The invention relates to amino acid derivatives for use in bioconjugation processes.

I NTRODUCTION

Pyrrolysine is a natural amino acid, the only one that is authentically specified by an amber codon. It uses a 21st aminoacyl-tRNA synthetase (PylRS), naturally evolved to be orthogonal to all other amino acids and tRNAs. Blight et AL, 2004 showed that PylRS and its counterpart tRNA (tRNApyl) can incorporate pyrrolysine at amber codons in E. coli. They also showed that the wt PylRS is naturally promiscuous and can incorporate analogs of lysine.

Yokoyama et al (EP1911840) demonstrated that the PylRS/tRNApyl system is orthogonal in eukaryotic cells and showed the incorporation of several non natural amino acids (nnAAs) into a target proteins encoded by amber codons in bacterial cells. These authors also identified key amino acid residues in pylRS that form the amino acid binding pocket and function in selecting pyrrolysine over other canonical amino acids. M utations at this site generated mutants able the recognize and aminoacylate the tRNApyl with AzZ-lys (Yanagisawa 2008).

This orthogonality extends to bacteria and eukaryotic cells.

PylRS is a naturally promiscuous synthetase that has naturally evolved to exclude lysine, but will incorporate lysine analogs without mutation, including azides, alkynes and alkenes, (Yanagisawa et a I, 2008; Neumann et al. 2008; Mukai et al., 2008; Nguyen et al., 2009). The basis of this specificity is dependent on hydrophobic interactions between amino acid residues of the pylRS binding pocket with the pyrrole ring of pyrrolysine that stabilizes and correctly positions the amino acid in the active site of the synthetase (Kavran et al., 2007). This RS/tRNA pair has been introduced via transient transfection into bacterial, yeast and mammalian cells and shown to be effective for incorporation of a number of non-natural amino acids into target proteins.

For instance, EP 1911840 demonstrates incorporation of Ν-ε-boc-Lysine into a target protein in E. coli cells.

Pyrrolysine analogs, defined as amino acid derivatives recognized by either native or genetically evolved PylRS and incorporated into proteins at a mber codon sites, have been disclosed in the past few years and reviewed, for instance, by Feckner et. al (Fekner, Li, & Chan, 2010) and Liu et al. Analogs bearing functional groups or post translational modifications have been site- specifically incorporated into proteins using pylRS-tRNApyl systems. Several studies, see e.g.

Yanagisawa et al , focused on mutations within the PylRS enzyme in order to accommodate analogs in which the N6 substituent were an aromatic ring within the binding pocket pyrrolysine. Others, for instance Nguyen et al (also in WO2010/139948) , and Li et al (also in WO2011/044255) focused on identification of pyrrolysine analogs which do not carry a bulky N6 substituent, with the result of obtaining simpler analogs which would be simple to synthesize and interact with native pylRS/tRNApyl pairs. Furthermore, Chin et al developed two analogs with terminal alkyne and azide groups, amenable to use for protein labeling via copper catalyzed click chemistry (CUAAC).

There remains a need to develop further pyrrolysine analogs. Whilst pyrrolysine analogs made thus far have been restricted to those evolved from a lysine backbone, the present inventors have generated pyrrolysine analogs successfully incorporated into proteins with native pylRS/tRNApyl pairs starting from a variety of amino acid structures.

SUMMARY OF THE INVENTION :

According to the invention there are provided pyrrolysine analogues of formulae V and VII as described herein.

There is also provided a mutant protein containing as non-natural amino acid one or more (e.g. one) pyrrolysine analogues of formulae V and VII as described herein.

There is also provided an antibody containing as non-natural amino acid one or more (e.g. one) pyrrolysine analogues of formulae V and VII as described herein in each heavy and/or light chain.

There is also provided a mutant protein or antibody as aforesaid which is conjugated via the one or more (e.g. one) non-natural amino acids to one or more (e.g. one) moieties selected from proteins, cytotoxic agents, drugs and polymers.

There is also provided use of a pyrrolysine analogue as aforesaid in the manufacture of a mutant protein e.g. antibody containing one or more non-natural amino acids.

Amino acid analogs described in the present invention are new and useful and have the merit of being straightforward to prepare, in being readily incorporated into proteins (typically without loss of bioactivity when used appropriately) and in providing useful means for bioconjugation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. PEGylation of azide containing monoclonal antibodies. Lane 1: Untreated Antibody, Lane 2: Antibody with pyrrolysine analog Formula V.l incorporated into heavy chain and subjected to PEGylation conditions; Lane 3: Antibody with pyrrolysine analog Formula VI.1 incorporated into heavy chain and subjected to PEGylation conditions.

Figure 2. HIC chromatogram of a 4D5-Auristatin F antibody drug conjugate with the antibody originally containing the pyrrolysine analog Formula VI.1, incorporated into the heavy chain.

BRIEF DESCRIPTION OF THE SEQUENCES OF THE SEQUENCE LISTING

SEQ ID No l:PylRS Methanosarcina mazei WT nucleotide sequence

SEQ ID No 2: PylRS Methanosarcina mazei W\ amino acid sequence SEQ ID No 3: PylRS Methanosarcina mazei, Y384F mutant nucleotide sequence

SEQ ID No 4: PylRS Methanosarcina mazei, Y384F mutant amino acid sequence

SEQ. ID No 5: tRNApyl Methanosarcina mazei

SEQ. ID No 6: U6 snRNA Promoter

SEQ ID No 7: U6-tRNApyl construct

SEQ ID No 8: GFP nucleotide sequence

SEQ ID No 9: GFP amino acid sequence

SEQ ID No 10: GFPY40 nucleotide sequence

SEQ ID No 11: GFPY40 amino acid sequence

SEQ ID No 12: anti-Her2 (4D5) gamma nucleotide sequence

SEQ ID No 13: anti-Her2 (4D5) gamma amino acid sequence

SEQ ID No 14: anti-Her2 (4D5) gamma_K274amber nucleotide sequence

SEQ ID No 15: anti-Her2 (4D5) gamma_K274amber amino acid sequence

SEQ ID No 16: anti-Her2 (4D5)Kappa nucleotide sequence

SEQ ID No 17: anti-Her2 (4D5)Kappa amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "amide" refers to a -C(=0)-NH- linkage.

The term "carbamate" refers to a -0-C(=0)-NH- linkage.

The term "ester" refers to a -C-C(=0)-0-C linkage

The term "alkyl" refers to an aliphatic linkage or substituent, typically containing 1-6 e.g. 1-4 carbon atoms and can be straight chain or branched. Examples include methyl, ethyl, n-propyl, i- propyl, n-butyl and t-butyl.

The term "alkoxy" refers to the group -O-alkyl.

The term "alkenyl", "alkene" or "olefin" refers to an aliphatic linkage or substituent, typically containing 2-6 e.g. 2-4 carbon atoms and can be straight chain or branched and which is unsaturated in respect of containing at least one C=C moiety. Examples include ethenyl, propen- 1-yl, propen-2-yl, and 2-methyl-propen-2-yl. An alkenyl group may be optionally substituted e.g. by one or more (e.g. 1) substituents such as halogen (e.g. CI) or an ether group (e.g. -0-Ci_ ₆ alkyl) although suitably it is not substituted. The term "alkynyl" or "alkyne" refers to an aliphatic linkage or substituent, typically containing 2-6 e.g. 2-4 carbon atoms and can be straight chain or branched and which is unsaturated in respect of containing at least one C≡C moiety. Examples include -C≡CH and -C≡C-CH ₃ . An alkynyl group may be optionally substituted e.g. by one or more (e.g. 1) substituents such as halogen (e.g. CI) or an ether group (e.g. -0-Ci_ ₆ alkyl) although suitably it is not substituted.

The term "cycloalkyi" refers to a n alicyclic and unsaturated compound typically containing 3 to 8 cyclic carbon atoms. Cycloalkyi groups may containing branching. The total number of carbon atoms will typically be 3 to 10. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, 3- methyl-cyclopropyl and cyclohexyl.

The term "cycloalkenyl" refers to an alicyclic compound typically containing 5 to 8 cyclic carbon atoms and containing at last one C=C moiety. Cycloalkenyl groups may containing branching. The total number of carbon atoms will typically be 5 to 10. Exemplary groups include cyclopentenyl, 3-methyl-cyclopropenyl and cyclohexenyl.

The term "heterocyclyl" refers to a cycloalkyi or cycloalkenyl moiety in which the ring contains one or more (e.g. one, two or three, such as one or two, especially one) heteroatom selected from O, N and S. Examples include azetidine, pyrrolidine, piperidine, piperazine, N-methylpiperazine, morpholine and thiomorpholine.

The term "aryl" refers to an aromatic ring structure that can be part of a linkage or part of a substituent. Aryl moieties may contain one ring (e.g. phenyl) or two rings (e.g. naphthyl). Aryl groups may be substituted e.g. by one or more (e.g. one or two, such as one) substituent selected from alkyl, alkenyl, alkynyl, fluoroalkyl, halogen, alkoxy, nitro and cyano. An exemplary aryl is phenyl.

The term "heteroaryl" refers to a heteroaromatic ring structure that can be part of a linkage or part of a substituent. The heteroaromatic ring may contain 1-4 (more usually 1-3 e.g. one or two) heteroatoms selected from O, N and S. Heteroaryl moieties may contain one ring or two rings.

Example groups containing one 6 membered ring include pyridine and pyrimidine. Example groups containing one 5 membered ring include pyrrole, furan, thiophene, oxazole, thiazole, diazole, thiadiazole and tetrazole. Heteroaryl moieties that contain two rings may contain heteroatoms in one or both rings. Examples include quinoline and isoquinoline. Heteroaryl groups may be substituted e.g. by one or more (e.g. one or two, such as one) substituent selected from alkyl, alkenyl, alkynyl, fluoroalkyl, halogen, alkoxy, nitro and cyano.

The term "aromatic halide" refers to an aromatic ring (typically phenyl) which is substituted by at least one (e.g. one) halo group such as fluorine, chloride, bromide or iodine. Said aromatic ring may contain further substituents e.g. those mentioned for a ryl.

The term "azide" and "azido" refers to a N=N(+)=N(-) functional group. The term "cycloalkyne" refers to a cyclic arrangement of carbon atoms (typically 6-9 membered, especially 8-9 membered) which includes a carbon-carbon triple bond captured in the ring structure. Examples include cyclooctyne and cyclononyne. A further example is benzyne.

Cycloalkyne groups may containing branching. The total number of carbon atoms will typically be 6 to 12 e.g. 6 to 10.

The term "ketone" refers to a C-C(=0)-C linkage.

The term "pyrrolysine analog" means an amino acid derivative recognized by either native or genetically evolved PylRS and incorporated into proteins at an amber codon site.

The expression "the side chain of one of the 20 natural amino acids" refers to the group R in the formula HOOC-CHR-NH ₂ relating to the 20 natural amino acids known by their single letter codes A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. Either L or S stereochemistry (or a mixture thereof) is intended, although L stereochemistry is preferred.

The present invention discloses pyrrolysine analogs.

Some pyrrolysine analogs of the resent invention have the structure of Formula V:

Formula V

wherein

Z = bond, CH ₂ , CH-NH ₂ , CH-OH, NH, O, S or CH-NH ₂ ;

b is 0 or an integer 1-7; and

FG = azide, alkene, alkyne, ketone, ester, aryl or cycloalkyne.

In formulae V when FG represents aryl, an example is aromatic halide e.g. 4-halo phenyl such as 4- iodo phenyl.

Moiety Z(CH ₂ ) _b FG may, for example, represent CO-aryl e.g. CO-phenyl or -COalkyl e.g. - COMe. Exemplary compounds of formula V are the following:

(2S)-2-amino-6-{[(2-azidoethoxy)carbonyl]amino}hexanoic acid

Formula V.l;

(2S)-2-amino-6-{[(prop-2-yn-l-yloxy)carbonyl]amino}hexanoic acid

Formula V.2;

(2S)-2-amino-6-{[(prop-2-en-l-yloxy)carbonyl]amino}hexanoic acid

Formula V.3;

(2S)-2-amino-6-(3-azidopropanamido)hexanoic acid

Formula V.4;

(2S)-2-amino-6-(pent-4-enamido)hexanoic acid

Formula V.5. -2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid

(S)-2-amino-6((2-oxo-2-propanamide)hexanoic acid HO

NH ₂ C

Formula V.7; and

(2S)-2-amino-6-(2-azidoacetamido)hexanoic acid

Formula V.8

Alternative pyrrolysine analogs suitable for use as non natural amino acids in the present invention have the structure of Formula VI:

Formula VI

wherein

Z = CH ₂ , CH-NH ₂ , CH-OH, NH, 0 or S;

FG = azide, alkene, alkyne, ketone, ester, aryl or cycloalkyne; and

b = an integer 1-4.

In formulae VI when FG represents aryl, an example is aromatic halide e.g.4-halo phenyl such as 4-iodo phenyl.

Suitbly Z represents NH. In another embodiment it represents CH.

b may, for example, represent 1 or 2.

FG may, for example, represent -N ₃ , -CH=CH ₂ , -C≡CH, -COCH ₃ , COOCH ₃ , phenyl substituted by halogen or cyclooctyne.

Exemplary compounds of Formula VI are:

(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoic acid Formula VI.1;

(2S)-2-amino-6-{[(prop-2-yn-l-yl)carbamoyl]oxy}hexanoic acid

Formula VI.2; and

(2S)-2-amino-6-{[(prop-2-en-l-yl)carbamoyl]oxy}hexanoic acid

Formula VI.3

In structures of formulae V and VI, when FG represents alkene, it suitably represents -CH=CH ₂ or - CH=CH-CH ₃ , preferably -CH=CH ₂ .

In structures of formulae V and VI, when FG represents alkyne, it suitably represents -C≡CH or - C≡C-CH ₃ , preferably -C≡CH.

In structures of formulae V and VI, when FG represents ketone, it suitably represents -C(=0)-CH ₃ or -C(=0)-CH ₂ -CH ₃ , preferably -C(=0)-CH ₃ .

In structures of formula VI, when FG represents ketone it may, for example, represent -C(=0)-aryl e.g. -C(=0)-phenyl.

In structures of formulae V and VI, when FG represents ester, it suitably represents -C(=0)-Oalkyl e.g. -C(=0)-Omethyl.

In structures of formulae V and VI, when FG represents aromatic halide, it suitably represents phenyl substituted by halogen, especially iodine (e.g. 4-iodo-phenyl).

In structures of formulae V and VI, when FG represents cycloalkyne, it suitably represents cyclooctyne, e.g. cyclooct-4,5-yne.

Advantageously, the nnAAs of formulas V and VI of the present invention have been shown to have good incorporation as demonstrated by GFP assay. Formula VI.1 had a similar level of translational compentency to Formula V.l in the GFP assay incorporation assay. Both the Formula V and VI are easily modified to incorporate a variety of useful functional groups which can be used for site selective post translational modification. Alkynes and alkenes are readily incorporated. The pyrrolysine analogs disclosed herein can be made using various methods. The reaction conditions can generally be determined by one of the ordinary skill in the art.

Formula V analogs are readily prepared by the addition of an activated carbonyl group, such as a chloroformate, activated carboxylic acid ester, isocyanate, activated carbonate or sulfonyl halide to a mono-protected diamino substrate of type 1, in which the a-amino group is protected by a protecting group ("PG") such as a Boc, Cbz, TFA, Acetyl or Fmoc group (see Scheme 1). The coupled product 3 can undergo further modifications, such as the displacement of halides with an azido nucleophile to install the desired functionality. Otherwise, the intermediate 3 is deprotected to remove the a-amino acid masking group to afford the desired Formula V analog. Scheme 1:

. or ac va e car oxy ac

1) Additional modificiations if necessary

Formula VI analogs were prepared by conjugation of hydroxyl amino acids 9 to substrates with activated carbonyls such as carboxylic acid ester, isocyanate, acid chlorides, activated carbonates or sulfonyl halides. The coupled product 11 can undergo further modifications, such as the installation of the azide functional group by displacement of leaving groups such as halides or activated alcohols. The desired amino acid analog 12 is obtained by final deprotection to remove the a-amino acid masking group. Protecting groups may be used as per Scheme 1. See Scheme 2

incorporation of non-natural amino acid into proteins

The pyrrolysine analogs disclosed herein can be incorporated into recombinant proteins. In particular, site specific incorporation of the analog into a recombinant protein can be achieved through amber suppression, wherein a nonsense (amber) codon is inserted within the nucleotide sequence encoding the recombinant protein, at a site where the pyrrolysine analog is to be inserted. The mutated nucleotide sequence, along with one or more plasmids encoding the PylRS and tRNApyl are inserted into a cell of a cell free expression system.

The host cell may be a eukaryotic cell line which is transformed with a vector comprising a DNA construct as aforesaid.

Alternatively, a cell-free expression system is provided, wherein a synthesis reaction lysate obtained from a host cell comprises at least one component required for the synthesis of polypeptides. The synthesis reaction lysate is obtained from bacterial or eukaryotic cells.

Preferably, the synthesis reaction lysate is obtained from eukaryotic cells, more preferably, from rabbit reticulocytes or wheat germ.

Preferably, the cell-free expression system is capable of expressing WT PylRS and tRNApyl of the present invention, wherein tRNApyl is introduced into the cells used to obtain the synthesis reaction lysate with DNA constructs of the invention.

Cell-free expression systems suitable for use in the present invention are described for instance in WO201008110, WO2010081111, WO2010083148, incorporated in their entirety herein by reference.

When the pyrrolysine analog is added to the cell or expression system, said analog is incorporated in the recombinant protein at the specified position. The nnAA and the tRNApyl are bound by the pylRS and the tRNApyl is subsequently aminoacylated with the nnAA. This tRNApyl containing an amber anticodon is released into the cytosol where in response to an amber stop codon can interact with the ribosome and the nnAA released to form a peptide bond with the growing polypeptide chain.

Recombinant proteins modified to incorporate a pyrroiysine analog of the invention include all recombinant proteins amenable to site specific post translational modifications, e.g. therapeutic proteins, for instance cytokines, antibodies and antibody derivatives (such as Fab fragments, or single chain antibodies, e.g. single chain variable fragments (scfvs)), peptides, enzymes, fusion proteins, decoy receptors, protein vaccines, protein hormones, e.g. insulin, growth factors,( e.g. human growth hormone, hGH, hGCSF, hFSH, hHCG). Further proteins modifiable with pyrroiysine analogs of the invention include diagnostic labels, imaging reagents.

Suitably, proteins may be modified site specifically to incorporate one or more than one nnAA (pyrroiysine analog) of the invention. For instance, an antibody may incorporate a nnAA of the invention at the heavy chain, or at the light chain, or at both light and heavy chain.

Site specific conjugation of proteins with incorporated non-natural amino acids

Proteins having incorporated pyrroiysine analogs of the present invention may be used for the preparation of functionalized protein conjugates. Molecules that may be conjugated to proteins having incorporated non-natural amino acids include (i) other proteins, e.g. antibodies especially monoclonal antibodies; (ii) polymers e.g. PEG groups or other groups that may cause half life extension in the system; (iv) cytotoxic agents e.g. Auristatin F; and (v) drug moieties e.g.

doxorubicin and moieties containing radioactive isotopes. Moreover these modified proteins can be conjugated to drugs or nucleotides for targeted delivery of these potent compounds.

More details of certain embodiments are given below in the discussion of antibody drug conjugates.

Pyrroiysine analogs may conveniently contain a unique chemical group permitting conjugation in a targeted fashion without risk of side reaction with other amino acids. For example non-natural amino acids may contain azide or alkyne groups permitting reaction with a molecule to be conjugated which contains a corresponding alkyne or azide group using the Huisgen 1,3-dipolar cycloaddition reaction.

Preferred conjugation chemistries of the invention include reactions which are orthogonal to the natural twenty amino acids. Such reactions do not interact or cause side reactions with the native 20 amino acids, they are specific to the functional groups associated with the reaction. Suitably the necessary functional groups are incorporated into the target protein via the pyrroiysine analogs of the present invention. Further, said reactions proceed under conditions which are not destructive to the protein, for instance aqueous solvents, with a pH range which is acceptable to the protein and maintains its solubility, at a temperature which does not lead to deleterious effects upon the protein.

Increasing the stability of the attachment moiety between the protein and the linker can be advantageous. Conventional methods conjugate to the thiol groups of cysteine by reaction with a maleimide forming a thiol ether. The thiol ether can undergo the reverse reaction releasing the linker drug derivative from the antibody. In an embodiment of the invention, the conjugation chemistry employed between an azide and an alkyne results in an aromatic triazole which is significantly more stable, and not as prone to reversibility.

In addition, the product of the reaction, the linkage between protein and payload, ought to be stable, equal to or greater than the stability associated with conventional linkages (amide, thiol ether). Though not an impediment to conjugation, it is often advantageous if the conjugation reactions can be done under native conditions, as this will eliminate an extra refolding processing step.

Preferred chemical conjugations for production of conjugates of the invention include : a 3+2 alkyne-azide cycloaddition, 3+2 dipolar cycloaddition, Husigen 3+2 cycloaddition, Copper promoted azide-alkyne cycloaddition (CuAAC), Ruthenium promoted azide alkyne cycloaddition (RAAC), metal promoted azide alkyne cycloaddition (MAAC), and strain promoted azide alkyne cycloaddition (SPAAC), palladium based couplings including the Heck reaction, Sonogashira reaction Suzuki reaction Sti lie coupling Hiyama/Denmark reaction olefin metathesis Diels-alder reaction carbonyl condensation with hydrazine, hydrazide, alkoxy amine or hydroxyl amine; strain promoted cycloadditions with nitriles and nitrile oxides,; electron promoted cycloaddition;

fragment extrusion cycloaddition; alkene cycloaddition followed by a β-elimination reaction.

According to one preferred embodiment, the incorporated amino acid contains an azide or an alkyne group and the process of chemical modification comprises reacting said azide or alkyne group with a reagent comprising an alkyne or azide group. The envisaged reaction is a Huisgen 1,3-dipolar cycloaddition reaction which leads to production of a triazole linkage. The reagent comprising an alkyne or azide group may be a protein (e.g. an antibody) or a cytotoxic agent or a drug or a substance suitable for half life extension (e.g. a PEG group) which carries an alkyne or azide group optionally via a linker.

Optionally, the Huisgen 1,3-dipolar cycloaddition reaction can be performed in the presence of Cu(l) catalysis.

Preferably, copper catalyzed cycloaddition reactions are carried at room temperature, in acqueous solution in presence of cysteine and tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine (TBTA). Alternatively, the copper catalyzed cycloaddition reactions are carried out from 4°C to 50°C in aqueous solution in the presence of sodium ascorbate and tris(3- hydroxypropyltriazolylmethyl)amine (THPTA). The reactions can also be carried out in mixed aqueous/organic solution with the organic component consisting of DMSO, DMF, methanol, ethanol, i-butanol, trifluoroethanol, propylene glycol, ethylene glycol and hexylene glycol.

In a variant reaction, the incorporated amino acid contains an azide or an alkene group and the process of chemical modification comprises reacting said azide or alkene group with a reagent comprising an alkene or azide group. The reagent comprising an alkene or azide group may be a protein (eg an antibody) or a toxin or a substance suitable for half life extension (eg a PEG group) which carries an alkyne or alkene group optionally via a linker.

The site specific conjugations between the incorporated nnAA and the target payioad can be done with fully folded proteins such as antibodies, antibody fragments, and cytokines. Alternatively, the conjugation can be done on denatured proteins in the presence of denaturants such as sodium dodecylsulfate and urea. The copper catalyzed azide alkyne addition can be done in the presence of denaturants and reducing agents such as dithiothreitol and 2-mercaptoethanol.

When more than one nnAA is incorporated into a target protein (e.g. an antibody), the chemical modification may be the same or different. For example if two nnAAs are incorporated, one may be modified to be conjugated to a drug moiety and one may be modified to be conjugated to a PEG moiety.

Conveniently, upon incorporation of more than one nnAA of the invention bearing different but complementary reactive groups, said nnAAs can react with each other to generate an

intramolecular link.

In an embodiment, conjugation chemistry of the invention is used for preparing an antibody drug conjugate. The conjugation chemistry may also be used to assemble antibody-protein conjugates, protein protein conjugates such as bispecifics composed of antibody fragments. The conjugation chemistry may also be used to conjugate polymer bond drug conjugates to targeting agents such antibodies and antibody fragments. The conjugation chemistry can also be used to attach polymers such as PEG to proteins to manipulate pharmacokinetic properties.

PEG moieties

Target proteins may be conjugated to PEG moieties. PEG moieties may be incorporated into antibody drug conjugates. The PEG moiety may typically have a molecular weight ranging between 0.5 kDa and 40 kDa e.g. 5kDa and 40 kDa. More preferably, the PEG moiety may have a molecular weight of around 20 kDa. In addition, the PEG moieties can have a molecular weight range from 100 - 2000 Da. PEG moieties may be straight chain or branched or multi armed

The PEG moieties can be functionalized with terminal alkynes, azides, cyanides, cycloalkynes, alkenes, aryl halides. The PEG can be functionalized in such as way as to be monofunctional, homobifunctional, heterobifunctional, and multi-homofunctional. Antibody Drug Conjugates (ADCs)

Pyrrolysine analogs according to the invention are particularly useful for production of Antibody Drug Conjugates (recombinant antibody covalently bound by a synthetic linker to a given drug, typically a cytotoxic drug, or else a protein or a PEG group) which are homogeneous nature, in which the number of drugs (or other conjugated molecule) per antibody and position of those drugs upon the antibody are explicitly controlled, whereby monoclonal antibodies containing incorporated non-natural amino acids are obtained and site specifically conjugated to a linker carrying a drug moiety (or other conjugated molecule) through orthogonal chemistry.

ADCs obtained with pyrrolysine analogs of the present invention may be manufactured following methods including the following steps:

1. Introducing into a stable cell line of the invention one or more plasmids carrying the DNA sequence coding for a full length antibody, whereby a stop codon is introduced at specific positions within the sequence

2. Purify the modified antibody with the pyrrolysine analog (nnAA) installed at desired position(s).

3. React a cytotoxin-linker derivative modified to include a functional group complimentary to the nnAA installed in the antibody with the modified antibody containing a complementary reactive group through an orthogonal chemistry

4. Purify the resulting ADC

Thus, the present invention also provides ADCs whereby the antibody component has been modified to incorporate non natural amino acids bearing a unique reactive functional group at desired positions, whereby such functional group allows conjugation to a drug moiety (or protein or PEG group).

In an embodiment the present invention provides an antibody conjugate comprising an anti-Her-2 antibody which is conjugated to one or more moieties (e.g. one, two, three or four, preferably one or two, especially one) selected from protein, drug and PEG moieties via linkers comprising a triazole moiety.

In particular, the triazole moiety may be formed by reaction of an azide or alkyne moiety in the side chain of a non-natural amino acid incorporated into the sequence of the anti-Her-2 antibody and an alkyne or azide moiety attached to the protein, drug or PEG moiety.

In one embodiment, the triazole moiety is formed by reaction of an azide or alkyne moiety in the side chain of a non-natural amino acid incorporated into the sequence of the anti-Her-2 antibody and an alkyne or azide moiety attached to the protein, drug or PEG moiety under conditions of Cu(l) catalysis. Cu(l) catalysis is accomplished by using either a native Cu(l) source such as copper iodide, copper bromide, copper chloride, copper thiolate, copper cyanide. The Cu(l) species can also be generated in situ by using a copper (II) source and a reducing agent. The copper (II) source can be copper sulfate, copper (II) chloride, or copper acetate. The reducing agent can be sodium ascorbate, dithiothreitol, TCEP, b-mercaptoethanol, hydrazine, hydroxylamine, sodium bisulfite, cystamine and cysteine.

Suitably, Cu(l) catalyzed cycloaddition are carried out in presence of ligands to stabilize the Cu(l) species present at the start of the reaction or generated in situ by reduction of a Cu(ll) source such as sodium sulfate with sodium ascorbate, including TBTA, THPTA, phenanthroline derivatives, pyridylmethanimine derivatives, diethylenetriamine, bipyridine derivatives, TMEDA, N,N-bis(2- pyridylmethyl)amine (BPMA) derivatives,N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediami ne (TPEN) derivatives, trialkylamines such as triethylamine, diisopropyl ethylamine, HEPES and MES.

In one embodiment a copper azide alkyne cycloaddition is used for the conjugation. Suitably, the reaction utilizes a a cytotoxic agent such as auristatin, amanitin, taxol or doxorubicin bearing a terminal alkyne. Further, the reaction employs a copper source such as copper sulfate, copper acetate, copper iodide or copper bromide; a reducing agents such as sodium ascorbate, hydrazine, hydroxylamine, sodium bisulfite, dithiothreitol, cysteine, b-mercaptoethanol;a copper chelating ligand such as Tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine (TBTA) or Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA). Suitably, the reaction may be performed at 4 - 50°C. Suitably, the reaction time ranges from 0.5 to 48 hrs. In an alternative embodiment, a strain promoted azide alkyne cycloaddition is used for conjugation. Suitably, the reaction utilizes a dye, a PEG polymer, or cytotoxic agent such as auristatin bearing a cycloocytne group. Suitably, the reaction is allowed to incubate at room temperature for 0.5 - 48h.

Drug moieties

Drug moieties of the present invention, such as cytotoxin drug moieties, include small molecules, natural products, synthetically derived drugs, proteins such as immunotoxins, and radionuclides.

In an embodiment, the drug moietyis an auristatin moiety eg auristatin or a derivative thereof such as monomethyl auristatin E (MMAE)(Vedotin) or monomethyl auristatin F (MMAF),

Auristatin F (AF), amanitin, Paclitaxel and doxorubicin.

Other drug moieties include maytansine, paclitaxel, doxorubicin and immunotoxins such as exotoxin or bouganin as well as radionuclides such as lodine-131, Yttrium-90,Samarium-135, and Strontium-89 which may also be incorporated into organic molecules. (see for instance: MMAE: Senter, PE, et. a I, BLOOD, 102, 1458-1465. MMAF : Senter, PE, et. al., Bioconj. Chem. 2006, 17, 114-124. Maytansine : Lewis-Phillips GD, Cancer Res., 63, 9280-9290, 2008. Bouganin :MacDonald GC, et. al, J. lmmunotherapy,32 574-84, 2009. Most suitably the drug moiety is a moiety selected from a doxorubicin, paclitaxel and auristatin moiety.

Salts

Pyrrolysine analogues described herein may optionally be employed in the form of a salt. Any such salts form an aspect of the invention. Salts of carboxylic acids may include salts formed with Group 1 and Group 2 metals, especially soluble salts such as sodium and potassium salts. Salts of amines may include salts formed with weak and strong acids, such as HCI, HBr or acetic acid.

EXAMPLES

Example 1. Preparation of Formula V and VI analogs

Preparation of (2S)-2-amino-6-(pent-4-enamido)hexanoic acid (Formula V.5)

In a 25mL roundbottomed flask was placed N-Boc-Lysine (500mg, 2.0mmol) suspended in dioxane (lOmL). 1M K ₂ C0 ₃ was added (5mL) and the solution was cooled to OC. 4-pentenoyl chloride (224uL, 2.0mmol) in dioxane (2mL) was added slowly. The solution was allowed to stir at OC for lh and then at room temperature for 4h. The solution was transferred to a extraction funnel and partitioned between water and ether. The organic layer was removed and the aqueous layer made acidic (pH=2) with citric acid. The aqueous layer was extracted with ethyl acetate (3x50mL), the organic layers combined and dried over sodium sulfate, filtered and concentrated. The resulting residue was carried forward into the next step.

The crude N-Boc-s-N-4-pentenoyl amide-lysine was placed in a 50mL round bottomed flask with acetonitrile (5mL) and TFA (2mL) and magnetically stirred for 2h. The mixture was concentrated. The solution was treated with toluene (lOmL) and concentrated (2X) and acetonitrile (lOmL) and concentrated (2X). The residue was dried overnight under vacuum. The residue was taken up in MeOH and precipitated with methyl-t-butyl ether. The viscous oil was isolated by centrifugation, the supernatant was disposed. Analytical MS : m/z (ES+) expected 229.2 (M+H)+, found 230.3. Preparation of Preparation of (S)-2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid (Formula V.6)

In a 50mL round bottomed flask with magnetic stirrer was dissolved pyruvic acid (3.5g, 23.3mmol) in a 2:1 mixture of dichloromethane and DMF (20mL). To this mixture was added DCC (5.7g, 27.6mmol) and NHS (3.2g, 27.6mmol). The mixture was heated to 50C for 30min with stirring. The solution was allowed to cool and then added through a filter to a suspension of N-Boc-Lysine (5.2g, 21.2mmol) in DMF (20mL) in a separate lOOmL round bottomed flask with magnetic stirrer. Triethyl amine (8.8mL, 63.6mmol) was added after addition of the activated ester, and the mixture was stirred overnight. The mixture was partitioned between ethyl acetate and citric acid. The layers were separated and the aqueous layer was extracted 4 times with ethyl acetate. The organic layers were combined, dried over sodium sulfate and concentrated. The resulting residue was further purified by flash chromatography to afford the final N-Boc lysine derivative as an oil. In a lOOmL roundbottomed flask was placed the keto-N-Boc lysine derivative (4g, 10.6mmol) in acetonitrile (50mL). To this was added a solution of hydrochloric acid (15mL, 4N in dioxane). The solution was stirred for 2h and concentrated. Final purification by flash chromatography afforded the target amino acid. Analytical MS : m/z (ES+) expected 278.1 (M+H)+, found 279.2.

Preparation of (2S)-2-amino-6-(2-azidoacetamido)hexanoic acid (Form

In a 25mL roundbottomed flask was placed N-Boc-Lysine (500mg, 2.0mmol) suspended in dioxane (5mL). Saturated NaHC0 ₃ was added (2mL) and the solution was cooled to 0°C. Bromoacetyl chloride (169uL, 2.0mmol) in dioxane (2mL) was added slowly. The solution was allowed to stir at 0C for lh and then at room temperature for 4h. The solution was transferred to a extraction funnel and partitioned between water and ether. The organic layer was removed and the aqueous layer made acidic (pH=2) with citric acid. The aqueous layer was extracted with ethyl acetate (3x50mL), the organic layers combined and dried over sodium sulfate, filtered and concentrated. The resulting residue was carried forward into the next step.

In a 50mL round bottomed flask was placed the crude N-Boc-£-2-bromoacetyl-lysine (740mg, 2.0mmol) in dioxane (lOmL). To this was added a solution of sodium azide (lOmL, 1M). The solution was stirred at 60°C overnight. The mixture was partitioned between citric acid (1M, 50mL) and ethyl acetate (lOOmL). The organic layer was retained, and the aqueous layer extracted 3 additional times. The organic layers were combined, dried over sodium sulfate and concentrated to an oil.

The crude N-Boc-£-2-azido-acetyl-lysine was dissolved in acetonitrile (lOmL) and TFA (2mL) was added. The mixture was stirred for 2h and then concentrated. The solution was treated with toluene (lOmL) and concentrated (2X) and acetonitrile (lOmL) and concentrated (2X). The residue was dried overnight under vacuum. The residue was taken up in MeOH and precipitated with methyl-t-butyl ether. The viscous oil was isolated by centrifugation, the supernatant was disposed. Analytical MS : m/z (ES+) expected 229.1 (M+H)+, found 230.2.

Preparation of Hydroxy - Norleucine derivatives (Formula VI.1 and Formula VI.2) In a lOOmL roundbottomed flask with magnetic stirring was placed N-Boc-Hydroxyl Norleucine (lg, 4.1mmol) and acetonitrile (50mL). The mixture was cooled to 0°C and p- nitrophenylchloroformate (979mg, 4.9mmol) and Pyridine (2mL) was added and the mixture stirred overnight. The mixture was concentrated and purified by flash chromatography. (Silica, DCM/MeOH gradient).

H

In a lOOmL roundbottomed flask with magnetic stirring was placed 2-N-Boc-ethylbromide (lg, 4.4mmol) in 25mL of dioxane. To this was added a solution of sodium azide (1M, 22.2mmol). The solution was stirred at 60C overnight. The mixture was partitioned between water and ethyl acetate. The ethyl acetate layer was retained and the aqueous layer was extracted with ethyl acetate three additional times. The organic layers were combined, dried over sodium sulfate and concentrated to an oil.

The oil was taken up in acetonitrile (35mL) and HCL in dioxane was added (4M, lOmL). The mixture was stirred for two hours and concentrated under vacuum.

Preparation of (2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoic acid (Formula VI.1)

In a 50mL round bottomed flask was placed the N-Boc-Norleucine p-nitrophenyl carbonate (503mg, 1.2mmoL) in dioxane (lOmL). To this was added a solution of the amino-azide (105mg, 1.2mmol) in dioxane (5mL) and pyidine (lmL). The solution was stirred overnight. The mixture was partitioned between ethyl acetate and 500mM citric acid. The ethyl acetate layer was retained and the aqueous layer was extracted with ethyl acetate three additional times. The organic layers were combined, dried over sodium sulfate and concentrated to an oil. The oil was further purified by flash chromatography.

The isolated Boc-protected amino acid was taken up in acetontirile (15mL) and treated with HCI in dioxane (4M, 5mL). The mixture was stirred for two hours and concentrated under vacuum.

Alternative Preparation of (2S)-2-amino-6-[[(2-azidoethyl)carbamoyl]oxy]hexanoic acid, Formula VI.1. Step 1 : In a 4mL vial with magnetic stirrer was placed Boc-N-6-hydroxynorleucine (50mg, leq) and DMF (ImL). To this was added 2-chloroethyl isocyanate (17.3mg, l.Oeq) and pyridine (32.3uL, 2 eq). The vial was capped and allowed to stir for 5h. The solution was transferred to a extraction funnel, diluted with ethylacetate and lOOmM citric acid. The mixture shaken and the layers separated. The aqueous layer was extracted with ethyl acetate two additional times. The organic layers combined, washed with 5% lithium chloride, dried with sodium sulfate, filtered and concentrated. The product was identified by mass spectrometry and taken forward into the next step directly.

Step 2 : In a 4mL vial with magnetic stirrer was placed the crude chloro derivative from above and DMSO (ImL). Sodium azide (130mg, 5eq) and pyridine (32.3uL, 2 eq) were added to the mixture and the vial was capped. The mixture was stirred overnight at 60°C. The mixture was transferred to an extraction funnel and diluted with lOOmM citric acid and ethyl acetate. The mixture was shaken and the layers separated. The aqueous layer was extracted with ethyl acetate two additional times. The organic layers combined, washed with 5% lithium chloride, dried with sodium sulfate, filtered and concentrated. The product was carried on to the next step.

Final Step : In a 20mL vial was placed the crude Boc protected amino acid and acetonitrile (2mL). To this was added a solution of hydrochloric acid in dioxane (4N, 2.5mL). The solution was stirred for 2h and then concentrated under reduced pressure. The mixture was lyophilized to a semi solid and used in translational testing. Analytical MS : m/z (ES+) expected 259.1 (M+H)+, found 260.2. Preparation of (2S)-2-amino-6-{[(prop-2-yn-l-yl)carbamoyl]oxy}hexanoic acid (Formula VI.2)

In a 50mL round bottomed flask was placed the N-Boc-Norleucine p-nitrophenyl carbonate (337mg, 0.8mmoL) in dioxane (lOmL). To this was added a solution of the amino-azide (135mg, 2.4mmol) in dioxane (5mL). The solution was stirred overnight. The mixture was partitioned between ethyl acetate and 500mM citric acid. The ethyl acetate layer was retained and the aqueous layer was extracted with ethyl acetate three additional times. The organic layers were combined, dried over sodium sulfate and concentrated to an oil. The oil was further purified by flash chromatography.

The isolated Boc-protected amino acid was taken up in acetontirile (15mL) and treated with HCI in dioxane (4M, 5mL). The mixture was stirred for two hours and concentrated under vacuum Preparation of (2S)-2-amino-6-{[(prop-2-en-l-yl)carbamoyl]amino}hexanoic acid, Formula VI.3.

In a 4mL vial with magnetic stirrer was placed Boc-N-6-hydroxynorleucine (50mg, leq) and DMF (1.5mL). To this was added allyl isocyanate (18.0uL, l.Oeq) and pyridine (32.3uL, 2 eq). The vial was capped and allowed to stir for 4h. The solution was transferred to an extraction funnel, diluted with ethylacetate and lOOmM citric acid. The mixture shaken and the layers separated. The aqueous layer was extracted with ethyl acetate two additional times. The organic layers were combined, washed with 5% lithium chloride, dried with sodium sulfate, filtered and concentrated. The product was identified by mass spectrometry and taken forward into the next step directly. In a 20mL vial was placed the crude hydroxyl leucine - allyl carbamate derivative in acetonitrile (2mL). To this was added a solution of hydrochloric acid in dioxane (4N, 2.5mL). The solution was stirred for 2h and then concentrated under reduced pressure. The mixture was lyophilized to a semi solid and used in translational testing. The product was confirmed by mass spectrometry. Additional purification could be done with ion exchange chromatography (DOWEX-50). Analytical MS : m/z (ES+) expected 230.1 (M+H)+, found 231.2.

Example 2. Translational testing of novel pyrrolysine analogs as nnAAs with a GFP assay.

An in vitro cell based assay was developed to assess the compatibility of the pylRS/tRNA pair and the the pyrrolysine analogs of the present invention (nnAAs) by and the efficiency of nnAAs integration into a target protein. For this, HEK293 cells stably expressing pylRS (3H7) were transiently transfected with plasmids for the expression of tRNApyl and a reporter construct encoding GFPY40 (containing amber codon in place of tyrosine at amino acid residue number 40 (where 1 is the initiator methionine)) using standard transfection protocols. Transfected cells were incubated with nnAAs at 2mM for 2-3 days GFP production was analyzed qualitatively by visual inspection under the microcope. The GFP fluorescence was quantified by flow cytometry using an Accuri flow cytometer and the geometric mean of the fluorescent cells determined.

This cell based assay was used to determine whether the different nnAAs were suitable substrates for the pylRS and allowed its translation into a target protein. Cells expressing the PylRS/tRNApyl pair and containing a vector encoding the GFPY40 reporter genewere incubated in the presence of the nnAAs. nnAAs that are readily utilized by the PylRS/tRNApyl pair support the translation of the nnAA into the amber site of GFP and allow read-through of the gene producing full length GFP (fluorescent protein). The fluorescence intensity of the cells depends on the efficiency of nnAA incorporation. Thus, nnAAs that are poorly utilized produce weakly fluorescent or non-fluorescing cells. Microscopic observation identified a number of nnAAs usable by the pylRS (Table 1, Positive GFP). Furthermore, the relative expression levels in each sample was compared to those generated by substrates known to be efficiently utilized by pylRS. Formula V.l (MFI = 931,289), Formula V.2 (MFI=1,676,250) and Formula V.3 (MFI=2,250,000) (see Table 1) supported high levels of GFP expression with a geometric mean.

Analog Formulae VI.1 and VI.3 and of the present invention were found by the inventors to be incorporated in the GFP reporter gene and yield green cells under the experimental conditions used. Among these, the analog of Formula VI.1 supported high levels of GFP expression (MFI 904206) and represents an analogue that is efficiently utilized by the pylRS/tRNA pair under the experimental conditions tested (see Table 2).

Table 1 : Formula V analog GFP results

Table 2 : Formula VI analog GFP results

Construction and expression of anti-Her2 antibody

A full length anti-Her2 antibody containing two non natural amino acids (one in each heavy chain)

(4D5-2AZ ab) was expressed in mammalian cells. A nnAA, containing an azide moiety, was incorporated at the selected sites and purified by affinity chromatography using either protein A resin (GE Healthcare) or by IgSelect (GE Healthcare, 17096901). The purified material was then concentrated and subjected to a conjugation reaction.

An antibody directed to the extracellular domain of Her2/neu was generated by cloning the variable regions of both the heavy and light chains of the mouse antibody 4D5 into vectors containing genes encoding human IgG. The variable regions of 4D5 were generated by gene synthesis using overlapping oligomers and cloned into the human IgGl frameworks encoded by pFUSE-CHIg-hGl (IgGl heavy chain;gamma) and pFUSE-CHLIg-hK (light chain; kappa; Invivogen) to generate a mouse-human hybrid. Amber codons were introduced into the heavy chain (gamma) at positions K274 by site directed mutagenesis. Clones containing the amber codon were identified by DNA sequencing. To generate an integrating construct the promoters and ORF for the heavy chain was amplified by PCR and cloned by restriction enzyme digestion and ligation into pOptivec (Life Technologies). The light chain and a single copy of the tRNA were joined by two step PCR method using overlapping oligomers and cloned into available sites into the pOptivec plasmid containing the heavy chain. The construct was then transfected into a CHO cell line containing the pylRS/tRNA pair and stably transfected cell lines showing high expression of the IgG selected. This represents a second example of a cell line stably expressing a mAb containing a nnAA indicating that the process has wide applicability for the use in the expression of mAbs. This cell line was utilized to generate IgG containing the nnAAs described above. The cells were grown to a density of 1-2 x 10 ⁶ cells/mL in Excel DHFR- medium (Sigma-Aldrich) and nnAA added to culture to a final concentration of 1 mM. Cells were incubated for 5 days and IgG purified from the growth medium. Supernatants were harvested and subjected to centrifugation to collect suspended cells and other debris. The supernatant was then filtered through a 0.22um filter to remove any particulate material prior to application to a chromatography column. The filtered supernatant was applied to a lmL -5mL prepacked HiTrap protein A Sepharose at 1-5 mL/min flow rate using an AKTA chromatography system. The bound material and resin were washed with PBS to remove loosely bound proteins and the bound material eluted with lOOmM glycine (pH 3.0) at a flow rate of 1 mL/min. Peak fractions containing the target protein were neutralized with 0.1 fraction volumes of 1M Tris-HCI (pH8.0). All constructs were dialyzed to PBS at 4°C for 16 hours into the final phosphate buffer. The antibody with Formula VI.1 as nnAA incorporated into both of its heavy chains at position 274 was called "4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2- azidoethyl)carbamoyl]oxy}hexanoic acid".

PEGylation of 4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy }hexanoic acid

In a 200uL PCR tube was placed phosphate buffer (5uL, 500mM, pH=7.4). A solution of 4D5- 2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hex anoic acid (Formula VI.1). (IOUL, 0.55 mg/mL) was added followed by a solution of 20KPEG cyclooctyne (3.3, 60mg/mL). The solution was mixed vigorously on a vortexer. The mixture was allowed to stand overnight. The mixture was diluted to 200uL and applied to Protein-A magnetic beads. The mixture was vortexed and allowed to rotate to mix the beads for 90min. The beads were immobilized and the run through material disposed. The beads were washed with PBS (2X) and then suspended in reducing gel buffer. Vortexed and then heated to 95C for 3min. The suspension was loaded directly onto an SDS-PAGE gel. Commassie staining of the SDS-PAGE gel indicated the selective PEGylation of the Heavy chain (Figure 1, Lane 3).

Conjugation of 4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy }hexanoic acid with Fluoroscene dye by SPAAC.

I n a 200uL PCR tube was placed phosphate buffer (65uL, 50mM, pH=7.4). A solution of 4D5- 2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hex anoic acid (30uL, 0.55 mg/mL) was added followed by a solution DMCO-Fluor 488 cyclooctyne (5.4, 5mM in DMSO, click chemistry tools). The solution was mixed vigorously on a vortexer. The mixture was allowed to stand for 24h. The mixture was analyzed by HIC chromatography (Tosoh TSKgel Butyl NPR with a gradient of 1M Sodium sulfate to phosphate buffer) showing the conjugation had occurred and resulted in a mixture wherein conjugation had occurred at one or two sites (generally referred to as DAR1 and DAR2 species; wherein DAR is defined as drug-to-antibody ratio) (Figure 2).

SEQUENCE LISTI NG SEQ ID NO 1

PylRS Methanosarcina mazei WT nucleotide sequence

ATGGATAAAAAACCACTAAACACTCTGATATCTGCAACCGGGCTCTGGATGTCCAGGACC GGAACAATTC ATAAAATAAAACACCACGAAGTCTCTCGAAGCAAAATCTATATTGAAATGGCATGCGGAG ACCACCTTGT TGTAAACAACTCCAGGAGCAGCAGGACTGCAAGAGCGCTCAGGCACCACAAATACAGGAA GACCTGCAAA CGCTGCAGGGTTTCGGATGAGGATCTCAATAAGTTCCTCACAAAGGCAAACGAAGACCAG ACAAGCGTAA AAGTCAAGGTCGTTTCTGCCCCTACCAGAACGAAAAAGGCAATGCCAAAATCCGTTGCGA GAGCCCCGAA ACCTCTTGAGAATACAGAAGCGGCACAGGCTCAACCTTCTGGATCTAAATTTTCACCTGC GATACCGGTT TCCACCCAAGAGTCAGTTTCTGTCCCGGCATCTGTTTCAACATCAATATCAAGCATTTCT ACAGGAGCAA CTGCATCCGCACTGGTAAAAGGGAATACGAACCCCATTACATCCATGTCTGCCCCTGTTC AGGCAAGTGC CCCCGCACTTACGAAGAGCCAGACTGACAGGCTTGAAGTCCTGTTAAACCCAAAAGATGA GATTTCCCTG AATTCCGGCAAGCCTTTCAGGGAGCTTGAGTCCGAATTGCTCTCTCGCAGAAAAAAAGAC CTGCAGCAGA TCTACGCGGAAGAAAGGGAGAATTATCTGGGGAAACTCGAGCGTGAAATTACCAGGTTCT TTGTGGACAG GGGTTTTCTGGAAATAAAATCCCCGATCCTGATCCCTCTTGAGTATATCGAAAGGATGGG CATTGATAAT

GATACCGAACTTTCAAAACAGATCTTCAGGGTTGACAAGAACTTCTGCCTGAGACCC ATGCTTGCTCCAA ACCTTTACAACTACCTGCGCAAGCTTGACAGGGCCCTGCCTGATCCAATAAAAATTTTTG AAATAGGCCC ATGCTACAGAAAAGAGTCCGACGGCAAAGAACACCTCGAAGAGTTTACCATGCTGAACTT CTGCCAGATG GGATCGGGATGCACACGGGAAAATCTTGAAAGCATAATTACGGACTTCCTGAACCACCTG GGAATTGATT TCAAGATCGTAGGCGATTCCTGCATGGTCTATGGGGATACCCTTGATGTAATGCACGGAG ACCTGGAACT TTCCTCTGCAGTAGTCGGACCCATACCGCTTGACCGGGAATGGGGTATTGATAAACCCTG GATAGGGGCA GGTTTCGGGCTCGAACGCCTTCTAAAGGTTAAACACGACTTTAAAAATATCAAGAGAGCT GCAAGGTCCG AGTCTTACTATAACGGGATTTCTACCAACCTGTAA SEQ ID NO 2

PylRS, Methanosarcina mazei WT amino acid sequence

M DKKPLNTLISATGLWMSRTGTIHKI KHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCK RCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAM PKSVARAPKPLENTEAAQAQPSGSKFSPAI PV STQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEV LLNPKDEISL NSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEI KSPILI PLEYIERMGI DN DTELSKQI FRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKI FEIGPCYRKESDGKEHLEEFTM LNFCQM GSGCTRENLESI ITDFLNHLGI DFKIVGDSCMVYGDTLDVM HGDLELSSAVVGPIPLDREWGIDKPWIGA GFGLERLLKVKHDFKN IKRAARSESYYNGISTNL SEQ ID NO 3

PylRS Methanosarcina mazei Y384F mutant nucleotide sequence

ATGGATAAAAAACCACTAAACACTCTGATATCTGCAACCGGGCTCTGGATGTCCAGGACC GGAACAATTC ATAAAATAAAACACCACGAAGTCTCTCGAAGCAAAATCTATATTGAAATGGCATGCGGAG ACCACCTTGT TGTAAACAACTCCAGGAGCAGCAGGACTGCAAGAGCGCTCAGGCACCACAAATACAGGAA GACCTGCAAA CGCTGCAGGGTTTCGGATGAGGATCTCAATAAGTTCCTCACAAAGGCAAACGAAGACCAG ACAAGCGTAA AAGTCAAGGTCGTTTCTGCCCCTACCAGAACGAAAAAGGCAATGCCAAAATCCGTTGCGA GAGCCCCGAA ACCTCTTGAGAATACAGAAGCGGCACAGGCTCAACCTTCTGGATCTAAATTTTCACCTGC GATACCGGTT TCCACCCAAGAGTCAGTTTCTGTCCCGGCATCTGTTTCAACATCAATATCAAGCATTTCT ACAGGAGCAA CTGCATCCGCACTGGTAAAAGGGAATACGAACCCCATTACATCCATGTCTGCCCCTGTTC AGGCAAGTGC CCCCGCACTTACGAAGAGCCAGACTGACAGGCTTGAAGTCCTGTTAAACCCAAAAGATGA GATTTCCCTG AATTCCGGCAAGCCTTTCAGGGAGCTTGAGTCCGAATTGCTCTCTCGCAGAAAAAAAGAC CTGCAGCAGA TCTACGCGGAAGAAAGGGAGAATTATCTGGGGAAACTCGAGCGTGAAATTACCAGGTTCT TTGTGGACAG GGGTTTTCTGGAAATAAAATCCCCGATCCTGATCCCTCTTGAGTATATCGAAAGGATGGG CATTGATAAT GATACCGAACTTTCAAAACAGATCTTCAGGGTTGACAAGAACTTCTGCCTGAGACCCATG CTTGCTCCAA ACCTTTACAACTACCTGCGCAAGCTTGACAGGGCCCTGCCTGATCCAATAAAAATTTTTG AAATAGGCCC ATGCTACAGAAAAGAGTCCGACGGCAAAGAACACCTCGAAGAGTTTACCATGCTGAACTT CTGCCAGATG GGATCGGGATGCACACGGGAAAATCTTGAAAGCATAATTACGGACTTCCTGAACCACCTG GGAATTGATT TCAAGATCGTAGGCGATTCCTGCATGGTCTTTGGGGATACCCTTGATGTAATGCACGGAG ACCTGGAACT TTCCTCTGCAGTAGTCGGACCCATACCGCTTGACCGGGAATGGGGTATTGATAAACCCTG GATAGGGGCA GGTTTCGGGCTCGAACGCCTTCTAAAGGTTAAACACGACTTTAAAAATATCAAGAGAGCT GCAAGGTCCG AGTCTTACTATAACGGGATTTCTACCAACCTGTAA SEQ ID NO 4

PylRS Methanosarcina mazei Y384F mutant amino acid sequence

M DKKPLNTLISATGLWMSRTGTIHKI KHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCK RCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAM PKSVARAPKPLENTEAAQAQPSGSKFSPAI PV STQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEV LLN PKDEISL NSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEI KSPILI PLEYIERMGI DN DTELSKQI FRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKI FEIGPCYRKESDGKEHLEEFTM LNFCQM GSGCTRENLESI ITDFLN HLGI DFKIVGDSCMVFGDTLDVM HGDLELSSAVVGPIPLDREWGIDKPWIGA GFGLERLLKVKHDFKNI KRAARSESYYNGISTNL SEQ ID NO 5

tRNApyl Methanosarcina mazei Gol

GGAAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAGCCGGGTTAGATTCCCG GGGTTTCCGCC A

SEQ ID NO 6

U6 snRNA promoter

Agagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagaga gataattagaattaatttgactgtaaacacaaag atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta aaattatgttttaaaatggactatcatatgcttacc gtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc SEQ ID NO 7

U6-tRNApyl construct

Aaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatac aaggctgttagagagataattagaattaatttg actgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttggg tagtttgcagtttttaaaattatgttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttg tggaaaggacgaaacaccgaattctctagactcga gggaaacctgatcatgtagatcgaatggactctaaatccgttcagccgggttagattccc ggggtttccggacaagtgcggtttttgttt

SEQ ID NO 8

GFP nucie!otide sequence

ATGGCCTCCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT GGTGATGTTAATGGGCACAAATTTTCTGTCA GTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTA CTGGAAAACTACCTGTTCCATGGCCAACAC TTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAAC GGCATGACTTTTTCAAGAGTGCCATGCCCGAA GGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCT GAAGTCAAGTTTGAAGGTGATACCCTTGTT AATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA CTCGAGTACAACTATAACTCACACAATGTAT ACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGTCACAACA TTGAAGATGGATCCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTG G CGATG G CCCTGTCCTTTTACCAG ACAACCATTACCTGTCG ACACAATCTG CCCTTTCG AAAG ATCCC AACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACAT GGCATGGATCAGGCCAAGCCTTTGTCTCAA GAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAA GACTACAGCGTCGCCAGCGCAGCTCTCTCTA GCGACGGCCG CATCTTCACTG GTGTCAATGTATATC ATTTTACTG G G G G ACCTTGTG CAG AACTCGTG GTG CTG G G CACTG CTG CTG CTG CGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGA GCCCCTGCGGACGGTGCCGACAGGTGCTT CTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCA GTTGGGATTCGTGAATTGCTGCCCTCTGG TTATGTGTG G G AG G G CTAA

SEQ ID NO 9

GFP amino acid sequence

MASKGEELFTGVVPILVELDGDVNGH KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRH DFFKSAMPEGYV QERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGH KLEYNYNSH NVYITADKQKNGIKANFKIRH NIEDGSVQLADHYQQNTPIG DGPVLLPDNHYLSTQSALSKDPN EKRDH MVLLEFVTAAGITHGMDQAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIF TGVNVYH F TGGPCAELVVLGTAAAAAAGNLTCIVAIGN EN RGILSPCGRCRQVLLDLH PGIKAIVKDSDGQPTAVGIRELLPSGYVWEG

SEQ ID NO 10

GFPY40 nucleotide sequence

ATGGCCTCCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTA GATGGTGATGTTAATGGGCACAAATTTTCTGTCA GTGGAGAGGGTGAAGGTGATGCTACATAGGGAAAGCTTACCCTTAAATTTATTTGCACTA CTGGAAAACTACCTGTTCCATGGCCAACAC TTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAAC GGCATGACTTTTTCAAGAGTGCCATGCCCGAA GGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCT GAAGTCAAGTTTGAAGGTGATACCCTTGTT AATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA CTCGAGTACAACTATAACTCACACAATGTAT ACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGTCACAACA TTGAAGATGGATCCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTG G CGATG G CCCTGTCCTTTTACCAG ACAACCATTACCTGTCG ACACAATCTG CCCTTTCG AAAG ATCCC AACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACAT GGCATGGATCAGGCCAAGCCTTTGTCTCAA GAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAA GACTACAGCGTCGCCAGCGCAGCTCTCTCTA GCGACGGCCG CATCTTCACTG GTGTCAATGTATATC ATTTTACTG G G G G ACCTTGTG CAG AACTCGTG GTG CTG G G CACTG CTG CTG CTG CGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGA GCCCCTGCGGACGGTGCCGACAGGTGCTT CTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCA GTTGGGATTCGTGAATTGCTGCCCTCTGG TTATGTGTG G G AG G G CTAA

SEQ ID N0 11

GFPY40 amino acid sequence

MASKGEELFTGVVPILVELDGDVNGH KFSVSGEGEGDAT*GKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDH MKRH DFFKSAMPEGY VQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSH NVYITADKQKNGIKAN FKIRH NIEDGSVQLADHYQQNTPI GDG PVLLPDNHYLSTQSALSKDPN EKRDHMVLLEFVTAAGITHGMDQAKPLSQEESTLIERATATINSIPISEDYSVASAALSS DGRIFTGVNVYH FTGGPCAELVVLGTAAAAAAGN LTCIVAIGNENRGILSPCGRCRQVLLDLH PGIKAIVKDSDGQPTAVGIRELLPSGYVWEG SEQ ID NO 12

anti-Her2 (4D5) gamma nucleotide sequence

ATGGAGGCTCCCGCCCAGCTGCTCTTTCTGCTCCTTCTCTGGCTTCCCGACACAACCGGT GAGGTGCAGCTGGTGGAGTCTGGCGGTGGC TTGGTACAGCCGGGCGGGTCCCTGCG CCTCTCCTGTG CCG CTTCCG G ATTCAACATCAAAG ACACGTATATTCACTG G GTCCGTCAG G CAC CTGGCAAGGGTCTGGAGTGGGTGAGCCGCATTTATCCTACCAATGGTTACACTCGCTACG CCGACTCTGTGAAGGGCCGCTTCACCATCA GCGCCGACACGTCCAAGAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACA CCGCGGTGTATTACTGCAGCCGCTGGGGC G GTG ATG G CTTTTACG CGATG GACTACTG G G G CCAG G G CACCCTG GTCACCGTCTCG AGTG CTAG CACCAAG G G CCCATCG GTCTTCCCC CTG G CACCCTCCTCCAAG AG CACCTCTG G G G G CACAG CG G CCCTG G G CTG CCTG GTCAAG G ACTACTTCCCCG AACCG GTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAAGTTGAGC CCAAATCTTGTG ACAAAACTCACACATG CCC ACCGTG CCCAG CACCTG AACTCCTG G G G G G ACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA ACAGCACGTACCGTGTGGTCAGCGTCCT CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGA CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAATGA

SEQ ID NO 13

anti-Her2 (4D5) gamma amino acid sequence MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVSRIYPTNGYTRYADSVKGRFTISADT SKNTLYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN H KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHN HYTQKSLSLSPG K

SEQ ID NO 14

anti-Her2 (4D5) gamma_K274amber nucleotide sequence

ATGGAGGCTCCCGCCCAGCTGCTCTTTCTGCTCCTTCTCTGGCTTCCCGACACAACCGGT GAGGTGCAGCTGGTGGAGTCTGGCGGTGGC TTGGTACAGCCGGGCGG GTCCCTG CG CCTCTCCTGTG CCG CTTCCG G ATTCAACATCAAAG ACACGTATATTCACTG G GTCCGTCAG G CAC CTGGCAAGGGTCTGGAGTGGGTGAGCCGCATTTATCCTACCAATGGTTACACTCGCTACG CCGACTCTGTGAAGGGCCGCTTCACCATCA GCGCCGACACGTCCAAGAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACA CCGCGGTGTATTACTGCAGCCGCTGGGGC GGTGATGG CTTTTACG CG ATG G ACTACTG G G G CCAG G G CACCCTG GTCACCGTCTCG AGTG CTAG CACCAAG G G CCCATCG GTCTTCCCC CTG G CACCCTCCTCCAAG AG CACCTCTG G G G G CACAG CG G CCCTG G G CTG CCTG GTCAAG G ACTACTTCCCCG AACCG GTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAAGTTGAGC CCAAATCTTGTG ACAAAACTCACACATG CCC ACCGTG CCCAG CACCTG AACTCCTG G G G G G ACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCTAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGAC CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA GGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGA SEQ ID NO 15

anti-Her2 (4D5) gamma_K274amber amino acid sequence

MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVSRIYPTNGYTRYADSVKGRFTISADT SKNTLYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEV* FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH N HYTQKSLSLSPG K

SEQ ID NO 16

anti-Her2 (4D5) Kappa nucleotide sequence ATGGAGGCTCCCGCCCAGCTGCTCTTTCTGCTCCTTCTCTGGCTTCCCGACACAACCGGT GACATCCAGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTGGGAGACCGTGTCACAATCACTTGCCGTGCTAGCCAGGATGTGAATAC AGCGGTGGCCTGGTATCAGCAGAAACCTG GCAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTTTTTGTACAGCGGCGTGCCGAGCC GCTTCAGCGGCAGCCGTTCTGGTACCGATTT CACTCTCACC ATCAG CTCTCTG CAACCG G AAG ATTTTG CAACTTACTACTGTCAACAG CACTACACCACTCCTCCG ACGTTCG G CCAAG G G ACCAAGGTGGAAATCGAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCT GATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA ACGCCCTCCAATCGGGTAACTCCCAGGAGA GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAA

SEQ ID O 17

anti-Her2 (4D5) Kappa amino acid sequence

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTIS SLQPEDFATYYCQQHYTTPPTFGQGTKVEIERTVAAPSVFIFPPSDEQLKSGTASVVCLL N NFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYS LSSTLTLSKADYEKH KVYACEVTH QG LSSPVTKSFNRGEC

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All patents and patent applications referred to herein are incorporated by reference in their entirety.