A genetically encoded photocaged cysteine and its use in bioconjugation

Abstract

A genetically-engineered secretory protein, antibody, or antibody fragment, wherein the protein, antibody or antibody fragment comprises one, or more, photocaged noncanonical (NcAA) cysteine analogs incorporated into the protein antibody, or antibody fragment, at specific, pre-determined amino acid residue sites; methods of making the protein or antibody and methods of using the protein or antibody are described.

Description

A GENETICALLY ENCODED PHOTOCAGED CYSTEINE AND ITS USE IN BIOCONJUGATIONRELATED APPLICATIONS[ o o o i ] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 548,935, filed on February 2, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with Government support under contract number R35 GM136437, awarded by National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on January 16, 2025, is entitled “0342.0017W01_ST26. xml”, and is 39,317 bytes in size.BACKGROUND OF THE INVENTION

[0004] Secretory proteins, such as cytokines, growth factors, hormones, serum proteins, coagulation factors, recombinant fusion proteins and multi-specific antibodies contain native disulfide bonds that are critical for proper folding and functionality.72Antibodies are one example of a class of secreted / secretory proteins that contain native disulfide bonds that are integral to maintain their structure and function in the Fab (antigen binding) and Fc (cellular receptor binding) regions of the antibody. Because of their unique function of binding antigens and cellular receptors, antibody-drug conjugates (ADCs) have revolutionized anti-cancer therapies in recent years by allowing targeted, selective delivery of cytotoxic agents to antigen-overexpressing cancer cells, increasing the efficacy while reducing off-target effects of such cytotoxic drugs.1'4The first-generation strategies to prepare ADCs relied on nonspecific chemistries such as lysine conjugation, leading to heterogeneous product mixtures with suboptimal therapeutic properties.5'9Effort has since shifted to the development of methods that allow construction of morehomogenous, site-specific ADCs, and offer a toolbox of available attachment sites to finetune the properties of such conjugates.10'19This led to the development of THIOMAB technology, in which uniquely reactive cysteine residues are engineered at permissive sites within the antibody that can be selectively conjugated to cytotoxic agents, creating ADCs with superior site-specificity and homogeneity.20'25Despite the success of the THIOMAB technology, there are some key limitations which still exist with this approach. The engineered cysteine residues in antibodies are frequently ‘disulfide-capped’ (i.e., oxidized to form a disulfide bond) with small thiol-containing cellular metabolites (e.g., cysteine or glutathione) during secretion, making it necessary to regenerate the thiol through reduction prior to conjugation.

[0005] However, this reductive process also disrupts key interchain disulfide bonds, which must be regenerated after conjugation through a reoxidation step. This cumbersome multi-step process can reduce the efficiency of conjugation, as well as cause “disulfide scrambling”, rendering the antibody nonfunctional.21’25Disulfide scrambling, or disulfide shuffling, refers to the incorrect formation or rearrangement of disulfide bonds within a protein. This can occur during protein synthesis, folding, or storage, leading to the formation of non-native disulfide bonds that can disrupt the protein's native structure and function. Additionally, certain capped engineered cysteine sites are resistant to reduction, which may limit the scope of this technology.26’27Because of these challenges, there are a limited number of sites that are compatible with the THIOMAB technology. Since the site of attachment is known to significantly affect the pharmacological properties of the resulting ADC,26’28'30it would be beneficial to have access to a broader set of potential sites for drug conjugation. Additionally, the ability to introduce conjugation-ready cysteine-thiols at predefined sites of an antibody in a manner that does not suffer from disulfide capping, and would not require the cumbersome reduction-conjugation- reoxidation workflow, would significantly improve the scope of this technology.SUMMARY OF THE INVENTION

[0006] Disulfide bonds are covalent bonds between the thiol groups of two cysteine residues formed post-translationally by the oxidation of a pair of cysteines. Disulfide bonds or disulfide bridges can greatly increase the stability of a protein and are primarily found in proteins that reside outside the chaperone rich protective environment of the cytoplasm, e.g., secretory or secreted proteins and peptides.72Examples of secretory proteins includeantibodies, hormones, interferons, extracellular enzymes, cytokines, growth factors, serum proteins, coagulation factors and fusion proteins.

[0007] Due to its unique reactivity, the cysteine side chain is frequently targeted for site-specific conjugation of important proteins such as antibodies. However, when a protein or an antibody is expressed / purified, the engineered cysteine residue undergoes oxidative capping (i.e., forming disulfide bonds) with other thiols in the protein making them unavailable for conjugation. A reductive treatment can regenerate them, but it also reduces structurally important internal disulfide bonds. Consequently, these disulfide bonds must be regenerated through an oxidative treatment. This cumbersome reduction / reoxidation process employed in the industry reduces conjugation yield, and makes many desirable site on the antibody inaccessible to this approach.

[0008] Using a photocaged noncanonical thiol-containing amino acid analog, such as a cysteine analog, when incorporated into the genetically-engineered protein, solves these two key issues. It avoids disulfide scrambling during protein folding, and disulfide capping during secretion. Afterward, it can be readily converted to a conjugation -ready thiol by light. Importantly, the genetically-engineered protein retains its biological functions such as secretability (for example, the ability to be secreted from cells or retaining the biological activity of being secreatable) and ability to bind to cognizant receptors or target proteins, cells or tissues.

[0009] Described herein is a method to site-specifically incorporate (chemoseletively incorporate or incorporate into a specific, designated site of the protein of interest) a chemically protected / photocaged analog of the thiol-containing amino acid cysteine into a secreted proteins that contain / comprise native disulfide bonds. As used herein, amino acid analog refers to a noncanonical (ncAA) amino acid, also referred to as an unnatural amino acids (UAA) or a non-naturally-occurring amino acid. This photocaged cysteine analog can be subsequently uncaged with high efficiency using irradiation / light resulting in conjugation-ready proteins and antibodies produced by these methods. The photocaged cysteine is efficiently incorporated into the protein and removed in a site-specific manner, and does not get capped, thus enabling site-specific conjugation of a biomolecule or chemical compound, such as a warhead or cargo, onto recombinant antibodies. This approach has tremendous potential for developing site-specific protein conjugates that are valuable as research reagents as well as biotherapeutics. Most importantly, theincorporation of this photocaged cysteine into proteins and antibodies containing native disulfide avoids disulfide scrambling and oxidative thiol capping during secretion.

[0010] Specifically described herein are compositions comprising a genetically- engineered, recombinantly-expressed secreted proteins containing native disulfide bonds, wherein the protein comprises one, or more, photocaged noncanonical (also referred to herein as ncAAs, non-naturally occurring amino acids or amino acid analogs), cysteine analogs incorporated into the protein at specific, pre-determined amino acid residue sites. The present invention encompasses any secretory protein with native disulfide bonds, and includes, for example, cytokines, growth factors, hormones, serum proteins, coagulation factors and fusion proteins. Specifically encompassed herein is any antibody, or antibody fragment containing disulfide bonds. Such proteins are characterized by their native disulfide bonds as well as secretory properties.

[0011] The incorporation of a photocaged ncAA residue such as a cysteine analog into the secreted protein or antibody introduces a photocaged / photoreactive thiol residue into the protein or antibody that can then be decaged resulting in a conjugation-ready free thiol residue (e.g. a free cysteine) in the protein or antibody, resulting in a protein, antibody or antibody fragment that has a conjugation-ready site for further modification, for example, by the conjugation of a biomolecule or chemical compound. Such proteins or antibodies comprising the photocaged ncAA are also referred to herein as functionalized or reactive proteins, or antibodies, as the caged sites are decaged and, thus, rendered available for additional reaction with chemical compounds or molecules to result in conjugated proteins, antibodies or antibody fragments. Some examples of suitable cysteine analogs that can be modified for photoreactivity are: N-acetyl-cysteine (NAC), S-sulfocysteine, N,N'-diacetyl- 1-cystine, N,N'-diacetyl-l-cystine dimethylester (DACDM), Fmoc-DL-buthionine, Fmoc-L- cysteic acid, Fmoc-L-selenomethionine or Fmoc-S-trityl-L-homocysteine. In a particular embodiment, the noncanonical cysteine analog is a Nvoc (4, 5-dimethoxy-2-nitrobenzyl) protected cysteine incorporated into the protein or antibody.

[0012] Any protein or antibody, or fragment thereof, and in particular any secreted protein or antibody, is suitable for use in the present invention. In particular, the secretory proteins of the present invention contain native internal disulfide bonds that must remain intact for functional biological activity such as the binding of an antibody to its binding partner (e.g., an antibody binding to / interacting with a receptor site or antigenic site) Moreparticularly the secretory protein is an antibody, for example, the anti-Her2 antibody (also referred to herein as Trastuzumab). Trastuzumab is a monoclonal antibody used in cancer therapy that targets (or binds to) a protein called human epidermal growth factor receptor 2 (HER2).

[0013] More specifically, the genetically-engineered anti-Her2 antibody, or fragment thereof, of the present invention comprises one or more photocaged (also referred to as photoreactive, photo-inducible or light-activated) cysteine analogs incorporated into the antibody at specific amino acid residue sites of the protein: at the aspartic acid at position 122 of the light chain (LC), the glycine at position 197 of the heavy chain (HC), or the alanine at position 121 of the heavy chain (HC), wherein the photocaged cysteine analog is a Nvoc protected-cysteine analog incorporated into the antibody, or fragment thereof.

[0014] As described herein, the photocaged noncanonical cysteine analog is decaged, resulting in a free cysteine amino acid residue, wherein the free cysteine is conjugated to a biomolecule or chemical compound such as a linker, warhead or cargo molecule.

[0015] The biomolecule or chemical compound can be any suitable biomolecule or chemical compound and is typically a cytotoxic or therapeutic molecule or chemical compound. The biomolecule or chemical compound can be, for example, a therapeutic or cytotoxic drug, a small organic molecule, a nucleic acid, a protein, a polypeptide, a peptide or a biophysical probe.

[0016] Examples of such biomolecules or chemical compounds are a chemical or peptide linker (which can be cleavable) for attachment of biomolecules or chemical compounds to the decaged thiol / cysteine; a warhead, for example, is a biological entity such as a chemical compound that can interact with, or bind to, or otherwise attach to a specific targeted protein often with a cytotoxic result or the inhibition of a cellular process as a result; or a cargo molecule, such as a cytotoxic drug that can be conjugated directly to the decaged cysteine, or can be attached to a warhead that is conjugated to the decaged thiol / cysteine. As described herein carbonylacrylic acid (CCA) and monomethyl auristatin (MMAF) have been conjugated to the antibody Trastuzmab as a chemical compound or biomolecule at the reactive cysteine sites.

[0017] Another embodiment of the present invention is a method of producing a protein or an antibody conjugate, wherein the protein or antibody is a secreted protein orantibody containing native disulfide bonds, wherein the method uses a photocaged noncanonical (ncAA) cysteine analog, comprising the steps of:

[0018] a.) providing a genetically-engineered secreted / secretory ’ secreatable protein containing native disulfide bonds such as an antibody, or fragment thereof, and further comprising one, or more noncanonical amino acid residues incorporated into specific sites of the protein, antibody, or fragment thereof, wherein the noncanonical residue is a photocaged cysteine analog and the specific site is not a native cysteine / disulfide site;

[0019] b.) irradiating the protein, antibody, or fragment thereof, of step a) under suitable conditions (e.g., exposing the protein, antibody or antibody fragment to a suitable light source / illumination source capable of decaging the photocaged ncAA) to decage the protected cysteine analog to expose a cysteine with a free thiol group, thereby producing a protein, an antibody, or fragment thereof, with a free, reactive cysteine residue;

[0020] c.) reacting the free cysteine residue of the protein, antibody, or fragment thereof of step b) with a cargo molecule comprising a therapeutic or cytotoxic drug, a biophysical probe, a peptide, or a small molecule, or nucleic acid under suitable conditions for the cargo molecule to conjugate to the free cysteine residue;

[0021] thereby producing a protein or an antibody conjugate.

[0022] In a particular embodiment the one, or more, noncanonical photocaged cysteine residues are exposed on the surface of the protein. In a further embodiment the photocaged noncanonical (ncAA) cysteine analog is Nvoc-cysteine residue. As described herein, the irradiation of the Nvoc-cysteine residue is at about 365 nm for about 10 minutes. Other suitable conditions for irradiation can be determined by one skilled in the art.

[0023] More specifically, the present method further encompasses conjugating a biomolecule or chemical compound to the genetically-engineered (mutated) secreteable protein, antibody, or fragment thereof, at the sites of the freed / reactive cysteine. Examples of such biomolecules or chemical compounds are a chemical or peptide linker (which can be cleavable) for attachment of biomolecules or chemical compounds to the decaged thiol / cysteine; a warhead, for example, a biological entity such as a chemical compoundthat can interact with, or bind to, or otherwise attach to a specific targeted protein often with a cytotoxic result or the inhibition of a cellular process as a result; or a cargo molecule, such as a cytotoxic drug that can be conjugated directly to the decaged cysteine or can be attached to a warhead that is conjugated to the decaged thiol / cysteine. As specifically described herein, carbonylacrylic acid (CCA) and monomethyl auristatin (MMAF) have been conjugated to the anti-Her2 antibody Trastuzmab as a chemical compound or biomolecule at the reactive cysteine sites.

[0024] Also encompassed by the present invention is a method of treating cancer in a patient, comprising administering to the patient in need thereof a protein-drug conjugate, or an antibody-drug conjugate produced by the method described herein, under conditions suitable for the protein or antibody-drug conjugate to react with the targeted cellular receptors, tissues or cancer cells and selectively kill the targeted cancer cells, thereby treating the cancer patient. In a particular embodiment wherein the targeted cancer cells are Her2 receptor-positive cancer cells, and the antibody-drug conjugate comprises the anti- Her2 antibody Trastuzmab comprising carbonylacrylic acid attached to monomethyl auristatin conjugated to the Trastuzmab at an amino acid residue position selected from the group consisting of aspartic acid at position 122 of the light chain, glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain.

[0025] Further encompassed by the present invention is a method of producing a conjugation-reactive secretor / secreted protein, or antibody, or antibody fragment, wherein the protein, antibody or antibody fragment contains native disulfide bonds, and wherein the antibody, or antibody fragment comprises a photocage noncanonical cysteine analog, comprising the steps of a.) co-transfecting mammalian cells with two plasmids comprising;1.) a nucleic acid encoding a variant antibody, or antibody fragment, mutated at one, or more, specific amino acid residue sites to incorporate a TAG stop codon; and2.) EcLeuRS / tRNA translation machinery components; b.) introducing a photocaged noncanonical cysteine analog to the mammalian cells of step a.) and incubating the cells with the plasmids and photocage noncanonical cysteine analog under conditions suitablefor the incorporation of the photocaged noncanonical cysteine analogs at the specific site of the TAG stop codon; and c.) purifying the antibody, or antibody fragment comprising the photocaged noncanonical cysteine analog, thereby, producing a conjugation-reactive antibody, or antibody fragment, wherein the antibody, or antibody fragment comprises a photocage noncanonical cysteine analog.

[0026] In a particular embodiment of the method, the photocaged noncanonical cysteine analog is Nvoc cysteine and the antibody is Trastuzmab, wherein the Trastuzmab comprises a photocaged noncanonical cysteine analog at an amino acid residue position selected from the group consisting of: aspartic acid at position 122 of the light chain, glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain.

[0027] Given the high efficiency, excellent chemoselectivity and scalability, the methods of the present invention are ideally suited for developing homogeneous, sitespecific protein bioconjugates. This technology will enable site-specific labeling of any recombinant protein. In addition, this reaction is compatible with existing bioconjugation reactions such as azide-alkyne cycloaddition. Together these two reactions can be used for the attachment of multiple different entities onto one protein with precise site control. Finally, cysteine targeted conjugation is the most popular bioconjugation in the industry, and many such conjugates have been evaluated in clinical settings. The compositions and methods described herein will address a critical limitation of cysteine conjugation, but benefit from established large-scale processes and clinical understanding of the ultimate conjugate.

[0028] The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:

[0030] FIG. 1A-B: FIG. 1A is a depiction of the currently used THIOMAB process as compared to the process of the present invention (FIG. IB) which shows the single-step generation of a conjugation-ready protein and site-specific conjugation.

[0031] FIG. 2 A-B show using engineered cysteine residues for site-specific labeling of antibodies. A) Engineered cysteine thiols on antibodies get disulfide-capped during secretion, and must be regenerated through reduction. This step also reduces interchain disulfide bonds that need to be reformed using an oxidative reaction. B) The strategy reported here avoids this problem through the incorporation of a photocaged cysteine residue, which can be readily decaged with light to reveal a conjugation-ready thiol functionality.

[0032] FIG. 3A-B show site-specific incorporation of a photocaged cysteine, and its use for bioconjugation following photo-decaging. A) Representative fluorescence images of HEK293T cells transfected with plasmids containing an engineered EcLeuRS / tRNA pair selective for photocaged cysteine and EGFP-40-TAG, 48 h post-transfection. Robust reporter expression was observed in the presence of 1 mM photocaged cysteine in the media, but not in its absence. B) ESI-MS analysis of purified EGFP mutant containing photocaged cysteine shows a mass consistent with incorporation of the desired ncAA. Following irradiation, the photocaged cysteine residue was successfully decaged. The resulting free cysteine residue could be efficiently conjugated to a mal eimide probe (100 pM, 2 h RT).

[0033] FIG. 4 A-B show incorporation of photocaged cysteine into Trastuzumab and its decaging post-purification. A) SDS-PAGE analysis (reducing and non-reducing) of three different mutants of Trastuzumab, containing photocaged cysteine at either HC-197, HC-121, or LC-122. Expi293 cells were transfected with plasmids containing the EcLeuRS / tRNA selective for photocaged cysteine and the appropriate antibody genes. The ncAA was provided in the media at the time of transfection at a final concentration of 1 mM. Secreted recombinant antibodies were purified from the growth medium. B) Successful incorporation of photocaged cysteine at the desired site of Trastuzumab, andsuccessful light-mediated decaging of this residue post-purification, were confirmed by ESI-MS analysis for all three mutants.

[0034] FIG. 5A-B show efficient synthesis of a functional antibody-drug conjugate using a photocaged cysteine residue. A) Trastuzumab mutant containing photocaged cysteine residue at HC-121 was decaged using light, and the resulting thiol was efficiently labeled using a MMAF-carbonylacrylic acid conjugate, as confirmed by ESI-MS analysis. B) The antibody-drug conjugate prepared in this manner showed selective toxicity toward SK-BR-3 (HER2+) cells relative to MDA-MB-231 (HER2-) as shown by MTT cellviability assay.

[0035] FIG. 6A-D show labeling reactions of Trastuzumab mutants containing decaged cysteine-thiol residues at various sites with a carbonylacrylic acid probe. A) Scheme describing the reaction of 3-benzoylacrylic acid with decaged antibody for cysteine conjugation. B) Reaction of the mutant antibody containing decaged cysteine-thiol at position 122 of the light chain resulted in an incomplete conversion to the conjugated product (denoted as +1). C) Reaction of the mutant antibody containing decaged cysteine- thiol at position 197 of the heavy chain resulted in a partial but incomplete conversion to the conjugated product. D) Reaction of the mutant antibody containing decaged cysteine- thiol at position 121 of the heavy chain resulted in complete conversion to the conjugated product. An additional small peak consistent with the second addition of the probe was also observed, possibly due to the presence of a small fraction of reduced cysteines elsewhere on the heavy chain.

[0036] FIG. 7A-B are plasmid maps and sequences. FIG. 7 A is the map of Plasmid pAcBacl-NVocRS-4xLtR. The NVocRS sequence is highlighted in blue, LtR sequences are highlighted in purple, and U6 promoter sequences are highlighted in orange. FIG. 7B shows the nucleic acid sequence of pAcBacl -NVocRS -4xLtR (SEQ ID NO: 1).

[0037] FIG. 8A-B are plasmid maps and sequences. FIG. 8 A describes Plasmids pcDNA3.1 AntiHer2-121HC-TAG, pcDNA3.1 AntiHer2-198HC-TAG and pcDNA3.1 AntiHer2-198HC-TAG. The pcDNA3.1 AntiHer2-121HC-TAG plasmid map is shown with the antibody expression cassette colored in blue on the map. The nucleic acid sequence of pcDNA3.1 AntiHer2-121HC-TAG is shown in FIG. 8B. The sequence of Plasmid pcDNA3.1 AntiHer2-198HC-TAG is the same as shown in FIG. 8B, except TAG is at position 198 in the HC instead of position 121. The nucleic acid sequence of PlasmidpcDNA3.1 AntiHer2-122LC-TAG is the same as shown in FIG. 8B, except TAG is at position 122 of the LC, and HC is wild-type sequence. FIG. 8B The nucleic acid sequence of pcDNA3.1 AntiHer2-121HC-TAG (SEQ ID NO:2).

[0038] FIG. 9 Amino Acid Sequence of Anti-Her2 Antibody (SEQ ID NO:3) Heavy Chain and (SEQ ID NO:4) Light chain.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0040] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Also, all conjunctions used are to be understood in the most inclusive sense possible. Thus, the word "or" should be understood as having the definition of a logical "or" rather than that of a logical "exclusive or" unless the context clearly necessitates otherwise. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and / or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and / or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[0041] It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an element discussed below could be termed a second element, and similarly, a secondelement may be termed a first element without departing from the teachings of the present invention.

[0042] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0043] Antibody-drug conjugates (ADCs) have emerged as a powerful class of anticancer therapeutics that enable selective delivery of toxic payloads into target cells. There is increasing appreciation for the importance of synthesizing such ADCs in a defined manner, where the payload is attached at specific permissive sites on the antibody with a particular drug to antibody ratio. Additionally, the ability to systematically alter the site of attachment is important to fine-tune the therapeutic properties of the ADC. Engineered cysteine residues have been used to achieve such site-specific programmable attachment of drug molecules onto antibodies. However, engineered cysteine residues on antibodies often get ‘disulfide-capped’ (e.g., formation of disulfide bridges) during secretion, and require reductive regeneration prior to conjugation. This reductive step also reduces structurally important disulfide bonds in the antibody itself, which must be regenerated through oxidation. This multistep, cumbersome process reduces the efficiency of conjugation and presents logistical challenges. Additionally, certain engineered cysteine sites are resistant to reductive regeneration, limiting their utility and the overall scope of this conjugation strategy.

[0044] As described herein, genetically encoded photocaged cysteine residues were site-specifically / chemoselectively incorporated into an antibody. This photocaged amino acid was efficiently decaged using light, revealing a free cysteine residue available for conjugation without disrupting the functional activity (binding activity) of the antibody structure. As described herein, one, or more ncAAs can be incorporated at several positions within full-length recombinant Trastuzumab and decaged efficiently. Functional antibodydrug conjugates (ADC) site-specifically modified with monomethyl auristatin F (MMAF) were also generated.

[0045] Described herein are compositions a genetically-engineered antibody, orantibody fragment, wherein the antibody or antibody fragment comprises one, or more, photo-caged noncanonical (NcAA) cysteine analogs incorporated into the antibody, or antibody fragment, at specific, pre-determined amino acid residue sites. The present invention specifically encompasses any secretable / secretory protein with native disulfide bonds, and in particular any antibody, or antibody fragment containing disulfide bonds. Most importantly, the methods of the present invention do not compromise or interfere with the structure of the native disulfide bonds -these native bonds remain structurally intact during the claimed process. In a particular embodiment, the noncanonical cysteine analog is a Nvoc (4, 5-dimethoxy-2-nitrobenzyl) protected cysteine incorporated into the protein or antibody, and more particularly the antibody is, for example, Trastuzmab.

[0046] Also described herein are methods of making the conjugation-ready proteins and methods of using the antibody-conjugated compositions in methods of treating cancers.

[0047] The limitations of using the current methods to conjugate proteins containing native disulfide bonds are detailed above. Described herein is an attractive strategy to overcome these limitations with the use of a photocaged cysteine residue, which can be site-specifically incorporated into the antibody at virtually any position through the genetic code expansion (GCE) technology, using an engineered aminoacyl tRNA synthetase (aaRS) / tRNA pair.31'37(See also U.S. Patent No. 10,717, 975 and 11,760,990 and WO 2020 / 219708, the teachings of which are incorporated herein in their entirety by reference). This caged cysteine residue can be subsequently decaged using light to reveal a free cysteine residue, which can then serve as a conjugation site without the need for reduction and reoxidation (Figures 1 and 2).

[0048] As described herein, a mutant E. coli leucyl-tRNA synthetase (EcLeuRS) / tRNA pair was used to incorporate a photocaged cysteine noncanonical amino acid (ncAA) into full-length human antibody with excellent efficiency. The ncAA- containing antibody can be fully decaged after just 10 min of irradiation, followed by direct conjugation to a cytotoxic payload to create a functional antibody-conjugate or antibody- drug-conjugate (ADC). Although the GCE technology has been used to site-specifically engineer other bioorthogonal reactive handles within an antibody structure for use with other bioconjugation techniques such as SPAAC38'40, IEDDA41’42, oxime43, 44, CRACR45'47, and others48, 49, the reagents needed for these methods are often less accessible than theirthiol-reactive counterparts.15’50By contrast, the approach described herein benefits from the expansive thiol conjugation toolbox, as well as the popularity of thiol -targeted chemistries in the industry, where these processes have been optimized in scale, and the resulting conjugates have been validated in clinical settings. In addition, this strategy can be potentially combined with other bioorthogonal ncAA handles for site-specific antibody labeling at multiple distinct sites to create more complex next-generation ADCs with multiple small molecule attachments.46’47’51-56

[0049] GCE technology enables the site-specific incorporation of ncAAs into proteins using an engineered orthogonal aaRS / tRNA pair capable of incorporating the ncAA of interest at an introduced nonsense codon.31-37EcLeuRS has been engineered to incorporate a range of ncAAs in eukaryotes (yeast as well as mammalian cells), including those with photocaged groups.37’51> 57-60One particular engineered EcLeuRS has been developed to allow the incorporation of Nvoc (4, 5 -dimethoxy -2-nitrobenzyl) protected serine, cysteine, and selenocysteine into proteins, which are readily deprotected upon irradiation.37’58-60As shown herein, this system can be used to incorporate photocaged Nvoc-cysteine (Figure 3A) into proteins in mammalian cells. To this end, HEK293T cells were co-transfected with two plasmids; one containing EcLeuRS expressed from a CMV promoter and 4 copies of enhanced tRNAEcLeu, each driven from a U6 promoter, and another containing an EGFP expressed from a CMV promoter harboring a TAG stop codon at position40. Only when the photocaged cysteine (ImM) was supplied in the media did we observe robust expression of the EGFP reporter (Figure 3 A), indicating successful incorporation of the ncAA. Purification of the mutant EGFP using a C-terminal polyhistidine tag followed by ESI-MS analysis confirmed the desired mass corresponding to the incorporation of the photocaged cysteine at the desired site (Figure 3B). The Nvoc functionality is known to be readily decaged when irradiated with 365 nm light. To confirm that this residue can be decaged in our recombinant reporter protein, the purified EGFP-40-NvocCys was irradiated using a 365 nm LED array for 10 min in the presence of ascorbic acid (included to scavenge potential reactive oxygen species generated during the photodecaging process). Subsequent ESLMS analysis indeed demonstrated that, under these conditions, the protein decaged cleanly to reveal free cysteine at the engineered position. To demonstrate that the released cysteine residue could be used for bioconjugation, the protein after photodecaging was treated with 100 pM ethyl maleimide for 2 h (Figure 3B). Subsequent ESI-MS showed clean modification of the cysteine residue to generate the desired conjugate.

[0050] After confirming that Nvoc-cysteine can be efficiently incorporated into proteins expressed in mammalian cells, and subsequently decaged and used for bioconjugation, the strategy was extended to a full-length human antibody. Three different TAG mutants of Trastuzumab, a monoclonal antibody targeted to the HER2 receptor overexpressed in a variety of cancer cells.61One of the TAG mutations was introduced at the aspartic acid at position 122 of the light chain (LC) (SEQ ID NO:4), while two others were placed at glycine at position 197 or alanine at 121 of the heavy chain (HC) (SEQ ID NO:3).

[0051] Importantly, the first two sites were chosen because they were previously found to be incompatible with the THIOMAB technology due to incomplete reoxidation25, and the last was chosen as a known site for successful stop codon suppression and conjugation.47

[0052] Plasmids encoding one of these mutants of Trastuzumab was co-transfected into Expi293 suspension cells with another plasmid containing the EcLeuRS / tRNA machinery (components required for translation and expression of the protein), and Nvoc- cysteine was supplied in the media at the time of transfection at a final concentration of 1 mM. The expression was allowed to continue for 7 days, and afterwards the mutant antibodies were purified using protein-G-affinity chromatography. For all three sites, the mutant full-length antibody was able to be expressed / purified in high yield (~4-6 mg / L); for reference, under identical expression conditions, the wild-type Trastuzumab was obtained at 13 mg / L yield (Figure 4A). ESI-MS analysis of the purified antibody mutants following deglycosylation and reduction confirmed heavy and light chain masses consistent with the site-specific incorporation of the ncAA at the desired sites (Figure 4B). To demonstrate that the photocage can be cleanly removed with light, the mutant antibodies were irradiated at 365 nm for 10 min. Subsequent MS analysis, following reduction and deglycosylation, revealed all three mutant antibodies underwent complete decaging to generate a free cysteine residue (Figure 4B-C). These observations show that the Nvoc-cys residue can be incorporated into different sites within an antibody with good efficiency and selectively, and subsequently converted to a free thiol through irradiation.

[0053] Next, the feasibility of utilizing the free thiol uncaged through this process for bioconjugation applications was demonstrated. Although thiol-maleimide chemistry hasbeen widely used for the preparation of therapeutic antibody conjugates in the past1’50’62, it is now well-established that the stability of these thiol-maleimide linkages are suboptimal due to their tendency to undergo thiol-exchange reactions in vivo.29’63Previous work from Bernardes et al. has demonstrated that carbonylacrylic acid derivatives can undergo an irreversible thiol-Michael addition with cysteine residues to generate ADCs with more stable linkages.64’65Thus, it was decided to use a carbonylacrylic acid derivative as the warhead for conjugation to the decaged antibodies. First, all three mutant antibodies, produced as described above and containing a decaged thiol residue, were allowed to react with the carbonylacrylic acid warhead alone (Figure 5 A), and the efficiency of conjugation was evaluated by ESI-MS analysis. Incubation of the mutants with 250 pM warhead for 16 h resulted in complete modification of the HC-121 mutant, while mutants LC-122 and HC- 197 afforded only partial conversion (Figure 6A-D). It is possible that the different degrees of conversion of these mutants reflect their respective accessibility within the antibody structure, dictated by their local microenvironments. Nonetheless, efficient incorporation of Nvoc-cys at LC-122 and HC-197 sites, and their subsequent photo-decaging to generate clean thiol residues, showcase the broad scope of this method. It is worth noting that these two particular sites were previously found to be incompatible with the THIOMAB technology, due to unsuccessful reoxidation of these antibodies following conjugation.25

[0054] Importantly, the antibody containing the decaged cysteine at position 121 of the heavy chain was successfully conjugated to the carbonylacrylic warhead with complete conversion, as confirmed by ESI-MS analysis (Figure 6D). To establish that functional ADCs could be generated, the carbonylacrylic acid warhead was attached to monomethyl auristatin F (MMAF), a well-established cytotoxic tubulin inhibitor that has been used for generating ADCs used to generate functional ADCs.66Incubation of the MMAF- carbonylacrylic acid (2 mM) with the mutant Trastuzumab antibody containing the decaged thiol at HC-121 position resulted in its full conversion to the corresponding ADC, as revealed by ESI-MS analysis (Figure 5A). To assess the functional efficacy of the resulting ADC, SK-BR-3 (HER2 overexpressing) and MDA-MB-231 (HER2-negative) cells were treated with it at increasing concentration, and after incubation for 5 days the viability of the cells was analyzed using the MTT assay. The ADC exhibited potent cytotoxicity towards the HER2+ SK-BR-3 cell line, but not towards the HER2- MDA-MB- 231 line. The IC50 of the ADC was found to be approximately 0.2 nM (Figure 4B), whichis in agreement with similar ADCs produced with comparable drug-to-antibody ratio.15’38>43, 67-69

[0055] In summary, described herein is a facile method to prepare site-specific antibody drug conjugates using a genetically encoded photocaged cysteine residue. The photocaged group can be rapidly removed using light, followed by direct conjugation to thiol reactive probes without the need for cumbersome reduction and subsequent reoxidation of the antibody. This technology may also allow the introduction of a conjugation-ready thiol residue at a wider variety of positions across the antibody, relative to what is accessible through direct cysteine mutations. Given that the site of conjugation is known to affect the therapeutic behavior of these conjugates, maximizing the number of available sites for possible attachment is highly valuable to fine-tune their efficacy. Although the GCE technology has been used to incorporate additional ncAAs with bioorthogonal conjugation handles, the strategy reported here can take advantage of the broad array of thiol-targeted bioconjugation techniques that have been developed and are widely accessible.

[0056] Furthermore, it has been previously demonstrated that the EcLeuRS / tRNA pair is orthogonal to both the pyrrolysyl (Pyl) and E. coli tryptophanyl-tRNA synthetase / tRNA pairs, and can be used in conjunction for successful incorporation of multiple different ncAAs into one protein in mammalian cells, including in full-length antibodies.47’51-53This technology has been used to site-specifically incorporate multiple different bioconjugation handles that are mutually compatible to allow precise labeling of a protein with multiple, distinct cargos. Given that the thiol -targeted bioconjugation chemistries are compatible with other bioorthogonal conjugation reactions accessible through ncAA methodology (e.g., SPAAC, IEDDA, CRACR, etc.), it should be possible to combine the photocaged cysteine platform with other ncAA incorporation systems for introducing multiple reactive handles into one antibody, integrating site-specific cysteine chemistry with other bioconjugation strategies.

[0057] Finally, this strategy could be extended for the modification of other types of excreted therapeutic proteins where capping would prevent the direct modification of engineered cysteines in a similar manner, such as for the preparation of site-specifically PEGylated cytokines.70’71Overall, this work should expand the scope of the widely used thiol-targeted conjugation approaches by making it simpler to introduce a conjugation-ready cysteine-thiol into secreted therapeutic proteins.

[0058] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments and examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0059] Exemplification:

[0060] Materials and methods:

[0061] General methods :

[0062] All cloning and plasmid propagation were performed in E. coli strain DHIOb. Antibiotics were supplied in the bacterial culture media at the following concentrations: 150 pg / mL ampicillin. Oligonucleotides were purchased from Azenta Life Sciences. Phusion polymerase, restriction enzymes, and T4 DNA ligase were obtained from Fisher Scientific. Sanger sequencing of DNA was performed by Azenta Life Sciences. HEK293T cells were maintained at 37 C and 5% CO2 using DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin at a final concentration of 100 U / mL. High glucose DMEM, trypsin 0.25%, penicillin-streptomycin (10,000 U / mL), and fetal bovine serum (FBS) were obtained from Fisher Scientific. Polyethylenimine (PEI MAX) from Polysciences (Warrington, PA) was used for transient transfection. Sodium butyrate used for transfection was obtained from Fisher Scientific. Expi293F cells were grown at 37 C, 8% CO2, using an orbital shaker at 125 rpm in Expi293 expression media supplemented with 0.5x antibiotic-antimycotic (Fisher). Cells were seeded at a density of 0.3 million cells / mL and sub-cultured every four days.

[0063] Plasmid construction:

[0064] pAcBacl plasmid containing the E. coli leucyl aaRS / tRNA pair and pAcBacl plasmid containing EGFP-40TAG were prepared as reported previously.52For expression of Trastuzumab, pcDNA3.1 -Her2 plasmid (gift from Prof. Han Xiao) was mutagenized using PrimeSTAR MAX DNA polymerase (Takara) and primer HugI-121 A-TAG. For the HC-197 mutant, pcDNA3.1-Her2-HC197-TAG was constructed by overlap extension PCR using primers AntiHer2-HC-SbfI-F, AntiHer2-HC197TAG-iF, AntiHer2-HC197TAG-iR, and AntiHer2-NotI-R, followed by digestion with Sbfl / Notl and insertion into pcDNA3.1- Her2 vector.

[0065] Analysis of EGFP reporter expression in HEK293T cells:

[0066] For small-scale EGFP expression, HEK293T cells were seeded at a density of 600,000 cells / well in a 12-well plate one day before transfection. DNA, PEI, and DMEM were pre-mixed and allowed to sit for 10 min prior to additional to cells. A total amount of1 pg DNA + 4 pL PEI (1 mg / mL) + 20 pL DMEM was used for transfection of each well. For two-plasmid transfection, 0.5 pg of each plasmid was used. The appropriate ncAA was supplied to each well at a final concentration of 1 mM. 48 h post-transfection, fluorescence images were taken using a Zeiss Axio Observer fluorescence microscope.

[0067] Expression / Purification, decaging, and labeling of EGFP:

[0068] For larger scale protein expression, HEK293T cells were seeded in 100 mm cell culture dishes (8 million cells per dish) one day prior to transfection. DNA, PEI, and DMEM were pre-mixed and allowed to sit for 10 min prior to additional to cells. A total amount of 12 pg DNA (6 pg of each plasmid) + 48 pl PEI (1 mg / mL) + 240 pl DMEM was used to transfect each dish. The ncAA was supplemented at 1 mM final concentration at the time of transfection. Sodium butyrate was also supplied in the transfection media at a final concentration of 2 mM. Cells were harvested 48 h post-transfection, lysed with CelLytic M, and protein was purified from the clarified lysate via the C-terminal polyhistidine tag using HisPur Ni-NTA resin following the manufacturer’s protocol. Purified EGFP was subjected to SDS-PAGE analysis and ESLMS (Agilent 1260 Infinity ESI-TOF).

[0069] For decaging of mutant EGFP proteins, the protein was first diluted using 100 mM HEPES pH 7 to a final protein concentration of ~3 pM and ~50 mM HEPES. Next, ascorbic acid (5 mM final concentration) was added and the solution was exposed to 365 nm light for 10 min on ice with a 120 W LED-array; Larson Electronics, 120 Watt Ultraviolet LED Light Emitter- 24 UV (365 nm) LEDs (part # LEDLB-24E-UV-365NM). For subsequent bioconjugation onto the resulting protein, N-Ethylmal eimide (Thermo Scientific) was added to a final concentration of 100 pM and the solution was incubated for2 h at room temperature. The reaction mixture was then directly analyzed by ESI-MS analysis (Agilent 1260 Infinity ESI-TOF).

[0070] Antibody expression and purification:

[0071] Antibody expression and purification was performed as described previously.47Briefly, when Expi293F cells reached a density of 3 million cells / mL (at least 95% viability), they were spun down and resuspended in media to a density of 20 million cells / mL for transfection. An equal ratio of the two plasmids were added to the cells at a final DNA concentration of 25 pg / mL. PEI MAX (40 mg / mL stock, 50 pg / mL final concentration) was then added to the cells. After the addition of DNA and PEI, the cells were incubated for 3 h under standard conditions with shaking, then diluted back to their original volume. At that time, 1 mM of the appropriate ncAA was supplied as well as 2 mM valproic acid.

[0072] After 7 days of expression, the cells were pelleted, and the media was collected. The media was passed through a 0.22 pm sterile filter and adjusted to pH 5.4 and 50 mM final concentration of NaOAc. For antibody purification, 1 mL of Pierce™ Protein G Agarose resin (Thermo Scientific) was equilibrated with wash buffer (50 mM NaOAc pH 5.4), and the media containing antibody was allowed to passed through it by gravity flow. The column was washed with 50 mM NaOAc pH 5.4, with a volume equal to about half the volume of media passed through the column. The antibody was then eluted in 1 mL fractions of 100 mM glycine (pH 2.7) into tubes containing IM phosphate pH 8 for neutralization. The fractions were concentrated using an Amicon Ultra 15 centrifugal filter unit and buffer exchanged to PBS. For ESLMS analysis, the antibody was reduced with 10 mM TCEP at 55 C for 15 min, followed by treatment with Remove-iT PNGaseF (New England BioLabs) for 1 h at 37 C. The PNGase was removed by incubation with chitin resin (New England BioLabs) for 30 min at RT, the sample was centrifuged to remove the resin, and the supernatant was subjected to ESLMS analysis (Agilent 1260 Infinity ESL TOF).

[0073] Antibody decaging and conjugation:

[0074] For decaging / conjugation of the antibody mutants containing photocaged cysteine, the protein was first diluted using 100 mM HEPES pH 7 to a final protein concentration of ~10 pM and ~50 mM HEPES. Next, ascorbic acid (5 mM final concentration) was added and the solution was exposed to 365 nm light for 10 min on ice with a 120 W LED-array (Larson Electronics, 120 Watt Ultraviolet LED Light Emitter- 24 UV LEDs- 365 nm- 9-42 VDC). The mixture was then buffer exchanged using a 10 kDa Amicon centrifugal filter unit to 20 mM phosphate pH 8. For subsequent labeling just with the carbonylacrylic acid warhead, 2.5 mM 3-benzoylacrylic acid (Thermo Scientific) inDMF was added to the antibody solution at a final concentration of 250 pM (10% DMF final concentration). For the labeling reaction using the MMAF-Caa conjugate to generate the ADC, MMAF-Caa (dissolved in DMSO) was added to the antibody solution at a final concentration of 1 mM (-10% DMSO final concentration). The solution was shaken at 250 rpm at 37 C for 24 h. The reaction mixture was then buffer exchanged with 20 mM phosphate pH 8. To prepare for ESI-MS analysis, the antibody was first reduced with 10 mM TCEP at 55 C for 15 min, followed by treatment with Remove-iT PNGaseF (New England BioLabs) for 1 h at 37 C. The PNGase was removed by incubation with chitin resin (New England BioLabs) for 30 min at RT, the same was centrifuged to remove the resin, and the supernatant was subjected to ESLMS analysis (Agilent 1260 Infinity ESL TOF).

[0075] Assessment of cytotoxicity:

[0076] To assess the cytotoxicity of the prepared ADC toward cancer cell lines, Vybrant MTT Cell Proliferation Assay Kit (Invitrogen) was used following the manufacturer’s instructions. Briefly, SK-BR-3 and MDA-MB-231 cells were cultured at 37 C and 5% CO2 using DMEM supplemented with 10% fetal bovine serum. Cells were seeded into a 96-well plate at a density of 5,000 cells / well one day prior to the experiment. A 100 nM solution of ADC or WT Trastuzumab was prepared using the culture media, sterile filtered using a 0.22 pm syringe filter, and 4x serial dilutions were prepared down to 0.005 nM. The media was aspirated out from each well of the 96-well plate, and 50 pL of antibody solution was added to each well. The cells were incubated with the antibody solutions for 5 days under standard culture conditions. Following incubation, the media was removed from the wells and replaced with 100 pL of fresh DMEM + FBS (no phenol red). 20 pL of a 2.5 mg / mL solution of MTT in PBS was added to each well, and incubated for 4 h at 37 C. Next, 100 pL of SDS-HC1 solution (1 g SDS / 10 mL 0.01M HC1) was added to each well, mixed thoroughly, and incubated at 37 C for 6 h. Following incubation, the wells were mixed again and the absorbance at 570 nm was recorded using a BioTek Synergy Neo2 microplate reader (Absorbance Endpoint, Wavelength: 570 nm, Read Speed: Normal, Delay: 50 msec, Measurements / Data Point: 8).

[0077] Synthesis of Nvoc-cysteine: Nvoc-cysteine was prepared as described previously.37

[0078] Synthesis of MMAF-Caa conjugate:

[0079] Synthetic route for synthesis of MMAF probe.

[0080] Synthesis of intermediate (2): A-hydroxysuccinimide (0.75 g, 6.52 mmol, 1.14 eqv.) and traw -4-oxo-4-phenyl-2-butenoate 1 (1.01 g, 5.7 mmol, 1.0 eqv.) were dissolved in 15 mL THF. The resulting yellow solution was brought to 0 °C using an ice- water / sodium chloride bath under a nitrogen atmosphere. After cooling, DCC (1.35 g, 6.54 mmol, 1.15 eqv.) was added. After stirring for 15 minutes the resulting orange colored heterogeneous mixture was kept at -20 °C overnight. The precipitate was filtered off using a fritted funnel using ~30 mL ethyl acetate. The filtrate was concentrated in a rotary evaporator to get an oily residue. Addition of isopropanol (~25 mL) resulted in a precipitate which was filtered off. To the filtrate hexane (~50 mL) was added to initiate crystallization. Resulted orange color crystals were isolated by filtration, dried to get the expected product (2). 'H-NMR (600 MHz, Acetone-D6): 5 8.27 (d; 1H; J = 12 Hz), 8.13- 8.14 (m; 2H), 7.73-7.75 (m; 2H), 7.61-7.64 (m; 2H), 7.02 (d;1H; J = 12 Hz), 2.95 (s; 4H); ESI-TOF-MS Calculated for C14H12NO5 ([M+H]+): 273.24, found: 273.24

[0081] Synthesis of MMAF -warhead (3):

[0082] MMAF.TFA salt (14 mg, 0.013 mmol, 1 eqv.) (BroadPharm) was dissolved in 0.3 mL anhydrous dimethyformamide (DMF) in a screw cap glass vial fitted with a magnetic stir bar and anhydrous K2CO3 (8 mg, 0.058 mmol, 4.5 eqv.) was added to the mixture followed by the addition of intermediate 2 (6 mg, 0.022 mmol, 1.7 eqv.). The vial was flushed with argon, sealed, and stirred overnight at room temperature (~22 °C). Total consumption of the MMAF and formation of a new molecular species corresponding to the molecule 3 was confirmed by HPLC-MS analysis of the crude reaction mixture. A mixture of water / acetonitrile (1 : 1, 6 mL) was added to dilute the reaction mixture. The expectedproduct 3 was purified by revere phase HPLC (RP-HPLC; Waters) analysis using a semipreparative C-18 column (Phenomenex, Jupiter® 10 pm, C-18, 300 A, 250 x 10 mm) utilizing mobile phases solvent-A (water-MeCN-0.1% trifluoroacetic acid in 95:5:0.1 ratio) and solvent B (MeCN-water-0.1% trifluoroacetic acid in 95:5:0.1 ratio); flow rate 6 mL / min. The following elution conditions was used for RP-HPLC: 0-10 min 100% A, 10- 40 min 100% A to 100% B, 40-45 min 100% B, 45-48 min 100% B to 100% A, 48-50 min 100% A). Fractions containing the expected product (judged by HPLC-MS analysis) were pooled together, concentrated in a rotary evaporator and lyophilized; RP-HPLC retention time of the product = 15.7 min. The expected product was obtained as a white solid; ESI- TOF-MS (m / z): [M+Na]+Calculated for CeoHssNeOisNa 1159.6199, obtained. 1159.4922

[0083] Table 1. Oligonucleotide sequences (SEQ ID NOS: 5-11, respectively)

[0084] The plasmid map of Plasmid pAcBacl -NVocRS-4xLtR is shown in Figure 7A.

[0085] The plasmid nucleic acid sequence of pAcBacl -NVocRS-4xLtR is shown inFigure 7B. The NVocRS sequence is highlighted in blue, LtR sequences are highlighted in purple, and U6 promoter sequences are highlighted in orange.

[0086] The plasmid map of Plasmid pcDNA3.1 AntiHer2-121HC-TAG is shown in Figure 8A and the antibody expression cassette is colored in blue.

[0087] The nucleic acid sequence of pcDNA3.1 AntiHer2-121HC-TAG is shown in Figure 8B. The TAG position is at position 121 of the heavy chain of the amino acid sequence of expressed anti-Her2 antibody.

[0088] The nucleic acid sequence of Plasmid pcDNA3.1 AntiHer2-198HC-TAG is the same as shown in Figure 8B, except TAG is at position 198 in the HC instead of position121 of the amino acid sequence of expressed anti-Her2 antibody.

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[0090] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A recombinantly-expressed secreted protein, polypeptide or peptide containing native disulfide bonds, wherein the protein comprises one, or more, photocaged noncanonical (NcAA) cysteine analogs incorporated into the protein, polypeptide or peptide at pre-determined specific amino acid residue sites.

2. The protein, polypeptide or peptide of claim 1, wherein the protein, polypeptide or peptide is selected from the group consisting of: an antibody or antibody fragment, a cytokine, a growth factor, a hormone, a serum protein, a coagulation factor, a fusion protein or a multi-specific antibody or fragment.

3. The protein, polypeptide or peptide of either claim 1 or 2, wherein noncanonical cysteine analog is a Nvoc (4, 5-dimethoxy-2-nitrobenzyl) protected cysteine.

4. The protein, polypeptide or peptide of any of claims 1 through 3, wherein the protein is an antibody, or antibody fragment, comprising one, or more, photo-caged noncanonical (NcAA) cysteine analogs incorporated into the antibody, or antibody fragment, at pre-determined specific amino acid residue sites.

5. The antibody, or antibody fragment, of claim 4, wherein the antibody is anti- Her2 antibody.

6. The antibody, or antibody fragment, of claim 5, wherein the one, or more, specific amino acid residue sites of anti-Her2 antibody determined for incorporation of the photo-caged noncanonical cysteine analog is selected from the group consisting of: aspartic acid at position 122 of the light chain (SEQ ID NO:4), glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain (SEQ ID NO:3) of the antibody.

7. The protein, polypeptide or peptide of any of claims 1 to 6, wherein the photocaged noncanonical cysteine analog is decaged, resulting in a free cysteine amino acid residue.

8. The protein, polypeptide or peptide of claim 7, wherein the free cysteine is conjugated to a biomolecule or chemical compound such as a linker, warhead or cargo molecule.

9. The protein, polypeptide or peptide of claim 8, wherein the biomolecule or chemical compound is selected from the group consisting of: a therapeutic or cytotoxic drug, a small organic molecule, a nucleic acid, a protein, a polypeptide, a peptide or a biophysical probe.

10. A genetically-engineered anti-Her2 antibody, or fragment thereof, comprising one or more Nvoc protected-cysteine analogs incorporated into the antibody, or fragment thereof, at one, or more amino acid residues selected from the group consisting of: aspartic acid at position 122 of the light chain (SEQ ID NO:4) , glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain (SEQ ID NO:3).

11. The genetically-engineered antibody, or fragment thereof, of claim 10 wherein the Nvoc protected-cysteine analog is decaged resulting in a free cysteine amino acid residue.

12. A method of producing a secretory protein, polypeptide or peptide conjugate wherein the protein, polypepide or peptide contains native disulfide bonds, the method using a photocaged noncanonical (ncAA) cysteine analog, comprising the steps of: a.) providing a genetically-engineered secretory protein, polypeptide or peptide, comprising native disulfide bonds, further comprising one, or more noncanonical amino acid residues in specific sites of the protein, polypeptide or peptide, wherein the noncanonical residue is a photocaged cysteine analog; b.) irradiating the protein, polypeptide or peptide of step a.) under suitable conditions to decage the protected cysteine analog, thereby producing an protein, polypeptide or peptide with a free, reactive cysteine residue; c.) reacting the free cysteine residue of the protein, polypeptide or peptide of step b) with a cargo molecule comprising a therapeutic or cytotoxicdrug, a biophysical probe, a peptide, or a small molecule, or nucleic acid under suitable conditions for the cargo molecule to conjugate to the free cysteine residue; thereby producing a secretory protein, polypeptide or peptide conjugate.

13. The method of claim 12, wherein the protein, polypeptide or peptide is an antibody, or antibody fragment.

14. The method of either claim 12 or 13, wherein the one, or more, noncanonical photocaged cysteine residues are exposed on the surface of the protein.

15. The method of any of claims 12 through 14, wherein the photocaged noncanonical (ncAA) cysteine analog is Nvoc-cysteine residue.

16. The method of claim 15, wherein the irradiation of the Nvoc-cysteine residue is at about 365 nm for 10 minutes.

17. The method of any of claims 12 through 15, wherein in step c.) the cargo molecule is carbonylacrylic acid.

18. The method of any of claims 12 through 17, wherein the cargo molecule is a cytotoxic drug.

19. The method of claim 18, wherein cytotoxic drug is monomethyl auristatin (MMAF).

20. A method of treating cancer in a patient, comprising administering to the patient in need thereof protein-drug or an antibody-drug conjugate produced by the method of any of claims 12-19, under conditions suitable for the protein-drug or antibody-drug conjugate to react with the targeted cancer cells and selectively kill the targeted cancer cells, thereby treating the cancer patient.

21. The method of claim 20, wherein the targeted cancer cells are HER2 receptorpositive cancer cells.

22. The method of either claim 20 or 21, wherein the antibody-drug conjugate comprises the antibody anti-Her2 antibody comprising carbonylacrylic acidattached to monomethyl auristatin conjugated to the anti-Her2 antibody at a position selected from the group consisting of: aspartic acid at position 122 of the light chain (SEQ ID NO:4), glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain (SEQ OID NO:3).

23. A method of producing a conjugation-reactive secretory protein, antibody, or antibody fragment, wherein the protein, antibody, or antibody fragment comprises native disulfide bonds, and wherein the protein or antibody or antibody fragment comprises a photocage noncanonical cysteine analog, comprising the steps of: a.) co-transfecting mammalian cells with two plasmids comprising;1.) a nucleic acid encoding a secretory protein, an antibody, or antibody fragment, mutated at one, or more, specific amino acid residue sites to incorporate a TAG stop codon; and2.) EcLeuRS / tRNA translation machinery components; and b.) introducing a photocaged noncanonical cysteine analog to the mammalian cells of step a.) and incubating the cells with the plasmids and photocaged noncanonical cysteine analog under conditions suitable for the incorporation of the photocaged noncanonical cysteine analogs at the specific site of the TAG stop codon; and c.) purifying the protein, antibody, or antibody fragment comprising the photocaged noncanonical cysteine analog, thereby, producing a conjugation-reactive secretory protein, antibody, or antibody fragment, wherein the antibody, or antibody fragment comprises a photocage noncanonical cysteine analog.

24. The method of claim 23, wherein the photocaged noncanonical cysteine analog is Nvoc cysteine.

25. The method of either of claim 23 or 24, wherein the antibody is anti-Her2 antibody.

26. The method of any claim 25, wherein the anti-Her2 antibody comprises a photocaged noncanonical cysteine analog at a position selected from the groupconsisting of: aspartic acid at position 122 of the light chain (SEQ ID NO:4), glycine at position 197 of the heavy chain, or alanine at position 121 of the heavy chain (SEQ ID NO:3).

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