Field of the Invention

The invention relates to photoactive photoreactive antibodies.

Introduction

Applications of antibodies depend upon their specific binding to antigens; binding that is mediated by interactions such as electrostatics, van der Waals, hydrophobic, and hydrogen bonding, that are susceptible to changes in microenvironment (Reverberi, R.; Reverberi, L., Factors affecting the antigen-antibody reaction. Blood Transfus. 2007, 5, 227-40), (Chmura, A. J.; Orton, M.S.; Meares, C.F. Antibodies with infinite affinity. Proc. Natl. Acad. Sci. U S A, 2001 , 98, 8480-4) and (Butlin, N.G.; Meares C.F., Antibodies with infinite affinity: origins and applications. Acc. Chem. Res., 2006, 39, 780-7). Replacing these non-covalent interactions with a covalent bond, while concurrently modulating the binding in response to an external stimulus, can further expand the applications of antibodies.

Several antibodies and antibody fragments have been previously developed for the treatment of various diseases, including cancer (Jovcevska, I; Muyldermans, S., The Therapeutic Potential of Nanobodies. BioDrugs, 2020, 34, 11-26), (Scott, A.M., Wolchok, J.D. and Old, L.J. Antibody therapy of cancer, Nat. Rev. Cancer, 2012, 12, 278-87) and (Lu, R.M., et al., Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci, 2020, 27, 1). These antibodies bind to cell surface receptors expressed at higher levels on cancer cells, addressing a major challenge of selective cell targeting in cancer therapy. Though full-length antibodies have shown promise for treatment of several cancers, limited success has been demonstrated in eliminating solid tumours. Due to their large size, full-length antibodies are unable to diffuse deep into solid tumours (Thurber, G.M.; Dane Wittrup, K., A mechanistic compartmental model for total antibody uptake in tumors. J. Theor. Biol., 2012. 314, 57-68). In addition, it has been shown that high affinity antibodies bind to the periphery of the tumour tissues forming a barrier and preventing their further penetration (Juweid, M., et al., Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier. Cancer Res., 1992. 52, 5144-53) and (Adams, G.P., et al., High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules. Cancer Res., 2001. 61 , 4750-5). Some studies in cancer patients estimate that only 0.01% of the injected antibodies accumulate per gram of solid tumour tissue Epenetos, A. A., et al., Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms. Cancer Res., 1986, 46, 3183-91). Small antibody fragments with low molecular weight have smaller Stokes radii and can thus diffuse much deeper into the tissues, presenting an excellent alternative to full-length antibodies. However, small antibody fragments have a low residence time in the body and often have a higher rate of dissociation (k _O fr) from the target compared to full length antibodies, limiting their clinical utility. To address these challenges, antibody fragments are often multimerised (van de Water, J. A., et al., Therapeutic stem cells expressing variants of EGFR-specific nanobodies have antitumor effects. Proc. Natl. Acad. Sci. U S A, 2012. 109, 16642-7) and (Roovers, R.C., et al., A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth. Int. J. Cancer, 2011. 129, 2013-24) and/or conjugated to larger proteins (Tijink, B.M., et al., Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol. Cancer Then, 2008. 7, 2288-97), which increases the size of antibody fragments; again, reducing their ability to penetrate into the tumour.

One of the methods to overcome the limitation of low residence time would be to replace the non-covalent interactions between the antibody fragment and its antigen with a covalent bond. In one such effort, an affibody that can form a covalent bond with its antigen upon irradiation with 365 nm light was developed by introducing a photocrosslinker attached via a cysteine residue in the antigen binding region of the affibody (Brasino, M., et al., Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors. J. Am. Chem. Soc., 2018. 140, 11820- 11828). Affibodies containing a latent bioreactive amino acid in its antigen binding region that forms a covalent bond with the target antigen by proximity-dependent reaction without any external impetus have also been developed (Wang, N., et al., Genetically Encoding Fluorosulfate-l-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc., 2018. 140, 4995-4999). The former had up to 100-fold lower binding affinity compared to its wild-type counterpart and thus requires using a high concentration for initial binding, while the latter could react with target antigen expressed on healthy cells causing side effects.

Though antibody-based therapeutics are more selective than several cytotoxic small molecule drugs used for the treatment of cancer, they can cause cardiac toxicity and skin reactions (Hansel, T.T., et al., The safety and side effects of monoclonal antibodies. Nat. Rev. Drug Discov., 2010. 9, 325-38). These side effects are due to the binding of antibody to its receptor antigen expressed on healthy cells. This challenge could be addressed by activating antibodyantigen binding in the tumour microenvironment. One notable example in this direction is the development of antibodies containing an inhibitory N-terminal domain that is removed by tumour-specific proteases (Desnoyers, L.R., et al., Tumor-specific activation of an EGFR- targeting probody enhances therapeutic index. Sci. Transl. Med., 2013. 5, 207ra144). However, this approach would be difficult to extend to antibody fragments whose N-terminus is not involved in binding with the antigen. We and others have also developed light-activated antibody fragments either by site-specific installation of photocaged functional groups or by introducing light-responsive proteins into antibodies (Self, C.H.; Thompson, S., Light activatable antibodies: models for remotely activatable proteins. Nat. Med., 1996. 2, 817-20), (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993), (Jedlitzke, B., et al., Photobodies: Light-Activatable Single-Domain Antibody Fragments. Angew. Chem. Int. Ed. Engl., 2020. 59, 1506-1510), and (Yu, D., et al., Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nat. Methods, 2019. 16, 1095-1100). In principle, such antibodies could be activated at the site of tumours using surgically implanted biocompatible light emitting diodes (LEDs) (Zhang, H., Rogers, J. A., Recent Advances in Flexible Inorganic Light Emitting Diodes: From Materials Design to Integrated Optoelectronic Platforms. Adv. Opt. Mater., 2019. 7,1800936-1800936), thereby reducing the side effects of antibody-based therapeutics.

A methodology that could improve the binding affinity of antibody fragments to specific tumour cells, while activating the binding at the site of tumour using one external impetus, such as light, without substantial change in the size of the antibody fragment could: 1) reduce the side effects of antibody fragments, 2) reduce the k _O ff of antibody fragments, 3) make the binding between the antibody fragment and the receptor less sensitive to the dynamic environment of tumour cells, 4) retain the high penetration of antibody fragments into tumour tissues, and 5) allow user defined control over antibody-antigen binding and its affinity.

Thus, there is a need for controlling the direct binding of an antibody to its target and catalysing the formation of a covalent bond between them, at the site of the tumour. The invention is aimed at addressing this need, in particular by engineering a photoreactive amino acid and a photocaged amino acid in the antigen binding region of an antibody fragment. The photoreactive amino acid is positioned to allow light-promoted covalent bond formation with the antigen without inhibiting binding, whereas the photocaged amino acid is positioned such that it inhibits antibody-antigen binding in absence of light and the binding is restored upon irradiation with light. Though genetic code expansion has allowed site-specific incorporation of multiple distinct noncanonical amino acids in model proteins (de la Torre, D.; Chin, J.W., Reprogramming the genetic code. Nat. Rev. Genet., 2021. 22, 169-184), site-specific incorporation of multiple noncanonical amino acids into antibody fragments remains challenging with only a few examples known (Oiler-Salvia, B.; Chin, J.W., Efficient Phage Display with Multiple Distinct Non-Canonical Amino Acids Using Orthogonal Ribosome- Mediated Genetic Code Expansion. Angew. Chem. Int. Ed. Engl., 2019. 58, 10844-10848). W02020193981 discloses antibodies with a photocaged amino acid. However, to the best of the inventor’s knowledge, there is no known example of a protein that has been conferred with concurrent photoactivity and photoreactivity.

Summary of the invention

We provide an effective control of antibody binding and the replacement of non-covalent interactions between the antibody and its antigen with a covalent bond. This has been achieved by the inclusion of a photoreactive amino acid and a photocaged amino acid in the antigen binding region of an antibody fragment. The photocaged amino acid hinders function of the antibody, that is binding of the antibody to its target antigen, until the molecule is liberated, or ‘decaged’, by exposure to light. The photoreactive amino acid catalyses the formation of a covalent bond between the antibody and target antigen upon exposure to light.

In one embodiment, incorporation of the photoreactive amino acid and a photocaged amino acid into the antibody is achieved by genetic site-specific incorporation. Thus, the primary antibody sequence is modified by direct incorporation of the photoreactive and photocaged amino acid. Peptide or chemical linkers are not used to modify the antibody. In other words, the photoreactive and photocaged amino acid are not conjugated to the antibody but are integrated by site specific modification of the antibody sequence as explained herein.

In one aspect, the invention relates to an antibody or fragment thereof wherein said antibody or fragment comprises a photocaged amino acid and a photoreactive amino acid in its antigen binding region. In one embodiment, the photocaged amino acid and photoreactive amino acid are genetically encoded. The photocaged amino acid prevents or reduces binding of the antibody to its target antigen in the absence of light. Exposure to light leads to the release of the caging group from the antibody, and the removal of the photocaging group results in binding of the antibody to its target. The photoreactive amino acid is capable of forming a covalent bond between the antibody and target antigen upon exposure to light.

The photocaged amino acid is present at a site in which the photocaged amino acid inhibits interaction with and/or binding of the antibody to its antigen in the absence of light.

The photoreactive amino acid is present at a site in which the photoreactive amino acid does not significantly inhibit or reduce interaction with and/or binding of the antibody to its antigen in the absence of light.

The photoreactive amino acid is capable of forming a covalent bond with the antigen target in the presence of light.

The photocaged amino acid may include a photoactive group selected from an o-nitrobenzyl functional group and variants thereof.

The photoreactive amino acid may be selected from p-benzoyl-L-phenylalanine, p-Azido-L- phenylalanine, trifluoromethyl phenyl diazirine, alkyl diazirine, and variants thereof.

The fragment may be selected from a F(ab')2, Fab, Fv, sFv, scFv, dAb, affibody or any part of the antibody retaining binding specificity to the target antigen.

The antibody or fragment thereof may be conjugated to another moiety.

The moiety may be selected from a half-life extension moiety, label or toxic moiety.

The moiety may be attached via an unnatural amino acid.

The antibody or fragment thereof may bind to a cell surface antigen.

The antibody or fragment thereof may bind to a tumor associated antigen, optionally selected from PSMA, Her2, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF, CECAM6, CAXII, CD24, CEA, Mesothelin, cMet, TAG72, MUC1 , MUC16, STEAP, Ephvlll, FAP, GD2, IL-13Ra2, L1-CAM, PSCA, GPC3, Her3, gpA33, CA-125, gangliosides G(D2), G(M2) and G(D3), Ep-CAM, bombesin-like peptides, PSA, HER2/neu, epidermal growth factor receptor, erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFpi , IGF-1 receptor, EGFa, c-Kit receptor, transferrin receptor, IL-2R and CO17-1A.

The antibody or fragment thereof may bind to an immune checkpoint molecule, optionally selected from LAG-3, PD-1 , PD-L1 , PD-L2, CTLA-4, TIM-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 or TGFR beta. The antibody may be 7D12.

The invention relates to a pharmaceutical composition comprising an antibody or fragment thereof and optionally a pharmaceutically acceptable carrier. the invention relates to an antibody or fragment thereof or a pharmaceutical composition according for use in the treatment of cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer.

The invention relates to an antibody or fragment thereof or a pharmaceutical composition further comprising activating the antibody or fragment thereof with a light source by irradiating a target tissue, for example at a wavelength of about 365nm.

Activating the antibody or fragment may comprise irradiating the cell, tissue, subject and/or irradiating a tumor in the subject; optionally comprising applying a suitable light source selected from the group consisting of a filtered conventional light source, a diode array and a laser.

The cancer may be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, leukemias such as ALL, CLL, AML, urothelial carcinoma leukemia and multiple myelomas.

The invention relates to a method for treating and/or preventing cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer comprising administering to a subject an antibody or fragment thereof or a pharmaceutical composition as described above wherein said antibody is conjugated to a label. The method may further comprise activating the antibody or fragment thereof with a light source by irradiating a target tissue, for example at a wavelength of about 365nm.

Activating the antibody or fragment may comprise irradiating the cell, tissue, subject and/or irradiating a tumor in the subject; optionally comprising applying a suitable light source selected from the group consisting of a filtered conventional light source, a diode array and a laser We also provide a method for diagnosing a disease comprising administering to a subject an antibody or fragment as described above or a pharmaceutical composition as described above wherein said antibody is conjugated to a label.

The invention relates to a method of reducing tumor cell growth and/or proliferation in a cell, tissue or subject comprising: a. administering a therapeutically effective amount of an antibody or fragment thereof as described above or a pharmaceutical composition as described above, wherein the antibody targets an antigen present on the surface of a tumor cell and exerts an inhibitory effect on growth and/or proliferation of the tumor cell when bound to the antigen and/or wherein the antibody that targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b. localizing the antibody or fragment to a tumor cell; c. irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and catalyse the covalent photocrosslinking between the antibody and target antigen; and d. inhibiting growth and/or proliferation of the tumor cell.

We also provide a method of killing a cell comprising: a. contacting a cell comprising a cell surface protein with a therapeutically effective amount of an antibody or fragment thereof as described above or a pharmaceutical composition as described above, wherein the antibody that targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b. irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and catalyse the covalent photocrosslinking between the antibody and target antigen; and c. killing the cell.

The invention relates to a nucleic acid comprising a nucleic acid encoding an antibody or antibody fragment as described above.

The invention relates to a vector comprising a nucleic acid as described above.

The invention relates to a host cell comprising a nucleic acid as described above or a vector as described above.

The invention relates to a method for producing an antibody or antibody fragment as described above, comprising a) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photocaged group at said position and b) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position.

The invention relates to a kit comprising the antibody or fragment thereof as described above, optionally comprising a LED wearable device or an LED implantable device.

We also provide an isolated protein comprising the SEQ ID NO. 1 or a sequence with at least 75% sequence identity thereto and comprising the following mutations compared to the wildtype Mb PyIRS: N311Q, C313T, W382A and Y349F.

The invention relates to a nucleic acid encoding the amino acid sequence as described above. The nucleic acid sequence as described above wherein said sequence comprises SEQ ID NO. 2 or a sequence with at least 75% sequence identity thereto.

The invention relates to a vector or plasmid comprising a nucleic acid as described above.

The invention relates to a host cell comprising a nucleic acid of claim as described above or a vector as described above.

The invention relates to a method for producing an antibody or antibody fragment having a photoreactive amino acid wherein the photoreactive group is p-benzoyl-L-phenylalanine comprising identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position wherein said is introduced using the isolated protein as described above.

The invention relates to a plasmid for use in the production of a photoactive photoreactive antibody or fragment thereof containing genetic components to assemble an orthogonal ribosome, an orthogonal aminoacyl-tRNA synthetase /tRNA pair, and a gene encoding an antibody or fragment thereof on an orthogonal ribosome binding site.

Brief Description of Figures

Figure 1. Site-specific incorporation of p-benzoyl-L-phenylalanine (Bpa) in a single chain antibody fragment, 7D12. (A) Crystal structure of 7D12-EGFR domain III complex (PDB ID: 4KRL). Residues Y32, Y109, and Y113 in the antigen binding pocket of 7D12 were replaced with Bpa. (B) Expression of wt-7D12 (WT) and its three mutants, viz. 32TAG, 109TAG and 113TAG without and with Bpa. Comparison of band intensities for amber mutants with wt-7D12 shows efficient incorporation of Bpa. Full-length protein expressed for negative samples (-Bpa) indicate that /MjRS(Bpa) might incorporate canonical amino acid in the absence of Bpa. (C) ESI-MS results for wt-7D12, 7D12-32Bpa, 7D12-109Bpa and 7D12-113Bpa demonstrate sitespecific incorporation of Bpa for expression of amber mutants of 7D12 with Bpa. Figure 2. Development of high affinity photoreactive 7D12 mutant. (A) On-cell binding assay to measure the binding between 7D12 and EGFR on the surface of A431 cells. The assay demonstrates that 7D12-109Bpa mutant binds to EGFR, whereas 7D12-32Bpa and 7D12- 113Bpa show near background binding. These experiments were performed in triplicates. (B) Chemiluminescence intensities obtained from on-cell binding experiments were quantified using CLARIOstar plate reader. The normalised intensities were plotted against log(concentration of 7D12). The data was fitted to sigmoidal nonlinear equation using GraphPad to obtain binding affinity values (K _D ). For wt-7D12 and 7D12-109Bpa, KD was estimated to be 27(±1.5) nM, and 48 (±7.2) nM, respectively (Supplementary Figure S4). For wt-7D12 and 7D12-109Bpa, lines show the fitting trace. For 7D12-32Bpa and 7D12-113Bpa, lines show connection between individual points. (C) Effect of incubation time prior to irradiation, and irradiation time on photocrosslinking between 7D12-109Bpa and EGFR. Band higher than EGFR indicates photocrosslinked 7D12-EGFR complex. Incubation time prior to irradiation has little effect on photocrosslinking efficiency. With increase in irradiation time, photocrosslinking efficiency increases from 17% at 5 min to 46% at 15 min irradiation. (D) Effect of relative amount of 7D12-109Bpa to sEGFR on photocrosslinking efficiency. The amount of sEGFR was fixed at 10 picomoles and amount of 7D12-109Bpa is changed from 0 to 200 picomoles. Photocrosslinking efficiency saturates above 100 picomoles of 7D12- 109Bpa. (E) Photocrosslinked product observed only with 7D12-109Bpa for samples irradiated with 365 nm light demonstrating that covalent bond formation between 7D12 and EGFR requires irradiation with 365 nm light, and Bpa at position 109 in 7D12. (F) No photocrosslinking observed between bovine serum albumin (BSA) and Bpa-containing 7D12 mutants, demonstrating that photocrosslinking requires specific binding between 7D12 and EGFR. (G) Photocrosslinking of 7D12-109Bpa to sEGFR performed in DMEM media containing 10% serum. Left panel shows Coomassie stained gel demonstrating successful photocrosslinking of 7D12-109Bpa to sEGFR in the control reaction performed in phosphate buffered saline (PBS). For same reaction performed in serum-containing media, bands corresponding to sEGFR and photocrosslinked product are not clear on Coomassie stained gel due to the presence serum proteins. The right panel shows anti-Hise antibody western blot of the photocrosslinking reactions that detects the C-terminal Hise tag on 7D12-109Bpa. The bands show sEGFR-7D12-109Bpa complex demonstrating successful photocrosslinking in serum containing media. Figure 3. Effect of 365 nm irradiation on the viability of A431 cells assessed using alamarBlue cell viability assay. Six replicates of each experiment were performed. The results demonstrate that greater than 90% of the cells are viable upon 10 min irradiation with 365 nm light.

Figure 4. Liquid chromatography tandem mass spectrometry (MS/MS) analysis of the photocrosslinked 7D12-109Bpa-sEGFR complex (SEQ ID NO. 5) demonstrates the presence of 7D12 in the crosslinked product. The 7D12 sequence is covered by 14 unique peptides (73% of the sequence) highlighted in grey. Location of 109Bpa is denoted as B. Representative MS/MS spectra of detected peptides matching the 7D12 sequence are also shown. Assigned b- and y-ions are annotated in red and blue, respectively. Ions belonging to other series, e.g. a-ions, are shown in green. Data was visualised using Scaffold 5.

Figure 5. Liquid chromatography tandem mass spectrometry (MS/MS) analysis of the photocrosslinked 7D12-109Bpa-sEGFR complex (SEQ ID NO. 6) demonstrates presence of EGFR in the crosslinked product. Protein sequence of EGFR showing detected peptides (49 unique peptides, covering 62% of the sequence; highlighted in grey). Representative MS/MS spectra of detected peptides matching the EGFR sequence are also shown. Assigned b- and y-ions are annotated in red and blue, respectively. Ions belonging to other series, e.g. a-ions, are shown in green. Data was visualised using Scaffold 5.

Figure 6. Assessing the selectivity of MjRS(pcY)/MjtRNACUA, and MjRS(Bpa)/MjtRNACUA at site-specific incorporation of pcY and Bpa at position 109 in 7D12. For MjRS(pcY)/MjtRNACUA, band corresponding to full-length 7D12 is only observed when the expression is performed in the presence of pcY and no full-length protein is observed when expression is performed with Bpa, demonstrating that MjRS(pcY) is specific for pcY. For MjRS(Bpa)/MjtRNACUA, band corresponding to full 7D12 is observed when the expression is performed in the presence of pcY or Bpa or without any non-canonical amino acid, demonstrating that MjRS(Bpa) is promiscuous. These gel images are obtained after Coomassie staining.

Figure 7. Development of efficient and selective M. barkeri Pyrrolysyl-tRNA synthetase (MbPyIRS)/ tRNA pair for site-specific incorporation of p-benzoyl-L-phenylalanine (Bpa). (A) Crystal structure of PylRS with adenylated pyrrolysine (PDB ID: 2Q7H). Residues N311 , C313, Y349 and W382 in the pyrrolysine binding pocket of MbPyIRS were mutated to all combinations of amino acids for directed evolution experiments. (B) To isolate Bpa-specific mutants of MbPyIRS, three rounds of directed evolution were performed and subsequently, 192 clones were screened. Two clones, A2 and E10 survived on chloramphenicol concentration up to 300 pg/ml in the presence of 1 mM Bpa (A2 +Bpa and E10 +Bpa), but did not survive in the absence of Bpa on chloramphenicol concentration at and above 100 pg/ml (A2 -Bpa and E10 -Bpa). Both these clones were the same and had following mutations compared to the wild-type MbPyIRS: N311Q, C313T and W382A, in addition to the Y349F pre-programmed mutation. (C) Expression of gst-1TAG-cam using newly evolved MbPyl(Bpa)RS without any ncAA, or with 1 mM Bpa, 1 mM O-(2-Nitrobenzyl)-L-tyrosine (photocaged tyrosine, pcY), 1 mM p-Azido- L-phenylalanine (AzF) or 1 mM N6-(tert-butoxycarbonyl)-L-lysine (BocK). Control expression using wt-MbPyIRS that is known to efficiently incorporate BocK is also performed. Comparison of band intensities of full length Gst-CaM obtained using MbPyl(Bpa)RS for expression with Bpa, pcY, AzF and BocK, and using wt-MbPyIRS for expression with BocK demonstrates highly efficient and specific incorporation of Bpa using MbPyl(Bpa)RS (Figure 4). (D) Selective incorporation of Bpa at positions 32 and 109 in 7D12 using MbPyl(Bpa)RS. Comparison of band intensities for expression with Bpa (1 mM), pcY (1 mM), and both Bpa (1 mM) and pcY (1 mM), indicate selective incorporation of Bpa in the presence of pcY. (E) ESI-MS of amber mutants of 7D12 expressed using MbPyl(Bpa)RS in the presence of both Bpa (1 mM) and pcY (1 mM) demonstrate that Bpa is selectively incorporated. Dotted lines with arrow indicates the expected molecular weight if pcY was incorporated.

Figure 8. Assessing the efficiency and substrate specificity of newly evolved MbPyl(Bpa)RS and previously known MmPyl(Bpa)RS, BpaRSI (Chembiochem, 2013. 14(16): p. 2100-5), at site-specific incorporation of Bpa. Expression of gst-1TAG-cam is performed with MbPyl(Bpa)RS, and BpaRSI either without any noncanonical amino acid (ncAA), or with p- benzoyl-L-phenylalanine (Bpa), O-(2-Nitrobenzyl)-L-tyrosine (photocaged tyrosine, pcY), p- Azido-L-phenylalanine (AzF) or N6-(tert-butoxycarbonyl)-L-lysine (BocK). As a control, expression of gst-1TAG-cam was also performed with wt-MbPyIRS that is known to efficiently incorporate BocK. Site-specific incorporation of amino acid in response to TAG stop codon will result in the full-length Gst-CaM (top band). A) Comparison of band intensities of full length Gst-CaM for expressions performed with Bpa, pcY, AzF and BocK using MmPyl(Bpa)RS and newly evolved MbPyl(Bpa)RS demonstrates that latter is highly efficient and specific at incorporating Bpa in Gst-CaM. This gel image is obtained after Coomassie staining. B) ncAA incorporation efficiency is calculated by taking the ratio of intensity of the top band (Gst-CaM) to the sum of intensities of top band (Gst-CaM) and lower band (Gst). The efficiency of MbPyl(Bpa)RS at incorporating Bpa and that of wt-MbPyIRS at incorporating BocK are similar.

Also, MbPyl(Bpa)RS is 5-fold selective for Bpa over pcY.

Figure 9. Expression of wt-7D12 using pSANG-oR-o7D12 plasmid. Coomassie stained gel image demonstrates that the expression of 7D12 is efficient and dependent on addition of IPTG.

Figure 10. Development of photoactive photoreactive 7D12 mutant. (A) A single plasmid containing genetic components to assemble the orthogonal ribosome, our newly evolved Bpa- specific MbPylRS(Bpa), AGTA-decoding evolved MbPyltRNAUACU, and 7D12 gene on orthogonal RBS was constructed. We named this plasmid: pSANG-oR-o7D12-Dual-Pyl(Bpa). Co-transformation of this plasmid with pULTRA-pcY allowed expression of 7D12-32pcY- 109Bpa. (B) Expression of 7D12-32pcY-109Bpa without and with pcY and Bpa. (C) ESI-MS of 7D12-32pcY-109Bpa is consistent with site-specific incorporation of pcY and Bpa in 7D12. Note that we observe a minor peak at 14537, which is a mass gain of 72 Da. This peak cannot be explained by dual incorporation of pcY or Bpa, and we are unsure of its origin. (D) On-cell binding assay demonstrates that 7D12-32pcY-109Bpa is a photoactive antibody. (E) The normalised intensities from on-cell binding assay were plotted against log(concentration of 7D12) . The data was fitted to sigmoidal nonlinear equation using GraphPad to obtain binding affinity values (KD). Before irradiation, the KD ofwt-7D12 and 7D12-109Bpa were 23(±2.6) nM and 54 (±14) nM, respectively. After irradiation, the KD of wt-7D12, 7D12-32Bpa, 7D12- 109Bpa and 7D12-32pcY-109Bpa were 22(±1.5) nM, 42(±5.4) nM, 35(±6) nM and 103 (±25) nM, respectively. For 7D12-32Bpa and 7D12-32pcY-109Bpa before irradiation, lines show connection between individual points. For all other experiments, lines show the fitting trace. F) Photocrosslinked product observed only with 7D12-109Bpa and 7D12-32pcY-109Bpa for samples irradiated with 365 nm light demonstrating that 7D12-32pcY-109Bpa gets activated and then forms a covalent bond with EGFR upon irradiation with 365 nm light. (G) Photocrosslinking of 7D12-32pcY-109Bpa to sEGFR performed in DM EM media containing 10% serum. Left panel shows Coomassie stained gel demonstrating photocrosslinking of 7D12-32pcY-109Bpa to sEGFR in the control reaction performed in phosphate buffered saline (PBS). For same reaction performed in serum containing media, bands corresponding to sEGFR and photocrosslinked product are not clear on Coomassie stained gel due to the presence of serum proteins. The right panel shows anti-His6 western blot of the photocrosslinking reactions that detects the C-terminal His6 tag on 7D12. The bands show SEGFR-7D12 complex demonstrating successful photocrosslinking of 7D12-32pcY-109Bpa in serum containing media.

Figure 11. On-cell binding assay to measure the binding of 7D12-109Bpa and 7D12-32pcY- 109Bpa towards MDA-MB-231 and SW-620 cells. MDA-MB-231 and SW-620 cells are used as negative control cell lines to access the specificity of 7D12-109Bpa and 7D12-32pcY- 109Bpa for EGFR. A) Near background binding was observed for 7D12-109Bpa and 7D12- 32pcY-109Bpa towards the control cell lines, MDA-MB-231 and SW-620, both before and after irradiation with 365 nm light. Binding assay of 7D12-109Bpa towards EGFR-positive, A431 cells, was performed as a control experiment. These results demonstrate that 7D12-109Bpa and 7D12-32pcY-109Bpa specifically bind to EGFR on cell surface. To ensure reproducibility, experiments were performed in duplicates represented as REP 1 and REP 2. These images were acquired using GE ImageQuant™ LAS 4000 gel imager. B) Chemiluminescence intensities obtained from on-cell binding experiments were quantified using CLARIOstar plate reader. For each lane (i.e. each row), the intensity from each well was subtracted from intensity for zero concentration in that lane, i.e. I-IO, and plotted against concentration of 7D12. This normalisation is performed to ensure that data between different cell lines, and between replicates could be compared. The data was plotted using GraphPad. The line shows connection between individual points.

Detailed Description

The present disclosure will now be further described. In the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols. 1 and 2, Ontermann and Duebel, eds., Springer-Verlag, Heidelberg (2010).

Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

In one aspect, we provide an antibody or fragment thereof wherein said antibody or fragment comprises a photocaged amino acid and a photoreactive amino acid in its antigen binding region.

In one embodiment, the antibody is thus modified by site specific modification to include an amino acid in its sequence which is linked to a photocaging group and an amino acid in its sequence which is linked to a photoreactive group. The photocaged group and photoreactive group are not conjugated to the native amino acid sequence by using a chemical or peptide linker. Instead, the photocaged amino acid and photoreactive amino acid are genetically encoded and therefore form part of the resulting amino acid sequence.

Genetic code expansion has enabled site-specific incorporation of several unnatural amino acids, including amino acids containing bio-orthogonal functional groups, photoreactive amino acids and photocaged amino acids, into proteins (Chin, J. W., Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 2014, 83, 379-408) and (Wang, L.; Xie, J.; Schultz, P. G., Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 225-249). Photocaged unnatural amino acids have been employed to control the activity of several biomolecules including transcription factors, kinases, proteases and inteins. Photoreactive unnatural amino acids within antibody fragments have been used in the context of cancer to form a covalent bond with its antigen upon irradiation with 365 nm light. This was developed by introducing a photocrosslinker attached via a cysteine residue in the antigen binding region of the affibody (Brasino, M. et al. Affibodies with Site-Specific Photo-Cross- Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors. J. Am. Chem. Soc., 2018. 140,37, 11820-11828). Methods to encode unnatural amino acids can use orthogonal amino acyl tRNA synthetase/tRNA pairs and orthogonal quadruplet decoding ribosomes. In the orthogonal tRNA synthetase/tRNA approach the unnatural amino acid is detected by the altered specificity of the orthogonal synthetase and used to amino acylate the orthogonal tRNA. During translation the orthogonal tRNA will then introduce the unnatural amino acid in response to a unique or “non-sense” codon. A non-sense codon may be an amber stop codon (UAG), an opal stop codon (UGA) or an ochre stop codon (UAA) or quadruplet codons such as AGLIA, AGGA, LIAGA. Multiple orthogonal tRNA synthetase/tRNA pairs have been developed including but not limited to; tyrosyl-tRNA synthetase (TyrRS)/tRNA ^Tyr from Methanocaldococcus jannaschii, lysine- RS/tRNA ^Lys pair from Pyrococcus horikoshii, a glutamine-RS/tRNA ^GIU pair from Methanosarcina mazei, pyrrolysine-RS/tRNA ^Pyl pair from Methanosarcina species, a pyrrolysyl-tRNA synthetase pair from Methanosarcina barkeri, a heterologous pair consisting of a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and a mutant tRNA ^Leu derived from Halobacterium sp., and pair consisting of P. horikoshii proline-RS and three engineered suppressors tRNA ^Pro from Archaeoglobus fulgidu.

Here, we genetically encode photocaged tyrosine and p-benzoyl-L-phenylalanine at specific locations in an exemplary antibody fragment, 7D12. The identity of each modified protein was confirmed by mass spectrometry. We have shown that unnatural amino acids, i.e. a photocaged amino acid and a photoreactive amino acid, can be concurrently genetically incorporated into the same antibody molecule at specific locations. The site-specific incorporation of the photocaged amino acid is present at a site in which the photocaged amino acid inhibits interaction and/or binding of the antibody to its antigen in the absence of light. The photocaged amino acid is present at a site in which the photocaged amino acid inhibits interaction and/or binding of the antibody to its antigen, wherein binding and/or interaction of the antibody to its antigen can be induced upon activation with a light source. The photoreactive amino acid is present at a site in which the photoreactive amino acid does not significantly inhibit/reduce interaction with and/or binding of the antibody to its antigen in the absence of light. The photoreactive amino acid allows covalent bond formation with the antigen target in the presence of light.

Photoremovable protecting groups, or photocages, also referred to as light-removable protection groups, are groups that are covalently linked to a target molecule to inhibit its activity. The addition of ‘caging’ group in a key position in the antibody sequence important for antigen binding, can achieve effective spatiotemporal control of activity of the antibody molecule. The caging group hinders binding of the antibody to its target until the molecule is liberated, or ‘decaged’, by exposure to a specific wavelength of light.

Thus, the antibody of interest is rendered biologically inactive (caged) by the chemical modification with a protecting group within its sequence as it can no longer bind to its target antigen. The chemical modification can be removed (decaged) with light by irradiation of a suitable wavelength. This leads to the release of the caging group from the antibody, and the removal of the photocaging group results in binding of the antibody to its target. In an embodiment the photocaged amino acid is a light-removable protection group

In one embodiment, the functional photocaging group, also referred to herein as a photoactive group, is selected from an o-nitrobenzyl group, or variants thereof. The photocaging group, or photoactive group may be selected from a nitrophenethyl group, a nitroveratryl group, an arylcarbonylmethyl group, a phenylacyl group, an o-alkylphenacyl group, a p-hydroxyphenacyl group, a nitrobenzyl group, a coumarin-4-ylmethyl group. The functional group may be light responsive and selected from terazine, azobenzene, diazirine or benzophenone. In another embodiment, the group is selected from tert-butoxycarbonyl (Boc), alkyne or azide. In one embodiment, the group is an o-nitrobenzyl group.

Photoreactive amino acids are chemically inert compounds that become reactive when exposed to a specific wavelength of light. Upon exposure to light, usually UV light, photoreactive amino acids are capable of forming a covalent bond with other proteins in close proximity in a photocrosslinking reaction. Photoreactive amino acids are also termed photo cross linkers. Thus, a photoreactive amino acid comprises at least one functional group which forms a reactive species upon irradiation with light, and thus is capable of forming a covalent bond to cross link proteins. Photoreactive amino acids are amino acid analogs.

Exemplary functional groups include, but are not limited to, an azide (e.g., aryl azides, azido- methyl-coumarins, and the like), benzophenones, anthraquinones, diazo compounds, diazirines, and psoralen derivatives. In certain embodiments, the photoreactive functional group comprises a benzophenone, an azide or a diazirine. In one embodiment, the photoreactive amino acid is selected from p-benzoyl-L-phenylalanine, benzofuranylalanine; benzotriazolylalanine, p-Azido-L-phenylalanine, trifluoromethyl phenyl diazirine, alkyl diazirine, and variants thereof. In another embodiment, the photoreactive amino acid is p-benzoyl-L-phenylalanine.

The presence of the photoreactive amino acid in the antibody molecule does not significantly inhibit or reduce the specific non-covalent binding between the antibody and its antigen in the absence of light. The ability to catalyse the formation of a covalent bond between the antibody and antigen in the presence of light is dependent on this non-covalent binding in the absence of light. For example, antigen binding is reduced by no more than 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

For example, antigen binding is reduced by no more than 10-fold, or less than 9-fold, or less than 8-fold, or less than 7-fold, or less than 6-fold, or less than 5-fold, or less than 4-fold, or less than 3-fold, or less than 2-fold, or less than 1.5-fold, or less than 1.4-fold, or less than 1.3- fold, or less than 1.2-fold, or less than 1.1-fold. In one embodiment, the photoreactive amino acid is present at a site in which the photoreactive amino acid reduces the interaction and/or binding of the antibody to its antigen less than 2-fold in the absence of light. Affinity to the antigen can be measured as explained herein.

In the absence of light, the antibody of interest is rendered biologically inactive (caged) by the chemical modification with a protecting group within its sequence as it can no longer bind to its target antigen. The irradiation with light by irradiation of a suitable wavelength activates the antibody, wherein a dual event occurs involving both the release of the photocaging group from the antibody allowing the liberation of the molecule, and the catalysation of a covalent bond between the antibody and its target antigen. The irradiation with light therefore acts as an activation switch where the antibody molecule is both liberated and covalently binds to its target antigen.

Antibodies that comprise both a photocaged amino acid in their antigen binding region and a photoreactive amino acid in their antigen binding region, are capable of specific binding to the target antigen and covalent bond formation with the target antigen, when activated with light. This concurrent activity can be exploited in therapeutic applications as disclosed herein. The inventors have shown that modification of only two residues in this way leads to an antibody molecule whose binding to the antigen can be controlled by light. As the modification affects only two residues, the modified antibody only differs by about 223.1 Da in molecular weight from the parent i.e. non-modified antibody.

The term “antibody” as used herein expressly includes antibody fragments. The term "antibody" broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass.

The term "CDR" refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1 -113 of the heavy chain).

The system described by Kabat is used herein unless otherwise specified. The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. , hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.

The term "antigen binding site" refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. Preferably, an antigen binding site is comprised within the associated VH and VL of an antibody or antibody fragment.

An antibody or fragment thereof, "which binds" or is “capable of binding” an antigen of interest” is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen. “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g, antibody or antigen -binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).

Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. Kon refers to the association rate constant of, e.g, an antibody or antigenbinding fragment thereof to an antigen, and koff refers to the dissociation of, e.g, an antibody or antigen-binding fragment thereof from an antigen.

The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10 ^-4 M, alternatively at least about 10 ^-5 M, alternatively at least about 10 ^-6 M, alternatively at least about 10 ^-7 M, alternatively at least about 10 ^-8 M, alternatively at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, alternatively at least about 10 ^-11 M, alternatively at least about 10 ^-12 M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The affinity can be determined by techniques known to one of ordinary skill in the art, such as surface plasmon resonance (SPR) or KinExA.

In one embodiment, the binding affinity is assessed by an on-cell assay. In another embodiment, the binding affinity is assessed by another technique known to one of ordinary skill in the art, such as surface plasmon resonance (SPR) or KinExA. The skilled person would be able to identify a suitable binding assay technique. The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen (to which an immunoglobulin, antibody or antibody fragment, specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment. An antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in different formats, using either labelled antigen or labelled antibody.

An antibody fragment is a portion of an antibody, for example as F(ab')2, Fab, Fv, sFv, single domain antibodies (dAbs) and the like. Functional fragments of a full length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs. scFv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

The smallest antigen binding fragment is the single variable fragment, namely the VH or VL domain. Binding to a light chain/heavy chain partner respectively is not required for target binding. Such fragments are termed single domain antibodies. A single domain antibody (~12 to 15 kDa) therefore has either the VH or VL domain and does not comprise other parts of an antibody. VH and VL domains respectively are capable of binding to an antigen. The antigenbinding entity of an antibody, reduced in size to one single domain (corresponding to the VH or VL domain), is generally referred to as a “single-domain antibody” or “immunoglobulin single variable domain”. Single domain antibodies derived from camelid heavy chain only antibodies that are naturally devoid of light chains as well as single domain antibodies that have a human heavy chain domain have been described (Muyldermans J Biotechnol. 2001 Jun; 74(4): 277- 302; Holliger Nat Biotechnol. 2005 Sep; 23(9): 1126-36).

Antigen binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341 : 544-546). Ward et al. named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb" generally refers to a single immunoglobulin variable domain (human VH, VHH - e.g. from camelid species- or VL) polypeptide that specifically binds antigen. The terms “single domain antibody (sdAb), variable single domain or immunoglobulin single variable domain (ISV)” are thus all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen.

Thus, the various aspects described herein relate to a full length antibody, such as a monoclonal antibody, and also relate to an antibody fragment which may be selected from a F(ab')2, Fab, Fv, sFv, scFv, dAb, affibody or any peptide derived from a full length antibody that retains specific binding affinity to its antigen.

The term "isolated" protein or polypeptide refers to a protein or polypeptide that is substantially free of other proteins or polypeptides, having different antigenic specificities. Moreover, protein or polypeptide may be substantially free of other cellular material and/or chemicals. Thus, the protein, nucleic acids and polypeptides described herein are preferably isolated. "Isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.

In one embodiment, the antibody or antibody fragment includes only two modifications in its amino acid sequence, that is it includes one amino acid that has a photocaged group and one amino acid that has a photoreactive group. In another embodiment, the antibody or antibody fragment includes more than one amino acid with a photocaged group and/or more than one amino acid with a photoreactive group. The modifications are located in the antigen binding site of the antibody. In other words, the residue selected contributes to or mediates antigen binding. However, as mentioned above, the presence of the photoreactive group does not significantly reduce or inhibit binding to the target antigen.

In one embodiment, the residue is located in CDR1 , CDR2 or CDR3. In another embodiment, the residue is not located in a CDR region, but mediates binding or contributes to specific binding to the antigen. Residues can be identified using structural analysis as described in the examples. Such methods are known in the art.

In one embodiment, the photoreactive group is located in CDR1 , CDR2 or CDR3. In another embodiment, the photoreactive group is not located in a CDR region, but mediates binding or contributes to specific binding to the antigen. In one embodiment, the photoreactive group is located in a framework region for example FR1 , FR2, FR3, FR4. Suitable positions for the photoreactive group to be introduced can be identified using structural analysis as described in the examples. Such methods are known in the art.

Thus, according to the embodiments described herein, two residues that interact with the binding to the target antigen are modified, and these modifications, by genetic introduction of one amino acid that has a photocaged group and one amino acid that has a photoreactive group, are positioned to inhibit or reduce binding of antibody to its target antigen in the absence of light. Exposure to light of a suitable wavelength, results in decaging of the photocaged residue and the antibody forms a covalent bond with the antigen via the photoreactive amino acid.

In one embodiment, the antibody or fragment is conjugated to another moiety. Said moiety is selected from a half life extension moiety, label or therapeutic moiety, such as a drug, an enzyme or a toxin. In one embodiment, the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin. The label may any label to aid in detection or isolation/purification of the antibody, such as radioisotopes, enzymatic proteins, fluorophores and fluorescent dyes.

The moiety can be linked to the heterodimeric protein, e.g. antibody, using linkers known in the art, e.g. via a chemical or peptide linker. The linkage can be covalent or non-covalent. An exemplary covalent linkage is via a peptide bond. In some embodiments, the linker is a polypeptide linker (L). Suitable linkers include for example a linker with GS residues such as (Gly4Ser)n. Other linkage/conjugation techniques include cysteine conjugation, e.g. for cysteine-based site specific antibody conjugation to a toxic payload.

The antibody or fragment may be modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome or using a serum albumin protein, or by Fc modification.

The antibody or fragment may be modified to alter other properties, for example biophysical performance or effector function. Modifications include but are not limited to glycosylation.

In one embodiment, the moiety may be conjugated to the antibody or fragment via an unnatural amino acid. The unnatural amino acid may be site specifically genetically encoded. It may comprise a functional group such as an azide group, an alkyne group, a tetrazine group and/or a strained alkene. The unnatural amino acid may be selected from one or more of p- acetylphenylalanine, alkyne lysine, p-azidomethyl-L-phenylalanine, p-azido-L-phenylalanine A/6-((2-azidoethoxy)carbonyl)-L-lysine. The skilled person would be able to determine a suitable site for incorporation of the unnatural amino acid depending on the moiety to be attached.

The antibody or fragment may be a therapeutic antibody or a diagnostic antibody (i.e. an antibody that does not have a therapeutic effect, but can be used for diagnostic purposes).

In one embodiment, the antibody or fragment binds to a cell surface antigen.

In one embodiment, the antibody or fragment binds to a tumor associated antigen (TAA). Tumor antigens can be loosely categorized as oncofetal (typically only expressed in fetal tissues and in cancerous somatic cells), oncoviral (encoded by tumorigenic transforming viruses), overexpressed/ accumulated (expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer-testis (expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted (expressed largely by a single cancer histotype), mutated (only expressed by cancer as a result of genetic mutation or alteration in transcription), posttranslationally altered (tumor-associated alterations in glycosylation, etc.), or idiotypic (highly polymorphic genes where a tumor cell expresses a specific “clonotype”, i.e., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies).

In one embodiment, the tumor associated antigen is selected from PSMA, Her2, Her3, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF, CECAM6, CAXII, CD24, ETA, MAGE, Mesothelin, cMet, TAG72, MUC1 , MUC16, STEAP, Ephvlll, FAP, GD2, IL-13Ra2, L1-CAM, PSCA, GPC3, gpA33, CA-125, gangliosides G(D2), G(M2) and G(D3), Ep- CAM, CEA, bombesin-like peptides, PSA, HER2/neu, epidermal growth factor receptor (EGFR), erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFβ1 , IGF-1 receptor, EGFa, c-Kit receptor, transferrin receptor, IL-2R, TAG-72 and CO17-1A.

In one embodiment, the antibody or fragment binds to EGFR. In one embodiment, the fragment is a VH or VHH and binds to EGFR. In one embodiment, the fragment is 7D12. In one embodiment, 7D12 includes a modified tyrosine and p-benzoyl-L-phenylalanine. In one embodiment, 7D12 includes a modified tyrosine at position 32 and p-benzoyl-L-phenylalanine at position 109.

In yet another embodiment, the antibody is an antibody that inhibits tumor cell growth and/or proliferation through binding to its antigen. For example, such an antibody may be selected from IMC-C225, EMD 72000, OvaRex Mab B43.13, anti-ganglioside G(D2) antibody ch14.18, CO17-1A, trastuzumab®, cetuximab®, rhuMAb VEGF, sc-321 , AF349, BAF349, AF743, BAF743, MAB743, AB1875, Anti-Flt-4AB3127, FLT41-A, rituximab, 2C3, CAMPATH 1 H, 2G7, Alpha IR-3, ABX-EGF, MDX-447, anti-p75 IL-2R, anti-p64 IL-2R, and 2A11.

In one embodiment, the antibody or fragment is an inhibitor of an immune checkpoint molecule. This may be selected from an inhibitor of one or more of PD-1 , PD-L1 , PD-L2, CTLA-4, TIM- 3, CEACAM, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 or TGFR beta. In another embodiment, the antibody may be an activator of a costimulatory molecule selected, for example, from an agonist of one or more of 0X40, OX40L, CD2, CD27, CDS, ICAM-1 , LFA-1 (CD11a/CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

The antibody may target tumor cell(s). A tumor cell comprises one or more cancer cells, or a mass of cancer cells, and can also encompass cells that support the growth and/or propagation of a cancer cell, such as vasculature and/or stroma, but not necessarily macrophages. For instance, therefore, the present invention envisages compositions and methods for reducing growth and/or proliferation of a tumor cell in a subject, wherein tumoricidial antibodies bind with specificity to cell surface epitopes (or epitopes of receptor-binding molecules) of a cancer cell or a cell that is involved in the growth and/or propagation of a cancer cell such as a cell within the vasculature of a tumor or blood vessels that supply tumors and/or stromal cells.

Pharmaceutical composition

In another aspect, the disclosure relates to a pharmaceutical composition comprising an antibody or fragment thereof described herein and optionally a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the antibody or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier where the drug antibody conjugates are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. In one embodiment, the carrier includes excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical composition can be in the form of a liquid, e.g., a solution, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously. When intended for oral administration, the composition can be in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Compositions can take the form of one or more dosage units.

Treatment of disease

In another aspect, we provide a method for treating a disease in a subject selected from a cancer, an immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease comprising administering an effective amount of an antibody or fragment thereof or a pharmaceutical composition as described herein to a subject in need thereof.

We also provide an antibody or fragment thereof as described above or a pharmaceutical composition as described above for use in the treatment of a disease, for example cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer. We also provide an antibody or fragment thereof as described above or a pharmaceutical composition as described above for use in the manufacture for of a medicament for the treatment of a disease, for example cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer.

As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disease. For example, treatment can include a postponement of development of the symptoms associated with a disease or disease, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

As used herein, the term "effective amount" means an amount of an antibody, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.

In one embodiment, the disease is cancer. The cancer can be selected from a solid or nonsolid tumor. For example, the cancer may be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, leukemias such as ALL, CLL, AML, urothelial carcinoma leukemia and multiple myelomas.

In one embodiment, the tumor is a solid tumor. Examples of solid tumors which may be accordingly treated include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS, neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma. Examples of vascularized skin cancers for which the antagonists of this invention are effective include squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by suppressing the growth of malignant keratinocytes, such as human malignant keratinocytes.

In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma.

In one embodiment, the cancer is locally advanced unresectable, metastatic, or recurrent cancer. In one embodiment, the cancer has progressed after another treatment, for example chemotherapy.

Cancers include those whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer).

The immune disease can be selected from graft vs. host disease, arthritis, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg- Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, Neuromyelitis optica (NMO), type 1 or immune -mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/ giant cell arteritis, transverse myelitis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

In one embodiment, the antibody, antibody fragment or pharmaceutical composition described herein is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the antibody is used in combination with surgery.

In one embodiment, we provide a photoactive photoreactive antibody drug conjugate comprising an antibody or fragment described herein conjugated to a toxic payload. Such conjugate can be used to kill tumor cells by antibody cell targeting upon light activation, internalization by the cells (generally cancerous), and payload release to expose the highly potent cytotoxic drugs to the tumor for toxin-mediated tumor cell killing. The toxic payload may be selected from small molecules (e.g. maytansanoid, auristatin), a protein toxin (e.g. Pseudomonas exotoxin, diphtheria toxin), a cytolytic immunomodulatory protein (e.g. Fas ligand) to kill targeted cells, a biologically active peptide (e.g. GLP-1) to extend the pharmacological half-life of the natural peptide, an enzymes (e.g. urease) to modify the biochemistry of the targeted microenvironment or radionuclides (e.g. 90Y, 1111n) for either killing or imaging of tumor cells.

The toxic pay load may be conjugated to the antibody using a number of chemical linkers including cleavable linkers such as disulfides, hydrazones, peptides, or non-cleavable linkers such as thioethers. In an embodiment the toxic payload is conjugated to the antibody by an unnatural amino acid. In this embodiment a further unnatural amino acid is site specifically genetically incorporated into the antibody sequence, this unnatural amino acid may be different to the photocaged amino acid. The unnatural amino acid for attaching the toxic payload may be positioned away from the antigen binding portion of the antibody, it may be present at a site within the constant region or the Fc region of the antibody. Further there maybe multiple attachment sites for the toxic payload each comprising an unnatural amino acid which may be the same or different.

The unnatural amino acids which may be used to attach the toxic payload include p- acetylphenylalanine, alkyne lysine, p-azidol-L-phenylalanine, p-azidol-L-methylphenylalanine A/6-((2-azidoethoxy)carbonyl)-L-lysine. The carbonyl group of p-acetylphenylalanine can react with alkoxyamine-functionalised linkers to produce oxime-conjugated ADCs. The alkyne group of alkyne lysine can react with azide functionalised linkers through copper-catalysed Huisgen cycloaddition (“click” reaction). The azide groups of p-azidomethyl-L-phenylalanine, A/6-((2- azidoethoxy)carbonyl)-L-lysine can react with alkyne-functionalized linkers through the copper- catalyzed Huisgen cycloaddition (“click” reaction).

In one embodiment, the antibody or composition is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition disclosed herein, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

The antibody, antibody fragment or pharmaceutical composition described herein can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. In another embodiment, delivery is of the nucleic acid encoding the drug, e.g. a nucleic acid encoding the molecule of the invention is delivered.

Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

In some embodiments, it can be desirable to administer the composition locally to the area in need of treatment, for example the tumor tissue, or by intravenous injection, infusion or topical administration. Such selective activation near the tumor site can lead to fewer side effects compared to conventional therapy based on antibody therapeutics. In this way, potentially higher dosages can be employed for the treatment.

The amount of the composition described herein that is effective/active in the treatment of a particular disease or condition will depend on the nature of the disease or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each subject's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

Typically, the amount is at least about 0.01 % of an antibody by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the antibody of the present invention by weight of the composition. Compositions can be prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the antibody. For administration by injection, the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

In one embodiment, the method comprises activating the antibody or fragment thereof by irradiating a target tissue or a subject. As used herein, “activating” means that due to exposure to light, a dual event occurs involving both the release of the photocaging group from the antibody allowing the liberation of the molecule, and the catalysation of a covalent bond between the antibody and its target antigen. The irradiation with light therefore acts as an activation switch where the antibody molecule is both liberated and covalently binds to its target antigen. The caging group is removed from the antibody and the antibody is now free to bind to the target antigen. The wavelength is chosen dependent on the photocaging and photocrosslinking group used. For example, it can be 365nm. Long wavelength UV radiations of 365 nm used for activation of photoactive photoreactive antibodies in this study are known to cause less direct DNA damage and toxicity when compared to short wavelength UV radiations (Kielbassa, C., Roza, C., Epe, B. Wavelength dependence of oxidative DNA damage induced by UV and visible light. Carcinogenesis, 1997. 18, 811-6) and are thus considered much safer for clinical use. In another embodiment, the light is in the infrared region of the light spectra. For example, when used in combination with upconverting nanoparticles, the photocage group can be decaged using 975 nm light, in the Infrared region of the light spectra.

Using the genetic incorporation approach the unnatural amino acid can be incorporated at any site within the antibody sequence. Routine mutagenesis techniques can be used to incorporate a nonsense codon which is then recognised by the orthogonal tRNA synthetase/tRNA pair and the unnatural amino acid is introduced. The photocaged amino acid may be introduced at a site in which it inhibits interaction and/or binding of the antibody to its antigen. In particular, the photocaged amino acid may be introduced at the antigen binding site or the CDRs of the antibody. The skilled person would be able to identify suitable sites for inhibiting interaction and/or binding using routine techniques including analysis of X-ray crystallographic structures of the antibody and antigen interaction, molecular dynamic simulations of the antibody and antigen interaction, or a combination of these techniques. The photoreactive amino acid may introduced at a site in which it catalyses the formation of a covalent bond between the antibody and the antigen. In particular, the photoreactive amino acid may be introduced at the antigen binding site or the CDRs of the antibody. The skilled person would be able to identify suitable sites for the insertion of a photoreactive amino acid using routine techniques including analysis of X-ray crystallographic structures of the antibody and antigen interaction, molecular dynamic simulations of the antibody and antigen interaction, or a combination of these techniques.

It is also possible to incorporate one or more unnatural amino acids into the antibody sequence. This may comprise one or more of the same unnatural amino acids or one or more of different unnatural amino acids. By using multiple orthogonal tRNA synthetase/tRNA pairs it is possible to incorporate different unnatural amino acids into the antibody sequence. In an embodiment the antibody comprises one or more photocaged amino acids and one or more photoreactive amino acids in the antigen binding site. In an embodiment, the antibody comprises one or more photocaged amino acids and one or more photoreactive amino acids in the antigen binding site and one or more further unnatural amino acids. The further unnatural amino acid/s may comprise a bio-orthongonal functional group which can be used to attach a further molecule to the antibody, this may be particularly useful for antibody drug conjugates (ADCs). Functional groups such as an azide group, an alkyne group, a tetrazine group and or a strained alkene group. A strained alkene group may be for example a cyclopropane group. The unnatural amino acid/s may comprise a functional group such as a thiol group, and/or a carbonyl group which may be used to attach a further molecule to the antibody.

We also provide an in vitro, ex vivo or in vivo method for diagnosing a disease comprising administering to a subject an antibody or fragment thereof as disclosed herein wherein said antibody is conjugated to a label. The method further comprises activating the antibody or fragment thereof by irradiating a target tissue or a subject.

We also provide an in vitro, ex vivo or in vivo method of reducing tumor cell growth and/or proliferation in a subject, cell, tissue or cell culture comprising administering a therapeutically effective amount of an antibody, antibody fragment or pharmaceutical composition described herein. In one embodiment, we provide an in vitro, ex vivo or in vivo method of reducing tumor cell growth and/or proliferation in a subject, cell, tissue or cell culture comprising a) administering a therapeutically effective amount of an antibody, antibody fragment or pharmaceutical composition described herein, wherein the antibody targets an antigen present on the surface of a tumor cell and exerts an inhibitory effect on growth and/or proliferation of the tumor cell when bound to the antigen and/or wherein the antibody directs the immune response to the tumor cell; b) localizing the antibody composition to a tumor cell; c) irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen; and d) inhibiting growth and/or proliferation of the tumor cell.

In one embodiment, we provide an in vitro, ex vivo or in vivo method of reducing tumor cell growth and/or proliferation in a subject, cell, tissue or cell culture comprising a) administering a therapeutically effective amount of an antibody, antibody fragment or pharmaceutical composition described herein, wherein the antibody that targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b) irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen wherein said antibody is internalised upon binding to the antigen; and c) killing the cell.

We also provide a method of killing a cell, comprising administering a therapeutically effective amount of an antibody, antibody fragment or pharmaceutical composition described herein. In one embodiment, we provide a method of killing a cell comprising: a) contacting a cell comprising a cell surface protein with a therapeutically effective amount of an antibody, antibody fragment or pharmaceutical composition described herein, wherein the antibody targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b) irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen wherein said antibody is internalised upon binding to the antigen; and c) killing the cell.

The method may be an in vitro, ex vivo or in vivo method for application in a subject, cell, tissue or cell culture.

In the methods above, irradiating the antibody or fragment comprises irradiating the subject, cell, tissue or cell culture; and/or irradiating a tumor in the subject, cell, tissue or cell culture.

We also provide a method for light dependent delivery of cytotoxic drugs to a cell, tissue or subject comprising providing an antibody, antibody fragment or pharmaceutical composition described herein conjugated to a toxic payload. In one embodiment, we provide a method for light dependent delivery of cytotoxic drugs comprising: a) contacting a cell or tissue an antibody, antibody fragment or pharmaceutical composition described herein wherein said antibody or fragment is conjugated to a toxic payload, wherein the antibody targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b) irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen wherein said antibody drug conjugate is internalised upon binding to the antigen; and c) killing the cell.

The method may be an in vitro, ex vivo or in vivo method for application in a subject, cell, tissue or cell culture.

We also provide a method for imaging of tumor cells comprising providing an antibody, antibody fragment conjugated to a label and delivering said antibody or fragment to a target cell or tissue. The method may be an in vitro, ex vivo or in vivo method for application in a subject, cell, tissue or cell culture.

We also provide a method of reducing the side effects associated with targeting cancer and/or tumour cells, comprising administering to a subject in need thereof the antibody, antibody fragment or pharmaceutical composition described herein. The method further comprises irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen.

The methods may further comprise applying a suitable light source selected from a filtered conventional light source, a diode array and a laser. For example, by using fiber- coupled laser diodes with diffuser tips, light, such as NIR, light can be delivered within several centimetres of otherwise inaccessible tumors located deep to the body surface. In addition to treating solid cancers, circulating tumor cells can be targeted since they can be excited when they traverse superficial vessels (for example using the NIR LED wearable devices disclosed herein).

Depending on the part of the body being treated, the photoactive antibodies can be either injected intravenously into the diseased area or applied topically to the skin.

We also provide a nucleic acid encoding an antibody or antibody fragment as described herein.

We also provide a vector comprising a nucleic acid encoding an antibody or antibody fragment as described herein. Furthermore, we provide an isolated nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette. Thus, the invention also relates to a plasmid, vector, transcription or expression cassette comprising a nucleic acid of the invention.

The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid molecule plasmid, vector, transcription or expression cassette as described above. The host cell may be a bacterial, viral, plant, fungal, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell, HeLa cell or other cell that would be apparent to the skilled person.

We also provide a method for producing an antibody or antibody fragment as described above, comprising a) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photocaged group at said position and b) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position. In one embodiment, the photocaged group is introduced using a /W/RS(photocaged tyrosine)/ /MjtRNAcuA pair and the photocrosslinking group is introduced using a Mb PylRS(p-benzoyl-L- phenylalanine)/ evolved Mb PyltRNAuAcu pair.

We also provide a kit comprising the antibody or fragment thereof as described herein. The kit may comprise a LED wearable device or an LED implantable device. A LED wearable device devices can include an article of clothing, jewellery, or a covering, and a near infrared (NIR) light emitting diode (LED) that is incorporated into the article of clothing, jewellery, or covering. Such devices can further include power and/or cooling sources. This permits the patient to wear the device (or be covered by the device) for extended periods of time, thus permitting treatment of tumor cells present in the blood or circulatory system.

We also provide an isolated protein comprising SEQ ID NO. 1. In one embodiment, the protein comprises at least 75%, at least 80%, at least 85%, at least 90% or at least 95% homology thereto and comprising the following mutations compared to the wild-type Mb PyIRS: N311Q, C313T, W382A and Y349F.

As used herein, the terms sequence "homology" or “identity” generally refer to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Altschul et al (1997) Nucl. Acids Res. 25 3389-3402) may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments were performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log- Expectation) algorithms (Edgar (2004a) Nucleic Acids Res 32: 1792-7; Edgar (2004b) BMC Bioinformatics 5:113.).

The isolated protein is an evolved Methanosarcina barkeri Pyrrolysyl-tRNA synthetase that is highly specific and efficient at incorporating p-benzoyl-L-phenylalanine into proteins. In one embodiment, in the presence of p-benzoyl-L-phenylalanine, azidophenylalanine, Boc- protected lysine, and photocaged tyrosine, said isolated protein does not significantly incorporate azidophenylalanine, Boc-protected lysine and/or photocaged tyrosine. In a further embodiment, in the presence of p-benzoyl-L-phenylalanine, azidophenylalanine, Boc- protected lysine, and photocaged tyrosine, said isolated protein does not incorporate azidophenylalanine and/or Boc-protected lysine. In a further embodiment, in the presence of p-benzoyl-L-phenylalanine and photocaged tyrosine, said isolated protein does not significantly incorporate photocaged tyrosine.

The term ‘significantly incorporate’ as used herein refers to the ability of azidophenylalanine, Boc-protected lysine, and photocaged tyrosine to outcompete p-benzoyl-L-phenylalanine as assessed by mass spectrometry, western blot or another method of protein characterisation that would be considered routine by a person skilled in the art.

We also provide a nucleic acid encoding the sequence of SEQ ID NO. 1 , and the embodiments of SEQ ID NO. 1. In one embodiment, said nucleic acid comprises SEQ ID NO. 2. In a further embodiment, the nucleic acid is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% homology thereto, but include codons that are required for the mutations as set out above. In a yet further embodiment, there is 100% homology thereto.

We also provide a vector or plasmid comprising a nucleic acid as described above. Furthermore, we provide an isolated nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette. Thus, the invention also relates to a plasmid, vector, transcription or expression cassette comprising the nucleic acid as described above. The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid molecule plasmid, vector, transcription or expression cassette as described above. The host cell may be a bacterial, viral, plant, fungal, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell, HeLa cell or other cell that would be apparent to the skilled person.

We also provide a method for producing an antibody or antibody fragment having a photoreactive amino acid wherein the photoreactive group is for example p-benzoyl-L- phenylalanine comprising identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position wherein said is introduced using the isolated protein as described above.

The photoreactive amino acid as described above becomes reactive when exposed to a specific wavelength of light, for example at a wavelength of about 365nm. Upon exposure to light, a covalent bond is formed between proteins in close proximity to one another in a photocrosslinking reaction.

We also provide a plasmid containing genetic components to assemble an orthogonal ribosome, an orthogonal aminoacyl-tRNA synthetase /tRNA pair, and a gene encoding an antibody or fragment thereof of interest on an orthogonal ribosome binding site.

As used herein, the term “genetic components to assemble an orthogonal ribosome” refers to an evolved orthogonal 16S ribosomal RNA, 23S ribosomal RNA and 5S ribosomal RNA genes. As used herein, the term “orthogonal ribosome” refers to an engineered ribosome which is able to selectively translate mRNA not recognized by their natural counterparts. For example, orthogonal ribosome may be optimized for the recognition of quadruplet codons. As used herein, the term “orthogonal aminoacyl-tRNA synthetase /tRNA pair” refers to (1) a synthetase that efficiently aminoacylates its cognate tRNA, but minimally aminoacylates endogenous tRNAs in the host organism, and (2) a tRNA that is a substrate for its cognate synthetase but is a poor substrate for endogenous synthetases. As used herein, the term “orthogonal ribosome binding site” refers to a binding site exclusively recognised by its cognate orthogonal ribosome. In one embodiment, the plasmid system can be adapted to express other antibody fragments containing distinct site-specifically incorporated noncanonical amino acids. For example, said gene encoding an antibody or fragment thereof on an orthogonal ribosome binding site may be swapped with another gene encoding an antibody or fragment thereof on an orthogonal ribosome binding site. For example, the 7D12 gene can be cut with the following restriction enzymes: SgrAI and Notl. The double stranded gene fragment corresponding to the alternative double stranded gene fragment can be inserted into the plasmid by a suitable molecular cloning methodology such as Gibson Cloning. The skilled person would be able to identify such suitable molecular cloning methodologies. Specific positions must be mutated (e.g. 32 and 109 in 7D12 to a TAG and an AGTA codon, respectively) to enable site-specific installation of photocaged tyrosine and p-benzoyl-L-phenylalanine, respectively.

In another embodiment, the plasmid system can be adapted to site-specifically install other pairs of noncanonical amino acids into 7D12 or other antibody fragments. This can be performed by digesting the plasmid with restriction enzymes (for example Ndel and Spel) and replacing the said orthogonal aminoacyl-tRNA synthetase /tRNA pair with another orthogonal aminoacyl-tRNA synthetase /tRNA pair.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The invention is further described in the non-limiting aspects.

Aspects

1. An antibody or fragment thereof wherein said antibody or fragment comprises a photocaged amino acid in its antigen binding region and a photoreactive amino acid in its antigen binding region.

2. The antibody or fragment thereof according to aspect 1 wherein the photocaged amino acid is present at a site in which the photocaged amino acid inhibits interaction and/or binding of the antibody to its antigen in the absence of light.

3. The antibody or fragment thereof according to aspect 1 or aspect 2 wherein the photoreactive amino acid is present at a site in which the photoreactive amino acid does not significantly inhibit or reduce interaction and/or binding of the antibody to its antigen in the absence of light.

4. The antibody or fragment thereof according to a preceding aspect wherein the photoreactive amino acid allows covalent bond formation with the antigen target in the presence of light.

5. The antibody or fragment thereof according to a preceding aspect wherein the photocaged amino acid includes a photoactive group selected from an o-nitrobenzyl functional group and variants thereof.

6. The antibody or fragment thereof according to a preceding aspect wherein the photoreactive amino acid is selected from p-benzoyl-L-phenylalanine, p-Azido-L- phenylalanine, trifluoromethyl phenyl diazirine, alkyl diazirine and variants thereof.

7. The antibody or fragment thereof according to a preceding aspect wherein said fragment is selected from a F(ab')2, Fab, Fv, sFv, scFv, dAb, affibody or any part of the antibody retaining binding specificity to the target antigen.

8. The antibody or fragment thereof according to a preceding aspect wherein the antibody or fragment thereof is conjugated to another moiety.

9. The antibody or fragment thereof according to aspect 8 wherein said moiety is selected from a half life extension moiety, label or toxic moiety. 10. The antibody or fragment thereof according to aspect 8 or 9 wherein said moiety is attached via an unnatural amino acid.

11. The antibody or fragment thereof according to a preceding aspect wherein said antibody or fragment thereof binds to a cell surface antigen.

12. The antibody or fragment thereof according to a preceding aspect wherein said antibody or fragment thereof binds to a tumor associated antigen, optionally selected from PSMA, Her2, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF, CECAM6, CAXII, CD24, CEA, Mesothelin, cMet, TAG72, MUC1 , MUC16, STEAP, Ephvlll, FAP, GD2, IL-13Ra2, L1-CAM, PSCA, GPC3, Her3, gpA33, CA-125, gangliosides G(D2), G(M2) and G(D3), Ep-CAM, bombesin-like peptides, PSA, HER2/neu, epidermal growth factor receptor, erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFβ1 , IGF-1 receptor, EGFα, c- Kit receptor, transferrin receptor, IL-2R and CO17-1A.

13. The antibody or fragment thereof according to any of aspects 1 to 11 wherein said antibody or fragment thereof binds to an immune checkpoint molecule, optionally selected from LAG-3, PD-1 , PD-L1 , PD-L2, CTLA-4, TIM-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 or TGFR beta.

14. The antibody or fragment thereof according to a preceding aspect wherein said antibody is 7D12.

15. A pharmaceutical composition comprising an antibody or fragment thereof according to a preceding aspect and optionally a pharmaceutically acceptable carrier.

16. The antibody or fragment thereof according to any of aspects 1 to 14 or a pharmaceutical composition according aspect 15 for use in the treatment of cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer.

17. The antibody or fragment thereof or a pharmaceutical composition according to aspect

16 further comprising activating the antibody or fragment thereof with a light source by irradiating a target tissue.

18. The antibody or fragment thereof or a pharmaceutical composition according to aspect

17 wherein the wavelength is 365nm.

19. The antibody or fragment thereof or a pharmaceutical composition according to any of aspects 17 or 18 wherein activating the antibody or fragment comprises irradiating the cell, tissue, subject and/or irradiating a tumor in the subject; optionally comprising applying a suitable light source selected from the group consisting of a filtered conventional light source, a diode array and a laser. 20. The antibody or fragment thereof or a pharmaceutical composition according to any of aspects 16 to 19 wherein the cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer .

21. A method of treating and/or preventing cancer, immune disease, neurological disease, inflammatory disease, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disease cancer comprising administering to a subject an antibody or fragment thereof according any of aspects 1 to 14 or a pharmaceutical composition according to aspect 15.

22. The method according to aspect 21 further comprising activating the antibody or fragment thereof with a light source by irradiating a target tissue.

23. The method according to aspect 22 wherein the wavelength is 365nm.

24. The method according to aspect 22 or 23 wherein activating the antibody or fragment comprises irradiating the cell, tissue, subject and/or irradiating a tumor in the subject; optionally comprising applying a suitable light source selected from the group consisting of a filtered conventional light source, a diode array and a laser

25. The method according to any of aspects 21 to 24 wherein the cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer . 26. A method for diagnosing a disease comprising administering to a subject an antibody or fragment thereof according any of aspects 1 to 14 or a pharmaceutical composition according to aspect 15 wherein said antibody is conjugated to a label.

27. A method of reducing tumor cell growth and/or proliferation in a cell, tissue or subject comprising: a. administering a therapeutically effective amount of an antibody or fragment thereof according to any of aspects 1 to 14 or a pharmaceutical composition according to aspect 15, wherein the antibody targets an antigen present on the surface of a tumor cell and exerts an inhibitory effect on growth and/or proliferation of the tumor cell when bound to the antigen and/or wherein the antibody that targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b. localizing the antibody or fragment to a tumor cell; c. irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen; and d. inhibiting growth and/or proliferation of the tumor cell.

28. A method of killing a cell comprising: a. contacting a cell comprising a cell surface protein with a therapeutically effective amount of an antibody or fragment thereof according to aspect 1 to 14 or a pharmaceutical composition according to aspect 15, wherein the antibody that targets an antigen present on the surface of a tumor cell is internalised when bound to the antigen; b. irradiating the antibody or fragment to release the photocaging group and induce specific binding of the antibody to the target antigen, and to catalyse the covalent photocrosslinking between the antibody and target antigen wherein said antibody is internalised upon binding to the antigen; and c. killing the cell.

29. The method of aspects 27 or 28 wherein irradiating the antibody or fragment comprises irradiating the cell, tissue, subject and/or irradiating a tumor in the subject; optionally comprising applying a suitable light source selected from the group consisting of a filtered conventional light source, a diode array and a laser.

30. A nucleic acid comprising a nucleic acid encoding an antibody or antibody fragment according to any of aspects 1 to 14.

31. A vector comprising a nucleic acid of aspect 30.

32. A host cell comprising a nucleic acid of aspect 30 or a vector of aspect 31. 33. A method for producing an antibody or antibody fragment according to any of aspects 1 to 14, comprising a) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photocaged group at said position and b) identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position.

34. A kit comprising the antibody or fragment thereof according to any of aspects 1 to 14, optionally comprising a LED wearable device or an LED implantable device.

35. An isolated protein comprising the SEQ ID NO. 1 or a protein with at least 75% sequence identity thereto and comprising the following mutations compared to the wild-type MbPyIRS: N311Q, C313T, W382A and Y349F.

36. A nucleic acid encoding the amino acid sequence according to aspect 35.

37. The nucleic acid sequence according to aspect 36 wherein said sequence comprises SEQ ID NO. 2.

38. A vector or plasmid comprising a nucleic acid of aspect 37.

39. A host cell comprising a nucleic acid of aspect 38 or a vector of aspect 37.

40. A method for producing an antibody or antibody fragment having a photoreactive amino acid wherein the photoreactive group is p-benzoyl-L-phenylalanine comprising identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position wherein said photoreactive group is introduced using the isolated protein of aspect 35.

41. A plasmid containing genetic components to assemble an orthogonal ribosome, an orthogonal aminoacyl-tRNA synthetase /tRNA pair, and a gene encoding an antibody or fragment thereof on an orthogonal ribosome binding site.

42. A plasmid according to aspect 41 wherein the aminoacyl-tRNA synthetase is the isolated protein of aspect 35.

43. A method for producing an antibody or antibody fragment having a photoreactive amino acid wherein the photoreactive group is p-benzoyl-L-phenylalanine comprising identifying an amino acid residue in the antigen binding region for site specific modification and introducing a photoreactive group at said position wherein said photoreactive group is introduced using the plasmid of aspect 41 or 42.

44. A method according to aspect 43 further comprising introducing a photoactive group into said antibody or antibody fragment.

45. The use of a plasmid of aspect 41 for use in the production of a photoreactive antibody or fragment thereof. The invention is further described in the non-limiting examples.

Examples

Here, we first identified a position for introducing photoreactivity into 7D12 by site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (Bpa).

Subsequently, we developed a generalised approach to site-specifically incorporate pcY and Bpa into proteins. Finally, we demonstrated that the site-specific incorporation of pcY and Bpa into 7D12 can allow its activation and covalent ligation to its target, EGFR, upon irradiation with 365 nm light.

Example 1 : Development of a high affinity photoreactive 7D12 mutant

Site-specific incorporation of ncAAs into proteins is achieved by assigning stop or quadruplet codons to ncAAs and supplying the cells with orthogonal aminoacyl-tRNA synthetase (aaRS)/ tRNA pairs. The orthogonal aaRS specifically charges the corresponding tRNA with the ncAA. The charged tRNA then binds to its cognate codon, generally an amber stop codon (TAG codon) or a quadruplet codon, on the messenger RNA allowing site-specific incorporation of a ncAA into the protein in a reaction catalysed by the ribosome (Young, D.D., et al., Playing with the Molecules of Life. ACS Chem. Biol., 2018. 13, 854-870).

In E. coli, evolved mutants of Methanocaldococcus jannaschii Tyrosyl-tRNA synthetase (/MjRS)/ /MjtRNA pair, and Methanosarcina Pyrrolysyl-tRNA synthetase (PylRS)/ tRNA pair have been employed extensively to genetically encode several ncAAs (Young, D.D., et al., Playing with the Molecules of Life. ACS Chem. Biol., 2018. 13, 854-870, Wan, W., et al., Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta, 2014. 1844, 1059-70). A MjRSI MjtRNA pair has been previously evolved for site-specific incorporation of Bpa (Chin, J.W., et al., Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Proc. Natl. Acad. Sci. U S A, 2002. 99, 11020-4). To develop photoreactive 7D12, we first used this mutant MjRS(Bpa)/ MjtRNA pair to incorporate Bpa in 7D12. The MjRS(Bpa)/ MjtRNA _CU a pair was cloned into a suppressor plasmid, pULTRA (Chatterjee, A., et al., A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry, 2013. 52, 1828-37), forming pULTRA-Bpa. Three tyrosine residues in 7D12, viz., 32, 109 and 113, at the binding interface of 7D12 and EGFR were targeted for replacement with Bpa (Figure 1A). If Bpa is accommodated at any of these positions without inhibiting 7D12-EGFR binding, it should be close enough to EGFR to allow light-dependent covalent bond formation between 7D12 and EGFR. To express these 7D12 mutants, the 7D12 gene was provided by the pSANGIO plasmid as our previous investigation (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993). Protein expression was performed in the presence and absence of Bpa (Figure 1 B). For the amber stop codon (TAG) mutants of 7D12, we observed a marked difference in the level of protein expressed with and without Bpa. Furthermore, electrospray ionization mass spectrometry (ESI-MS) analysis is consistent with site-specific incorporation of Bpa in 7D12 at positions 32, 109, and 113 (Figure 1C). 2.6 mg of wt-7D12, 2.26 mg of 7D12-32Bpa, 0.57 mg of 7D12-109Bpa, and 0.35 mg of 7D12-113Bpa, per litre of culture were obtained after purification.

Next, we assessed the binding of Bpa-containing mutants of 7D12 to EGFR expressed on the surface of cancer cells using our previously developed on-cell assay (Bridge, T., et al., Site- Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993). Briefly, EGFR- positive A431 cancer cells were incubated with varying concentrations of 7D12 and its Bpa- containing mutants in DM EM media containing 10% serum in a 96-well plate. After incubation, unbound 7D12 was removed, cells were fixed to the surface, and 7D12 bound to the surface of A431 cells was estimated by detecting the hexa-histidine (His ₆ ) tag at the C-terminus of 7D12. Our on-cell assay demonstrates that the presence of Bpa at positions 32 and 113 in 7D12 inhibits its binding to EGFR (Figure 2A). However, Bpa at position 109 decreases the binding affinity by only two-fold and the dissociation constant (K _D ) of wt7D12 and 7D12-109Bpa to EGFR were estimated to be 27 (±1.5) nM and 48 (±7.2) nM, respectively (Figure 2B). Previous investigations focused on introducing photoreactive functional groups at the antibodyantigen binding interface have often led to 10- to 100-fold decease in K _D values (Brasino, M., et al., Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors. J. Am. Chem. Soc., 2018. 140, 11820-11828, Islam, M., et al., Chemical Diversification of Simple Synthetic Antibodies. ACS Chem. Biol., 2021. 16, 344-359). Subsequently, we evaluated if Bpa at position 109 allows light-dependent covalent bond formation between 7D12 and EGFR. In vitro experiments were performed by incubating 7D12- 109Bpa with the extracellular domain of EGFR (sEGFR), followed by irradiation with 365 nm light. Samples were analysed by denaturing SDS-PAGE and a band higher than sEGFR indicates the covalently linked 7D12-EGFR complex. For these experiments, the amount of sEGFR was fixed at 10 picomoles, to be able to see a clearly defined band on SDS-PAGE gels.

We assessed the effect of incubation time, and irradiation time, on photocrosslinking efficiency. sEGFR was incubated with 10-fold excess of 7D12-109Bpa for 5 and 15 minutes, and the samples were irradiated for 0, 4, and 15 minutes. Incubation time prior to irradiation was seen to have a negligible effect on photocrosslinking efficiency. In contrast, as the irradiation increased from 4 to 15 minutes, the percentage of photocrosslinked product increased from 17% to 46%, respectively (Figure 2C). As long irradiation times could be toxic to cells, we also assessed the viability of A431 cells irradiated with 365 nm light for 0 to 15 minutes (Figure 3). Greater than 90% of A431 cells were found to be viable after a 10-minute exposure to 365 nm light. Thus, the irradiation time was fixed to 10 minutes. Next, we evaluated the effect of the relative amounts of 7D12-109Bpa to sEGFR, on the photocrosslinking efficiency. 10 picomoles of sEGFR was incubated with 0 to 200 picomoles of 7D12-109Bpa for 5 minutes, and samples were irradiated with 365 nm for 10 minutes. Photocrosslinking efficiency saturates at 43% when the concentration of 7D12-109Bpa is 10-fold or higher compared to sEGFR (Figure 2D). The photocrosslinking efficiency observed with the 7D12-109Bpa is similar to other photoreactive proteins (Brasino, M., et al., Anti-EGFR Affibodies with Site-Specific Photo- Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors. J. Am. Chem. Soc., 2018. 140, 11820-11828, Chin, J.W., et al., Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Proc. Natl. Acad. Sci. U S A, 2002. 99, 11020-4). Taking these results together, we concluded that the optimal conditions to assess photocrosslinking are, in a 10 μl reaction, 10 picomoles of sEGFR, 100 picomoles of 7D12 mutants, 5 minutes of incubation time prior to irradiation, and irradiation time of 10 minutes.

Next, we demonstrated that the light-dependent covalent bond formation between 7D12- 109Bpa and EGFR is antibody-specific, and antigen-specific. Photocrosslinking of sEGFR was performed with wt-7D12, 7D12-32Bpa, 7D12-109Bpa, and 7D12-113Bpa using our optimised conditions as described above. In these experiments, the crosslinked product was observed only for 7D12-109Bpa when irradiated with 365 nm light (Figure 2E). The results demonstrate that light-dependent covalent bond formation between 7D12 and EGFR: 1) requires site- specifically incorporated Bpa, as no covalent bond formation was observed between wt-7D12 and EGFR when irradiated with 365 nm light, and 2) requires specific binding between 7D12 and EGFR. 7D12-32Bpa and 7D12-113Bpa that do not bind to EGFR (Figure 2A), are unable to form a covalent bond with EGFR upon irradiation with light. To test that 7D12-109Bpa- EGFR bond formation is antigen-specific, we also irradiated 7D12-109Bpa with an unrelated protein, bovine serum albumin (BSA) that resulted in no crosslinked product (Figure 2F).

Furthermore, we demonstrated that photocrosslinking of 7D12-109Bpa can be performed in serum containing media (Figure 2G). 7D12-109Bpa was incubated with sEGFR in phosphate buffered saline (PBS) as a control similar to previous in vitro photocrosslinking experiments or DM EM media containing 10% serum, then irradiated with 365 nm light and subsequently analysed by denaturing SDS-PAGE. In the resulting Coomassie stained gel the crosslinked product is observed for reactions carried out in PBS but is masked by other proteins for reactions performed in serum containing DMEM media. However, a western blot detecting the 7D12-109Bpa C-terminal Hise tag shows a band corresponding to the sEGFR-7D12-109Bpa complex in both reactions, performed in PBS and in serum, but only after irradiation with 365 nm demonstrating light-mediated crosslinking of 7D12-109Bpa to sEGFR in serum containing media. We thus developed a high affinity photoreactive 7D12 mutant, 7D12-109Bpa, that selectively forms a covalent bond with EGFR upon irradiation with light under biologically relevant conditions.

We also characterised the photocrosslinked 7D12-109Bpa-sEGFR complex using mass spectrometry. The band corresponding to the photocrosslinked 7D12-109Bpa-sEGFR complex was excised from SDS-PAGE, destained, and digested with trypsin/chymotrypsin. The digested sample was then analysed by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Peptide fragments corresponding to 7D12 (73% coverage) and sEGFR (62% coverage) were observed in the photocrosslinked 7D12-109Bpa-sEGFR complex analysed by MS/MS (Figure 4 and 5).

Next, we aimed to develop a photoactive photoreactive mutant of 7D12. We have earlier demonstrated that site-specific incorporation of photocaged tyrosine (pcY) at position 32 in 7D12 inhibits its binding to EGFR, and irradiation with 365 nm light restores this binding (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993). Hence, we examined whether site-specific incorporation of Bpa at position 109, and pcY at position 32 could confer concurrent photoreactivity and photoactivity to 7D12.

Expression of proteins containing two distinct site-specifically incorporated ncAAs in live cells requires two mutually orthogonal aaRS/ tRNA pairs, which are also orthogonal to the host aaRS/ tRNA pairs. In addition, these aaRSs should be able to charge the tRNA exclusively with their corresponding ncAA in the presence of other ncAAs and canonical amino acids, which could be a potential challenge for structurally similar pcY and Bpa (Guo, L.T., et al., Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proc. Natl. Acad. Sci. II S A, 2014. 111 , 16724-9, Hohl, A., et al., Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen. Sci. Rep., 2019. 9, 11971). In E. coli, Methanosarcina barkeri Pyrrolysyl-tRNA synthetase (Mb PyIRS)/ Mb PyltRNA, and MjRS/ MjtRNA pairs have been shown to be orthogonal to each other and have enabled site-specific incorporation of several pairs of ncAAs into model proteins (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403, Sachdeva, A., et al, Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc., 2014., 136, 77785-7788). We decided to use these two aaRS/ tRNA pairs for site-specific incorporation of pcY and Bpa in 7D12. In order to select which of the two pairs to employ for site-specific incorporation of pcY/ Bpa, we first assessed the selectivity of the known MjRS(Bpa) and MjRS(pcY) that have been used for site- specific incorporation of Bpa and pcY, respectively (Chin, J.W., et al., Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Proc. Natl. Acad. Sci. II S A, 2002. 99, 11020-4, Deiter, A., et al., A genetically encoded photocaged tyrosine. Angew. Chem. Int. Ed. Engl., 2006. 45, 2728-31). We employed both these aaRSs for site-specific incorporation of pcY and Bpa in response to a TAG stop codon at position 109 in 7D12. Protein expression was performed in the absence and presence of pcY or Bpa (Figure 6). For theMjRS(pcY), the full-length 7D12 was only observed when expression was performed with pcY and no protein was observed for expression with Bpa. However, theMjRS(Bpa) appears to be promiscuous, and incorporates both Bpa and pcY into 7D12. Thus, for dual incorporation of pcY and Bpa in 7D12, we decided to employ the highly selective MjRS(pcY) for the site-specific incorporation of pcY, and the Mb PyIRS for site-specific incorporation of Bpa into 7D12. Next, we embarked upon evolving an efficient and selective Mb PyIRS for the site-specific incorporation of Bpa. Example 2: Directed evolution of M. barken Pyrrolysyl-tRNA synthetase (PylRS)ZtRNA pair to allow efficient and selective site-specific incorporation of p-benzoyl-L- phenylalanine (Bpa)

To evolve an efficient and specific Mb PyIRS for site-specific incorporation of Bpa, we generated a library of Mb PyIRS mutants. Four amino acid residues in the amino acid binding pocket of Mb PyIRS, viz. N311 , C313, W382 and W386, were randomized to all combinations of amino acids (Figure 7A). In addition, the Y349F mutation that is known to improve the aminoacylation efficiency of MbPyIRS was introduced in the library ^[39] . Using this library, three rounds of alternating positive and negative selections were carried out to isolate Bpa-specific Mb PyIRS mutants. Positive selection was performed in the presence of Bpa and allowed survival of cells containing Mb PyIRS mutants that incorporate any amino acid in response to a TAG stop codon in the chloramphenicol acetyltransferase (CAT) gene. The negative selection was performed in the absence of Bpa and assisted in eliminating Mb PyIRS mutants from the library that incorporate canonical amino acids. After the final positive selection, 192 clones were screened at various concentrations of chloramphenicol in the presence and absence of Bpa. Two clones, viz. A2 and E10, survived at concentrations of chloramphenicol up to 300 μg/ml in the presence of Bpa, where no growth was observed in the absence of Bpa (Figure 7B), indicating that these two clones specifically incorporate Bpa into the chloramphenicol acetyltransferase in response to a TAG stop codon. Both these clones were sequenced and had the following identical mutations compared to the wild-type Mb PyIRS: N311Q, C313T and W382A, in addition to the Y349F pre-programmed mutation, whereas position 386 remained unchanged. We named this mutant aaRS, Mb Pyl(Bpa)RS.

Next, we assessed the specificity and efficiency of the Mb Pyl(Bpa)RS by expressing a fusion protein of glutathione-S-transferase and calmodulin (Gst-CaM), where the first amino acid in calmodulin is incorporated in response to a TAG stop codon (gst-1TAG-cam). These experiments allowed us to estimate ncAA incorporation efficiency by measuring the relative amount of full-length Gst-CaM to Gst. Cells were supplied with the gst-1TAG-cam gene, Mb PyltRNAcuA and Mb Pyl(Bpa)RS. Protein expression was performed with Bpa, pcY, azidophenylalanine (AzF), Boc-protected lysine (BocK) and without any ncAA. In parallel, we also performed a similar expression experiment with wt-Mb PylRS in the presence and absence of BocK. Cells were lysed and protein purification was performed using glutathione sepharose beads to isolate Gst-tagged proteins (Figure 7C). Using the ratio of band intensities for full- length Gst-CaM to the sum of intensities of Gst-CaM and Gst, the efficiency of Mb Pyl(Bpa)RS and wt-MbPylRS at incorporating Bpa and BocK was estimated to be 49% and 40%, respectively (Figure 8). It may be noted that ncAA incorporation efficiency is not an absolute number and can vary widely with different plasmid systems (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993, Chatterjee, A., et al., A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry, 2013. 52, 1828-37). It is the comparison between Mb Pyl(Bpa)RS and wt- Mb PylRS which is more important, with the latter known to efficiently incorporate BocK, which is why it is often used as a benchmark (Wan, W., et al., Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta, 2014. 1844, 1059-70). These results demonstrate that the newly evolved Mb Pyl(Bpa)RS/MbPyltRNAcuA pair is highly efficient at site-specific incorporation of Bpa into proteins. This newly evolved Mb Pyl(Bpa)RS was also found to be more efficient than the previously knownMmPyIRS mutant, BpaRSI (Lacey, V.K., et al., Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings. Chembiochem, 2013. 14, 2100-5) (Figure 8). Furthermore, expression of Gst-CaM using the MbPyl(Bpa)RS/ Mb PyltRNAcuA pair in the presence of AzF, BocK and pcY demonstrated that Mb Pyl(Bpa)RS does not incorporate AzF or BocK and is 5-fold more specific for Bpa compared to pcY.

Subsequently, we assessed the specificity of the newly evolved Mb Pyl(Bpa)RS at incorporating Bpa into 7D12 in the presence of pcY. A single plasmid containing all three genetic elements, viz. 7D12-32TAG or 7D12-109TAG, MbPyltRNAcuA and Mb Pyl(Bpa)RS, required for the site-specific incorporation of Bpa into 7D12 using Mb Pyl(Bpa)RS was constructed. We named these plasmids as: pSANG_7D12-32TAG_Mb Pyl(Bpa)_tRNAcuA and pSANG_7D12-109TAG_MbPyl(Bpa)_tRNAcuA. BL21(pLysS) cells were supplied with pSANG_7D12-32TAG_MbPyl(Bpa)_tRNAcuA or pSANG_7D12-

109TAG_Mb Pyl(Bpa)_tRNAcuA, and protein expression was performed without any ncAA, with Bpa, with pcY, or with equimolar amounts of Bpa and pcY (Figure 7D). Though in the absence of Bpa, Mb Pyl(Bpa)RS catalyses some incorporation of pcY, mass spectrometry results confirm that Bpa outcompetes pcY when the expression of 7D12 was performed in the presence of equimolar amounts of Bpa and pcY (Figure 7E). Using Mb Pyl(Bpa)RS, we did not observe molecular weight corresponding to incorporation of pcY at either of the two positions, 32 or 109, in 7D12 when protein expression was performed in the presence of Bpa and pcY. Thus, we evolved a highly efficient and specific Mb PylRS(Bpa)/ tRNA pair that exclusively incorporates Bpa in the presence of pcY, and could be employed for dual incorporation of Bpa and pcY in 7D12.

Example 3: Development of a photoactive photoreactive 7D12 mutant by site-specific incorporation of pcY and Bpa in 7D12

Two distinct ncAAs have been site-specifically incorporated into proteins expressed in E. coli either by assigning: i) two distinct stop codons to ncAAs (Italia, J.S., et al., Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites. J. Am. Chem. Soc., 2019. 141 , 6204-6212), ii) a stop codon and a quadruplet codon to ncAAs (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403), and iii) two distinct quadruplet codons to ncAAs (Dunkelmann, D.L., et al., A 68- codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat. Chem., 2021. 13(11): p. 1110-1117). Of these, the expression system that utilizes a stop and a quadruplet codon, a quadruplet decoding evolved orthogonal ribosome and a quadruplet decoding evolved Mb PyltRNAs has demonstrated good protein yields (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403, Sachdeva, A., et al, Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc., 2014., 136, 77785-7788). We decided to employ this system for dual incorporation of pcY and Bpa at positions 32 and 109, respectively, in 7D12.

First, we developed a plasmid for expressing wt-7D12 using the orthogonal ribosome. The ribosome binding site (RBS) of 7D12 was changed to an orthogonal RBS (oRBS), forming the pSANG-o7D12 plasmid. Subsequently, a rRNA operon containing an evolved orthogonal 16S ribosomal RNA gene, as well as a 23S and 5S ribosomal RNA genes was cloned into the pSANG-o7D12 plasmid, forming pSANG-oR-o7D12. 0.7 mg of wt-7D12 per litre of culture was obtained using the pSANG-oR-o7D12 plasmid (Figure 9). Next, we mutated positions 32 and 109 in 7D12 to a TAG and an AGTA codon, respectively, forming pSANG-oR-o7D12-Dual plasmid. Our newly evolved Bpa-specific Mb PylRS(Bpa) and an AGTA-decoding evolved Mb PyltRNAuAcu were then transplanted into the pSANG-oR-o7D12-Dual plasmid, creating a single plasmid that contains genetic components to assemble an orthogonal ribosome for the expression of 7D12, and allows for the encoding of Bpa in response to an AGTA codon. We named this plasmid: pSANG-oR-o7D12-Dual-Pyl(Bpa) (Figure 10A). pSANG-oR-o7D12-Dual- Pyl(Bpa) plasmid was co-transformed with pULTRA-pcY plasmid into BL21 cells for sitespecific incorporation of pcY at position 32 using /MjRS(pcY)/ /MjtRNAcuA pair and Bpa at position 109 using Mb PylRS(Bpa)/ evMb PyltRNAuAcu pair, in 7D12. (Figure 10A).

Protein expression was performed without the addition of any ncAA, with pcY, with Bpa, and with pcY and Bpa (Figure 10B and Figure 11). Full-length 7D12 was observed when the expression was performed with both pcY and Bpa. In addition, some full-length protein was also obtained for expression with only pcY. This might be due to the undesired incorporation of pcY by Pyl(Bpa) in the absence of Bpa, which is consistent with our previous data shown and discussed in Figures 7C, 7D, and 7E. Mass spectrometry results for expression products obtained in the presence of both ncAAs, pcY and Bpa, prove dual incorporation of pcY and Bpa into 7D12 (Figure 10C). Taken together, these results demonstrate site-specific incorporation of pcY at position 32 and Bpa at position 109 in 7D12, forming 7D12-32pcY- 109Bpa.

Next, we measured the binding affinity of 7D12-32pcY-109Bpa to EGFR before and after 365 nm irradiation using our on-cell assay. As a control, we also measured the binding affinity of wt-7D12, 7D12-32pcY and 7D12-109Bpa. Without irradiation, near-background binding was observed for 7D12-32pcY-109Bpa and 7D12-32pcY. This is due to pcY at position 32 that inhibits 7D12-EGFR binding, consistent with our previous observations (Bridge, T., et al., Site- Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993) (Figure 10D). The binding of 7D12-32pcY-109Bpa to EGFR was restored when the sample was irradiated with 365nm light for 10 minutes. Also, as demonstrated in the previous section, Bpa at position 109 does not inhibit binding. The binding affinity of 7D12-32pcY-109Bpa to EGFR after irradiation was estimated to be 103 (±25) nM (Figure 10E). These results demonstrate that 7D12-32pcY- 109Bpa is a photoactive antibody.

We also demonstrate that both 7D12-32pcY-109Bpa and 7D12-109Bpa show near background level binding to MDA-MB-231 and SW620 cells when compared to A431 cells, before or after irradiation with 365 nm light (Figure 11). MDA-MB-231 cell line expresses significantly lower levels of EGFR than A431 cells, and SW620 cells have been previously used as EGFR-negative cell line (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993, Ross, S.L., et al., Bispecific T cell engager (BiTE(R)) antibody constructs can mediate bystander tumor cell killing. PLoS One, 2017. 12, e0183390, Kruwel, T., In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor. Sci. Rep., 2016. 6, 21834). These results further demonstrate that the binding between 7D12 mutants containing site-specifically incorporated Bpa/pcY remain specific to EGFR-expressing cells before/after irradiation with 365 nm light.

Next, we assessed the photoreactive property of 7D12-32pcY-109Bpa. 100 picomoles of 7D12-32pcY-109Bpa were incubated with 10 picomoles of sEGFR and subsequently irradiated with 365 nm light. As a control, we performed similar experiments with wt-7D12, 7D12-32pcY and 7D12-109Bpa (Figure 10F and Figure 9). A higher molecular weight band than that of sEGFR, indicating EGFR covalently linked to 7D12, was observed only for samples containing 7D12-109Bpa and 7D12-32pcY-109Bpa after 365 nm irradiation. 33.4% of 7D12-32pcY- 109Bpa was estimated to crosslink to EGFR. Additionally, photocrosslinking of 7D12-32pcY- 109Bpa was assessed in serum containing media. Comparison of a Coomassie stained gel and an anti-His ₆ western blot for detecting crosslinked product demonstrates successful light- mediated crosslinking of 7D12-32pcY-109Bpa to sEGFR in serum-containing media (Figure 10G). These results demonstrate that 7D12-32pcY-109Bpa is a photoreactive antibody. Taken together, results from both the binding and the photocrosslinking assays demonstrate that 7D12-32pcY-109Bpa is a photoactive photoreactive antibody fragment.

Materials and methods

Cell lines and noncanonical amino acids and general methods: Human epithelial squamous carcinoma cell line, A-431 ; Human breast adenocarcinoma cell line, MDA-MB-231 and Human colon adenocarcinoma cell line, SW620 were purchased from Sigma-Aldrich. All mammalian cell lines were cultured in DMEM (Gibco, Invitrogen) containing L-glutamine, 4.5 g/L D-Glucose, 110 mg/L Sodium pyruvate, 10% (v/v) foetal bovine serum (FBS), and a cocktail of penicillin and streptomycin (PEN/STREP). This medium will be referred to as “complete medium”. Mammalian cells were grown in humidified atmosphere in 5% CO2 at 37°C. p-benzoyl-L-phenylalanine (Bpa) was purchased from Fluorochem, and p-Azido-L- phenylalanine (AzF) and N6-(tert-butoxycarbonyl)-L-lysine (BocK) were purchased from BACHEM. O-(2-Nitrobenzyl)-L-tyrosine (photocaged tyrosine, pcY) was synthesized using the procedures similar to reported earlier (Deiter, A., et al., A genetically encoded photocaged tyrosine. Angew. Chem. Int. Ed. Engl., 2006. 45, 2728-31).

Routine liquid chromatography-mass spectrometry (LC-MS) analysis was carried out as previously described on a Bruker microQTOF-QIII mass spectrometer (Keller, A., et al., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem., 2002. 74, 5383-92).

Construction of pULTRA_MjRS(Bpa)/MjtRNAcuA: pULTRA-CNF plasmid (Addgene plasmid #48215) was digested with Not\ and the reaction mixture was run on a 1% agarose gel. The band corresponding to the plILTRA backbone was cut out and extracted using the QIAquick Gel Extraction Kit (QIAGEN). The MjRS(Bpa) was amplified using PCR from pSUP/MjRS(Bpa)/tRNAcuA plasmid (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403) and subsequently cloned into the plILTRA backbone using Gibson cloning (New England Biolabs). The identity of pULTRA_MjRS(Bpa)//MjtRNA _CU a plasmid was confirmed by Sanger sequencing. This plasmid is referred to as pULTRA-Bpa in the text.

Expression and purification of amber mutants of 7D12 with site-specifically incorporated p-benzoyl-L-phenylalanine (Bpa): Chemically competent BL21 (DE3)pLysS cells containing pULTRA-Bpa were transformed with pSANG10_7D12, pSANG10_7D12- 32TAG, pSANG10_7D12-109TAG, or pSANG10_7D12-113TAG plasmids. After transformation, cells were recovered in 1 ml SOB medium for one hour at 37°C. 50 pl of recovered cells were transferred onto LB-agar plates supplemented with 50 pg ml ^-1 kanamycin and 100 pg ml ^-1 spectinomycin. The plates were incubated overnight (37°C, 16 h). A single colony from each plate was used to inoculate 50 ml of 2xTY-GKS media (2xTY media with 4% glucose, 50 pg ml ^-1 kanamycin and 100 pg ml ^-1 spectinomycin) and incubated overnight (37°C, 220rpm, 16 h). The next day, this culture was used to inoculate fresh 500 ml 2xTY-GKS media to an OD ₆₀₀ of 0.1. This was then incubated until OD ₆₀₀ reached 0.4-0.6 (37°C, 220rpm, 2-3 h), at which point IPTG (1 mM final concentration) was added to induce the expression of 7D12 and the culture was split into two, one half supplemented with 2 mM Bpa (positive samples in Figure 1 B) and to the other half nothing was added (negative samples in Figure 1 B). The cultures were incubated overnight (30°C, 160rpm, 16 h). The following day, cells were pelleted (3,200g, 4°C, 10 minutes), the supernatant was discarded, and the cells were resuspended in 25 ml periplasmic extraction buffer-1 (20% sucrose, 100 mM Tris-HCI, 1 mM EDTA, pH 8.0). The resuspended cells were incubated on ice for 30 minutes and then centrifuged (10,000g, 4°C, 10 minutes). The supernatant was removed and stored at 4°C (periplasmic fraction-1). The resulting pellet was resuspended in 25 ml periplasmic extraction buffer 2 (5 mM MgCl ₂ and incubated on ice for 20 minutes. The samples were centrifuged (10,000g, 4°C, 10 minutes) and the supernatant was collected (periplasmic fraction-2). Both the periplasmic fractions were combined, passed through a 0.2-pm filter, and dialyzed overnight at 4°C against 1x phosphate buffered saline (PBS). The following day, 500pl of Ni-NTA resin (ThermoFisher Scientific) was added to the dialyzed solution and mixed gently on a rocker (4°C, 1 h). This was transferred into a gravity-flow column and washed three times with 10 ml PBS buffer each time. The resin was then washed twice with 8 ml Ni-NTA wash buffer (50 mM Tris-HCI, 300 mM NaCI and 20 mM imidazole, pH 8.0). To elute the bound 7D12, 500 pL Ni-NTA elution buffer (50 mM Tris- HCI, 300 mM NaCI and 200 mM imidazole, pH 8.0) was added and incubated for 15 minutes at room temperature, and the elution fraction was collected. This process was repeated 8 times, the elution fractions were pooled together and dialyzed overnight at 4°C against PBS buffer. Dialyzed samples were then concentrated using a Vivaspin 500 column with 3 kDa molecular weight cut-off (GE Healthcare) and the yields were determined using a colorimetric Pierce BCA protein assay (Thermo Fisher Scientific) measured at 562 nm. After protein purification and concentration, the samples were subsequently resolved by SDS-PAGE. To this end, Nu-PAGE LDS loading buffer (Invitrogen) was added to 20 pl of protein samples and heated at 95°C for 15 minutes, then centrifuged (13,000 g, 15 minutes, 4°C) and loaded on a 4-12% Bis-Tris gel (Invitrogen) along with SeeBlue Plus2 protein ladder (ThermoFisher Scientific). The gel was then stained with Coomassie Blue (InstantBlue, Abeam) and the identity of the protein was further confirmed by electrospray ionization mass spectrometry coupled with liquid chromatography, LC-MS. The molecular weights determined through SDS- PAGE and mass spectrometry were in good agreement with the expected molecular weight of 7D12 and its mutants with site-specifically incorporated Bpa (Figure 1 B). Similar method was employed for the expression and purification of 7D12 mutants containing pcY.

On-cell assay for measuring the binding of His-tagged antibody fragments: All cell lines were grown in Dulbecco’s modification of Eagle medium (DMEM; Gibco, Invitrogen) containing L-glutamine, 4.5 g/L D-Glucose, 110 mg/L Sodium pyruvate, 10% (v/v) foetal bovine serum (FBS), and 1% (v/v) cocktail of penicillin and streptomycin (PEN/STREP, Sigma-Aldrich), which will be referenced as “complete medium” in the text. Cells were grown in a T-75 flask in complete medium (DMEM, 10% FBS, 1% PEN/STREP) using standard tissue culture procedures until 80-90% confluence. After washing with 1x phosphate buffered saline (PBS) and trypsinising, cells were pelleted (300g, 5 minutes) and resuspended in 10 ml fresh complete medium. The cells were then counted on a hemocytometer and diluted to 200,000 cells/ml. 200 pl of this solution was dispensed into each well (40,000 cells/well) of a white 96- well plate (Corning) and grown overnight. After 12-16 h, the medium was replaced with 200 pl of complete medium supplemented with 7D12 or its mutants at the desired concentration. The plate was incubated for 5 minutes (37°C, 5% CO2). If the binding affinity is measured after 365 nm irradiation, this plate was placed on a transilluminator (GelDocMega; BioSystematica) and irradiated with a photon flux and the intensity of 33 mW/cm ² and 14 mW, respectively, at 365 nm for 10 minutes. Note that the photon flux and the intensity of 365 nm light from the UV transilluminator were measured using a laser power meter (FieldMate; Coherent) at the surface of the transilluminator where the samples were placed. After removing medium, the cells were washed once with complete medium (150pl) and then fixed using formaldehyde; 150 pl of 3.7% formaldehyde solution in sterile Mili-Q water was added to each well and incubated for 20 min at room temperature. The formaldehyde solution was removed and cells were washed three times (150 pl, 5 minutes, gentle rocking) with PBST (1X PBS supplemented with 1% Tween-20). After removing the wash buffer, 100 pl of blocking buffer (10% milk in PBST) was added and cells were incubated at room temperature for 1 h with gentle rocking. The blocking buffer was removed. 50 pl solution containing primary anti-6x-His tag antibody was added to each well and the plate was incubated at room temperature for 1 h. The primary antibody solution contained mouse anti-6x-His tag antibody (ThermoFisher Scientific) at 1 :1000 dilution and 1% milk in PBST. After incubation with the primary antibody, cells in each well were washed three times with PBST (150 pl, 5 minutes, gentle rocking). Subsequently, 50 pl of HRP-linked secondary antibody solution was applied to each well and incubated at room temperature for 1 h. The HRP-linked secondary antibody solution contained anti-mouse-HRP- linked antibody (Cell Signaling Technology) at 1 :1500 dilution and 1 % milk in PBST. After incubation with secondary antibody, the cells were washed six times with PBST (150 pl, 5 minutes, gentle rocking). Finally, 150 pl of SuperSignal chemiluminescent Substrate (ThermoFisher Scientific) was added to each well and the plate was imaged using GelDoc XR+ (Bio-Rad). The chemiluminescence intensity in each well was further quantified by measuring the signal using a CLARIOstar plate reader (BMG labtech). At least three replicates were performed for each on-cell assay.

In vitro experiments to assess light-dependent covalent bond formation between 7D12 mutants and sEGFR: Various volumes of 50 pM stock of 7D12 mutants were mixed with 1 pl of 10 pM sEGFR (PeproTech) and 1 pl of 10X PBS in a final volume of 10 pl. The reaction mixture was incubated for 5 minutes at 37°C. Samples were aliquoted on to a cover slip (VWR, Catalog No. 631-0153) and then irradiated with 365 nm light using a transilluminator (GelDocMega; BioSystematica) with a photon flux and the intensity of 33 mW/cm ² and 14 mW, respectively, at 365 nm for 10 minutes. Note that the photon flux and the intensity of 365 nm light from the UV transilluminator were measured using a laser power meter (FieldMate; Coherent) at the surface of the transilluminator where the samples were placed. sEGFR in the reaction mixture and the crosslinked product was then enzymatically deglycosylated using PNGase F (New England Biolabs) before analysis using SDS-PAGE. This was achieved by adding 1 pl of glycoprotein denaturing buffer (10X) to the above reaction mixture, followed by incubation at 95°C for 10 minutes. Denatured samples were then transferred to ice and incubated for 5 minutes followed by centrifugation at 13,000g for 5 minutes at 4°C. This was followed by addition of 2 pl GlycoBuffer 2 (10X), 2 pl 10% NP-40, 5 pl H2O and 1 pl PNGase F, and incubation at 37°C for 1 hour. After addition of Nu-PAGE LDS loading buffer (Invitrogen) the samples were run on a 4-12% Bis-Tris gel (Invitrogen) along with protein ladder (SeeBlue Plus2 Prestained standard from ThermoFisher Scientific) as a marker. The gel was then stained with Coomassie Blue (InstantBlue, Abeam) and imaged using GelDoc (Bio-Rad).

In vitro experiments to assess light-dependent covalent bond formation between 7D12 mutants and sEGFR in serum containing media: 2 pl of 50 pM stock of 7D12 mutants were mixed with 2 pl of 5 pM sEGFR (PeproTech) and 5 pl of DMEM media containing 20% (v/v) serum in a final volume of 10 pl. DMEM media is Dulbecco’s modification of Eagle medium (DMEM; Gibco, Invitrogen) containing L-glutamine, 4.5 g/L D-Glucose, 110 mg/L Sodium pyruvate. Serum is foetal bovine serum (FBS). The reaction mixture was incubated for 5 minutes at 37°C. Samples were aliquoted on to a cover slip (VWR, Catalog No. 631-0153) then irradiated with 365 nm light using a transilluminator (GelDocMega; BioSystematica) with a photon flux and the intensity of 33 mW/cm ² and 14 mW, respectively, at 365 nm for 10 minutes. Note that the photon flux and the intensity of 365 nm light from the UV transilluminator were measured using a laser power meter (FieldMate; Coherent) at the surface of the transilluminator where the samples were placed. sEGFR in the reaction mixture and the crosslinked product was then enzymatically deglycosylated using PNGase F (New England Biolabs) before analysis using SDS-PAGE. This was achieved by adding 1 pl of glycoprotein denaturing buffer (10X) to the above reaction mixture, followed by incubation at 95°C for 10 minutes. Denatured samples were then transferred to ice and incubated for 5 minutes followed by centrifugation at 13,000g for 5 minutes at 4°C. This was followed by addition of 2 pl GlycoBuffer 2 (10X), 2 pl 10% NP-40, 5 pl H2O and 1 pl PNGase F, and incubation at 37°C for 1 hour. After addition of Nu-PAGE LDS loading buffer (Invitrogen) the samples were run on a 4-12% Bis-Tris gel (Invitrogen) along with protein ladder (SeeBlue Plus2 Prestained standard from ThermoFisher Scientific) as a marker. The gel was then either stained with Coomassie Blue (InstantBlue, Abeam) and imaged using GelDoc (Bio-Rad), or the proteins were transferred onto a nitrocellulose membrane (Invitrogen™ iBIot™ 2 Transfer Stacks, nitrocellulose) using an iBIot 2 Dry Blotting System (Thermo Fisher Scientific) for western blot. After transferring the proteins onto the nitrocellulose membrane, the membrane was incubated with the blocking buffer (10% milk in phosphate buffered saline (PBS) containing 0.1 % Tween- 20 (called PBS-T later in the text)) for 1 hour at room temperature. Subsequently, the blocking buffer was removed and the membrane was washed with PBS-T. The membrane was then incubated with the primary antibody (6x-His Tag mAb (HIS.H8) mouse (Thermo Fisher Scientific) at 1 :2000 dilution in 1 % milk, PBS-T) overnight at 4°C. Subsequently, the membrane was washed three times with PBS-T. The membrane was then incubated with the secondary antibody (Anti-mouse IgG, HRP linked antibody (Cell Signaling Technology) at 1 :3000 dilution in 1 % milk, PBS-T) for 1 hour at room temperature. Subsequently, the membrane was washed three times with PBS-T. Finally, for signal development, the membrane for incubated with the substrate for Horse Radish Peroxidase, SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). The membrane was then imaged using a GE ImageQuant™ LAS 4000 gel imager.

Effect of 365 nm irradiation on A431 cells assessed using cell viability assay: A431 cells were grown in a T-75 flask in complete medium using standard tissue culture procedures until 80-90% confluence. Cells were then washed once with Dulbecco's phosphate-buffered saline (DPBS), detached using Trypsin-EDTA, pelleted (300g, 5 minutes) and resuspended in 10 ml fresh complete medium. The resuspended cells were then counted using hemocytometer, and diluted to 200,000 cells/ml of complete medium. 200 pl of these cells were seeded (40,000 cells/well) into each well of a white 96-well plate (Corning). The plates were incubated overnight, 12-16 hours (37°C, 5% CO2). The following day medium in each well was replaced with fresh pre-warmed complete medium. These plates were then irradiated with 365nm light for 0, 5, 10 and 15 minutes using a transilluminator (GelDocMega; BioSystematica) at 32.8 mW/cm ² . After irradiation, medium in each well was removed and replaced with 90 pl fresh prewarmed complete medium and 10 pl alamarBlue reagent. This was incubated for 2 hours (37°C, 5% CO2). The fluorescence emission at 590 nm (with excitation at 560 nm) from each well was quantified using CLARIOstar plate reader (BMG labtech). The fluorescence intensity of a standard containing no cells was subtracted from the fluorescence intensity from each well and plotted as a bar graph. Six replicates of each experiment were performed. Mass spectrometry analysis of photocrosslinked 7D12-109Bpa-sEGFR complex: The band corresponding to photocrosslinked 7D12-EGFR was excised from SDS-gels, destained, and digested with sequencing grade trypsin/chymotrypsin, as previously described. ^[62] Aliquots of the peptides were used for LC-MS/MS analysis on an Orbitrap Eclipse™ Tribrid™ mass spectrometer (Thermo Fisher, Hemel Hempstead, UK equipped with an UltiMate™ 3000 RSLCnano System (Thermo Fisher) using a nanoEase M/Z column (HSS C18 T3, 100 A, 1.8 μm; Waters, Wilmslow, UK). The samples were loaded and trapped using a pre-column with 0.1 % TFA at 20 pl min ^-1 for 3 min. The trap column was then switched in-line with the analytical column for separation using the following long gradient of solvents A (water, 0.05% formic acid) and B (80% acetonitrile, 0.05% formic acid) at a flow rate of 0.2 pl min ^-1 : 0-4 min 3% B; 4-10 min linear increase B to 7%; 10-70 min increase B to 37%; 70-90 min increase B to 55%; followed by a ramp to 99% B and re-equilibration to 3% B, for a total running time of 122 minutes. The peak lists were used to search against a custom database containing the proteins of interest in a background of common contaminants (MaxQuant) using an in-house Mascot Server version 2.8.0 (Matrix Science, London, UK) with trypsin/chymotrypsin digestion and 2 missed cleavages. Oxidation (Met), acetylation (protein N-terminus), and deamidation (Asn, Gin) were used as variable modifications and carbamidomethyl (Cys) as a fixed modification. Mass tolerances were 6 ppm for precursor ions and 0.6 Da for fragment ions. Scaffold version 5.1.2 (Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction (Keller, A., et al., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem., 2002. 74, 5383-92). Peptide identifications of >95.0% probability and protein identifications >99.0% probability by the Peptide Prophet algorithm were accepted with at least 1 identified peptide in Scaffold 5.

M. barkeri Pyrrolysyl-tRNA synthetase library generation for directed evolution: We first constructed the Y349F mutant of PylRS. pBK_pylRS (Nguyen, D.P., et al., Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells. J. Am. Chem. Soc., 2014. 136, 2240-3) was PCR amplified using primer pair: Mb_pylRS_Y349F_F and Mb_pylRS_Y349F_R (Table 1), and Q5-DNA polymerase (New England Biolabs, NEB) according to the manufacturer’s instructions. The PCR product was subsequently purified using PCR purification kit (Qiagen). The purified PCR product was digested with Dpnl and Bsal (NEB), ligated using T4 DNA ligase (NEB), and transformed into electrocom petent E. coli GeneHog cells (ThermoFisher Scientific). Plasmid isolated from the respective transformant was checked for the presence of the desired mutation using Sanger sequencing. Hereafter, the mutation carrying plasmid, pBK_pylRS_349F, was used as template for construction of the library. The first round of inverse PCR was performed to mutate positions 311 and 313 to NNK codons. This was achieved using primer pair: N311_C313_F and N311_C313_R (Table 1), and amplification by Q5-DNA polymerase (NEB). The PCR product was subsequently purified using PCR purification kit (Qiagen). The purified PCR product was digested with Dpnl and Bsal (NEB), ligated using T4 DNA ligase (NEB) and transformed into electrocompetent E. coli GeneHog cells. The electroporated cells were recovered in 1 ml SOC medium for one hour at 37°C. To this, 6 ml of LB media supplemented with Kanamycin (50 pg ml ^-1 in the final volume) was added, a small aliquot (0.1 ml) was taken to generate a serial dilution series that was plated onto LB agar containing Kanamycin in order to determine the number of clones in the library. The rest of the culture was incubated overnight (37°C, 220rpm, 12-16 h). Next morning, the cells were pelleted by centrifugation (3200g, 4°C, 10 min), and the plasmid DNA was isolated using Qiagen plasmid miniprep kit (Qiagen). This plasmid DNA library was called: pBK_pylRS_349F_311 NNK_313NNK. Using the LB agar plates we determined that the library contained a total of 1 x 10 ⁶ clones. The isolated plasmid DNA was used as a template for the next round of inverse PCR to mutate positions 382 and 386 to NNK codons. The procedure of PCR, digestion and transformation was again repeated. Note that 3 independent 50 pl PCR reactions were performed using 4 ng of pBK_pylRS_349F_(311 ,313)X template DNA each, primer pair MbpylRS_W382_G386X_F/ MbpylRS_W382_G386X_R (Table 1) and Q5-DNA polymerase (NEB). To guarantee a sufficient number of transformants in the final library a total of 12 electroporations was carried out and after regenerating the cells for 1 hour in 12 ml SOC medium at 37°C, 38 ml of LB medium and Kanamycin for a final concentration of 50 pg ml ^-1 were added and the culture was incubated shaking overnight. The final library contained a total of 1.8 x 10 ⁸ clones and was assessed through Sanger sequencing of the pylRS gene to verify its quality. This plasmid DNA library was called: pBK_pylRS_349F_(311 ,313,382,386)X.

Directed evolution of M. barken Pyrrolysyl-tRNA synthetase for efficient site-specific incorporation of p-benzoyl-L-phenylalanine (Bpa) into proteins: Freshly prepared E. coli GeneHog cells (ThermoFisher Scientific) carrying the pREP-PyIT plasmid ^[47] were electroporated with the pBK_pylRS_349F_(311,313,382,386)X library DNA. The cells were recovered in SOC media containing 1mM Bpa at 37°C for 1 hour, and subsequently plated onto LB agar containing 25 pg ml ^-1 kanamycin, 12.5 pg ml ^-1 tetracycline, 50 pg ml ^-1 chloramphenicol and 1mM Bpa. These plates were incubated at 37°C for 36-48 hours and the resulting colonies were washed with LB broth. Plasmid DNA from the colonies was isolated using Qiagen plasmid miniprep kit (Qiagen). The isolated plasmid DNA was digested with Dral (NEB) in order to remove the pREP plasmid, purified using SureClean (Bioline) and subsequently electroporated into E. coli GeneHog cells containing the pYOBB2 plasmid ¹⁴⁷¹ for the negative selection. The cells were recovered in SOC media containing 0.2 % arabinose at 37°C for 1 hour and plated onto LB agar containing 50 pg ml ^-1 kanamycin, 50 pg ml ^-1 chloramphenicol and 0.2 % arabinose. The plates were incubated at 37°C for 14-18 hours. The surviving colonies were washed with LB broth and the plasmid DNA was isolated using Qiagen plasmid miniprep kit. The isolated plasmid DNA was digested with Dral (NEB) in order to remove the pYOBB2 plasmid, purified using SureClean and subsequently electroporated into E. coli GeneHog cells containing pREP plasmid for the final positive selection. Procedure similar to the first positive selection was followed. 192 colonies from the final positive selection plate were picked and transferred to two 96-deepwell plates (ThermoFisher Scientific), where each well contained 200 pl LB broth supplemented with 50 pg ml ^-1 kanamycin and 25 pg ml ^-1 tetracycline. The plates were incubated at 37°C with shaking at 220 rpm overnight (12-16 hours). Next morning, 1 pl of overnight culture from each well was used to inoculate two cultures, one without and the other with 1 mM Bpa (all cultures were supplemented with 25 pg ml ^-1 kanamycin and 12.5 pg ml ^-1 tetracycline) in four 96-deepwell plates. The plates were incubated at 37°C with shaking at 220 rpm for 3 h, and subsequently 2.5 pl from each well were spotted onto LB agar plates with and without 1 mM Bpa, at a range of chloramphenicol concentrations, viz. 0, 100, 200 or 300 pg ml ^-1 . All LB agar plates were supplemented with 25 pg ml ^-1 kanamycin, and 12.5 pg ml ^-1 tetracycline. The plates were incubated at 37°C for 12-16 hours and imaged using GelDoc (Bio-Rad).

Construction of pREP_gst-1TAG-cam plasmid: The gst-cam gene cassette encompassing the lac promoter and the gst-cam fusion gene was PCR amplified using Q ₅ -DNA polymerase and primers gst-cam_pREP_F and gst-cam_pREP_R (Table 1) with the pRSF_wtRibo_wtRBS-Gst-1TAG_CaM plasmid (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403) serving as template DNA. The resulting PCR product was cloned into pREP plasmid digested with BsaAl and Seal (NEB) using a Gibson Assembly Cloning Kit (NEB) according to the manufacturer’s instructions. The sequence of the resulting plasmid pREP_gst-1TAG-cam was verified through Sanger sequencing. Expression and purification of GST-CaM for assessing the specificity and efficiency of the Mb Pyl(Bpa)RS: E. coli GeneHog cells containing pREP_gst-1TAG-cam plasmid were transformed with pBK_Mb PylRS(Bpa) plasmid and plated onto LB agar-KT (LB agar containing 50 μg ml ^-1 kanamycin and 25 μg ml ^-1 tetracycline). The following day a single colony was inoculated into 5 ml LB-KT media (LB media containing 50 pg ml ^-1 kanamycin and 25 pg ml ^-1 tetracycline) and incubated overnight (37°C, 220 rpm, 14-16 hours). The overnight culture was diluted to OD ₆₀₀ of 0.1 in fresh 25 ml LB-KT media, incubated at 37°C and 220 rpm until the OD ₆₀₀ reached 0.4. The cultures were induced with IPTG (1 mM) split into two cultures of 10 ml each and Bpa (1 mM) was added to one of the cultures. The cultures were incubated overnight (14-16 hours, 37°C, 220 rpm), pelleted the next day (5,000g, 10 min, 4°C), washed with 1 ml PBS, and suspended in 800 pl of BugBuster protein extraction reagent (Novagen), supplemented with protease inhibitor cocktail tablets (Roche), 1 mg ml ^-1 lysozyme and 1 mg ml ^-1 DNase I. The cell suspension was incubated at 25°C for 1 hour on a ThermoMixer (Eppendorf) at 900 rpm. Cell debris was pelleted (17,000 g, 30 min, 4°C), the supernatant was transferred to a fresh tube and 50 pl of Glutathione Sepharose 4b beads (Cytiva) were added. The beads were incubated with the extracted proteins for 1 h at 4°C to bind Gst-tagged proteins. Subsequently, the beads were spun down at 500 g at 4°C and washed with 800 pl PBS (4 times). Finally, the bound protein was eluted by heating the beads in 1xLDS sample buffer (ThermoFisher Scientific) supplemented with 100 mM DTT at 95°C for 5 min. The beads were spun down (17,000 g for 10 min at 4°C) and the supernatant was analysed on a 4-12% Bis-Tris gel. In addition, SeeBlu Plus2 pre-stained protein standard (ThermoFisher Scientific) was also loaded on the gel as marker. The gels were subsequently stained with InstantBlue (Abeam) Coomassie protein stain.

Construction of pSANG_7D12-32TAG_Mb Pyl(Bpa)_tRNA _CU A and pSANG_7D12- 109TAG_Mb Pyl(Bpa)_tRNAcuA plasmids: First, Mb Pyl(Bpa)RS-A2 gene was cut out of pBK_ Mb Pyl(Bpa)RS-A2 plasmid with Nde\ and Pst\ and sub-cloned into two other plasmids pAS61 and pAS64. pAS61 and pAS64 plasmids contain a single copy of wt-Mb PylRS/ Mb PyltRNAcuA pair and wt-Mb PylRS/ evMb PyltRNAuAcu pair, respectively. The resulting pAS61 and AS64 derivatives were named pAS61-A2 and pAS64-A2, respectively. Next, the DNA fragments comprising of Mb Pyl(Bpa)RS-A2 I Mb PyltRNAcuA pair from pAS61-A2 was PCR amplified using primers AS61_to_pSang_F and AS61_to_pSang_R (Table 1), and Q ₅ DNA polymerase (NEB). This PCR product was cloned into pSANG10_7D12-32TAG and pSANG10_7D12- 109TAG plasmids that had been previously digested with Sphl, using the Gibson Assembly Cloning Kit (NEB) according to the manufacturer’s instructions. The integrity of the resulting plasmids pSANG_7D12-32TAG_Mb Pyl(Bpa)_tRNA _CU A and pSANG_7D12- 109TAG_Mb Pyl(Bpa)_tRNA _CUA was verified through Sanger sequencing.

Construction of pSANG-oR-o7D12 plasmid: We first changed the ribosome binding site (RBS) of 7D12 in pSANG10-wt7D12 plasmid (Bridge, T., et al., Site-Specific Encoding of Photoactivity in Antibodies Enables Light- Mediated Antibody-Antigen Binding on Live Cells. Angew. Chem. Int. Ed. Engl., 2019. 58, 17986-17993) to orthogonal RBS (Wang,K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403). pSANG10-wt7D12 plasmid was digested using Xbal and Sall (NEB). After digestion, the reaction mixture was run on a 1 % agarose gel. The band corresponding to the pSANG-10 backbone was extracted using QIAquick Gel Extraction Kit (QIAGEN). A gene fragment containing orthogonal RBS and part of 7D12 (obtained from Integrated DNA Technologies (IDT)) was cloned into the digested pSANG-10 backbone using Gibson cloning (New England Biolabs). We named this plasmid as: pSANG-o7D12. The integrity of pSANG-o7D12 plasmid was confirmed by Sanger sequencing. Next, the orthogonal- 16S ribosomal RNA (o-16S rRNA), 23S rRNA, and 5S rRNA genes were inserted into pSANG-o7D12. o-16S rRNA, 23S rRNA, and 5S rRNA genes were amplified from pRSF-riboQ1-o-gst-cam plasmid (Wang, K., et al., Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem., 2014. 6, 393-403) using following primers: orRNA_RSF_SANG_F and orRNA_RSF_SANG_R (Table 1), and Q ₅ -DNA polymerase (New England Biolabs) according to the manufacturer’s instructions. The PCR product was purified using PCR purification kit (QIAGEN). For insertion of this PCR product into pSANG-o7D12, pSANG-o7D12 plasmid was digested with Bgll (New England Biolabs). After digestion, the reaction mixture was run on a 1% agarose gel. The band corresponding to the linear pSANG-o7D12 plasmid was extracted using QIAquick Gel Extraction Kit (QIAGEN). The PCR product containing O-16S rRNA, 23S rRNA, and 5S rRNA was cloned into this linear plasmid using Gibson Assembly Cloning Kit (NEB). We named this plasmid as: pSANG-oR-o7D12. The integrity of pSANG-oR-o7D12 plasmid was confirmed by Sanger sequencing.

Construction of pSANG-oR-o7D12-Dual (pSANG-oR-o7D12-32 ^TAG 109 ^AGTA ) plasmid: pSANG-oR-o7D12 plasmid was digested using SgrA1 and Blpl (NEB) in order to remove the o7D12 DNA fragment from this plasmid. After digestion, the reaction mixture was run on a 1% agarose gel. The band corresponding to the pSANG-oR backbone was extracted using QIAquick Gel Extraction Kit (QIAGEN). A gene fragment containing orthogonal RBS and 7D12- 32TAG-109AGTA (obtained from IDT) was cloned into the digested pSANG-oR backbone using Gibson Assembly Cloning Kit (NEB). We named this plasmid as: pSANG-oR-o7D12- Dual. The integrity of pSANG-oR-o7D12-Dual plasmid was confirmed by Sanger sequencing the 7D12 region in the plasmid.

Construction of pSANG-oR-o7D12-Dual-Pyl(Bpa) (pSANG-oR-o7D12-32 ^TAG 109 ^AGTA - Mb Pyl(Bpa)-evtRNAuacu) plasmid: First, pSANG-oR-o7D12-Dual plasmid was digested with BamHI (NEB). After digestion, the linearized plasmid was purified using QIAquick PCR Purification Kit (QIAGEN). Next, the DNA fragment containing Mb Pyl(Bpa)RS-A2/ evMb PyltRNAuAcu pair from pAS64-A2 (For pAS64-A2, see section on “Construction of pSANG_7D12-32TAG_Mb Pyl(Bpa)_tRNAcuA and pSANG_7D12-

109TAG_Mb Pyl(Bpa)_tRNAcuA plasmids”) was PCR amplified using primers o- pSANG_AS61_BamHI_f and o-pSANG_AS61_BamHI_r (Table 1), and Q ₅ DNA polymerase (NEB). This PCR product was cloned into pSANG-oR-o7D12-Dual plasmid previously digested with BamHI, using the Gibson Assembly Cloning Kit (NEB) according to the manufacturer’s instructions. We named this plasmid as: pSANG-oR-o7D12-Dual-Pyl(Bpa). The identity of pSANG-oR-o7D12-Dual-Pyl(Bpa) plasmid was confirmed by Sanger sequencing.

Sequences

SEQ ID NO: 1

MMDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLWN NSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVS APKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPAS APAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRL YTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTEL SKQI FRVDKN LCLRPM LAPTLYNYLRKLDRI LPGPI KI FEVGPCYRKESD GKEHLEEFTMVQFTQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVF GDTLDIMHGDLELSSAWGPVSLDREWGIDKPAIGAGFGLERLLKVMHGF KNIKRASRSESYYNGISTNL

SEQ ID NO: 2

ATGATGGATAAAAAACCGCTGGATGTGCTGATTAGCGCGACCGGCCTGTG

GATGAGCCGTACCGGCACCCTGCATAAAATCAAACATCATGAAGTGAGCC

GCAGCAAAATCTATATTGAAATGGCGTGCGGCGATCATCTGGTGGTGAAC

AACAGCCGTAGCTGCCGTACCGCGCGTGCGTTTCGTCATCATAAATACCG

CAAAACCTGCAAACGTTGCCGTGTGAGCGATGAAGATATCAACAACTTTC

TGACCCGTAGCACCGAAAGCAAAAACAGCGTGAAAGTGCGTGTGGTGAGC

GCGCCGAAAGTGAAAAAAGCGATGCCGAAAAGCGTGAGCCGTGCGCCGAA ACCGCTGGAAAATAGCGTGAGCGCGAAAGCGAGCACCAACACCAGCCGTA

GCGTTCCGAGCCCGGCGAAAAGCACCCCGAACAGCAGCGTTCCGGCGTCT

GCGCCGGCACCGAGCCTGACCCGCAGCCAGCTGGATCGTGTGGAAGCGCT

GCTGTCTCCGGAAGATAAAATTAGCCTGAACATGGCGAAACCGTTTCGTG

AACTGGAACCGGAACTGGTGACCCGTCGTAAAAACGATTTTCAGCGCCTG

TATACCAACGATCGTGAAGATTATCTGGGCAAACTGGAACGTGATATCAC

CAAATTTTTTGTGGATCGCGGCTTTCTGGAAATTAAAAGCCCGATTCTGA

TTCCGGCGGAATATGTGGAACGTATGGGCATTAACAACGACACCGAACTG

AGCAAACAAATTTTCCGCGTGGATAAAAACCTGTGCCTGCGTCCGATGCT

GGCCCCGACCCTGTATAACTATCTGCGTAAACTGGATCGTATTCTGCCGG

GTCCGATCAAAATTTTTGAAGTGGGCCCGTGCTATCGCAAAGAAAGCGAT

GGCAAAGAACACCTGGAAGAATTCACCATGGTTCAGTTTACTCAAATGGG

CAGCGGCTGCACCCGTGAAAACCTGGAAGCGCTGATCAAAGAATTCCTGG

ATTATCTGGAAATCGACTTCGAAATTGTGGGCGATAGCTGCATGGTGTTT

GGCGATACCCTGGATATTATGCATGGCGATCTGGAACTGAGCAGCGCGGT

GGTGGGTCCGGTTAGCCTGGATCGTGAATGGGGCATTGATAAACCGGCGA

TTGGCGCGGGTTTTGGCCTGGAACGTCTGCTGAAAGTGATGCATGGCTTC

AAAAACATTAAACGTGCGAGCCGTAGCGAAAGCTACTATAACGGCATTAG

CACGAACCTGTAA

SEQ ID NO: 3

MMDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLWNNSRSCRTA RAFR

HHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPL ENSV

SAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAK PFREL EPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGIN NDTE

LSKQI FRVDKN LCLRPM LAPTLYNYLRKLDRI LPGPI KI FEVGPCYRKESDGKEH LEEFTM VN FCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAWGPVSL D REWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL

SEQ ID NO: 4

ATGATGGATA AAAAACCGCT GGATGTGCTG ATTAGCGCGA CCGGCCTGTG GATGAGCCGT ACCGGCACCC TGCATAAAAT CAAACATCAT GAAGTGAGCC GCAGCAAAAT CTATATTGAA ATGGCGTGCG GCGATCATCT GGTGGTGAAC AACAGCCGTA GCTGCCGTAC CGCGCGTGCG TTTCGTCATC ATAAATACCG CAAAACCTGC AAACGTTGCC GTGTGAGCGA TGAAGATATC AACAACTTTC TGACCCGTAG CACCGAAAGC AAAAACAGCG TGAAAGTGCG TGTGGTGAGC GCGCCGAAAG TGAAAAAAGC GATGCCGAAA AGCGTGAGCC GTGCGCCGAA ACCGCTGGAA AATAGCGTGA GCGCGAAAGC GAGCACCAAC ACCAGCCGTA GCGTTCCGAG CCCGGCGAAA

AGCACCCCGA ACAGCAGCGT TCCGGCGTCT GCGCCGGCAC CGAGCCTGAC CCGCAGCCAG CTGGATCGTG TGGAAGCGCT GCTGTCTCCG GAAGATAAAA TTAGCCTGAA CATGGCGAAA CCGTTTCGTG AACTGGAACC GGAACTGGTG ACCCGTCGTA AAAACGATTT TCAGCGCCTG TATACCAACG ATCGTGAAGA TTATCTGGGC AAACTGGAAC GTGATATCAC CAAATTTTTT GTGGATCGCG GCTTTCTGGA AATTAAAAGC CCGATTCTGA TTCCGGCGGA ATATGTGGAA CGTATGGGCA TTAACAACGA CACCGAACTG AGCAAACAAA TTTTCCGCGT GGATAAAAAC CTGTGCCTGC GTCCGATGCT GGCCCCGACC CTGTATAACT ATCTGCGTAA ACTGGATCGT ATTCTGCCGG GTCCGATCAA AATTTTTGAA GTGGGCCCGT GCTATCGCAA AGAAAGCGAT GGCAAAGAAC ACCTGGAAGA ATTCACCATG GTTAACTTTT GCCAAATGGG CAGCGGCTGC ACCCGTGAAA ACCTGGAAGC GCTGATCAAA GAATTCCTGG ATTATCTGGA AATCGACTTC GAAATTGTGG GCGATAGCTG CATGGTGTAT GGCGATACCC TGGATATTAT GCATGGCGAT CTGGAACTGA GCAGCGCGGT GGTGGGTCCG GTTAGCCTGG ATCGTGAATG GGGCATTGAT AAACCGTGGA TTGGCGCGGG TTTTGGCCTG GAACGTCTGC TGAAAGTGAT GCATGGCTTC AAAAACATTA AACGTGCGAG CCGTAGCGAA AGCTACTATA ACGGCATTAG CACGAACCTG TAA

Table 1