binding of recombinant human ErbB2 / HER2 protein and addition of anti-His tag-HRP.

[0245] It was observed that mouse anti-ADC antibodies were generated (due to ADA effects) on day 7 post-ADC injection (data not shown). Thus, plasma levels of the administered ADCs were analyzed from blood samples collected from 0 to72 hours. By comparing the mouse plasma concentrations of the intact ADC (that detects all conjugated species) and Total antibody (that detects all unconjugated, partially and fully conjugated antibodies), loss of drug-linker can be detected and ADC stability in mouse plasma monitored (Figure 14). Concentration-time curves reveal that plasma levels of intact SGZ-230 ADC are almost identical to its Total antibody levels (Figure 14A) This is also confirmed by the comparable half-lives and AUC values of intact SGZ-230 ADC and its Total antibody: 3.13 vs 2 14 day and 6,004 vs 5,836 pg*h / mL, respectively (Table 4A) This result clearly indicates that PAL-mediated conjugation of MMAE at the light chain C-terminus of Disitamab is highly stable. Stability of SGZ-230 is moderately improved over Aidixi as seen in the latter’s ADC / Total antibody concentration-time curves (Figure 14B) and is also supported by the shorter half-life and lower AUC of intact Aidixi ADC compared to its Total antibody: 2.54 vs 3 6 day and 5,393 vs 6,823 pg*h / mL respectively (Table 4A). Overall, half-life of intact Aidixi ADC is 30 % lower than its Total antibody and its AUC value is 21 % less than its Total antibody. Notably, half-life and AUC of intact SGZ-230 ADC are respectively 46 % and 3 % higher than its Total Antibody Better in vivo stability of SGZ-230 is likely the reason for its good in vivo anti-tumor effects when compared to Aidixi (Figure 14B).

[0246]

[0247] Table 4A. In vivo mouse PK parameters of Singzyme MMAE-ADCs & Disitamab-

[0248] Unlike SGZ-230, plasma stabilities of SGZ-022 and SGZ-024 resemble Aidixi, with greater variance between the plasma concentrations of their intact ADCs and Total antibodies in the concentration-time curves (Figures 14C and 14D) as well as their in vivo PK parameters (Table 4A). The half-lives of intact SGZ-022 and SGZ-024 ADCs are respectively 49 % and 21 % lower than their Total antibodies (3.78 vs 7.4 days and 1 97 vs 2.49 days, respectively), whilst the AUC values of their intact ADCs are 28 % and 23 % lowerthan their respective Total antibodies: 5,963 vs 8253.28 pg*h / mL and 4453.8 vs 5759 6 p,g*h / mL, respectively Overall, the in vivo mouse PK profiles of these two PAL-conjugated trastuzumab MMAE-ADCs are in line with their relatively lower % TGI against the PDX model HBCx-1 .

[0249] SGZ-026 is highly stable in mouse plasma, as the concentration-time curve of its intact ADC is almost identical to that of its total antibody (Figure 14E). Both the half-life and AUC of the intact SGZ-026 ADC are higherthan its Total antibody value:7.9 vs 4.9 days and 8,780 vs 7,627 p,g*h / mL (Table 4B)

[0250] Conversely, intact Enhertu ADC is slightly less stable than its Total antibody (Figure 14F), with the halflife and AUC of the former being lowerthan those of its Total antibody value by 6.4 % and 36 %, respectively (5.9 vs 6.3 days and 13,626 vs 20, 919 pg*h / mL (Table 4B). Despite the AUC value of intact SGZ-026 ADC being 36 % lower than that of intact Enhertu ADC, SGZ-026 performed as well as Enhertu in eradicating the in vivo growth of the HER2 (2+) breast cancer PDX model, HBCx-1.

[0251] Table 4B. In vivo mouse PK parameters of Singzyme T-DXd-ADC (SGZ-026) & Enhertu. Calculated from Total Antibody / ADC concentrations in mouse plasma at Days 0-3 post-ADC injection. Single i.v. 3mg / kg dose, 8 mice / group, BALB / c mice; T= trastuzumab.

[0252] Whereas the DAR values and in vivo mouse PK parameters of SGZ-026 are not very dissimilar from SGZ-024 and SGZ-230, it demonstrated superior in vivo anti-tumor effects in HBCx-1 , compared to the two latter ADCs It is possible that the different linker and payload in SGZ-026 (GGFG and DXd) compared to SGZ-024 and SGZ-230 (EVCit and MMAE) are the reasons for the striking difference in their in vivo anti-tumor properties In addition, the work described here highlights the disconnect between in vitro cytotoxic and in vivo anti-tumor effects of ADCS bearing the DXd payload directed against HER2 (2+) cells. This phenomenon is likely related to the slower cell killing action of topoisomerase inhibitors compared to microtubule inhibitors.

[0253] References

[0254] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains Full citations for these references are provided below. The entirety of each of these references is incorporated herein

[0255] 1 . Younes A et al (2010) Brentuximab (SGN-35) for relapsed CD30-positive lymphomas New England

[0256] J Med. 363(19), 1812-1821

[0257] 2. Von Minckwitz G et al (2019) Trastuzumab emtansine for residual invasive HER2-positive breast cancer New England J Med. 380(7), pp. 617-628

[0258] 3. Lambert, J.M., Chari, R.V J. (2014) Ado-trastuzumab emtansine (T-DM1): An antibody-drug conjugate (ADC) for HER2-positive breast cancer Journal of Medicinal Chemistry 57(16), pp. 6949- 6964 4. Carter P.J. & Lazar G A. (2018) Next generation antibody drugs: pursuit of the “high hanging fruit” Nat. Rev. Drug. Discov. 17, 197-223

[0259] 5. Chary R.V J., Miller M.L., Widdison W.C (2014) Antibody-drug conjugates: an emerging concept in cancertherapy. Angew Chem Int Ed 53, 3796-3827

[0260] 6. De Goeij B E.C G & Lambert J. (2016) New developments for antibody-drug conjugate-based therapeutic approaches Antibody-drug conjugates. Current Opinion in Immunology 40, 14-23

[0261] 7. Ducry L. & Stump B. (2010) Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chem 21 , 5-13

[0262] 8. Beck, A., et al (2017) Strategies and challenges forthe next generation of antibody-drug conjugates Nature Reviews Drug Discovery 16(5), pp. 315-337

[0263] 9. Sochaj A M , Swiderska K W., Otlewski J. (2015) Current methods forthe synthesis of homogeneous antibody-drug conjugates. Biotechnology advances 33, 775-784

[0264] 10. Kim CH, Axup JY, Schultz PG (2013) Protein conjugation with genetically encoded unnatural aminoacids. Curr Opin. Chem. Biol. 17, 412-419

[0265] 11. Axup JY et al (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. PNAS 109, 16101-16106

[0266] 12. Zhu Z et al (2014) Site specific antibody-drug conjugation through an engineered glycosyltransferase and a chemically reactive sugar. Mabs 6, 1190-1200

[0267] 13. Strop P (2014) Versatility of microbial transglutaminase Bioconjug Chem 25, 855-862

[0268] 14. Nguyen, G.K T., Wang, S , Qiu, Y., Hemu, X., Lian, Y , and Tam, J.P. (2014). Butelase 1 is an Asx- specific ligase enabling peptide macrocyclization and synthesis. Nature Chemical Biology 10: 732- 738.

[0269] 15. Hemu X., El Sahili A., Hu S., Wong K , Zhang X., Serra A , Wong Y.H., Chen Y., Goh B.C., Darwis D A., Chen M.W , Sze S.K., Liu C-F, Wu B., Lescar J, Tam J.P (2019) Structural determinants for peptide bond formation by asparaginyl ligases Proc. Natl. Acad. Sci. USA 116(24) :11737-11746. doi: 10.1073 / pnas.1818568116

[0270] 16. Yang, R., Wong, Y H., Nguyen, G.K T., Tam, J.P., Lescar, J., and Wu, B (2017). Engineering a Catalytically Efficient Recombinant Protein Ligase Journal of the American Chemical Society 139: 5351-5358.

[0271] 17. Hu S, El Sahili A, Kishore S, Wong YH, Hemu X, Goh BC, Wang Z, Tam JP, Liu C-F & Lescar J (2022) Structural basis for proenzyme maturation, substrate recognition and ligation by a hyperactive peptide asparaginyl ligase Plant Cell 34(12):4936-4949. doi: 10.1093 / plcell / koac281

[0272] 18. Xia, Y, Li, F, Zhang, X, Balamkundu, S, Tang, Z, Hu, S, Lescar, J, Tam J P, Liu, C-F (2023) A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation J Am Chem Soc. 143(23)

[0273] 19. Deeks, E.D (2021) Disitamab : First Approval Drugs (2021) 81 :1929-1935

[0274] 20. Hiromi Okamoto, Masataka Oitate, Katsunobu Hagihara, Hideyuki Shiozawa, Yoshitake Furuta, Yusuke Ogitani and Hiroshi Kuga (2020) Pharmacokinetics of trastuzumab (T-DXd), a novel anti- HER2 antibody-drug conjugate, in HER2-positive tumour-bearing mice XENOBIOTICA 50 (10), 1242-1250 21. Dorywalska M et al. (2016) Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site- Specific ADCs and Its Mitigation by Linker Design Mol Cancer Ther 15 (5): 958-970.

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Claims

Claims1 . An antibody payload conjugate comprising an antibody and a payload, the antibody payload conjugate comprising an antibody with a light chain and a heavy chain, wherein at least one of the light chain or the heavy chain is conjugated to the payload by a tripeptide PAL conjugation motif, the tripeptide PAL conjugation motif having the sequence P1-PTP2” wherein P1 is Asn or Asp, P1” is any amino acid and P2” is a hydrophobic amino acid or a p-branched amino acid.

2. The antibody payload conjugate according to claim 1 wherein the payload is conjugated to the light chain of the antibody.

3. The antibody payload conjugate according to claim 2 further comprising a spacer between the light chain and the tripeptide PAL conjugation motif.

4. The antibody payload conjugate according to claim 3 wherein the spacer consists of at least 2 amino acid residues.

5. The antibody payload conjugate according to claim 4 wherein the spacer consists of at least 5 amino acid residues.

6. The antibody payload conjugate according to claim 4 or claim 5 wherein the spacer consists of 2 to 20 amino acid residues, preferably 5 to 10 amino acid residues.

7. The antibody payload conjugate according to any one of claims 4 to 6 wherein the spacer comprises one or more Glycine residues8. The antibody payload conjugate according to claim 7 wherein the spacer comprises Gly-Ser, Gly-Gly- Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

9. The antibody payload conjugate according to any one of claims 1 to 5 wherein the tripeptide PAL conjugation motif has the sequence Asn-Gly-lle (NGI)10. The antibody payload conjugate according to any one of claims 2 to 9 wherein the payload is conjugated at the C terminus of the light chain.

11. The antibody payload conjugate according to any one of the preceding claims wherein payload further comprises a linker, the linker conjugated to the antibody light chain via the tripeptide PAL conjugation motif12. The antibody payload conjugate according to any one of the preceding claims wherein the payload is a cytotoxic payload, a non-cytotoxic immune modulator or a detectable label.

13. An antibody light chain or antibody heavy chain comprising a P1-P1 -P2’ tripeptide PAL motif, whereinP1 is Asn or Asp, P1 ’ is Gin or Glu and P2' is a hydrophobic amino acid or a -branched amino acid.

14. The antibody light chain or antibody heavy chain according to claim 13 wherein the PAL recognition motif is at the C-terminus of the antibody light chain.

15. The antibody light chain or antibody heavy chain according to claim 13 or claim 14 wherein the PAL recognition motif comprises or consists of the amino acid sequence Asn-GIn-Leu (NQL).

16. The antibody light chain or antibody heavy chain according to any one of claims 13 to 15 further comprising a spacer, wherein preferably the spacer is between the antibody light chain and the PAL recognition motif17. The antibody light chain or antibody heavy chain according to claim 16 wherein the spacer consists of at least 2 amino acid residues.

18. The antibody light chain or antibody heavy chain according to claim 17 wherein the spacer consists of at least 5 amino acid residues.

19. The antibody light chain or antibody heavy chain according to claim 17 or claim 18 wherein the spacer consists of 2 to 20 amino acid residues, preferably 5 to 10 amino acid residues20. The antibody light chain or antibody heavy chain according to any one of claims 17 to 19 wherein the spacer comprises one or more Glycine residues.

21. The antibody light chain or antibody heavy chain according to claim 20 wherein the spacer comprises Gly-Ser, Gly-Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.

22. Nucleic acid encoding an antibody light chain or an antibody heavy chain according to any one of claims 13 to 21.

23. A payload for conjugation to an antibody comprising a PAL recognition motif and optionally a linker, the linker arranged between the PAL recognition motif and the payload.

24. A method for preparing an antibody payload conjugate, the method comprising: providing an antibody comprising an antibody light chain and an antibody heavy chain, wherein at least one of the light chain or the heavy chain includes a tripeptide PAL motif; contacting the antibody with a payload, wherein the payload has a PAL recognition motif; incubating the antibody and the payload in the presence of a peptide asparaginyl ligase (PAL) and glutaminyl cyclase (QC);wherein the PAL ligates the tripeptide PAL motif to the PAL recognition motif, thereby generating an antibody payload conjugate wherein the antibody is conjugated to the payload via a PAL conjugation motif 25. The method according to claim 24 comprising a spacer between the antibody light chain or antibody heavy chain and the tripeptide PAL motif.

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