Antibodies and fragments thereof targeting psma

Abstract

The present disclosure relates to PSMA binding proteins and antibody-drug conjugates comprising such a PSMA binding protein. The disclosure further relates to the PSMA binding proteins or the antibody-drug conjugates for use for the treatment of cancer. The disclosure further relates to method of making such PSMA binding proteins and antibody-drug conjugates.

Description

[0001] ANTIBODIES AND FRAGMENTS THEREOF TARGETING PSMA CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit of European patent application No. EP25305271.6, filed February 28, 2025, the disclosure of which is incorporated herein by reference in its entirety.

[0003] REFERENCE TO A SEQUENCE LISTING

[0004] This application incorporates by reference the Sequence Listing submitted in XML format named 70714W001_seq_list 20Feb2026.xml, created on or about February 20, 2026 and containing about 46,885 bytes.

[0005] FIELD OF THE INVENTION

[0006] The present disclosure relates to a prostate-specific membrane antigen (PSMA) binding protein and antibody-drug conjugate comprising the PSMA binding protein, as well as methods of use thereof.

[0007] BACKGROUND TO THE INVENTION PSMA is a highly tumor-restricted cell-surface protein whose differential expressions correlate with cancer grades and stages. It served as a target for the development of tumorspecific monoclonal antibodies to deliver systemic, targeted radiation to cancer. Rosopatamab is a monoclonal antibody (mAb) against the external domain of PSMA and has demonstrated safety in numerous human-studies with anti-tumor activity when radiolabeled and conjugated with drug. After binding, the PSMA-antibody complex is internalized into the cell.

[0008] Several preclinical studies were performed employing PSMA-targeting ADCs for the treatment of metastatic castration-resistant prostate cancer (mCRPC). In preclinical studies, the respective ADCs showed strong and specific cytotoxicity. Unfortunately, in clinical trials, they show only limited anti-tumor activity, accompanied by common treatment-related adverse effects such as neutropenia and neuropathy.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention concerns anti-PSMA antibodies, or antigen binding fragments thereof, and antibody-drug conjugates comprising such antibodies or fragments. The invention further relates to the antibody, antigen binding fragment or the antibody-drug conjugates (ADC) for use for the treatment of cancer.

[0011] In some aspects, disclosed herein is a new anti-PSMA antibody binding to a different epitope than Rosopatamab and having different binding characteristics. This novel antibody binds to an epitope distinct from the one recognized by Rosopatamab and shows anexcellent cancer cell penetration capacity. Also disclosed herein is a new antibody-drug conjugate (ADC) from this antibody which demonstrated promising in vivo results.

[0012] The invention thus relates to an anti-PSMA antibody, or an antigen binding fragment thereof, comprising:

[0013] a. a variable heavy chain domain (VH) comprising:

[0014] i. a CDR1-H of sequence SEQ ID NO: 1,

[0015] ii. a CDR2-H of sequence SEQ ID NO: 2, and

[0016] iii. a CDR3-H of sequence SEQ ID NO: 3; and

[0017] b. a variable light chain domain (VL) comprising:

[0018] i. a CDR1-L of sequence SEQ ID NO: 4,

[0019] ii. a CDR2-L of sequence SEQ ID NO: 5, and

[0020] iii. a CDR3-L of sequence SEQ ID NO: 6,

[0021] or a variant thereof comprising up to five point mutations across said six CDR sequences, wherein said variant retains a binding affinity to PSMA.

[0022] In some embodiments the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof according to some embodiments of the present disclosure, comprises a Human IgGl Constant heavy chain (CH) and a Human IgGl Constant light chain (CL), in particular a human kappa constant domain.

[0023] The preferred antibody according to some embodiments of the present disclosure, named hereinafter " XT42," is an IgGl having for heavy chain the amino acid sequence SEQ ID NO: 11 and for light chain the amino acid sequence SEQ ID NO: 12.

[0024] The invention thus, in some aspects, relates to the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof according to some embodiments of the present disclosure, further comprising an agent selected from the group consisting of therapeutic agents, imaging agents, and theranostic agents, wherein the agent is conjugated to the antibody.

[0025] The invention, in some further aspects, relates to the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof according to some embodiments of the present disclosure, for use for the treatment, prevention and / or diagnostic of cancer, preferably selected from prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

[0026] The invention, in some aspects, also relates to a pharmaceutical composition comprising an anti-PSMA antibody, or an antigen binding fragment thereof, or a variantthereof according to some embodiments of the present disclosure, and a pharmaceutically acceptable carrier.

[0027] Also provided herein is a pair of isolated nucleic acids comprising a sequence encoding respectively the variable heavy chain domain (VH) and variable light chain domain (VL) of the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof according to some embodiments of the present disclosure.

[0028] Also provided herein is an antibody-drug conjugate (ADC) comprising (i) an anti-PSMA antibody, or an antigen binding fragment thereof, or a variant thereof according to some embodiments of the present disclosure, (ii) a cytotoxic payload, and (iii) a linker connecting the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof and the cytotoxic payload.

[0029] Further provided herein is a multispecific antibody construct that binds at least to PSMA and to one or more antigens on T cells, and an ADC comprising said multispecific antibody, a cytotoxic payload, and a linker connecting the bispecific antibody construct and the cytotoxic payload.

[0030] The invention, in some further aspects, relates to an ADC according to some embodiments of the present disclosure for use for the treatment, prevention and / or diagnostic of cancer, preferably selected from prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

[0031] Also provided herein is a pharmaceutical composition comprising an antibody-drug conjugate according to some embodiments of the present disclosure, and a pharmaceutically acceptable carrier.

[0032] DESCRIPTION OF FIGURES

[0033] Figure 1. Kinetic curves of binding of XT42 (antibody according to some embodiments of the present disclosure) and the control antibody (J591) to human PSMA.

[0034] Figure 2. Kinetic curves of binding of XT42 to different PSMA (Cross reactivity evaluation with human PSMA, rat PSMA, cynomolgus PSMA, and mouse PSMA).

[0035] Figure 3. Kinetic curves of binding of XT42 and J591 to study epitope binding and competition.

[0036] Figure 4. Binding curves of XT42 and an irrelevant antibody 13R4 on human PSMA-expressing LnCap clone FGC.Figure 5. Kinetic curves of internalization of XT42, 13R4 and J591 antibodies in human PSMA-positive LnCap cells.

[0037] Figure 6. Pharmacokinetics curves of XT42 and XT42-ADC in Sprague-Dawley rats. Figure 7. Evolution in time of the mean tumor volume in LnCaP-C4.2 xenograft model of prostate cancer, further to intravenous injection of XT42-ADC, J591-ADC, or vehicle (PBS).

[0038] Figure 8. Evolution in time of the mean tumor volume using the LuCap96CR PDX model comparing the antitumor efficacy of XT42-ADC and Enzalutamide.

[0039] Figure 9. Evolution in time of the mean tumor volume in LnCaP-C4.2 xenograft model of prostate cancer, dose-response study of XT42-ADC.

[0040] Figure 10. Dose-response curves demonstrating antitumor potency of Compound 10 and Compound 9 on PSMA-positive C4-2 cell line as measured by cell viability.

[0041] DETAILED DESCRIPTION DEFINITIONS

[0042] " Prostate-specific membrane antigen" or " PSMA" also known as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), NAAG peptidase, is a membrane protein encoded by the FOLH1 (folate hydrolase 1) gene. It is highly expressed in the prostate, and his expression is known to be upregulated in some prostate cancer. A reference sequence of human PSMA is available in Uniprot database, under accession number Q04609 entry version 232 of 5 February 2025).

[0043] The terms " PSMA binding protein" or "anti-PSMA antibody or antigen binding fragment thereof" as used herein interchangeably refer to antibodies and other protein constructs, such as domains, that are capable of binding to PSMA. " Antigen binding protein" is used interchangeably with " PSMA binding protein" in the present disclosure. This does not include the natural cognate ligand or receptor.

[0044] The term "antibody" is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., a domain antibody (DAB)), antigen binding antibody fragments, Fab, F(ab')2, Fv, disulphide stabilized Fv (dsFv), single chain Fv (scFv), disulphide-stabilized scFv, diabodies, tandem diabodies (TANDABS), (dsFv)2, sc(Fv)2, triabodies, tetrabodies, bispecific and multispecific antibodies, etc., and modified versions of any of the foregoing (for a summary of alternative "antibody" formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).The term, full, whole or intact antibody, used interchangeably herein, refers to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2structure folds to form three functional domains comprising two antigen-binding fragments, known as 'Fab' fragments, and a 'Fc' crystallisable fragment. The Fab fragment is composed of the variable domain at the aminoterminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CHI (heavy) and CL (light). The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding Clq, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences, which are called μ, α, γ, ε and δ respectively, each heavy chain can pair with either a K or A light chain. The majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG (IgGl, IgG2, IgG3 and IgG4), the sequences of which differ mainly in their hinge region.

[0045] Fully human antibodies can be obtained using a variety of methods, for example using yeast-based libraries or transgenic animals (e.g. mice) that are capable of producing repertoires of human antibodies. Yeast presenting human antibodies on their surface that bind to an antigen of interest can be selected using FACS (Fluorescence-Activated Cell Sorting) based methods or by capture on beads using labelled antigens. Transgenic animals that have been modified to express human immunoglobulin genes can be immunised with an antigen of interest and antigen-specific human antibodies isolated using B-cell sorting techniques. Human antibodies produced using these techniques can then be characterised for desired properties such as affinity, developability and selectivity.

[0046] Alternative antibody formats include alternative scaffolds in which the one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U. S. Patent Application Publication Nos. 2005 / 0053973, 2005 / 0089932, 2005 / 0164301) or an EGF domain.

[0047] Antigen binding site refers to a site on an antigen binding protein that is capable of specifically binding to an antigen, this may be a single variable domain, or it may be paired VH / VL domains as can be found on a standard antibody. Single-chain Fv (ScFv) domains can also provide antigen-binding sites.In some embodiments of the present invention, an "antibody" is a natural or conventional immunoglobulin molecule in which disulfide bonds (or disulfide bridge) link two heavy chains to each other and each heavy chain is linked to a light chain by a disulfide bond. In some cases, there are two types of light chain, lambda ( ) and kappa (K). In some cases, there are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. Preferably, an antibody as disclosed herein is an IgG antibody molecule.

[0048] The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or "complementarity determining regions" (CDRs). The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

[0049] " Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR-L1, FR-L2, FR-L3, FR-L4, and FR-H1, FR-H2, FR-H3, FR-H4, respectively.

[0050] In the context of the invention, CDR / FR definition in an immunoglobulin light or heavy chain is to be determined based on Kabat numbering, unless otherwise indicated.

[0051] The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter " Kabat et al."). This numbering system is used in the present specification unless otherwise indicated.

[0052] It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al. (1989) Nature 342: 877-883. The structure and protein foldingof the antigen binding protein may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person.

[0053] Other numbering conventions for CDR sequences available to a skilled person include " AbM" (University of Bath) and "contact" (University College London) methods.

[0054] Table 1 below represents one definition using each numbering convention for each CDR or binding unit. The Kabat numbering scheme is used in Table 1 to number the variable domain amino acid sequence. It should be noted that some of the CDR definitions may vary depending on the individual publication used.

[0055] Table 1: Numbering schemes

[0056] Kabat CDR Chothia CDR AbM CDR Contact CDR

[0057] Hl 31-35 / 35A / 35B 26-32 / 33 / 34 26-35 / 35A / 35B 30-35 / 35A / 35B

[0058] H2 50-65 52-56 50-58 47-58

[0059] H3 95-102 95-102 95-102 93-101

[0060] LI 24-34 24-34 24-34 30-36

[0061] L2 50-56 50-56 50-56 46-55

[0062]

[0063] L3 89-97 89-97 89-97 89-96

[0064] Accordingly, a PSMA binding protein (anti-PSMA antibody or antigen-binding fragment thereof) is provided, which comprises any one or a combination of the following CDRs:

[0065] CDR-H1 of SEQ ID NO: 1,

[0066] CDR-H2 of SEQ ID NO: 2,

[0067] CDR-H3 of SEQ ID NO: 3,

[0068] CDR-L1 of SEQ ID NO: 4,

[0069] CDR-L2 of SEQ ID NO: 5, and

[0070] CDR-L3 of SEQ ID NO: 6.

[0071] CDRs may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein, such as the specific binding of PSMA (e.g., human PSMA).

[0072] It will be appreciated that each of CDR-H1, H2, H3, LI, L2, L3 may be modified alone or in combination with any other CDR, in any permutation or combination. In one embodiment, a CDR is modified by the substitution, deletion or addition of up to 3 amino acids, for example 1 or 2 amino acids, for example 1 amino acid. Typically, the modification is a substitution, particularly a conservative substitution, for example as shown in Table 2 below.Table 2: Conventional amino acid substitutions

[0073] Side chain Members

[0074] Hydrophobic Met, Ala, Vai, Leu, He

[0075] Neutral hydrophilic Cys, Ser, Thr

[0076] Acidic Asp, Glu

[0077] Basic Asn, Gin, His, Lys, Arg

[0078] Residues that influence chain Gly, Pro

[0079] orientation

[0080]

[0081] Aromatic Trp, Tyr, Phe

[0082] For example, in a variant CDR, the flanking residues that comprise the CDR as part of alternative definition(s) e.g. Kabat or Chothia, may be substituted with a conservative amino acid residue.

[0083] Such antigen binding proteins comprising variant CDRs as described above may be referred to herein as "functional CDR variants".

[0084] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single primary structure that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.

[0085] Terms "antigen binding fragment" or " Fragments" of (conventional) antibodies refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen, in particular it comprises the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fab, F(ab')2, Fv, disulphide stabilized Fv (dsFv), single chain Fv (scFv), disulphide-stabilized scFv, diabodies, tandem diabodies (TANDABS), (dsFv)2, sc(Fv)2, triabodies, tetrabodies, bispecific and multispecific antibodies formed from antibody fragments.

[0086] The term " Fab" denotes an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papain, are bound together through a disulfide bond.

[0087] The term " F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.A single chain Fv ("scFv") polypeptide is a covalently linked VH:: VL heterodimer, which is expressed usually from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

[0088] "dsFv" is a VH:: VL heterodimer stabilized by a disulfide bond.

[0089] "(dsFv)2" denotes two dsFv coupled by a peptide linker.

[0090] The term "bispecific antibody" or " BsAb" denotes an antibody combining the antigenbinding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different epitopes simultaneously either on a single antigen or two different antigens. A bispecific antibody of the present invention may be bivalent, trivalent, or tetravalent. As used herein, "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen binding sites in an antibody molecule. These antigen recognition sites may recognize the same epitope or different epitopes.

[0091] The term "multispecific antibody" denotes an antibody combining the antigen-binding sites of two or more antibodies within a single molecule.

[0092] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a lightchain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0093] In some embodiments, the antigen-binding fragment provided herein is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, triabodies and tetra bodies.

[0094] The antibody or antibody fragment or variant can be an isotype or subtype; preferably is an IgG, eventually an IgG with mutation(s), still preferably an IgGl, or an IgG4, more preferably an IgGl.

[0095] The term "humanized antibody" refers to an antibody, which is initially wholly, or partially of non-human origin, and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most ofthe time human CH and CL domains. In an embodiment, a humanized antibody has constant domains of human origin. As used herein, the term "humanized antibody" refers to a chimeric antibody which contains minimal sequence derived from non-human immunoglobulin, e.g. the CDRs.

[0096] As used herein, the term "specificity" refers to the ability of an antigen binding protein to bind detectably an epitope presented on an antigen, such as PSMA, while having relatively little detectable reactivity with non-PSMA proteins or structures (such as other proteins presented on cancer cells, or on other cell types). Specificity can be relatively determined by binding or competitive binding assays, using, e.g., surface plasmon resonance, or biolayer interferometry. Specificity can be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is PSMA).

[0097] The term "epitope" as used herein refers to that portion of the antigen that makes contact with a particular binding domain of the antigen binding protein, also known as the paratope. An epitope may be linear or conformational / discontinuous. A conformational or discontinuous epitope comprises amino acid residues that are separated by other sequences, i.e. not in a continuous sequence in the antigen's primary sequence assembled by tertiary folding of the polypeptide chain. Although the residues may be from different regions of the polypeptide chain, they are in close proximity in the three-dimensional structure of the antigen. In the case of multimeric antigens, a conformational or discontinuous epitope may include residues from different peptide chains. Particular residues comprised within an epitope can be determined through computer modelling programs or via three-dimensional structures obtained through methods known in the art, such as X-ray crystallography. Epitope mapping can be carried out using various techniques known to persons skilled in the art as described in publications such as Methods in Molecular Biology 'Epitope Mapping Protocols,' Mike Schutkowski and Ulrich Reineke ( volume 524, 2009) and Johan Rockberg and Johan Niivebrant (volume 1785, 2018). Exemplary methods include peptide based approaches such as pepscan whereby a series of overlapping peptides are screened for binding using techniques such as ELISA or by in vitro display of large libraries of peptides or protein mutants, e.g. on phage. Detailed epitope information can be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy and cryogenic-electron microscopy (cryo-EM). Mutagenesis, such as alanine scanning, is an effective approach whereby loss of binding analysis is used for epitope mapping. Another method ishydrogen / deuterium exchange (HDX) combined with proteolysis and liquid-chromatography mass spectrometry (LC-MS) analysis to characterize discontinuous or conformational epitopes.

[0098] The term "affinity", as used herein, means the strength of the binding of an antigen binding protein to an epitope. The affinity of an antigen binding protein is given by the dissociation constant Kd, defined as [Ab] x [Ag] I [Ab-Ag], where [Ab-Ag] is the molar concentration of the antigen binding protein-antigen complex, [Ab] is the molar concentration of the unbound antigen binding protein and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N. Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance, or biolayer interferometry. The affinity of an antigen binding protein can also be defined as the ratio of the on-rate (kon) and off-rate (koff) constant. The association constant (kon) is used to characterize the rate at which an antigen binding protein binds to its target. The dissociation constant (Koff) is used to measure the rate at which the antigen binding protein dissociates from its target.

[0099] Avidity, also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g. the sum total of the strength of binding of two molecules to one another at multiple sites, e.g. taking into account the valency of the interaction.

[0100] As used herein, the term "cytotoxic agent" as used herein refers to a drug substance that has cytotoxic activity and causes destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic drugs, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0101] As disclosed herein, in some cases, by "substantially similar", it is meant not significantly different.

[0102] Throughout the instant application, the term "comprising" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of"). Furthermore, the indefinite article "a" or "an" does not exclude plurality.In one embodiment, PSMA antigen binding proteins of the present disclosure show cross-reactivity between human PSMA and cynomolgus PSMA. In an embodiment, the PSMA antigen binding proteins of the invention specifically bind human and cynomolgus PSMA. This is particularly useful, since drug development typically requires testing of lead drug candidates in cynomolguses before the drug is tested in humans. The provision of a drug that can bind human and mouse species allows one to test results in these systems and make side-by-side comparisons of data using the same drug. This avoids the complication of needing to find a drug that works against a cynomolgus PSMA and a separate drug that works against human PSMA, and also avoids the need to compare results in humans and cynomolguses using nonidentical drugs. Cross reactivity between other species used in disease models such as dog or monkey, such as cynomolgus monkey, is also envisaged.

[0103] Competition between the PSMA binding protein of the invention and a reference PSMA binding protein, e.g. a reference antibody (e.g., J591 as described below), may be determined by one or more techniques known to the skilled person such as ELISA, FMAT, Surface Plasmon Resonance (SPR) or FORTEBIO OCTET Bio-Layer Interferometry (BLI). Such techniques may also be referred to as epitope binning. In one embodiment, the competition assay is carried out by Example 3.

[0104] " Percent identity" or "% identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the " Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST / KERR) or software (e.g. DNASTAR Lasergene or GenePAST / KERR). Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence disclosed herein, in particular in one or more of the claims.

[0105] " Percent identity" or"% identity" between a query amino acid sequence and a subject amino acid sequence is the " Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST / KERR) or software (e.g. DNASTAR Lasergene or GenePAST / KERR). Importantly, a query amino acid sequence may be described by an amino acid sequencedisclosed herein, in particular in one or more of the claims. Preferably, in some embodiments disclosed herein, the percentage of identity is calculated after pairwise global sequence alignment with Smith-Waterman algorithm, for instance using EMBOSS Needle with default settings (matrix BLOSUM62, gap open 10, gap extend 0.5, end gap penalty false, end gap open 10, end gap extend 0.5).

[0106] The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. In the case of nucleic acid sequences, such alterations include at least one nucleotide residue deletion, substitution or insertion, wherein said alterations may occur at the 5'- or 3'-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the nucleotide residues in the query sequence or in one or more contiguous groups within the query sequence. In the case of amino acid sequences, such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.

[0107] For antibody sequences, the % identity may be determined across the entire length of the query sequence, including the CDRs. Alternatively, the % identity may exclude one or more or all of the CDRs, for example all of the CDRs are 100% identical to the subject sequence and the % identity variation is in the remaining portion of the query sequence, e.g. the framework sequence, so that the CDR sequences are fixed and intact.

[0108] The variant sequence substantially retains the biological characteristics of the unmodified protein.

[0109] The VH or VL (or HC or LC) sequence may be a variant sequence with up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s).

[0110] The HC sequence may be a variant sequence with up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s).The LC sequence may be a variant sequence with up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s).

[0111] The sequence variation may exclude one or more or all of the CDRs, for example the CDRs are the same as the VH or VL (or HC or LC) sequence and the variation is in the remaining portion of the VH or VL (or HC or LC) sequence, so that the CDR sequences are fixed and intact.

[0112] Typically, the variation is a substitution, particularly a conservative substitution, for example as shown in Table 2.

[0113] The variant sequence substantially retains the biological characteristics of the unmodified protein, such as the specificity binding of target PSMA.

[0114] PSMA binding protein as described herein may be incorporated into pharmaceutical compositions for use in the treatment of the human diseases described herein. In one embodiment, the pharmaceutical composition comprises a PSMA binding protein in combination with one or more pharmaceutically acceptable carriers and / or excipients.

[0115] Such compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice.

[0116] Pharmaceutical compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal). In one embodiment, the composition is suitable for intravenous administration. Pharmaceutical compositions may be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral, vaginal, or rectal administration).

[0117] The pharmaceutical composition may be included in a kit containing the antigen binding protein together with other medicaments, and / or with instructions for use. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use. The kit may also include devices used for administration of the pharmaceutical composition.

[0118] The terms "individual", "subject" and "patient" are used herein interchangeably. In one embodiment, the subject is an animal. In another embodiment, the subject is a mammal, such as a primate, for example a marmoset or monkey. In another embodiment, the subject is a human.The PSMA binding protein described herein may also be used in methods of treatment. It will be appreciated by those skilled in the art that references herein to treatment refer to the treatment of established conditions. However, compounds of the invention may, depending on the condition, also be useful in the prevention of certain diseases. The PSMA binding protein described herein is used in an effective amount for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of the PSMA binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of, or to prevent or cure, the disease.

[0119] The term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, a disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0120] The term "preventing" or "prevention", as used herein, means impeding or slowing the apparition of a disorder or condition to which such term applies.

[0121] The term "diagnosing" or "diagnostic", as used herein, means identifying a disorder or a condition or a characteristic thereof in a subject, and / or detecting the region of a subject affected by a disorder or a condition to which such term applies.

[0122] " Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable vehicle (or carrier or excipient) refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0123] The form of the pharmaceutical compositions including the PSMA binding protein or variant thereof of the invention and the route of administration naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc. The PSMA binding protein or the variant thereof, or the ADC of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. In a particular embodiment, the antibody of the invention, the antigen binding fragment thereof, the variant thereof or the ADC of the invention is administered intravenously.

[0124] PSMA binding proteins provided herein may be prepared by any of a number of conventional techniques. For example, the PSMA binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems.A number of different expression systems and purification regimes can be used to generate the antigen binding protein of the invention. Generally, host cells are transformed with a recombinant expression vector encoding the desired antigen binding protein. The expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system. A wide range of host cells can be employed, including Prokaryotes (including Gram negative or Gram positive bacteria, for example Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergilus sp.), or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, NSO, PER. C6, HEK293, HeLa).

[0125] The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., plant or animal). The host cell may be a non-human host cell.

[0126] Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0127] The cells can be cultured under conditions that promote expression of the antigen binding protein using a variety of equipment such as shake flasks, spinner flasks, and bioreactors. The polypeptide is recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprised of various filtration and chromatographic processes developed to selectively concentrate and isolate the antigen binding protein. The purified antigen binding protein may be formulated in a pharmaceutically acceptable composition.

[0128] Fc engineering methods can be applied to modify the functional or pharmacokinetics properties of an antibody. Effector function may be altered by making mutations in the Fc region that increase or decrease binding to Clq or Fey receptors and modify CDC or ADCC activity respectively. Modifications to the glycosylation pattern of an antibody can also be made to change the effector function. The in vivo half-life of an antibody can be altered by making mutations that affect binding of the Fc to the FcRn (Neonatal Fc Receptor).

[0129] As used herein, the term "alkyl" refers to a saturated hydrocarbon chain, straight or branched, having the specified number of carbon atoms. For example, " Ci-ioalkyl" refers to a straight or branched alkyl group having from 1 to 10 carbon atoms (i.e. having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms). The term "alkyl" includes, but is not limited to, methyl, ethyl, propyl (for example, ^propyl or isopropyl), butyl (for example, / ?-butyl, sec-butyl, isobutyl or te / t-butyl), pentyl, and hexyl. Examples of " Ci-ioalkyl" as used herein include, but are not limited to, methyl, ethyl, n-propyl and isopropyl, butyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, n-hexane, isohexane, 3-methylpentane, neohexane, 2,3-dimethylbutane, heptane, octane, nonane, and decane.

[0130] As used herein, the term "alkenyl" refers to an alkyl group as defined above that includes at least one double bond (for example one, two or three double bonds, preferably one double bond). Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, and hexenyl groups. The term "alkenylene" refers to a divalent radical derived from a straight or branched, unsaturated hydrocarbon group containing at least 1 double bond and, for example, 2 to 4 carbon atoms (C2-4alkenylene). Exemplary groups include, but are not limited to, -CH=CH-, -CH=CHCH2-, and -CH2CH=CH-.

[0131] The term "alkynyl" refers to a straight or branched hydrocarbon radical containing the specified number of carbon atoms and at least 1 triple bond. For example, " C2-6alkynyl" has 2 to 6 carbon atoms. Exemplary groups include, but are not limited to, ethynyl and propynyl.

[0132] As used herein, the term "alkylene" refers to a divalent or trivalent radical derived from a straight or branched, saturated hydrocarbon chain of carbon atoms. For example, C1-10alkylene refers to a divalent or trivalent radical derived from a straight or branched, saturated hydrocarbon chain having from 1 to 10 carbon atoms (i.e., having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms). Examples of C1-10alkylene include -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-and -CH2CH2CH2-, -CH2(CH2)2CH2-, -C(CH3)(CH3)CH2-, -CH2C(CH3)(CH3)-, -CH(CH2)CH(CH2)-, -C(CH2CH2CH3)-, -C(CH(CH3)(CH3))-, -CH2(CH2)3CH2-, -CH(CH3)(CH2)2CH2-, -(CH2)6, -(CH2)7, -(CH2)8-, -(CH2)9, -(CH2)10-.

[0133] As used herein, the term "cycloalkyl" refers to a saturated, monocyclic, hydrocarbon ring of carbon atoms. For example, the term "C3-10cycloalkyl" refers to a saturated, monocyclic, hydrocarbon ring having from 3 to 10 carbon atoms (i.e. having 3, 4, 5, or 6 carbon atoms), and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl groups. A "cycloalkylene" group has the same meaning, but refers to a divalent or trivalent radical derived from a saturated, monocyclic, hydrocarbon ring of carbon atoms.

[0134] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine. As used herein, the term "heterocycloalkyl" refers to a saturated or unsaturated 3 to 10 membered monocyclic or bicyclic ring, which must contain at least one heteroatom, which is selected from nitrogen, oxygen, and sulfur. Heterocycloalkyl groups may contain one or more C(O), S(O) or SO2groups. Bicyclic heterocycloalkyl groups may be bridged, fused orspiro bicyclic groups. However, heterocycloalkyl groups are not aromatic. Heterocycloalkyl groups containing more than one heteroatom may contain different heteroatoms. "5- or 6-membered heterocycloalkyl" refers to a saturated or unsaturated 5- or 6-membered monocyclic ring, which must contain 1 or 2 non-carbon atoms, which are selected from nitrogen, oxygen, and sulfur. Exemplary groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, pyrazolinyl, imidazolidinyl, imidazolinyl, oxazolinyl, thiazolinyl, piperidinyl, piperazinyl, tetra hydrofuranyl, di hydrofuranyl, 1,3-dioxolanyl, tetrahydro-2H-pyranyl, di hydropyranyl, morpholinyl, morpholinyl-3-one, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-oxathiolanyl, 1,3-oxathianyl, 1,3-dithianyl, 1,4-oxathiolanyl, 1,4-oxathianyl, 1,4-dithianyl, piperidyl-2-one, pyrimidinyl-2,4(lH,3H)-dione, thiomorpholinyl, and thiomorpholinyl 1,1-dioxide. The term "heterocyloalkylene" has the same meaning, but refers to a divalent or trivalent radical form derived from the heterocycloalkyl group.

[0135] The term "substituted" in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced by one of the defined substituents. In the case where groups may be selected from a number of alternative groups, the selected groups may be the same or different. Examples of substituted groups bears two or more substituents (e.g., one, two or three), each substituent is independently selected from the specified alternatives. The substituents may be identical, different, or a combination thereof (for example, in a group having three substituents, two may be the identical and one may be different). For example, the term "substituted" refers to the one, two, three or four groups selected from NH2,-NH-, Ci-3alkyl, Ci-3alkoxy, oxo, cyano, hydroxy, halogen, and -(CH2)3NHCONH2.

[0136] The term "aryl" refers to a monocyclic or bicyclic, hydrocarbon, aromatic radical. Aryl includes, for example, phenyl and naphthyl. An aryl group may contain 6 to 14 carbon atoms. The term "arylene" refers to the same groups but in divalent or trivalent forms.

[0137] The term "heteroaryl" refers to a group or moiety comprising an aromatic monovalent monocyclic or bicyclic radical, containing 5 to 14 ring atoms, including at least one (e.g., one, two, three or four) heteroatom independently selected from nitrogen, oxygen and sulfur. This term also encompasses bicyclic heterocyclic-aryl compounds containing an aryl ring moiety fused to a heterocycloalkyl ring moiety, containing 5 to 10 ring atoms, including at least one (e.g., one, two, three or four) Exemplary groups include, but are not limited to furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, benzofuranyl, isobenzofuryl, 2,3-dihydrobenzofuryl, 1,3-benzodioxolyl, dihydrobenzodioxinyl, benzothienyl, indolizinyl, indolyl, isoindolyl, dihydroindolyl, benzimidazolyl,dihydrobenzimidazolyl, benzoxazolyl, dihydrobenzoxazolyl, benzthiazolyl, benzoisothiazolyl, dihydrobenzoisothiazolyl, indazolyl, imidazopyridinyl, pyrazolopyridyl, benzotriazolyl, triazolopyridyl, purinyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, quinazolinyl, 1,5-naphthyridinyl, 1,6-naphthyridinyl, 1,7-naphthyridinyl, 1,8-naphthyridinyl, and pteridinyl. Examples of 5-membered "heteroaryl" groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, and isothiazolyl. Examples of 6-membered "heteroaryl" groups include oxo-pyridyl, pyridyl, pyridazinyl, pyrazinyl, and pyrimidinyl. Examples of 6,6-fused "heteroaryl" groups include quinolinyl, isoquinolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, quinazolinyl, 1,5-naphthyridinyl, 1,6-naphthyridinyl, 1,7-naphthyridinyl, 1,8-naphthyridinyl, and pteridinyl. Examples of 6,5-fused "heteroaryl" groups include benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl, indolizinyl, indolyl, isoindolyl, and indazolyl. "heteroarylene" groups refers to the same groups but in divalent or trivalent radical form.

[0138] As used herein, the term "natural amino acid" refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins, such as, glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gin), cysteine (Cys), methionine (Met) or a isomer thereof, e.g., isoglutamic acid (iGlu) or isoaspartic acid (iAsp).

[0139] As used herein, the term "linker" refers to a group (functional grouping) that joins the antigen binding protein to the payload(s) of the ADC molecule. In the present invention, the linker attaches to the antigen binding protein via covalent bonds formed between the antigen binding protein and functional groups (e.g. electrophilic functional groups) on the linker. As used herein, the term "tridentate linker" refers to a linker of an ADC molecule that forms covalent bonds with three atoms from the antigen binding protein and payload(s). In some cases, the tridentate linker forms covalent bonds with two different atoms of the antigen binding protein and one atom of a payload. In other cases, the tridentate linker forms covalent bonds with one atom of the antigen binding protein and two atoms of a payload, or one atom of each of two payloads.

[0140] The term "substituted" in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced by one of the defined substituents. Where a group bears two or more substituents (e.g., two or three), each substituent is independentlyselected from the specified alternatives. The substituents may be identical, different, or a combination thereof (for example, in a group having three substituents, two may be identical and one may be different).

[0141] The term "independently selected" means that where more than one substituent is selected from a number of possible substituents, those substituents may be the identical or different. Thus, each substituent is separately selected from the entire group of recited possible substituents.

[0142] The term "pharmaceutically acceptable" refers to those compounds (including salts), materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0143] The term "therapeutically effective amount" refers to the quantity of an antibody-drug conjugate, or an antigen binding protein, or a nucleic acid disclosed herein, or a pharmaceutically acceptable salt thereof, which will elicit the desired biological response in a human body. It may vary depending on the compound, the disease and its severity and the age and weight of the subject to be treated.

[0144] The three-letter codes and one-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem, 243: 3558 (1968).

[0145] The compounds e.g., antibody-drug conjugates) of the present disclosure, including salts of compounds of the present disclosure, or adducts of compounds of the present disclosure, contain multiple asymmetric centers (also referred to as a chiral center) and may, therefore, exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures. Chiral centers, such as chiral carbon atoms, may also be present in a substituent such as an alkyl group. Where the stereochemistry of a chiral center present in a compound of the present disclosure, or in any chemical structure illustrated herein, is not specified the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Where the stereochemistry of a chiral center present in a compound of the present disclosure, or in any chemical structure illustrated herein, is specified the structure is intended to encompass the individual stereoisomers specified.

[0146] The present invention includes all suitable isotopically-labelled compounds of the disclosure, for example all suitable isotopically-labelled compounds of Formula (I) (including Formulae (I), (I-C)), (I-A), (I-Al), (I-Al-iso), (I-A2), (I-A2-iso), (I-B), (I-Bl), (I-C), (I-DO), (I-D1), (I-D2), (I-EO), (I-El), (I-E2), (I-FO), (I-Fl), (I-F2), or (II), or Formula [Ab'Red-M]), or a salt thereof, or an adduct thereof, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure include isotopes of hydrogen, such as2H and3H, carbon, such as11C,13C and14C, chlorine, such as36CI, fluorine, such as18F, iodine, such as123I and125I, nitrogen, such as13N and15N, oxygen, such as15O,17O and18O, phosphorus, such as32P, and sulfur, such as35S. Certain isotopically-labelled compounds of the disclosure, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0147] Isotopically labelled compounds of the disclosure can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically labelled reagents in place of the non-labelled reagent previously employed.

[0148] The compounds of the disclosure may be in the form of a salt. Salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. Furthermore, pharmaceutically acceptable salts of the compounds of the present disclosure may be prepared during further processing of the free base form, for example in situ during manufacture into a pharmaceutical formulation.

[0149] Illustrative acid salts of the compounds of the present invention can be prepared from the following acids, including, without limitation: hydrochloric, mesylic, formic, acetic, benzoic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, nitric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, hydrobromic, hydroiodic, isocitric, pamoic, propionic, anthranilic, oxaloacetic, oleic, stearic, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, phosphoric, phosphonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, sulfuric, salicylic, cyclohexylaminoethanesulfonic acid (CHES), alginic, [3-hydroxybutyric, galactaric andgalacturonic acids. Preferred salts include the salts of hydrochloric acid and mesylic acid (i.e. preferred salts include HCI salts and mesylate salts).

[0150] All of the above salts can be prepared by those skilled in the art by conventional means from the corresponding compound of the present invention. For example, the salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the 30 solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990), the disclosure of which is hereby incorporated by reference with regards to the lists of suitable salts.

[0151] The compounds of the present disclosure may exist in both unsolvated and solvated forms. The term 'solvate' comprises the compound of the invention and one or more solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water. Solvates include hydrates and other solvates wherein the solvent of crystallization may be isotopically substituted, e.g., D2O, d6-acetone, d6-DMSO.

[0152] PSMA BINDING PROTEINS

[0153] The invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising: (a) (i) any one or a combination of CDRs selected from the group consisting of: CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 7, and CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 8; or (ii) a CDR variant of (i), wherein the variant has 1, 2, or 3 amino acid modifications; or (b) a VH region comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 7, and / or a VL region comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 8.

[0154] In some embodiments, the PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) provided herein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR-H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6. In some of these embodiments, the VH region of thePSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3; and the VL region of the PSMA binding protein comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6, with up to five amino acid modifications across said six CDR sequences. In some cases, the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR-H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6.

[0155] In some embodiments, the PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) provided herein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21, a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24. In some of these embodiments, the VH region of the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21; and the VL region of the PSMA binding protein comprises a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24, with up to five amino acid modifications across said six CDR sequences. In some cases, the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21, a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24.

[0156] In some embodiments, the PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) provided herein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27, a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30. In some of these embodiments, the VH region of the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27; and the VL region of the PSMA binding protein comprises a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30, with up to five amino acid modifications across said six CDR sequences. In some cases, the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27, a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30.In some embodiments, the PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) provided herein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33, a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36. In some of these embodiments, the VH region of the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33; and the VL region of the PSMA binding protein comprises a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36, with up to five amino acid modifications across said six CDR sequences. In some cases, the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33, a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36.

[0157] In some embodiments, the PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) provided herein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39, a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42. In some of these embodiments, the VH region of the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39; and the VL region of the PSMA binding protein comprises a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42, with up to five amino acid modifications across said six CDR sequences. In some cases, the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39, a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42.

[0158] In some aspects, the invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising:

[0159] a. a variable heavy chain domain (VH) comprising:

[0160] i. a CDR1-H of sequence NYHMN SEQ ID NO: 1,

[0161] ii. a CDR2-H of sequence DISGSSRYIHYADFVKG SEQ ID NO: 2, and iii. a CDR3-H of sequence SSGGYYYGYGMDV SEQ ID NO: 3; and

[0162] b. a variable light chain domain (VL) comprising:

[0163] i. a CDR1-L of sequence AGTSSDVGGYHYVS SEQ ID NO: 4,ii. a CDR2-L of sequence DDSDRPS SEQ ID NO: 5, and

[0164] iii. a CDR3-L of sequence SSGTYYSTRV SEQ ID NO: 6,

[0165] or a variant thereof comprising up to five point mutations across said six CDR sequences, preferably wherein said variant retains a binding affinity to PSMA, even more preferably wherein said variant retains a binding affinity to PSMA that is substantially similar to, or improve to that of the anti-PSMA antibody, or antigen binding fragment thereof lacking said mutations.

[0166] In some aspects, the invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising:

[0167] a. a variable heavy chain domain (VH) comprising:

[0168] i. a CDR1-H of the sequence of SEQ ID NO: 19,

[0169] ii. a CDR2-H of the sequence of SEQ ID NO: 20, and

[0170] iii. a CDR3-H of the sequence of SEQ ID NO: 21; and

[0171] b. a variable light chain domain (VL) comprising:

[0172] i. a CDR1-L of the sequence of SEQ ID NO: 22,

[0173] ii. a CDR2-L of the sequence DDS, and

[0174] iii. a CDR3-L of the sequence of SEQ ID NO: 24,

[0175] or a variant thereof comprising up to five point mutations across said six CDR sequences, preferably wherein said variant retains a binding affinity to PSMA, even more preferably wherein said variant retains a binding affinity to PSMA that is substantially similar to, or improve to that of the anti-PSMA antibody, or antigen binding fragment thereof lacking said mutations.

[0176] In some aspects, the invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising:

[0177] a. a variable heavy chain domain (VH) comprising:

[0178] i. a CDR1-H of the sequence of SEQ ID NO: 25,

[0179] ii. a CDR2-H of the sequence of SEQ ID NO: 26, and

[0180] iii. a CDR3-H of the sequence of SEQ ID NO: 27; and

[0181] b. a variable light chain domain (VL) comprising:

[0182] i. a CDR1-L of the sequence of SEQ ID NO: 28,

[0183] ii. a CDR2-L of the sequence of SEQ ID NO: 29, and

[0184] iii. a CDR3-L of the sequence of SEQ ID NO: 30,

[0185] or a variant thereof comprising up to five point mutations across said six CDR sequences, preferably wherein said variant retains a binding affinity to PSMA, even more preferablywherein said variant retains a binding affinity to PSMA that is substantially similar to, or improve to that of the anti-PSMA antibody, or antigen binding fragment thereof lacking said mutations.

[0186] In some aspects, the invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising:

[0187] a. a variable heavy chain domain (VH) comprising:

[0188] i. a CDR1-H of the sequence of SEQ ID NO: 31,

[0189] ii. a CDR2-H of the sequence of SEQ ID NO: 32, and

[0190] iii. a CDR3-H of the sequence of SEQ ID NO: 33; and

[0191] b. a variable light chain domain (VL) comprising:

[0192] i. a CDR1-L of the sequence of SEQ ID NO: 34,

[0193] ii. a CDR2-L of the sequence of SEQ ID NO: 35, and

[0194] iii. a CDR3-L of the sequence of SEQ ID NO: 36,

[0195] or a variant thereof comprising up to five point mutations across said six CDR sequences, preferably wherein said variant retains a binding affinity to PSMA, even more preferably wherein said variant retains a binding affinity to PSMA that is substantially similar to, or improve to that of the anti-PSMA antibody, or antigen binding fragment thereof lacking said mutations.

[0196] In some aspects, the invention relates to a PSMA binding protein (e.g., an anti-PSMA antibody, or antigen binding fragment thereof) comprising:

[0197] a. a variable heavy chain domain (VH) comprising:

[0198] i. a CDR1-H of the sequence of SEQ ID NO: 37,

[0199] ii. a CDR2-H of the sequence of SEQ ID NO: 38, and

[0200] iii. a CDR3-H of the sequence of SEQ ID NO: 39; and

[0201] b. a variable light chain domain (VL) comprising:

[0202] i. a CDR1-L of the sequence of SEQ ID NO: 40,

[0203] ii. a CDR2-L of the sequence of SEQ ID NO: 41, and

[0204] iii. a CDR3-L of the sequence of SEQ ID NO: 42,

[0205] or a variant thereof comprising up to five point mutations across said six CDR sequences, preferably wherein said variant retains a binding affinity to PSMA, even more preferably wherein said variant retains a binding affinity to PSMA that is substantially similar to, or improve to that of the anti-PSMA antibody, or antigen binding fragment thereof lacking said mutations.The PSMA binding protein binds specifically to PSMA, preferably specifically to human PSMA. In some cases, the PSMA binding protein provided herein binds to both human and cynomolgus PSMA. In some cases, the PSMA binding protein does not bind to mouse PSMA.

[0206] The PSMA binding protein has preferably a binding affinity to PSMA and in particular to hPSMA, that is substantially similar to XT42 or improved, preferably in the nanomolar range.

[0207] In a particular embodiment, the variant according to some embodiments of the present disclosure comprises up to four, up to three, up to two, or one point mutation(s) across said six CDR sequences. In an embodiment, said variant retains a binding affinity to PSMA that is substantially similar to, or improved compared to that of the PSMA binding protein according to some embodiments of the present disclosure. In an embodiment, said variant retains a binding affinity to PSMA that is substantially similar to, or improved compared to that of the antigen binding fragment of the PSMA binding protein according to some embodiments of the present disclosure. In other words, the point mutations in the variant according to some embodiments of the present disclosure do not provoke a significant loss of the binding affinity.

[0208] The antigen binding fragment or the variant may be any type of mAb fragment that keeps substantially the ability of the whole antibody to bind to PSMA, it can be for example a scFv, Fab or a F(ab')2.

[0209] According to some embodiments of the present disclosure, an antigen binding fragment of the invention has an amino acid sequence comprising at least 200 amino acids and binds to PSMA. In particular, an antigen binding fragment of the invention retains a binding affinity to PSMA, preferably that is substantially similar to, or improved compared to that of the PSMA binding protein of the invention.

[0210] Advantageously, the PSMA binding protein does not bind to PSMA through the same epitope as Rosopatamab.

[0211] Advantageously, the PSMA binding protein is internalized in PSMA expressing cancer cells after its binding to PSMA at their surface.

[0212] In an embodiment, the variant according to some embodiments of the present disclosure has a cancer cell internalization capacity that is substantially similar to or improved compared to that of the PSMA binding protein according to some embodiments of the present disclosure. In another embodiment, the variant according to some embodiments of the present disclosure has a cancer cell internalization capacity that is substantially similar to or improved compared to that of the antigen binding fragment of the anti-PSMA antibody according to some embodiments of the present disclosure. In other words, the point mutations

[0213] 1in the variant according to some embodiments of the present disclosure do not lead to a significant loss of the cancer cell internalization capacity.

[0214] By "cancer cell internalization," it is meant the capacity to penetrate cancer cells or to be internalized in cancer cells. According to some embodiments of the present disclosure the cancer cells used for evaluating this capacity are cancer cells (for example LnCap cells) expressing PSMA, preferably human PSMA, such as human PSMA-positive LnCap cells. Such capacity depends among others on the affinity and the speed of internalization of the antigen binding protein. Preferably, the cancer cell internalization capacity according to some embodiments of the present disclosure is measured by antibody internalization assays such as the Incucyte® Fabfluor-pH Antibody assay, as demonstrated in Example 5.

[0215] Preferably, the antibody, the antigen binding fragment thereof, or the variant thereof according to some embodiments of the present disclosure has a cancer cell internalization capacity that is substantially similar to that of XT42, or improved.

[0216] In an embodiment, the variant according to some embodiments of the present disclosure has half maximal effective concentration (EC50) for binding to PSMA-positive cells that is substantially similar or improved compared to that of the PSMA binding protein according to some embodiments of the present disclosure. In another embodiment, the variant according to some embodiments of the present disclosure has an EC50 for binding to PSMA-positive cells that is substantially similar to or improved compared to that of the antigen binding fragment of the PSMA binding protein according to some embodiments of the present disclosure. In other words, the point mutations in the variant according to some embodiments of the present disclosure do not lead to a significant increase of the EC50 for binding to PSMA positive cells.

[0217] By "half maximal effective concentration (EC50)," it is meant the concentration of PSMA binding protein, or antigen binding fragment thereof or variant thereof, which induces a binding response halfway between the baseline and maximum. According to some embodiments of the present disclosure, the cancer cells used for evaluating this capacity are PSMA-positive cells, preferably human PSMA-positive cells, and in particular cancer cells (for example LnCap cells), such as human PSMA-positive LnCap cells. Preferably, the EC50 for binding to PSMA-positive cells according to some embodiments of the present disclosure is measured by flow cytometry analysis, as demonstrated in Example 4.

[0218] Preferably, the PSMA binding protein according to some embodiments of the present disclosure has an EC50 for binding to PSMA-positive cells that is between 0.01nM and 20nM, more preferably between 0.01 and 1.5 nM.In a particular embodiment, the PSMA binding protein according to some embodiments of the present disclosure has an EC50 for binding to PSMA-positive cells comprised between 0.3 nM and 0.4 nM.

[0219] The PSMA binding protein may be a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody. Preferably, the PSMA binding protein or variant thereof is a monoclonal antibody.

[0220] In some embodiments, the PSMA binding protein comprises:

[0221] (a) a VHthat comprises sequence SEQ ID NO: 7 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, (b) a VLthat comprises sequence SEQ ID NO: 8 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, or

[0222] (c) a VHthat comprises sequence SEQ ID NO: 7 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a VLthat comprises sequence SEQ ID NO: 8 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0223] In some embodiments, the PSMA binding protein or variant thereof comprises a Humanized IgGl Constant heavy chain (CH) and a Humanized IgGl Constant light chain (CL).

[0224] In some embodiments, the PSMA binding protein or variant thereof comprises a Human IgGl Constant heavy chain (CH) and a Human IgGl Constant light chain (CL), in particular a human kappa constant domain.

[0225] In an embodiment, sequences of the human IgGl CH come from Genbank AEL33691.1 (as of 25 July 2016) modified R222K, E377D, M381L (according to Kabat numbering). Sequences of the human IgGl CL (Kappa) come from Genbank CAC20459.1 (as of 11 June 2015) modified 108R, A110V (according to Kabat numbering).

[0226] In some embodiments, the PSMA binding protein or variant thereof comprises:

[0227] (a) a heavy chain that comprises sequence SEQ ID NO: 9 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto,

[0228] (b) a light that comprises sequence SEQ ID NO: 10 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, or(c) a heavy chain that comprises sequence SEQ ID NO: 9 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a light chain that comprises sequence SEQ ID NO: 10 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0229] In some embodiments, the PSMA binding protein or variant thereof comprises:

[0230] (a) a heavy chain that comprises or consists in sequence SEQ ID NO: 11 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto,

[0231] (b) a light that comprises or consists in sequence SEQ ID NO: 12 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, or

[0232] (c) a heavy chain that comprises or consists in sequence SEQ ID NO: 11 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a light chain that comprises or consist in sequence SEQ ID NO: 12 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0233] In some aspects, the invention further relates to a pair of nucleic acids encoding respectively the VHand VLchain of a PSMA binding protein or a variant thereof as disclosed herein.

[0234] In some embodiments, the pair of nucleic acid comprises sequences SEQ ID NO: 13, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and SEQ ID NO: 14 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0235] In some aspects, the invention also relates to a pair of nucleic acids encoding respectively the heavy chain and light chain of a PSMA binding protein or a variant thereof as disclosed herein.

[0236] In some embodiments, the pair of nucleic acid comprises sequences SEQ ID NO: 17, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and SEQ ID NO: 18 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0237] In some aspects, the invention also relates to one or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein disclosed herein. In some cases, the one or more nucleic acids comprise a sequence having at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 14.

[0238] In some embodiments of the present invention, the one or more nucleic acids comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 14. In some of these embodiments, the one or more nucleic acids further comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 15, and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 16.

[0239] In some embodiments, the one or more nucleic acids comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 17, and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 18.

[0240] In some embodiments, the one or more nucleic acids comprise a sequence according to SEQ ID NO: 17, and a sequence according to SEQ ID NO: 18.

[0241] In yet another aspect, the present invention relates to a vector comprising nucleic acids disclosed herein, e.g., the one or more nucleic acids encoding polypeptides capable of forming a PSMA binding protein disclosed herein. The vector may be suitable for transfecting a host cell for production of the PSMA binding protein. Alternatively, the vector may be suitable for administration into a subject for expressing the PSMA binding protein in vivo, e.g., for therapeutic use in a subject. In some cases, the vector is a DNA vector. In other cases, the vector is an RNA vector.

[0242] In yet another aspect, the present invention relates to a host cell comprising the nucleic acids encoding polypeptides capable of forming a PSMA binding protein disclosed herein.

[0243] In yet another aspect, the present invention relates to a method of producing the PSMA binding protein disclosed herein. The method may comprise culturing a host cell comprising the nucleic acids encoding polypeptides capable of forming a PSMA binding protein disclosed herein, under conditions suitable for producing the PSMA binding protein.MULTISPECIFIC ANTIBODIES BINDING TO PSMA AND TO ANTIGENS ON IMMUNE CELLS

[0244] Also provided herein are PSMA binding proteins that are in the form of a multispecific (e.g. bispecific) antibody construct that binds at least to PSMA and another antigen. Such multispecific or bispecific antibody constructs typically comprise (a) a first antigen binding moiety that specifically binds PSMA, and (b) a second antigen binding moiety that specifically binds antigens on immune cells (e.g., T, NK), wherein the first and second antigen binding moieties are connected directly or by a linker.

[0245] Antigens on immune cells can be selected from selected but not limited to CD3, CD4, CD8, CD16A, CD19, CD22, CD25, CD27, CD28, CD134 (0X40), CD137(4-1BB), ICOS (CD278), LAG-3, TIM-3, TIGIT, VISTA, CTLA-4 and PD-1.

[0246] In some embodiments, the multispecific or bispecific antibody construct comprises a first antigen binding moiety that specifically binds PSMA (for example, one of the two paratopes of the monoclonal antibody), and a second antigen binding moiety (for example, the other paratope of the monoclonal antibody), which specifically binds to an antigen selected from the group consisting of CA 125, CA 15-3, CA 19-9, L6, Lewis Y, Lewis X, alphafetoprotein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUC1, CEA, gplOO, MART-1, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, Neu oncogene product, HER2, HER3, Nectin-4, tissue factor, Trop-2, FRo, ROR1, EGFR, c-MET, B7-H3, Ang-2, BCMA, CD3, CD19, CTLA-4, DLL3, EpCAM, Factor IXa, Factor X, Fey receptors, or any other T cell-associated antigen involved in immune activation or modulation, without limitation.

[0247] ANTIBODY DRUG CONJUGATES

[0248] The invention further relates to antibody-drug conjugates (interchangeably referred to as an " ADC," "antibody drug conjugate," "immunoconjugate," or "antigen binding proteindrug conjugate") that comprise a PSMA binding protein (e.g., an anti-PSMA antibody or an antigen binding fragment thereof) as described herein, and that further comprises an agent (also referred to as "payload") selected from the group consisting of therapeutic agents, imaging agents, diagnostic agents, and combinations thereof, wherein the agent is conjugated to the PSMA binding protein. In some embodiments, the agent is selected from the group consisting of cytotoxic agents, immunostimulatory agents, targeted protein degradation agents, immunocytokines and radiopharmaceutical agents. In some cases, the agent is a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, a growthinhibitory agent, a toxin (e.g., a protein toxin, such as an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), an antiviral agent, a radioactive isotope (i.e., a radioconjugate), an antibiotic, or a small interfering RNA (siRNA).

[0249] Antibody-drug conjugates have been used for the local delivery of cytotoxic payloads, e.g., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9): 1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev.

[0250] 26:151-172; U. S. Pat. No. 4,975,278). Antibody-drug conjugates allow for, inter alia, the targeted delivery of a drug moiety to a tumor, and intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Tsuchikama and An, Protein and Cell, (2018) 9: 33-46). Antibody-drug conjugates can enable selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK) / pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy (Tsuchikama and An 2018); Beck A. et al (2017) Nature Rev. Drug Disc. 16: 315-337).

[0251] An antibody-drug conjugate (ADC) typically comprises (i) an antibody or antigen binding fragment thereof, (ii) a cytotoxic payload, and (iii) a linker connecting the antibody or antigen binding fragment thereof and the cytotoxic payload.

[0252] In some embodiments, the ADC comprises (i) a PSMA binding protein as disclosed herein, (ii) a cytotoxic payload, and (iii) a linker connecting the PSMA binding protein and the cytotoxic payload.

[0253] In some aspects, provided herein is an antibody-drug conjugate having the following general structure:

[0254] (T)p-Ab-(L-(Payload)m)n

[0255] (I-C)),

[0256] where in formula (I-C)):

[0257] Ab is a PSMA binding protein according to some embodiments of the present disclosure;

[0258] n is 1 or 2;

[0259] m is an integer larger than 0 (e.g., 1, 2, 3, 4, 5 or 6);

[0260] p is 0 or 1;

[0261] L is an optional linker described herein; andPayload is a payload described herein (e.g., a molecule exerting a biological activity and selected from the group consisting of diagnostic agents, therapeutic agents and labelling agents, e.g., a cytotoxic payload).

[0262] The term "drug to antibody ratio" or " DAR" refers to the molar ratio of payload (e.g., cytotoxic agent) moieties per antibody. In some cases, the term refers to the weighted average molar ratio of payload molecules per antibody in a population of at least two ADC molecules. DAR can be measured using mass spectrometry. In some cases, when describing a single ADC molecule, the term refers to the molar ratio of payload moieties per antibody in the ADC molecule. For instance, in some cases, the compound of formula (I-C)) above has a controlled DAR value of m x n. In some of these cases, the payload is conjugated at one or two specific conjugation sites within the antibody.

[0263] In other cases, the ADC provided herein has a DAR that ranges from 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, or any number in between when averaged over a population of at least two ADC molecules. In some of these embodiments, the ADC does not have a controlled DAR value. In some cases, the payload may be conjugated at one or more conjugation sites of the antibody in a non-specific manner within a population of ADC molecules.

[0264] Suitable ADC linkers include cleavable and non-cleavable linkers. In some cases, the linker comprises a self-immolative linker. In some embodiments of the present disclosure, the linker comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein the linker comprises a cleavable linker that is a peptide linker. In some embodiments, the linker is a cleavable linker. Such likers may include chemical cleavage linkers (e.g. hydrazone bond and disulfide bond) and enzyme cleavage linkers (e.g. glucuronide bond and peptide bond). In some embodiments, the linker is a non-cleavable linker (e.g., a thioether or maleimidocaproyl group (MC)). Exemplary linkers may include 6-maleimidocaproyl, maleimidopropanoyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PABC), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC), and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

[0265] Non-limiting examples of cytotoxic payloads that may be incorporated in ADCs disclosed herein include tubulin inhibitors (e.g. auristatin derivatives such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), DNA damaging agents e.g. inducing DNA double strand break, such as calicheamicins; DNA alkylation, such as duocarmycins; DNA intercalation, such as topoisomerase I inhibitors; DNA crosslink, such as pyrrolobenzodiazepines (PBD)), and immunomodulators (such as TLR agonists). Suitablecytotoxic payloads in ADCs disclosed herein may also include radioligands such as225Actinium,212Lead,68Gallium,177Lutetium,223Radium,89Zirconium.

[0266] Payload

[0267] Payload may be chosen from drug or imaging agents, oligonucleotides, chelating ligands capable of complexing with radionuclides, cytotoxic drugs and antineoplastic agents. According to an embodiment, Payload may be chosen from drugs, such as cytotoxic drugs and antineoplastic agents.

[0268] In certain embodiments, an antibody-drug conjugate provided herein comprises an antigen binding protein, such as an antibody, and a drug, such as toxin, such as a chemotherapeutic agent. The drug can be modified (e.g., via standard synthetic chemistry) to allow its chemical attachment (e.g., to contain a reaction handle to allow its chemical attachment) to a reactive end of a linker that joins the drug to the antigen binding protein.

[0269] Drugs, such as chemotherapeutic agents, useful in the generation of immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93 / 21232 published Oct. 28, 1993.

[0270] In addition to toxins, a radioactive material, such as a radionucleotide, may be used as the drug in an ADC. A variety of radionucleotides are available for the production of radioconjugated antibodies. Examples include 212Bi, 1311, 131In, 90Y, and 186Re.

[0271] Antigen binding proteins (such as antibodies) of the present invention may also be conjugated to one or more toxins, including, but not limited to, a calicheamicin, a maytansinoid, a dolastatin, an aurostatin, a trichothecene, and CC1065, and a derivative of these toxins that have toxin activity. Suitable cytotoxic agents include, but are not limited to, an auristatin including monomethyl auristatin F (MMAF) and monomethyl auristatin E (MMAE) as well as an ester form of MMAE, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane (such as paclitaxel and docetaxel), a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, DM-4, and netropsin. Other suitable cytotoxic agents include anti-tubulin agents, such as an auristatin, avinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolastatin. Antitubulin agents include dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine (AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, DM-4, and eleutherobin.

[0272] Antibody drug conjugates can be produced by conjugating the anti-tubulin agent monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) to an antigen binding protein (such as an antibody). MMAE can be represented by the following structure, and has an IUPAC name of (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5-methyl-l-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide:

[0273]

[0274] MMAF can be represented by the following structure, and has an IUPAC name of (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid:

[0275]

[0276] In the case of MMAE, the linker can consist of a thiol-reactive maleimidyl, a caproyl spacer, the dipeptide valine-citrullinyl, or p-aminobenzyloxycarbonyl, a self-immolative fragmenting group. In the case of MMAF, a protease-resistant maleimidocaproyl linker can be used. The conjugation process leads to heterogeneity in drug-antibody attachment, varying in both the number of drugs bound to each antibody molecule (mole ratio [MR]), and the siteof attachment. The most prevalent species is the material with an MR = 4; less prevalent are materials with MR of 0, 2, 6, and 8. The overall average drug-to-antibody MR is approximately 4.

[0277] Suitable drugs include:

[0278] - dolastatins such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD);

[0279] - maytansines and maytansinoids such as DM1 and DM4;

[0280] - antracyclins such as doxorubicin, nemorubicin and PNU-159682;

[0281] - calicheamicins;

[0282] - etoposides;

[0283] - taxanes;

[0284] - duocarymycins such as CC-1065 and duocarmycin A;

[0285] - benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolobenzodiazepines; pyrrolobenzodiazepine dimers; indolino-benzodiazepines; indolino-benzodiazepine dimers; oxazolidinobenzodiazepines);

[0286] - vinca alkaloids;

[0287] - amanitins such as o-amanitin, [3-amanitin, y-amanitin, s-amanitin;

[0288] - irinotecan derivatives;

[0289] - exatecan derivatives;

[0290] - exotoxins such as diphtheria toxin, shiga toxin, or subunits thereof; and

[0291] - vincristine derivatives.

[0292] In some embodiments, each Payload in an antibody-drug conjugate disclosed herein is independently selected from dolastatins such as MMAE, MMAF, MMAD; maytansines and maytansinoids such as DM1 and DM4; antracyclins such as doxorubicin, nemorubicin and PNU-159682; calicheamicins; etoposides; taxanes; duocarymycins such as CC-1065 and duocarmycin A; benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolobenzodiazepines; pyrrolobenzodiazepine dimers; indolino-benzodiazepines; indolino-benzodiazepine dimers; oxazolidinobenzodiazepines); vinca alkaloids; amanitins such as o-amanitin, [3-amanitin, y-amanitin, s-amanitin; irinotecan derivatives; exatecan derivatives; exotoxins such as diphtheria toxin, shiga toxin, or subunits thereof; vincristine derivatives. In some embodiments, all the Payloads in an antibody-drug conjugate disclosed herein are the same, e.g., selected from dolastatins such as MMAE, MMAF, MMAD; maytansines and maytansinoids such as DM1 and DM4; antracyclins such as doxorubicin, nemorubicin and PNU-159682; calicheamicins; etoposides; taxanes; duocarymycins such as CC-1065 andduocarmycin A; benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolobenzodiazepines; pyrrolobenzodiazepine dimers; indolino-benzodiazepines; indolino-benzodiazepine dimers; oxazolidinobenzodiazepines); vinca alkaloids; amanitins such as o-amanitin, [3-amanitin, y-amanitin, s-amanitin; irinotecan derivatives; exatecan derivatives; exotoxins such as diphtheria toxin, shiga toxin, or subunits thereof; vincristine derivatives. In some embodiments, not all the Payloads in an antibody-drug conjugate provided herein are the same.

[0293] According to an embodiment, Payload is a dolastatin such as MMAE, MMAF, MMAD, and more particularly said payload is MMAE. In some embodiments, each Payload in an antibody-drug conjugate disclosed herein is independently MMAE, MMAF or MMAD. In some embodiments, each Payload in an antibody-drug conjugate disclosed herein is MMAE.

[0294] Suitable oligonucleotides include silencing RNA, microRNA, antisense oligonucleotides, DNA, or RNA oligomers. Typically they are between 10 and 5000 nucleotides in length, more particularly, between 20 and 200.

[0295] Imaging agents include detection agents, biological markers, tracing agents, such as fluorophores, dyes, radioactive tracers.

[0296] Auristatins and Dolastatins

[0297] In some embodiments, the immunoconjugate comprises an antigen binding protein (such as an antibody) conjugated to a dolastatin or a dolostatin peptidic analog or derivative, an auristatin (U. S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U. S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin (a pentapeptide derivative of dolastatin) drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02 / 088172).

[0298] Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, disclosed in " Monomethylvaline Compounds Capable of Conjugation to Ligands," U. S. Patent No. 7,498,298. As used herein, the abbreviation " MMAE" refers to monomethyl auristatin E. As used herein, the abbreviation " MMAF" refers to dovaline-valine-dolaisoleuine-dolaproine-phenylalanine.

[0299] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder andK. Lubke, " The Peptides," volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The auristatin / dolastatin drug moieties may be prepared according to the methods of: U. S. Pat. No. 5,635,483; U. S. Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863. See also Doronina (2003) Nat Biotechnol 21(7):778-784; " Monomethylvaline Compounds Capable of Conjugation to Ligands," U. S. Patent No.

[0300] 7,498,298, (disclosing, e.g., linkers and methods of preparing monomethylvaline compounds such as MMAE and MMAF conjugated to linkers). Biologically active organic compounds that act as cytotoxic agents, specifically pentapeptides, are disclosed in US Patent Nos. 6,884,869; 7,498,298; 7,098,308; 7,256,257; and 7,423,116.

[0301] Maytansine and Maytansinoids

[0302] Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U. S. Pat. No.

[0303] 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U. S. Pat. No. 4,151,042). Highly cytotoxic maytansinoid drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. Methods for isolating ansamitocins are described in U. S. Patent No. 6,573,074. Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U. S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0304] Antibody-maytansinoid conjugates are prepared by chemically linking an antigen binding protein (such as an antibody) to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U. S. Pat. No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin / antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U. S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or atother positions of the maytansinol molecule, such as various maytansinol esters. Methods for preparing maytansinoids for linkage with antibodies are disclosed, e.g., in U. S. Patent Nos.

[0305] 6,570,024 and 6,884,874.

[0306] Calicheamicin

[0307] The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, e.g., U. S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296. Structural analogues of calicheamicin that may be used include, but are not limited to, yll, a2I, a3I, N-acetylyll, PSAG and TII (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U. S. patents). Another anti-tumor drug that the antibody can be conjugated to is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

[0308] Other Cytotoxic Agents

[0309] Other cytotoxic agents, such as antitumor agents, that can be conjugated to an antigen binding protein (such as an antibody) include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U. S. Pat. Nos. 5,053,394 and 5,770,710, as well as esperamicins (U. S. Pat. No. 5,877,296).

[0310] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93 / 21232 published Oct. 28, 1993.

[0311] The present invention further contemplates an immunoconjugate formed between an antigen binding protein (such as an antibody) and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0312] For selective destruction of the tumor, the antigen binding protein (such as an antibody) may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphicstudies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0313] The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or 1123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. " Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0314] The -(L-(Payload)m)n fragments present in compounds of formula (I) may be diversely chosen and are not limited according to some embodiments of the present disclosure. Many such fragments have been described and developed in existing conjugates.

[0315] Linkers

[0316] In ADCs, the antigen binding protein (such as an antibody) can be conjugated directly to the payload (e.g., the cytotoxic agent) or via a linker. The linker covalently tethers the antibody and payload components. The linker contains a reactive end that allows the linker (e.g., a terminus of the linker) to chemically attach to an antigen binding protein. The linker can contain a reactive end that allows the linker (e.g., a terminus of the linker) to chemically attach to a drug, or the linker can be attached to the drug during synthesis.

[0317] A linker can contain a spacer region, e.g., between its reactive terminus that attaches to the antigen binding protein and its reactive terminus that attaches to the drug. The spacer, e.g., can serve to spatially separate the antigen binding protein from the drug so that the antigen binding protein does not interfere with the drug and / or to spatially separate the antigen binding protein from the drug so that the drug does not interfere with the antigen binding protein. In addition, or alternatively, the spacer can provide stability and / or solubility and / or hydrophilicity and / or otherwise improve the thermodynamic properties of the immunoconjugate, e.g., particularly if the drug is hydrophobic. A spacer region can contain, e.g., poly(ethylene glycol) (PEG) or hydrocarbon chains of varying lengths. A spacer region can contain elements to improve the properties of a given immunoconjugate, such as a sulphonamide (e.g., an acyl sulphonamide or carbamoyl sufonamide, e.g., a HYDRASPACE™ element), optionally in the context of a PEG element. See, e.g., U. S. Pat. No. 9,636,421 andVerkade et al. (2018), Antibodies 7:1-12. A spacer region can contain a cleavage site, discussed below.

[0318] Suitable linkers include, for example, cleavable and non-cleavable linkers. Non-cleavable linkers consist of stable bonds that are resistant to proteolytic degradation, ensuring greater stability than that of cleavable linkers. Examples of non-cleavable linkers are disclosed in WO 2018 / 002902. See also Tsuchikama and An (2018).

[0319] A cleavable linker (e.g., a linker with a cleavage site in the spacer region) is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can comprise or be a dipeptide linker, such as a vali ne-citrul line (Val-Cit) or a phenylalanine-lysine (Phe-Lys) linker. Other suitable linkers include linkers hydrolyzable at a pH of less than 5.5, such as a hydrazone linker. Additional suitable cleavable linkers include a glutathione sensitive linker, such as a disulfide linker. A pyrophosphate diester linker can also be employed. Upon internalization, the pyrophosphate diester gets promptly cleaved through the endosomal-lysosomal pathway (Kern et al., (2016) J Am Chem Soc 138:1430-1445). Bristol-Myers Squibb has described particular lysosomal enzyme-cleavable antitumor drug conjugates. See, for example, U. S. Pat. No. 6,214,345. Seattle Genetics has published applications U. S. Pat. Appl. 2003 / 0096743 and U. S. Pat. Appl. 2003 / 0130189, which describe p-aminobenzylethers in drug delivery agents. The linkers described in these applications are limited to aminobenzyl ether compositions.

[0320] As used herein, the term "self-immolative spacer" refers to a covalent linker engineered to break down spontaneously when exposed to specific stimuli (for example, lysosomal proteases, acidic 15 pH, or reducing environments). These spacers link a drug release trigger to its payload and undergo a multi-step disassembly (via an electronic cascade or cyclisation) to release the payload in its native, unmodified form.

[0321] Conjugates of the antigen binding protein and drug, such as a cytotoxic agent, may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2, 6-di isocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).Additionally, the linker may contain one or more linker components, e.g., in the spacer. Exemplary linker components include 6-maleimidocaproyl, maleimidopropanoyl (MP), valinecitrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PABC), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC), and N-Succinimidyl (4-iodo-acetyl)a mi nobenzoate (SIAB). Additional linker components are known in the art and some are described herein. See also " Monomethylvaline Compounds Capable of Conjugation to Ligands," U. S. Patent No. US7,498,298.

[0322] High concentrations of enzymes play a critical role in tumor cell growth, angiogenesis, invasion, and metastasis. There are several types of enzymes over-expressed locally and specifically in the extracellular environment of solid tumors, including proteases (e.g., matrix metalloproteinase and cathepsin B), peptidases (e.g., aminopeptidase), and lipases (e.g., phospholipase A2). Insertion of protease specific peptide cleavage sites into the linker enables release of the drug conjugate in the tumour extracellular milieu thus circumventing side effects mediated by target binding in healthy tissue (Chen B, et al., (2017) Theranostics 7:538-558).

[0323] Linkers (e.g., in the spacer region of a linker) may also comprise amino acids and / or amino acid analogs. Amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (VC or Val-Cit), alanine-phenylalanine (AF or Ala-Phe). Exemplary tripeptides include: glyci ne-val ine-citrul li ne (Gly-Val-Cit or GVC) and glycine-glycine-glycine (Gly-Gly-Gly or GGG). Amino acid residues that comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. Cathepsin B has a relatively broad scope of substrate, but it preferentially recognizes certain sequences such as phenylalanine-lysine (Phe-Lys) and valinecitrulline (Val-Cit) and cleaves a peptide bond on the C-terminal side of such sequences. In particular, Val-Cit and Val-Ala linkers coupled with p-aminobenzyloxycarbonyl (Val-Cit-PABC and Val-Ala-PABC) are successful cleavable linkers for ADCs (Dubowchik et al., (2002) Bioconjugate Chem 13:855-869; Hartley, (2011) Expert Opin Investig Drugs 20:733-744).

[0324] Antigen binding proteins such as antibodies may be made reactive for conjugation with linker reagents. Nucleophilic groups on antibodies include but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine,thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).

[0325] Antigen binding proteins such as antibodies may also be modified to introduce electrophilic moieties that can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups that may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U. S. Pat. No. 5,362,852). Such aldehydes can be reacted with a drug moiety or linker nucleophile.

[0326] Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0327] In some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyl linkers may be cleavable by enzymes that are present cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a Phe-Leu, a Gly-Phe-Leu-Gly (SEQ ID NO: 43) linker, Gly-Gly-Phe-Gly (SEQ ID NO: 44)). Other such linkers are described, e.g., in U. S. Pat. No. 6,214,345. In specific embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U. S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0328] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U. S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U. S. Pat. No. 5,622,929).

[0329] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a glutathione sensitive linker such as a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylth ioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U. S. Pat. No. 4,880,935.)In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.

[0330] 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0331] Typically, the linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment," in the context of a linker, means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of ADC or ADC derivative, are cleaved when the ADC or ADC derivative present in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating independently with plasma both (a) the ADC or ADC derivative (the " ADC sample") and (b) an equal molar amount of unconjugated antibody or therapeutic agent (the "control sample") for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with that present in control sample, as measured, for example, by high performance liquid chromatography methods.

[0332] In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the drug (i.e., in the milieu of the linker-drug moiety of the ADC or ADC derivate as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the drug and the antigen binding protein (such as an antibody) or derivative thereof (i.e., in the milieu of the ADC or ADC derivative as described herein).

[0333] A variety of linkers that can be used with the present compositions and methods are described in WO 2004010957 entitled " Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease".

[0334] Production of ADCs: Attachment to Antigen Binding Protein

[0335] A drug (e.g., via a linker) can be attached to an antigen binding protein (such as an antibody) through various techniques. See, e.g., Tsuchikama and An (2018). The antigen binding protein (such as an antibody) can be modified (e.g., via chemical conjugation, genetic engineering to introduce a non-naturally occurring amino acid, or enzymatic conjugation) to allow its chemical attachment (e.g., to contain a reaction handle to allow its chemical attachment) to a reactive end of a linker that joins the antigen binding protein to the drug. Chemical conjugationIn chemical conjugation, accessible amino acid residues on the surface of the antibody undergo a controlled reaction with a reaction handle installed on the linker.

[0336] Lysine amide coupling: Amide coupling is a major ADC conjugation method connecting a payload and solvent accessible lysine residues on the antibody using linkers containing activated carboxylic acid esters.

[0337] Cysteine coupling: Cysteine-based conjugation methods rely on a specific reaction between cysteine residues of an antibody and a thiol-reactive functional group installed on the drug. In general, antibodies do not possess free thiols, although they may be found in the variable domains of certain antibodies. Cysteine residues may form disulfide bonds with each other.

[0338] The points of attachment are cysteines produced by mild reduction of the interchain disulfides of the antibody that is carried out whilst antibodies are immobilised on Protein G affinity resin (thus enabling the use of large reagent excesses without intermediate purifications). While immobilized, a large excess of TCEP will fully reduce the interchain disulfides but has no impact upon the binding of the antibody to the resin.

[0339] The number of thiols per antibody generated by this procedure depends upon the source and isotype of the antibodies. For example, human (and mouse-human chimeric) IgGls have 4 reducible disulfides, and thus generate 8 thiols upon full reduction, whereas murine IgGls have 5 reducible disulfides and produce 10 thiols. In human IgGl, which is most commonly used in modern ADCs, the 4 interchain disulfides, which are generally not critical for structural stability of IgGl, can be selectively reduced under mild conditions to give 2, 4, 6, or 8 free thiols while keeping the 12 intrachain disulfides intact. If ADCs with the maximal drug loading (e.g., 10 drugs per antibody for the murine IgGls) are desired, then a maleimido-drug-linker can simply be added to the immobilized antibodies in sufficient excess to ensure complete conjugation. However, ADCs with fewer drugs per antibody can also be prepared from fully reduced antibodies by including a biologically inert capping agent such as N-ethyl maleimide (NEM), which occupies some of the available thiols on the antibody. When the maleimido-drug-linker and the capping agent are added simultaneously to the fully reduced antibody and in large excess (at least 3-fold), the two maleimide electrophiles compete for the limiting number of available thiols. In this fashion, the drug loading is determined by the relative thiol reaction rates of the drug-linker and capping agent, and thus can be considered to be under kinetic control. The relative reaction rates of maleimido-drug-linkers do vary significantly, and thus the molar ratio of drug-linker to NEM present in a reaction mix must be determined empirically to arrive at a panel of ADCs with a desired level of drug loading.THIOMAB: This technology introduces two new cysteine residues (one per heavy chain) for selective antibody attachment (Junutula et al., (2008) Nat Biotechnol 26:925-932). This engineered cysteine technology enables generation of highly homogeneous ADCs with a DAR (drug antibody ratio) of 2.

[0340] Cysteine rebridging: This is another strategy to better control DAR and heterogeneity of ADCs. Dibromomaleimide, dibromopyridazinediones, and a 1,3-bis (p-toluenesulfonyl) propane-based core can accept two reduced cysteines derived from interchain disulfide bonds to afford a rebridged antibody. See, e.g., Tsuchikama and An (2018).

[0341] Cysteine conjugation using aryl palladium complexes: In this method, the aryl palladium reagents are readily prepared by mixing active palladium-phosphine complexes and various aryl halides. The resulting complexes undergo a thiol arylation with reduced cysteine residues of the antibody (Vinogradova et al., (2015) Nature 526:687-691).

[0342] Non-natural amino acid incorporation by genetic engineering:

[0343] Installation of non-natural amino acid residues with a reaction handle is a strategy that allows for a site-specific chemical conjugation, leading to strictly controlled DARs (Kline T et al., (2015) Pharm Res 32: 3480-3493.

[0344] Oxime ligation: Protein expression systems (bacteria, yeast, and mammalian cells) have been developed where p-acetylphenylalanine containing a carbonyl group is genetically encoded by introducing a unique codon-tRNA synthetase (Axup et al., (2012) Proc Natl Acad Sci USA 109:16101-16106; Tian et al., (2014) Proc Natl Acad Sci. USA 111:1766-1771). Engineered antibodies containing p-acetylphenylalanine residues are produced using either of the expression systems, and the carbonyl groups introduced react with alkoxyamine-functionalized linkers to provide oxime-conjugated ADCs.

[0345] Copper-catalyzed azide-alkyne cyclization: p-azidomethyl-L-phenylalanine (Zimmerman et al., (2014) Bioconjugate Chem 25:351-361) and N6-((2-azidoethoxy)carbonyl)-L-lysine (VanBrunt et al., (2015) Bioconjugate Chem 26:2249-2260) can be used in this technique. The incorporated azide groups are used for conjugation with alkyne-functionalized linkers through the copper-catalyzed Huisgen cycloaddition (generally termed "click chemistry") to provide triazole-li nked ADCs.

[0346] Strain-promoted (copper-free) azide-alkyne cyclization: As another technique, bioorthogonal conjugation of azide-incorporated antibodies can be achieved by using strained cyclooctyne-functionalized linkers that do not require a cytotoxic, oxidative copper catalyst (Zimmermann E et al., (2014) Bioconj. Chem. 25: 351-361).

[0347] Enzymatic conjugationSeveral enzymes have been used for conjugating an antibody with a drug or for installing unique reaction handles on the antibody scaffold for chemical conjugation. These enzymes modify the antibody in a site- or amino acid sequence-specific manner (Tsuchikama and An (2018)).

[0348] Transpeptidation using sortase: Sortase A from Staphylococcus aureus recognizes the LPXTG (X: any amino acid) motif, cleaves the th reonine-g lycine (T-G) bond, and attaches an oligoglycine (oligo-G)-containing molecule. Various cargo can be fused to the oligo-G for sortase A-mediated conjugation, e.g., peptides, proteins, and nucleic acids (Witte et al., (2012) Proc Natl Acad Sci USA 109:11993-11998).

[0349] Transpeptidation using microbial transglutaminase: A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR of 2 (one conjugation site per heavy chain) (Jeger et al., (2010) Angew Chem Int Ed Engl 49:9995-9997; Dennler et al., (2014) Bioconjugate Chem 25:569-578). An N297Q mutation prior to this conjugation provides two more reaction sites (DAR = 4). An alternative version using a peptide sequencespecific transglutaminase has been developed (Strop et al., (2013) Chem Biol 20:161-167). This enzyme recognizes and utilizes LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation.

[0350] N-Glycan engineering: Incorporation of an aldehyde group on the N-glycan terminus using β-1,4-galactosyltransferase (GalT) and o-2,6-sialyltransferase (SialT) results in introduction of a sialic acid on each N-glycan terminus, which is subsequently converted into an aldehyde group using NaIO4 under mild oxidation conditions. The aldehyde groups generated are used to conjugate aminooxy-functionalized payloads (drugs) (Zhou et al., (2014) Bioconjugate Chem 25:510-520).

[0351] Non-natural saccharides: Another approach involves incorporating non-natural saccharides possessing orthogonal reaction handles into the antibody, such as by GlycoConnect technology (van Geel et al., (2015) Bioconjugate Chem 26:2233-2242). The glycan chain at Asn297 is trimmed using the endoglycosidase Endo S2 and azide groups are introduced using a mutant galactosyl transferase GalT(Y289L) and N-azidoacetylgalactosamine (GalNAz). The azide handles are used for a strain-promoted click reaction with payloads (drugs), resulting in stable and homogeneous ADCs with tightly controlled DARs.Compounds of formula (I) with site-specific conjugation and controlled DAR

[0352] In a particular embodiment, the antibody-drug conjugate (ADC) is of formula (I):

[0353] (T)p-Ab'-(L-(Payload)m)n

[0354] (I)

[0355] having:

[0356] a controlled drug-to-antibody ratio (DAR) of (m x n); and

[0357] a site-specific conjugation at one or two of a pair of interchain cysteine residues of Ab' defined as the conjugated cysteine residues;

[0358] where in formula (I):

[0359] Ab' is an anti-PSMA antibody, or antigen binding fragment thereof or variant thereof according to some embodiments of the present disclosure, comprising two heavy chains and two light chains, each chain comprising one or more interchain cysteine residues, such that each of said conjugated cysteines are bound to either -(L-(Payload)m or optional T fragment, and the remaining interchain cysteine residues form interchain disulfide bridges;

[0360] n is 1 or 2;

[0361] m is 1, 2, 3, 4, 5 or 6;

[0362] p is 0 or 1;

[0363] T, if present, is a terminal group covalently bound to one of said two reduced Sulfur atoms of said interchain cysteine residues of Ab' that is not covalently bound to L;

[0364] each L is independently a linear or branched di-, tri-, tetra-, penta-, hexa- or hepta-valent linker moiety covalently binding Payload to said Ab, where said L is attached to Ab' through one or two of said reduced Sulfur atoms of said interchain cysteine residues and binds said Ab' with one or more Payload; Payload is a molecule exerting a biological activity and selected from the group consisting of diagnostic agents, therapeutic agents and labelling agents. PSMA binding protein according to some embodiments of the present disclosure typically comprises one or more polypeptide chains made of amino acids, it being provided that the cysteine residues may form covalent bonds through the formation of disulphide bridges, typically between different peptide chains within Ab'. These are called herein "interchain cysteine residues".Typically, said Ab' comprises two heavy chains and two light chains, where preferably each heavy chain comprises three cysteine residues and each light chain comprises one cysteine residue in the hinge region.

[0365] These chains are covalently bridged together by disulfide bonds created between the sulfur atom of the cysteine residues.

[0366] As used herein, the hinge region refers to an amino acid stretch in the central part of the two heavy chains, which links these two chains by disulfide bonds, whose respective sulfur atoms belong to cysteine residues.

[0367] Typically, the heavy chains are bridged together by the two interchain disulfide bridges created between two pairs of cysteine residues (the inter (heavy chains) cysteine residues), and each heavy chain is further bridged to a light chain by a further disulfide bridge created between two cysteine residues (the inter (heavy-light chains) cysteine residues).

[0368] Typically, said Ab' thus comprises two pairs of inter (heavy chains) cysteine residues and two pairs of the inter (heavy-light chains) cysteine residues.

[0369] These 8 cysteine residues involved in interchain bridges are herein referred as "interchain cysteines".

[0370] Those interchain cysteines that are involved in the conjugation (i.e.) covalently attached to either -(L-(Payload)m or optional T fragments are herein referred to as "conjugated cysteines residues" or "cysteine residues of the conjugation site."

[0371] More specifically, said Ab' comprises two heavy chains and two light chains, each chain comprising one or more cysteine residue involved in inter chains disulfide bridges, and wherein each heavy chain comprises one N-proximal inter (heavy chains) cysteine residue that is located nearest to the N-terminal end of said heavy chain.

[0372] These two N-proximal inter (heavy chains) cysteine residues (also called herein " N-proximal cysteines") are a key site for site-specific conjugation according to some embodiments of the present disclosure.

[0373] Without being bound by theory, it is hypothesized that these N-proximal cysteines are adjacent to or close to histidine residues that may confer the appropriate reactivity for complex formation with a metal atom, that will then lead to a reactive position for conjugation.

[0374] In some embodiments, said N-proximal cysteines are also referred to as Cysteine-239 according to Kabat numbering. According to an embodiment, said conjugated cysteines residues are the two C-239 cysteines.

[0375] According to another embodiment, another key site for site-specific conjugation is anyone of the two pairs of inter (heavy-light chains) cysteine residues. According to analternative embodiment, said conjugated cysteines residues are any pair of the two pairs of inter (heavy-light chains) cysteine residues.

[0376] Ab' is preferably an Immunoglobulin G, such as an Immunoglobulin G of IgGl or IgG4 isotype, for example, IgGl isotype. In some cases, Ab' comprises sequence of a kappa type IgGl constant heavy (CH) chain.

[0377] According to an embodiment, Ab' may be a bispecific monoclonal antibody (BsMAb, BsAb).

[0378] In some cases, Ab' derives from an unmodified anti-PSMA antibody in that it comprises two sulfur atoms that would otherwise form an interchain disulphide bridge that are reduced.

[0379] Specifically, in Ab' the Sulfur atom of each N-proximal inter (heavy chains) cysteine residue is reduced, while the remaining interchain cysteine residues are unaffected and still form interchain disulfide bridges.

[0380] According to an embodiment, Ab' has its two N-proximal sulfur atoms reduced.

[0381] The conjugation site of said site-specific conjugation of compound (I) provided herein is at one or two of the interchain cysteine residues as defined above, e.g., the conjugated cysteine residues.

[0382] It is to be understood that conjugated cysteine residues are based on a pair of cysteine residue otherwise involved in a disulfide bridge, i.e., before reduction of the sulfur atoms.

[0383] Typically, said conjugated cysteine residues may be either N-proximal inter(heavy chains) cysteine residues or inter(heavy-light chains) cysteine residues.

[0384] It is also to be understood that when the conjugation is at one interchain cysteine residue, T is present, and when the conjugation is at two interchain cysteine residues, T is absent.

[0385] According to a first embodiment, the conjugation site is at one or two of the N-proximal inter(heavy chains) cysteine residues that are located nearest to the N-terminal end of the heavy chains, e.g., one or two of the two C-239 cysteines according to Kabat numbering.

[0386] According to a second embodiment, the conjugation site is at one or two of the inter(heavy-light chains) cysteine residues.

[0387] According to an illustration of any first or second embodiment, the conjugate of formula (I) may be a monoconjugate (DAR=1). In some instances of the embodiment, L is a trivalent linker covalently binding said Payload with the two sulfur atoms of the conjugated cysteine residues of Ab'.

[0388] Said conjugate of formula (I) may be of formula (I-A):

[0389] Ab'-L-(Payload)(I-A)

[0390] having a controlled drug-to-antibody ratio (DAR) of 1;

[0391] where in formula (I-A):

[0392] Ab' and Payload are defined as herein;

[0393] L is a trivalent linker moiety covalently binding together the two said reduced Sulfur atoms of Ab' with Payload.

[0394] Preferably, said conjugate of formula (I-A) comprises one of the following fragments: * — S - R’— L1 * — S

[0395] ~^X — L3 — Payload R" — L1 — Payload

[0396] * — S - R’ — L2 o

[0397]

[0398] r * —

[0399] wherein each S designates the reduced Sulfur atom of the conjugated cysteine residues of Ab' that are bound to Payload;

[0400] * designates the attachment of said Sulfur atom to the rest of Ab' at each conjugated cysteine residue, typically at each heavy chain through the corresponding N-proximal inter(heavy chains) cysteine residues or at a light chain and a heavy chain through the corresponding inter(heavy-light chains) cysteine residues;

[0401] Li, L2and L3identical or different are independently optional divalent Spacer moieties; preferably Li, L2are identical and L3is different;

[0402] X is chosen from linear, branched or cyclic trivalent moieties, comprising 0 to 9 nitrogen atoms and from 0 to 30 carbon atoms, preferably linear or branched moieties comprising 1 to 3 carbon atoms;

[0403] R' is a group formed as a result of a reaction between a thiol and a thiol-reactive group, such as maleimide, succinimide, 3-arylpropiolonitrile, bromo-maleimide, chloroacetamide, bromoacetamide, iodoacetamide, acrylamide, vinyl sulfone, pyridyl disulfide, alpha-halo carbonyl, vinyl pyridine, preferably maleimide;

[0404] R" is a group formed as a result of a reaction between two thiols and a bifunctional thiol-reactive group, such as 3,4-disubstituded maleimide, succinimide, 3,3'-(phenylene)dipropiolonitrile, dibromopyridazinedione, bissulfone, allyl sulfone, 3-bromomaleimide or arsenous acid.

[0405] According to the first embodiment above, the compounds of formula (I-A) are of formula (I'-A) where the conjugated cysteine residues are each said N-proximal inter(heavy chains) cysteine residues. According to this embodiment, the attachment * is located at each heavy chain through the corresponding N-proximal inter(heavy chains) cysteine residue, or located at a light chain and at a heavy chain,As an illustration, said conjugate of formula (I-A) may be schematically represented by the following formula (I-Al):

[0406]

[0407] where Payload, X, R', Li, L2and L3are defined as above.

[0408] As a further illustration, said conjugate of formula (I-A) may be schematically represented by the following formula (I-A2):

[0409]

[0410] where R", Payload, Li are defined as above.

[0411] According to the second embodiment, in the compounds of formula (I-A), the site of conjugation is anyone of the two pairs of said inter(heavy-light chains) cysteine residue.

[0412] According to this embodiment, the attachment * is located at the light chain and at the heavy chain through said one inter(heavy-light chains) cysteine residue.

[0413] As an illustration, said conjugate of formula (I-A) may be schematically represented by the following formula (I-Al-iso):

[0414]

[0415] where Payload, X, R', Li, L2and L3are defined as above.

[0416] As a further illustration, said conjugate of formula (I-A) may be schematically represented by the following formula (I-A2-iso):

[0417]

[0418] where Payload, R", Li are defined as above.

[0419] According to another illustration of any first or second embodiment, the conjugate of formula (I) may be an even multiple conjugate (DAR=2m).

[0420] Said antibody conjugate of formula (I) may be of formula (I-B):

[0421] Ab'-(L-(Payload)m)2

[0422] (I-B)

[0423] having a controlled drug-to-antibody ratio (DAR) of 2m;where in formula (I-B):

[0424] m is as defined above, preferably 1, 2, 3, 4, 5 or 6;

[0425] Ab' and Payload are defined as herein;

[0426] each of the two L is di-, tri-, tetra-, penta-, hexa- or hepta-valent linear or branched linker moiety covalently binding one of said reduced Sulfur atoms of said conjugated cysteine residues of Ab' with m Payload(s).

[0427] It is to be understood that when m > 1, then each Payload can be the same or different.

[0428] Preferably, said conjugate of formula (I-B) comprises two following fragments:

[0429] * — S - R' — L4 — (Payload)m

[0430] wherein

[0431] m and Payload are defined as herein;

[0432] S designates the reduced Sulfur atom of each of the two said conjugated cysteine residues, respectively;

[0433] * designates the attachment of said Sulfur atom to the rest of Ab' at the conjugated cysteine residue, typically at each heavy chain through the corresponding N-proximal inter(heavy chains) cysteine residues, or at a light chain and a heavy chain through the corresponding inter(heavy-light chains) cysteine residues;

[0434] each L4identical or different is an optional linear or branched Spacer moiety;

[0435] ' is a group formed as a result of a reaction between the reduced thiol group of Ab' and a thiol-reactive group, such as maleimide, succinimide, 3-arylpropiolonitrile, bromo-maleimide, chloroacetamide, bromoacetamide, iodoacetamide, acrylamide, vinyl sulfone, pyridyl disulfide, alpha-halo carbonyl, vinyl pyridine.

[0436] According to the first embodiment above, the compounds of formula (I-B) are such that the conjugated cysteine residues are each of the two said N-proximal inter(heavy chains) cysteine residues (C-239 cysteine, Kabat numbering).

[0437] According to this embodiment, the attachment * is located at each heavy chain through the corresponding N-proximal inter(heavy chains) cysteine residue.

[0438] As an illustration, said conjugate of formula (I-B) may be schematically represented by the following formula (I-Bl):

[0439]

[0440] where Payload, k, R', L4are defined as above and k is defined as m above.

[0441] According to the second embodiment, in the compounds of formula (I-B), the site of conjugation is anyone of the two pairs of said inter(heavy-light chains) cysteine residues.

[0442] According to this embodiment, an attachment * is located at the light chain and the other attachment * is at the heavy chain, through said one inter(heavy-light chains) cysteine residue.

[0443] Compounds (I-A) and (I-B) are representative compounds (I) where T is absent and there are two conjugated cysteine residues: either two N-proximal inter(heavy chains) cysteine residues are conjugated to Payload, or the two of a pair of inter(heavy-light chains) cysteine residues are conjugated to Payload.

[0444] According to another illustrative embodiment, the conjugate of formula (I) may be a multiple conjugate (DAR=m).

[0445] Said antibody conjugate of formula (I) may be of formula (I-C):

[0446] T-Ab'-L-(Payload)m

[0447] (I-C)

[0448] having a controlled drug-to-antibody ratio (DAR) of m;

[0449] where in formula (I-C):

[0450] m, Ab' and Payload are as defined herein;

[0451] L is di-, tri-, tetra- or penta-valent linear or branched linker moiety covalently binding one of said reduced Sulfur atoms of the two N-proximal inter(heavy chains) cysteine residues of Ab' with m Payload(s); andT is defined as herein and is covalently bound to Ab' through the other one of said reduced Sulfur atoms of the two N-proximal inter(heavy chains) cysteine residues of Ab'.

[0452] Preferably, said conjugate of formula (I-C) comprises each of the two following fragments:

[0453] * — S R' — L4 — (Payload)m

[0454] ! - S - *

[0455] Nd -

[0456]

[0457] o

[0458] wherein

[0459] Payload and m are defined as herein;

[0460] each S designates one reduced Sulfur atom of each of the two said N-proximal inter(heavy chains) cysteine residues;

[0461] each * designates the attachment of said Sulfur atom to the rest of Ab' at each heavy chain through the corresponding N-proximal inter(heavy chains) cysteine residue; each L4 identical or different is an optional linear or branched Spacer moiety;

[0462] R' is a group formed as a result of a reaction between the reduced thiol group of Ab' and a thiol-reactive group, such as maleimide, succinimide, 3-arylpropiolonitrile, bromo-maleimide, chloroacetamide, bromoacetamide, iodoacetamide, acrylamide, vinyl sulfone, pyridyl disulfide, alpha-halo carbonyl, vinyl pyridine;

[0463] Nu is a nucleophilic moiety chosen from OH, -NH2, -NH-NH2, -NH-OH, -NH-R1, - NR1R2, Where R1, R2and R3are chosen from -CH2-CONH2, -CH2-COOH, Ci-Ci2alkyl, C6-Ci2aryl, C5-Ci2heteroaryl; — (CH2— CH2— O— )r— CH3groups; in which r is an integer ranging from 1 to 24.

[0464] Compounds (I-C) are representative compounds (I) where only one N-proximal inter(heavy chains) cysteine residue is conjugated to Payload, and the other is covalently bound to T.

[0465] As an illustration, said conjugate of formula (I-C) may be schematically represented by the following formula:

[0466]

[0467] where R', L4are defined as above and k is defined as m above.

[0468] In the formulae (I), (I-C)), (I-A), (I-Al), (I-Al-iso), (I-A2), (I-A2-iso), (I-B), and / or (I-C) above and below:

[0469] According to an embodiment, the optional Li, L2and L3spacers may consist of one or multiple successive units selected from the group consisting of: a linear or branched, saturated or unsaturated, Ci-C6o alkylene group optionally interrupted and / or terminated on one or both sides by one or more chemical groups selected from -O-, -S-, -S(O)-, -SO2-, -O-P(O)(OH)-O-, -C(O)-, -NH-, -NMe-, -C(O)NH-, -NHC(O)-, -NH-C(O)-NH, -O-C(O)-NH-, -O-C(O)-O-; C3-C8cycloalkylene; C3-C8heterocyclylene; 5 to 12 membered heteroarylene; C6-Ci2arylene; amino acids; polypeptides; glycosylene; — (CH2— CH2— O— )r— groups; in which r is an integer ranging from 1 to 24 and / or optionally substituted by one or more of OH, CN, NH2, CF3, NHCONH2, CONH2, COOH, =0, NH2, glycosyl, Ci-C12alkyl, C3-C8cycloalkyl, C6-Ci2aryl, C5-C12 heteroaryl, — (CH2— CH2— O — )r — CH3;

[0470] Preferable units comprise in particular units known as valine-citrulline (VC) and / or PABC (p-aminobenzyl carbamate).

[0471] According to an embodiment, R' may include the following groups:

[0472]

[0473] According to an embodiment, R" may include the following groups:

[0474]

[0475] According to a particular embodiment, X may be chosen from the following groups:

[0476]

[0477] where R2is chosen from H, OH, CN, NH2, CF3, NHCONH2, CONH2, COOH, =0, NH2, Ci-Ci2alkyl, C6-Ci2aryl, C5-Ci2heteroaryl; — (CH2— CH2— O— )r— CH3groups; in which r is an integer ranging from 1 to 24;

[0478] According to an embodiment, the optional L4spacer may consist of one or multiple linear or branched units, which links interchain cysteine residue of the antibody with one or multiple Payload molecules.

[0479] Linear units of the L4include: saturated or unsaturated, Ci-C6o alkylene group optionally interrupted and / or terminated on one or both sides by one or more chemical groups selected from -O-, -S-, -S(O)-, -SO2-, -O-P(O)(OH)-O-, -C(O)-, -NH-, -NMe-, -C(O)NH-, -NHC(O)-, -NH-C(O)-NH, -O-C(O)-NH-, -O-C(O)-O-; C3-C8cycloalkylene; C3-C8heterocyclylene; 5 to 12 membered heteroarylene; C6-Ci2arylene; amino acids; polypeptides; glycosylene; — (CH2— CH2— O— )t— groups; in which r is an integer ranging from 1 to 24 and / or optionally substituted by one or more of OH, CN, NH2, CF3, NHCONH2, CONH2, COOH, =0, NH2, glycosyl, Ci-Ci2alkyl, C3-C8cycloalkyl, C6-Ci2aryl, C5-Ci2heteroaryl, — (CH2— CH2— O— )r— CH3.

[0480] Branched units of the L4may be chosen from branched or cyclic trivalent or tetravalent moieties, comprising 1 to 9 nitrogen atoms and from 0 to 30 carbon atoms, including the following groups:

[0481]

[0482] Representative known fragments for -S-L-(Payload) as described below include:

[0483]

[0484]

[0485] OH

[0486]

[0487] HO

[0488] °\

[0489]

[0490] HOHO

[0491]

[0492] HO

[0493]

[0494] pH

[0495]

[0496] IOM17IZ. OZ.

[0497]

[0498] where the

[0499]

[0500] indicates a bind site to the antibody (the S is from the cysteine residue of the antibody).

[0501] Preferably, the fragment for -S-L-(Payload) included in the ADC according to some embodiments of the present disclosure is the one represented just above.

[0502] According to some embodiments of the present disclosure, the conjugates of formula (I) comprise -(L-(Payload)m)n covalently and site-specifical ly linked at one or two of the specific interchain cysteine residues as defined above, e.g., the conjugated cysteine residues.

[0503] According to an embodiment, the conjugates of formula (I) may further comprise one or more identical or different -(L-(Payload)m)n covalently linked at one or more additional sites of conjugation. Such conjugates comprising additionally grafted -(L-(Payload)m) are encompassed by Formula (I) and are herein referred to as "further conjugated conjugates"." According to an embodiment, said additional sites of conjugation include cysteine residues of Ab, such as interchain cysteine residues different from the conjugated cysteine residues as defined above.

[0504] The compounds of formula (I) may also be used as substrates for further conjugation of payload fragments.

[0505] According to an embodiment, said additional sites of conjugation are interchain cysteine residues of Ab' different from the conjugated cysteine residues as defined above.

[0506] In some embodiments of the present disclosure, provided herein is an ADC of formula (I):(T)p-Ab'-(L-(Payload)m)n

[0507] (I),

[0508] wherein n is 2 and p is 0. In some of these embodiments, each L independently comprises a thio-reactive spacer, optionally a succinimidyl group, a maleimidyl group or an arylpropiolonitrile group. In some cases, each L independently comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein each L independently comprises a cleavable linker that is a peptide linker. In some cases, each L independently comprises a succinimidyl group, a maleimidyl group, a caproyl group, a va line-citrull inyl group, and / or a p-aminobenzyloxycarbonyl group. In some cases, each L independently comprises a succinimidyl group, a caproyl group, a valine-citrullinyl group, and / or a p-aminobenzyloxycarbonyl group. In other cases, each L independently comprises a maleimidyl group, a caproyl group, a valine-citrullinyl group, and / or a -a mi nobenzyloxycarbonyl group.

[0509] In some embodiments of the present disclosure, provided herein is an ADC of formula (I):

[0510] (T)p-Ab'-(L-(Payload)m)n

[0511] (I),

[0512] wherein n is 1, and p is 0. In some of these embodiments, L is a tridentate linker. In some cases, L comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein L comprises a cleavable linker that is a peptide linker. In some cases, L comprises a bis-succinimidyl group, a caproyl group, a valine-citrullinyl group, and a p-a mi nobenzyloxycarbonyl group. In some cases, L comprises a bis-maleimidyl group, a caproyl group, a valine-citrullinyl group, and a -a mi nobenzyloxycarbonyl group.

[0513] In some cases, L in formula (I) above is a tridentate linker such as one of those described in US Patent Publication No. 20160015832, which is incorporated herein by reference in its entirety.

[0514] In some cases, L comprises the following structure:

[0515]

[0516] where W is substituted aryl, heteroaryl, linear alkyl, cycloalkyl, heterocycloalkyl, or

[0517] any combination thereof. The left hand

[0518]

[0519] s indicate binding sites to the antibody

[0520] (via the cysteine residue), the right hand side

[0521]

[0522] indicates a binding site to the payload.

[0523] In some cases, L comprises the following structure:

[0524]

[0525] where W is substituted C6-i4ar lene, C5-14heteroarylene, Ci-3oalkylene, Ci-3oheteroalkylene, C3-iocycloalkylene, C3-i0heterocycloalkylene, or any combination

[0526] thereof. The left hand

[0527]

[0528] s indicate binding sites to the antibody (via the cysteine

[0529] residue), the right hand side

[0530]

[0531] indicates a binding site to the payload.

[0532] Unless otherwise indicated, in the structure of the tridentate linkers depicted herein that are suitable for the antibody-drug conjugate according to some embodiments of thepresent disclosure, such as the one above, the ~ depicted on the left hand side indicates

[0533] a binding site to a sulfur atom of a cysteine residue of the antibody, and the

[0534]

[0535] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0536] Unless specified otherwise, in the present disclosure,

[0537]

[0538] indicates a single or double bond.

[0539] In some cases, L comprises the following structure:

[0540]

[0541] where W is substituted C6-i4ar lene, C5-14heteroarylene, Ci-3oalkylene, Ci-3oheteroalkylene, C3-iocycloalkylene, C3-i0heterocycloalkylene, or any combination thereof.

[0542] In some cases, L comprises the following structure:

[0543]

[0544] where W is substituted C6-i4arylene, C5-14heteroarylene, Ci-3oalkylene, Ci-3oheteroalkylene, C3-iocycloalkylene, C3-i0heterocycloalkylene, or any combination thereof.

[0545] In some cases, L comprises a tridentate structure according to the following structure:

[0546]

[0547] wherein:

[0548] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0549] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl;

[0550] Rxis linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, poly(ethylene glycol) chain, or any combination thereof;

[0551] a is 0 or 1;

[0552] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0553] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—; R10, R11, and R12are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl;

[0554] R13and R14are each independently selected from linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, or any combination thereof; Q is N or CR15; R15is selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, or alkynyl; x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;the

[0555]

[0556] s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0557] cysteine residue of the antibody, and the

[0558]

[0559] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0560] In some cases, L comprises a tridentate structure according to the following structure:

[0561]

[0562] wherein:

[0563] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0564] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl;

[0565] Rxis linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, poly(ethylene glycol) chain, or any combination thereof;

[0566] a is 0 or 1;

[0567] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0568] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—; R10, R11, and R12are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl;

[0569] R13and R14are each independently selected from linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, or any combination thereof; Q is N or CR15; R15is selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, or alkynyl; x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;the s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0570] cysteine residue of the antibody, and the

[0571]

[0572] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0573] In some cases, L comprises a tridentate structure according to the following structure:

[0574] Z— N

[0575] O 1

[0576] O |

[0577] y— N

[0578]

[0579] wherein:

[0580] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0581] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl;

[0582] Rxis linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, poly(ethylene glycol) chain, or any combination thereof;

[0583] a is 0 or 1;

[0584] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0585] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[0586] R10, R11, and R12are each independently selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl;

[0587] R13and R14are each independently selected from linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, or any combination thereof; Q is N or CR15; R15is selected from H, linear alkyl, cycloalkyl, heterocycloalkyl, alkenyl, or alkynyl; x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;the

[0588]

[0589] s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0590] cysteine residue of the antibody, and the

[0591]

[0592] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0593] In some cases, L comprises a tridentate structure according to the following structure:

[0594]

[0595] wherein:

[0596] indicates a single or double bond,

[0597] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0598] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5-14heteroaryl;

[0599] R1is substituted or unsubstituted Ci-iOalkylene, Ci-wheteroalkylene, C3-locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-i4arylene, C5-i4heteroarylene, polyethylene glycol) chain, or any combination thereof;

[0600] a is 0 or 1;

[0601] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0602] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;R10, R11, and R12are each independently selected from H, Cuoalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-14aryl, or C5-14heteroaryl;

[0603] R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, Ci-ioheteroalkylene, C3-i0cycloalkylene, heterocycloalkylene, C1-10alkenylene, C6-14arylene, C1-14heteroarylene, or any combination thereof;

[0604] Q is N or CR15;

[0605] R15is selected from H, Ci-ioal kyl, C3-i0cycloalkyl, C3-i0heterocycloalkyl, C1-10alkenyl, or Ci-ioalkynyl;

[0606] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;

[0607] the

[0608]

[0609] s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0610] cysteine residue of the antibody, and the

[0611]

[0612] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0613] In some cases, L comprises a tridentate structure according to the following structure:

[0614]

[0615] wherein:

[0616] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0617] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-i0heterocycloalkyl, Ci-iOalkenyl, Ci-ioalkynyl, C6-i4aryl, C5-14heteroaryl;

[0618] R1is substituted or unsubstituted Ci-ioalkylene, Ci-ioheteroalkylene, C3-locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-^arylene,

[0619] C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[0620] a is 0 or 1;

[0621] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0622] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[0623] R10, R11, and R12are each independently selected from H, Cuoalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-14aryl, or C5-14heteroaryl;

[0624] R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, Cnoheteroalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci-ioalkenylene, C6-i4ar lene, C5-14heteroarylene, or any combination thereof;

[0625] Q is N or CR15; R15is selected from H, Ci-ioalkylene, C3-i0cycloalkylene, C3-ioheterocycloalkylene, Ci-ioalkenylene, or Cnoalkynylene;

[0626] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;

[0627] the

[0628]

[0629] s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0630] cysteine residue of the antibody, and the

[0631]

[0632] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0633] In some cases, L comprises a tridentate structure according to the following structure:

[0634] R14

[0635]

[0636] wherein:

[0637] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5-wheteroar l;

[0638] R1is substituted or unsubstituted Ci-iOalkylene, Ci-wheteroalkylene, C3-locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-warylene,

[0639] C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[0640] a is 0 or 1;

[0641] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0642] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[0643] R10, R11, and R12are each independently selected from H,

[0644] Ci-ioalkyl, C3-iocycloalkyl, C3-i0heterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-i4ar l, C5-14heteroaryl;

[0645] R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, Cnoheteroalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci-ioalkenylene, C6-i4ar lene, C5-14heteroarylene, or any combination thereof;

[0646] Q is N or CR15; R15is selected from H, Ci-ioalkyl, C3-i0cycloalkyl, C3-i0heterocycloalkyl, Ci-ioalkenyl, Cnoalkynyl;

[0647] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l;

[0648] the

[0649]

[0650] s depicted on the left hand side indicate binding sites to a sulfur atom of a

[0651] cysteine residue of the antibody, and the

[0652]

[0653] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0654] In some cases, L comprises one of the following tridentate structures:

[0655]

[0656] , wherein the

[0657]

[0658] s depicted on the left hand side indicate binding sites to a sulfuratom of a cysteine residue of the antibody, and the

[0659]

[0660] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0661] In some cases, L comprises one of the following tridentate structures:

[0662]

[0663] indicate binding sites to a sulfur atom of a cysteine residue of the antibody, and the

[0664]

[0665] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0666] In some cases, L comprises one of the following tridentate structures:

[0667]

[0668] indicate binding sites to a sulfur atom of a cysteine residue of the antibody, and the

[0669]

[0670] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0671] In some cases, L comprises one of the following tridentate structures:

[0672]

[0673] wherein the

[0674]

[0675] s depicted on the left hand side indicate binding sites to a sulfur

[0676] atom of a cysteine residue of the antibody, and the

[0677]

[0678] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0679] In some cases, L comprises one of the following tridentate structures:

[0680]

[0681] wherein the

[0682]

[0683] s depicted on the left hand side indicate binding sites to a sulfur

[0684] atom of a cysteine residue of the antibody, and the

[0685]

[0686] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0687] In some cases, L comprises one of the following tridentate structures:

[0688]

[0689]

[0690] wherein the

[0691]

[0692] s depicted on the left hand side indicate binding sites to a sulfur

[0693] atom of a cysteine residue of the antibody, and the

[0694]

[0695] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0696] In some cases, L comprises one of the following tridentate structures:

[0697]

[0698]

[0699] wherein the

[0700]

[0701] s depicted on the left hand side indicate binding sites to a sulfur

[0702] atom of a cysteine residue of the antibody, and the

[0703]

[0704] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0705] In some cases, L comprises one of the following tridentate structures:

[0706]

[0707]

[0708] wherein the

[0709]

[0710] s depicted on the left hand side indicate binding sites to a sulfur

[0711] atom of a cysteine residue of the antibody, and the

[0712]

[0713] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0714] In some cases, L comprises one of the following tridentate structures:

[0715]

[0716]

[0717] wherein the

[0718]

[0719] s depicted on the left hand side indicate binding sites to a sulfur

[0720] atom of a cysteine residue of the antibody, and the

[0721]

[0722] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0723] In some cases, L comprises one of the following tridentate structure:

[0724]

[0725] wherein the

[0726]

[0727] s depicted on the left hand side indicate binding sites to a sulfur

[0728] atom of a cysteine residue of the antibody, and the

[0729]

[0730] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0731] In some cases, L comprises one of the following tridentate structure:

[0732] H N N

[0733] H

[0734]

[0735] wherein the

[0736]

[0737] s depicted on the left hand side indicate binding sites to a sulfur

[0738] atom of a cysteine residue of the antibody, and the

[0739]

[0740] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0741] In some cases, L comprises one of the following tridentate structure:

[0742] H N N

[0743] H

[0744]

[0745] wherein the

[0746]

[0747] s depicted on the left hand side indicate binding sites to a sulfur

[0748] atom of a cysteine residue of the antibody, and the

[0749]

[0750] depicted on the right hand side indicates a binding site to other component of the linker-payload.

[0751] In one aspect, provided herein is an ADC of formula (I-D2):

[0752]

[0753] HO (I-D2), or a pharmaceutically acceptable salt thereof.

[0754] In one aspect, provided herein is an ADC of formula (I-DO):

[0755]

[0756] (I-DO), or a pharmaceutically acceptable salt thereof.

[0757] In one aspect, provided herein is an ADC of formula (I-Dl):

[0758]

[0759] (I-Dl), or a pharmaceutically acceptable salt thereof.

[0760] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-E2):

[0761]

[0762] (I-E2), or a pharmaceutically acceptable salt thereof.

[0763] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-EO):

[0764]

[0765] (I-EO), or a pharmaceutically acceptable salt thereof.

[0766] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-El):

[0767]

[0768] (I-El), or a pharmaceutically acceptable salt thereof.

[0769] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-F2):

[0770]

[0771] (I-F2), or a pharmaceutically acceptable salt thereof.

[0772] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-FO):

[0773]

[0774] (I-FO), or a pharmaceutically acceptable salt thereof.

[0775] In one aspect, provided herein is an antibody-drug conjugate (ADC) according to formula (I-Fl):

[0776]

[0777] (I-Fl), or a pharmaceutically acceptable salt thereof.

[0778] In each case of formulae (I-DO), (I-Dl), (I-D2), (I-EO), (I-El), (I-E2), (I-FO), (I-Fl), and (I-F2), Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and each S represents a sulfur atom of a reduced Cysteine residue of Ab'.

[0779] In some cases, in formula (I-DO), (I-Dl), (I-D2), (I-EO), (I-El), (I-E2), (I-FO), (I-Fl), and / or (I-F2), each S represents a sulfur atom of a reduced Cysteine residue of the heavy chain and the light chain, respectively. In other embodiments, each S represents a sulfur atom of a reduced Cysteine residue of the two heavy chains, respectively. In some of these emdodiments, Ab' can be a PSMA binding protein provided herein that comprises two heavy chains that comprise a kappa type IgGl Constant heavy (CH) chain region. For instance, the reduced Cysteine residue is Cysteine-239 of the two heavy chains according to Kabat numbering in the hinge region of the antibody. In some cases, the Ab' is a PSMA binding protein that comprises a CH region comprising the sequence of SEQ ID NO: 9, and each S represents a sulfur atom of Cysteine at position 109 of SEQ ID NO: 9, i.e., Cysteine-239 according to Kabat numbering. In some cases, the Ab' is a PSMA binding protein that comprises a heavy chain comprising the sequence of SEQ ID NO: 11, and each S represents a sulfur atom of Cysteine at position 231 of SEQ ID NO: 11, i.e., Cysteine-239 according to Kabat numbering.

[0780] The antibody-drug conjugate according to formula (I-D2) can also be depicted as the following structure, where the succinimidyl group is bound to a sulfur atom of a cysteine residue of Ab':

[0781]

[0782] The antibody-drug conjugate according to formula (I-E2) can also be depicted as the following structure, where the succinimidyl group is bound to a sulfur atom of a cysteine residue of Ab':

[0783]

[0784] The antibody-drug conjugate according to formula (I-E2) can also be depicted as the following structure, where the succinimidyl group is bound to a sulfur atom of a cysteine residue of Ab':

[0785]

[0786] Process of preparation of the compounds of formula (I) or mixtures thereof

[0787] Also disclosed herein is a process of preparation of an antibody conjugate of formula (I) as defined herein (including formulae (I), (I-C)), (I-A), (I-Al), (I-Al-iso), (I-A2), (I-A2-iso), (I-B), (I-Bl), (I-C), (I-DO), (I-Dl), (I-D2), (I-EO), (I-El), (I-E2), (I-FO), (I-Fl), (I-F2)), said process comprising complexing a metal atom from a compound of formula (II) as defined herein and conjugating said compound of formula (II) with 1 to n conjugating fragments L-(Payload)m as defined herein. The process may also comprise the additional step of purifying the intermediate of formula (II), prior to preparing compound (I).

[0788] The process may typically comprise reacting a compound of formula (II) with a metal complexing agent so as to form a de-complexed compound.

[0789] Said metal complexing agent may typically be chosen from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA).

[0790] The process also comprises the coupling of said compound with a conjugation agent. Said de-complexing step and conjugating step may be conducted separately in sequence (in any order) or as a one pot reaction.

[0791] In particular, the one pot reaction may comprise preparing a reactional mixture comprising said compound of formula (II), said metal complexing agent and said conjugation agent.

[0792] According to a first alternative, the preparation of said reactional mixture may comprise adding said conjugation agent to compound (II), followed by adding said complexing agent.

[0793] According to a second alternative, the preparation of said reactional mixture may comprise adding said complexing agent to compound (II), followed by adding said conjugation agent.Without being bound by theory, it has been observed that in either the first or second alternative, the de-complexing reaction typically occurs before the conjugation step, irrespective of the order of introduction of the complexing agent and conjugation agent.

[0794] Therefore, the preparation of conjugate (I) is herein referred as "1. complexing agent, 2. conjugation agent". This sequence is used to illustrate an example and is not necessarily the order of introduction of the reagents.

[0795] According to an embodiment, the process of preparation of the conjugates of formula (I) (I7) and / or (I") comprises preparing a mixture comprising a conjugation agent, a complexing agent and compound (II). Preferably it comprises adding said conjugation agent to compound (II) followed by adding said complexing agent to the mixture.

[0796] According to another embodiment, the process of preparation of the conjugates of formula (I) (I7) and / or (I77) preparing a mixture comprising conjugation agent, complexing agent and compound (II). Preferably it comprises adding said complexing agent to compound (II) followed by adding conjugation agent to the mixture.

[0797] The process of preparation of the conjugates of formula (I) (I7) and / or (I") may also comprise purifying compound of formula (II).

[0798] According to an embodiment, all steps are preferably conducted in a buffered reacting medium, selected from the group consisting of Hepes, Histidine buffer, PBS, and MES, having a pH value comprised between 5.5 and 8, and at a temperature of about -10°C to 37°C, for example, at about 0°C to 20°C.

[0799] Typically, reactions may be conducted in conditions compatible with Ab, such as pH comprised between 7 and 9, preferably between 7 and 8, at a temperature comprised between 20 and 40°C, preferably at room temperature.

[0800] Typically, the reaction may be conducted in a buffered medium such as TBS, PPB, PBS, or HEPES medium. Reaction time may be comprised between 1 and 10 hours, preferably between 1 and 5 hours.

[0801] The concentration of Ab in the reactional mixture may be adjusted; advantageously it may be comprised between 6.0 and 8.0 g / L.

[0802] The nature of the reagents and experimental conditions of the process may depend on the degree of conjugation that is desired.

[0803] As an illustration:

[0804] The process of preparation of a conjugate of formula (I-A) may be schematically represented by the following scheme:

[0805] Complex of formula (II) + (conjugation agent +complexing agent) compound (I-A)where Ab', L, M and Payload are defined as above.

[0806] The formation of a compound (I-Al) may be favored by adding first the conjugation agent to the compound of formula (II), followed by adding the complexing agent.

[0807] The formation of a compound (I-Al-iso) may be favored by adding first the complexing agent to the compound of formula (II), followed by adding the conjugation agent.

[0808] As an illustration, the process of preparation of a conjugate of formula (I-B) typically (I-Bl) may be schematically represented by the following scheme:

[0809] 1 Complexing agent

[0810] 2. Conjugation agent

[0811]

[0812] where k is defined as m above, and Payload and R' are defined as above.

[0813] The formation of a compound (I-B) may be favored by adding first the conjugation agent to the compound of formula (II), followed by adding the complexing agent.

[0814] The process of preparation of a conjugate of formula (I-C) may be schematically represented by the following scheme:1 Nucleophilic cleavage of thioester

[0815] 2 Thiol conjugation

[0816]

[0817] where k is defined as m above, and Payload, Nu and R' are defined as above.Conjugation agent

[0818] The term conjugation agent refers to any compound bearing at least one L-(Payload)mfragment and one or more reactive function capable of reacting with reduced sulfur atom(s) present in Ab, so as to lead to a -S-L-(Payload)mfragment as described above, ensuring the covalent binding to one or two of the conjugated cysteine residues.

[0819] According to an embodiment, said conjugation agent may be terminated by a thiolreactive function, such as a maleimide or APN group.

[0820] The conjugation agent may be chosen from thiol-reactive compounds, such as compounds bearing a maleimide or APN ((hetero)arylpropiolonitri le) terminal group.

[0821] Regioisomers may be formed because Ab generally comprises a number of cysteine residues that are available for reduction, coordination and subsequent conjugation. Nevertheless, it has been demonstrated that these regioisomers are formed in minor amounts as the conjugated cysteine residues as defined above are predominantly coordinated according to some embodiments of the present disclosure. Therefore, the compounds of formula (I), (II) are thus site specifically conjugated at the conjugated cysteine residues and as such differ from the mixtures typically obtained by state-of-the-art conjugation processes.

[0822] In the sense of the invention, the term "regioisomer" thus refers to isomeric coordination complexes or conjugates, where the site of reduction of sulfur atoms with cysteine residues may vary on the backbone of the Ab.

[0823] The mixtures comprising the compound of formula (I) may also comprise conjugates having different -(L-(Payload)m)n fragments, including conjugates having different antibodydrug ratio.

[0824] As discussed above, the conjugates according to some embodiments of the present disclosure may be "heterogeneous" in some extent, although enriched in one site-specific conjugate.

[0825] In some embodiments of the present disclosure, the mixtures comprising the conjugates of the invention alongside other compounds are also encompassed by the present invention.

[0826] If desired, the desired compounds of formula (I) or (II) may be isolated by known techniques, such as hydrophobic interaction chromatography (HIC).

[0827] Compounds of formula (II) and mixtures thereof

[0828] In complex of formula (II), the two said reduced sulfur atoms are involved in a coordination bond with said Metal atom, thus forming a group -S-M-S- that links together thetwo heavy chains. Typically, the two sulfur of the two C-239 cysteines (Kabat numbering) form together a -S-M-S- group.

[0829] According to an embodiment, the metal atom is a transition metal, in particular chosen from Zn, Co, Cd, Hg, or any combination thereof. Preferably, the metal atom (M) is Zn.

[0830] As an illustration, said complex of formula (II) may be schematically represented by the following formula:

[0831]

[0832] Hinge region

[0833] The compounds of formula (II) represent versatile key intermediates towards a diversity of antibody-drug conjugates (ADCs) of formula (I) as shown below.

[0834] Process of preparation of the compounds of formula (II) and mixtures thereof

[0835] As an illustration, said process of preparation of said complex of formula (II) may be schematically illustrated by the following scheme:Hinge region

[0836] Oxidizing

[0837] agent

[0838] I

[0839]

[0840] Hinge region

[0841] According to an embodiment, the oxidizing agent is dehydroascorbic acid (DHAA). According to an embodiment, the process of preparing a compound of formula (II) may be carried out on an isolated compound of [Ab'Red-M] or a mixture comprising the same, in particular a mixture where the compounds of formula [Ab'Red-M] are predominantly present, as defined herein.

[0842] Compounds of formula [Ab'RecrM] and mixtures thereof

[0843] In some embodiments, in Ab'Red-M each heavy chain comprises one N-proximal inter(heavy chains) cysteine residue that is located nearest to the N-terminal end of said heavy chain,and wherein each Sulfur atom of each said N-proximal inter(heavy chains) cysteine residue is reduced and the two reduced Sulfur atoms form together with the M atom the following group: -S-M-S- within the coordination complex;

[0844] and the Sulfur atoms of the remaining interchain cysteine residues are reduced in the form of thiol -SH groups.

[0845] Typically, in compound Ab'Red-M, the sulfur atoms of all interchain cysteine residues are reduced in the form of thiol -SH groups, except the two sulfur atoms of the two C-239 cysteines (Kabat numbering) residues that form together with the M atom the following group: -S-M-S- within the coordination complex.

[0846] A compound of formula Ab'Red-M differs from Ab' as defined herein in that apart from the two reduced sulfur atoms that are involved in the coordination complex with the Metal atom, the other sulfur atoms are also reduced.

[0847] As an illustration, said complex Ab'Red-M may be schematically represented by the following formula:

[0848]

[0849] Hinge region

[0850] Process of preparation of the complex Ab'Red-M and mixtures thereof

[0851] AbRed refers to reduced Ab and differs from Ab in that all Sulfur atoms of said interchain cysteine residues are reduced and in the form of thiol -SH groups.

[0852] The reducing agent for this step may be for example Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dithiothreitol (DTT) or 2-mercaptoethanol (BME).

[0853] For the reducing step, preferred conditions include 37 °C, 1.5 h, 5-10 equiv. TCEP. In some cases, the reaction lasts for about 1.5 h to about 2.5 h, e.g., about 1.5 h, about 2 h, or about 2.5 h.

[0854] According to an embodiment, the metal salt may be chosen from dihalogenide metal salts, such as ZnCl2, or other metal salts such as Zn(OAc)2.The step of reacting with the metal salt may be conducted in aqueous buffers, such as phosphate-buffered saline, HEPES, or BES buffers, at room temperature, over 5-10 minutes.

[0855] As an illustration, said process of preparation of said complex of formula Ab'Red-M may be schematically illustrated by the following scheme:

[0856] Reducing agent

[0857] Ab Ab'Red

[0858]

[0859] interchain disulfide bonds

[0860] Hinge region

[0861] Metal salt

[0862]

[0863] Hinge region THERAPEUTIC USES AND PHARMACEUTICAL COMPOSITIONS

[0864] The anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof according to some embodiments of the present disclosure are for use as a medicament, in particular for use for treating, preventing and / or diagnosing cancer, preferably selected from prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer. In a morepreferred embodiment they are used for prostate cancer, in particular castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer.

[0865] The invention further relates to a method of treatment, prevention and / or diagnostic of cancer in a subject in need thereof, which comprises administering to the subject an anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof as herein disclosed.

[0866] In one embodiment, an antibody or antigen-binding fragment thereof or variant thereof having applications in a use or method of the present invention is administered as a single dose, or an initial dose followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof. The dose may vary depending upon the age and the weight of a subject to be administered, target disease, conditions, route of administration, and the like.

[0867] The invention also relates to a pharmaceutical composition comprising an anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof as herein disclosed, and a pharmaceutically acceptable carrier.

[0868] In some embodiments, at least one prostate cancer therapeutic agent is used in combination with the anti-PSMA antibody, or antigen binding fragment thereof, or variant thereof. Said prostate cancer therapeutic agent may be selected from the group consisting of chemotherapeutic agents (such as but not limited to Docetaxel, or Enzalutamide), immune checkpoint inhibitors (such as but not limited to anti-PDl or anti-TIGIT antibodies), nextgeneration androgen receptor signaling inhibitors (such as Abiraterone, Apalutamide, or Darolutamide), radiopharmaceuticals (such as Lutetium-177-PSMA (Lu-177-PSMA-617), Radium-223), bone-targeted therapies (such as Denosumab or Zoledronic Acid), and PARP inhibitors (such as Olaparib or Rucaparib).

[0869] The ADCs according to some embodiments of the present disclosure are for use as a medicament, in particular for use for treating, preventing and / or diagnosing cancer, preferably selected from prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer. In a more preferred embodiment, they are used for prostate cancer, in particular castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer.

[0870] The invention further relates to a method of treatment, prevention and / or diagnostic of cancer in a subject in need thereof, which comprises administering to the subject an ADC as herein disclosed.In one embodiment, ADC having applications in a use or method of the present invention is administered as a single dose, or an initial dose followed by administration of a second ora plurality of subsequent doses of the antibody or antigen-binding fragment thereof. The dose may vary depending upon the age and the weight of a subject to be administered, target disease, conditions, route of administration, and the like.

[0871] The invention also relates to a pharmaceutical composition comprising an ADC as herein disclosed, and a pharmaceutically acceptable carrier.

[0872] The subject is a mammal, such as a primate (e.g. human, monkey), a rodent (e.g. rat, mouse), a canine (e.g. a dog), or a feline (e.g. a cat). Preferably, the subject is a human.

[0873] In some embodiments, at least one prostate cancer therapeutic agent is used in combination with the ADC. Said prostate cancer therapeutic agent may be selected from the group consisting of chemotherapeutic agents (such as Docetaxel, or Enzalutamide), immune checkpoint inhibitors (such as anti-PDl or anti-TIGIT antibodies), next-generation androgen receptor signaling inhibitors (such as Abiraterone, Apalutamide, or Darolutamide), radiopharmaceuticals (such as Lutetium-177-PSMA (Lu-177-PSMA-617), Radium-223), bone-targeted therapies (such as Denosumab or Zoledronic Acid), and PARP inhibitors (such as Olaparib or Rucaparib).

[0874] Embodiments of the Disclosure

[0875] This disclosure is further illustrated by the following paragraphs:

[0876] [1] A PSMA binding protein comprising:

[0877] (a) (i) any one or a combination of CDRs selected from the group consisting of:

[0878] CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 7, and CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 8; or (ii) a CDR variant of (i), wherein the variant has 1, 2, or 3 amino acid modifications; or

[0879] (b) a VH region comprising a sequence at least 80% identical to SEQ ID NO: 7, and / or a VL region comprising a sequence at least 80% identical to SEQ ID NO: 8.

[0880] [2] The PSMA binding protein of paragraph [1], wherein the PSMA binding protein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR-H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6.[3] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR-H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6, with up to five amino acid modifications across said six CDR sequences.

[0881] [4] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR-H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6.

[0882] [5] The PSMA binding protein of paragraph [1], wherein the PSMA binding protein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21, a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24.

[0883] [6] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21, a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24, with up to five amino acid modifications across said six CDR sequences.

[0884] [7] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, a CDR-H3 of sequence SEQ ID NO: 21, a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24.

[0885] [8] The PSMA binding protein of paragraph [1], wherein the PSMA binding protein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27, a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30.

[0886] [9] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27, a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30, with up to five amino acid modifications across said six CDR sequences.

[0010] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, a CDR-H3 of sequence SEQ ID NO: 27, a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30.

[0887]

[0011] The PSMA binding protein of paragraph [1], wherein the PSMA binding protein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33, a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36.

[0888]

[0012] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33, a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36, with up to five amino acid modifications across said six CDR sequences.

[0889]

[0013] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, a CDR-H3 of sequence SEQ ID NO: 33, a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36.

[0890]

[0014] The PSMA binding protein of paragraph [1], wherein the PSMA binding protein comprises at least one of a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39, a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42.

[0891]

[0015] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39, a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42, with up to five amino acid modifications across said six CDR sequences.

[0892]

[0016] The PSMA binding protein of paragraph [1] or [2], wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, a CDR-H3 of sequence SEQ ID NO: 39, a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42.

[0017] The PSMA binding protein of any one of paragraphs [1]-

[0016] , wherein the VH region of the PSMA binding protein comprises the sequence of SEQ ID NO: 7 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or the VL region of the PSMA binding protein comprises the sequence of SEQ ID No: 8 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0893]

[0018] The PSMA binding protein of any one of paragraphs [1]-

[0017] , wherein the VH region of the PSMA binding protein comprises the sequence of SEQ ID NO: 7, and the VL region of the PSMA binding protein comprises the sequence of SEQ ID NO: 8.

[0894]

[0019] The PSMA binding protein of any one of paragraphs [1]-

[0017] , wherein the sequence of the VH region of the PSMA binding protein is according to SEQ ID NO: 7, and the sequence of the VL region of the PSMA binding protein is according to SEQ ID NO: 8.

[0895]

[0020] The PSMA binding protein of any one of paragraphs [1]-

[0019] , wherein the PSMA binding protein binds to human PSMA.

[0896]

[0021] The PSMA binding protein of any one of paragraphs [l]-

[0020] , wherein the PSMA binding protein does not bind to PSMA through the same epitope as Rosopatamab (J591).

[0897]

[0022] The PSMA binding protein of any one of paragraphs [1]-

[0021] , which comprises one or more of:

[0898] (a) a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody; or

[0899] (b) Fv, Fab, F(ab')2, Fab', dAb, dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, triabodies, tetrabodies, bispecific antibody, or multispecific antibody.

[0900]

[0023] The PSMA binding protein of any one of paragraphs

[0001] -

[0022] , wherein the PSMA binding protein comprises a heavy chain and a light chain.

[0901]

[0024] The PSMA binding protein of any one of paragraphs [l]-

[0023] , wherein the PSMA binding protein comprises two heavy chains and two light chains.

[0902]

[0025] The PSMA binding protein of any one of paragraphs [l]-

[0024] , wherein the PSMA binding protein is an IgG, optionally an IgGl, optionally a human IgGl.

[0903]

[0026] The PSMA binding protein of any one of paragraphs [l]-

[0025] , wherein the PSMA binding protein comprises a Human IgGl Constant heavy chain (CH) and a Human IgGl Constant light chain (CL), optionally a human kappa constant domain.

[0027] The PSMA binding protein of any one of paragraphs

[0001] -

[0026] , wherein the PSMA binding protein comprises a Constant heavy chain (CH) region comprising the sequence SEQ ID NO: 9 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or a Constant light chain (CL) region comprising the sequence of SEQ ID NO: 10 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0904]

[0028] The PSMA binding protein of any one of paragraphs [l]-

[0027] , wherein the PSMA binding protein comprises a CH region comprising the sequence of SEQ ID NO: 9, and a CL region comprising the sequence of SEQ ID NO: 10.

[0905]

[0029] The PSMA binding protein of any one of paragraphs [l]-

[0027] , wherein the PSMA binding protein comprises a CH region according to the sequence of SEQ ID NO: 9, and a CL region according to the sequence of SEQ ID NO: 10.

[0906]

[0030] The PSMA binding protein of any one of paragraphs [l]-

[0029] , wherein the PSMA binding protein comprises a heavy chain comprising the sequence SEQ ID NO: 11 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or a light chain comprising the sequence of SEQ ID NO: 12 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0907]

[0031] The PSMA binding protein of any one of paragraphs [l]-

[0030] , wherein the PSMA binding protein comprises a heavy chain comprising the sequence of SEQ ID NO: 11, and a light chain comprising the sequence of SEQ ID NO: 12.

[0908]

[0032] The PSMA binding protein of any one of paragraphs [1]-

[0031] , wherein the PSMA binding protein comprises a heavy chain according to the sequence of SEQ ID NO: 11, and a light chain according to the sequence of SEQ ID NO: 12.

[0909]

[0033] A PSMA binding protein comprising a VH region comprising a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and a VL region comprising a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6.

[0910]

[0034] A PSMA binding protein comprising a VH region comprising a CDR-H1 of sequence SEQ ID NO: 19, a CDR-H2 of sequence SEQ ID NO: 20, and a CDR-H3 of sequence SEQ ID NO: 21, and a VL region comprising a CDR-L1 of sequence SEQ ID NO: 22, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 24.

[0035] A PSMA binding protein comprising a VH region comprising a CDR-H1 of sequence SEQ ID NO: 25, a CDR-H2 of sequence SEQ ID NO: 26, and a CDR-H3 of sequence SEQ ID NO: 27, and a VL region comprising a CDR-L1 of sequence SEQ ID NO: 28, a CDR-L2 of sequence SEQ ID NO: 29, and a CDR-L3 of sequence SEQ ID NO: 30.

[0911]

[0036] A PSMA binding protein comprising a VH region comprising a CDR-H1 of sequence SEQ ID NO: 31, a CDR-H2 of sequence SEQ ID NO: 32, and a CDR-H3 of sequence SEQ ID NO: 33, and a VL region comprising a CDR-L1 of sequence SEQ ID NO: 34, a CDR-L2 of sequence SEQ ID NO: 35, and a CDR-L3 of sequence SEQ ID NO: 36.

[0912]

[0037] A PSMA binding protein comprising a VH region comprising a CDR-H1 of sequence SEQ ID NO: 37, a CDR-H2 of sequence SEQ ID NO: 38, and a CDR-H3 of sequence SEQ ID NO: 39, and a VL region comprising a CDR-L1 of sequence SEQ ID NO: 40, a CDR-L2 of sequence DDS, and a CDR-L3 of sequence SEQ ID NO: 42.

[0913]

[0038] A PSMA binding protein comprising a VH region according to SEQ ID NO: 7, and a VL region according to SEQ ID NO: 8.

[0914]

[0039] A PSMA binding protein comprising a heavy chain according to SEQ ID NO: 11, and a light chain according to SEQ ID NO: 12.

[0915]

[0040] One or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein according to any one of paragraphs [l]-

[0039] .

[0916]

[0041] One or more nucleic acids according to paragraph

[0040] , comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 14.

[0917]

[0042] One or more nucleic acids comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 14.

[0918]

[0043] One or more nucleic acids according to paragraph

[0041] or

[0042] , further comprising: a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 15, and a sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 16.

[0919]

[0044] One or more nucleic acids according to any one of paragraphs

[0040] -

[0043] , comprising: a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 17, and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 18.

[0920]

[0045] One or more nucleic acids according to any one of paragraphs

[0040] -

[0043] , comprising: a sequence according to SEQ ID NO: 17, and a sequence according to SEQ ID NO: 18.

[0921]

[0046] A pair of isolated nucleic acids comprising a sequence encoding respectively the VH domain and VL domain of the PSMA binding protein according to any one of paragraphs [l]-

[0039] .

[0922]

[0047] A pair of isolated nucleic acids according to paragraph

[0046] , comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 14.

[0923]

[0048] A vector comprising the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] .

[0924]

[0049] A host cell comprising one or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein according to any one of paragraphs [l]-

[0039] .

[0925]

[0050] A host cell comprising the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] .

[0926]

[0051] A method of making a PSMA binding protein according to any one of paragraphs [l]-

[0039] , the method comprising culturing a host cell comprising one or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein, or one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] , or the pair of nucleic acids of paragraph

[0046] or

[0047] , under conditions suitable for producing the PSMA binding protein.

[0927]

[0052] An antibody-drug conjugate comprising the PSMA binding protein according to any one of paragraphs [l]-

[0039] and a payload, wherein the payload is conjugated tothe PSMA binding protein, optionally wherein the payload selected from the group consisting of therapeutic agents, imaging agents, diagnostic agents, and combinations thereof.

[0928]

[0053] The antibody-drug conjugate of paragraph

[0052] , wherein the payload is selected from the group consisting of cytotoxic agents, immunostimulatory agents, targeted protein degradation agents, immunocytokines, radiopharmaceutical agents, and combinations thereof.

[0929]

[0054] The antibody-drug conjugate of paragraph

[0052] , wherein the payload is a cytotoxic agent, optionally selected from the group consisting of: dolastatins, maytansines, maytansinoids, antracyclins, calicheamicins, etoposides, taxanes, duocarymycins, benzodiazepines, benzodiazepine containing drugs, vinca alkaloids, amanitins, exatecan derivatives, exotoxins, vincristine derivatives, and combinations thereof.

[0930]

[0055] The antibody-drug conjugate of paragraph

[0052] , wherein the payload comprises a dolastatin, optionally selected from the group consisting of: MMAE, MMAF, MMAD, a uri statin E, and combinations thereof.

[0931]

[0056] The antibody-drug conjugate of paragraph

[0052] , wherein the payload comprises MMAE.

[0932]

[0057] The antibody-drug conjugate of any one of paragraphs

[0052] -

[0056] , wherein the PSMA binding protein is directly conjugated to the payload.

[0933]

[0058] The antibody-drug conjugate of any one of paragraphs

[0052] -

[0056] , wherein the antibody-drug conjugate further comprises a linker connecting the PSMA binding protein and the payload.

[0934]

[0059] The antibody-drug conjugate of paragraph

[0058] , wherein the linker comprises a linear or branched di-, tri-, tetra-, penta-, hexa- or hepta-valent linker moiety.

[0935]

[0060] The antibody-drug conjugate of paragraph

[0058] or

[0059] , wherein the linker comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein the linker comprises a cleavable linker that is a peptide linker.

[0936]

[0061] The antibody-drug conjugate of any one of paragraphs

[0058] -

[0059] , wherein the linker comprises a linker component selected from the group consisting of: valine- citrullinyl, alanine-phenylalaninyl, glycine-valine-citrullinyl, glycine-glycine-glycinyl, alanine-alanine-asparaginyl, 6-maleimidocaproyl, maleimidopropanoyl (MP), arylpropiolonitrile, alanine-phenylalaninyl (Ala-Phe), p-a mi nobenzyloxycarbonyl (PABC), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC), N-Succinimidyl (4-iodo- acetyl)aminobenzoate (SIAB), and combinations thereof.

[0937]

[0062] The antibody-drug conjugate of any one of paragraphs

[0058] -

[0061] , wherein the linker comprises a thio-reactive spacer, optionally a maleimidyl group, a succinimidyl group, or an arylpropiolonitrile group.

[0938]

[0063] The antibody-drug conjugate of any one of paragraphs

[0058] -

[0062] , wherein the linker comprises a maleimidyl group, a succinimidyl group, a caproyl group, a valinecitrulline group, and / or a -a mi nobenzyloxycarbonyl group.

[0939]

[0064] The antibody-drug conjugate of any one of paragraphs

[0058] -

[0063] , wherein the linker comprises a tridentate linker.

[0940]

[0065] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises

[0941]

[0942] wherein

[0943]

[0944] ' indicates a single or double bond, and wherein W is substituted Ce- i4arylene, C5-14heteroarylene, Ci-3oalkylene, Ci-3oheteroalkylene, C3-i0cycloalkylene, C3-ioheterocycloalkylene, or any combination thereof.

[0945]

[0066] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises the following structure:

[0946]

[0947] wherein W is substituted C6-i4arylene, C5-14heteroarylene, Ci-3oalkylene, Ci- soheteroalkylene, C3-iocycloalkylene, C3-ioheterocycloalkylene, or any combination thereof.

[0948]

[0067] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises

[0949]

[0950] wherein W is C6-i4ar lene, C5-14heteroarylene, Ci-30alkylene, C3-i0cycloalkylene, C3- loheterocycloalkylene, or any combination thereof.

[0951]

[0068] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises the following structure:

[0952]

[0953] wherein:

[0954] ' indicates a single or double bond,

[0955] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0956] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5-14heteroaryl;

[0957] R1is substituted or unsubstituted Ci-iOalkylene, Ci-wheteroalkylene, C3-locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-i4arylene, C5-i4heteroarylene, polyethylene glycol) chain, or any combination thereof;

[0958] a is 0 or 1;

[0959] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0960] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[0961] R10, R11, and R12are each independently selected from H, Cuoalkyl, C3-i0cycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-i4aryl, or C5-i4heteroaryl;R13and R14are each independently selected from substituted or unsubstituted Ci- walkylene, Cnoheteroalkylene, C3-iocycloalkylene, heterocycloalkylene, Ci- walkenylene, C6-i4ar lene, Ci-wheteroarylene, or any combination thereof;

[0962] Q is N or CR15;

[0963] R15is selected from H, Ci-ioal kyl, C3-iocycloalkyl, C3-i0heterocycloalkyl, C1-10alkenyl, or Ci-ioalkynyl;

[0964] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[0965]

[0069] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises the following structure:

[0966]

[0967] wherein:

[0968] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0969] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-i0heterocycloalkyl, Ci-iOalkenyl, Ci-ioalkynyl, C6-i4aryl, C5- C5-14heteroaryl;

[0970] R1is substituted or unsubstituted Ci-iOalkylene, Cnoheteroalkylene, C3- locycloalkylene, C3-i0heterocycloalkylene, Ci-ioalkenylene, Cnoalkynylene, C6-14arylene, C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[0971] a is 0 or 1;

[0972] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0973] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;R10, R11, and R12are each independently selected from H, C1-10alkyl, C3-i0cycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-i4aryl, or C5-i4heteroaryl;

[0974] R13and R14are each independently selected from substituted or unsubstituted Ci- walkylene, Ci-ioheteroalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci- walkenylene, C6-i4arylene, C5-i4heteroarylene, or any combination thereof;

[0975] Q is N or CR15;

[0976] R15is selected from H, Ci-iOalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci- walkenylene, or Ci-iOalkynylene;

[0977] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[0978]

[0070] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises the following structure:

[0979] A— N

[0980] O 1

[0981] O |

[0982] y— N

[0983]

[0984] wherein:

[0985] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[0986] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5- C5-14heteroaryl;

[0987] R1is substituted or unsubstituted Ci-iOalkylene, Ci-ioheteroalkylene, C3- locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-14arylene, C5-i4heteroarylene, polyethylene glycol) chain, or any combination thereof;

[0988] a is 0 or 1;

[0989] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[0990] R10, R11, and R12are each independently selected from H,

[0991] Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, C1-10alkenyl, Ci-iOalkynyl, C6-i4ar l, C5- i4heteroaryl;

[0992] R13and R14are each independently selected from substituted or unsubstituted Ci- walkylene, Ci-wheteroalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci- walkenylene, C6-i4ar lene, C5-14heteroarylene, or any combination thereof;

[0993] Q is N or CR15;

[0994] R15is selected from H, Cuoal kyl, C3-i0cycloalkyl, C3-i0heterocycloalkyl, Ci-iOalkenyl, Ci- walkynyl;

[0995] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[0996]

[0071] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[0997]

[0998] where

[0999]

[1000] ' indicates a single or double bond.

[1001]

[0072] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1002]

[1003]

[0073] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1004]

[1005]

[0074] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1006]

[1007]

[1008] where indicates a single or double bond.

[1009]

[0075] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1010]

[1011]

[1012]

[0076] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1013]

[1014]

[0077] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises a bis-succinimidyl group, a bis-maleimidyl group, a caproyl group, a valine-citrulline group, and / or a -a mi nobenzyloxycarbonyl group.

[1015]

[0078] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1016]

[1017] where

[1018]

[1019] ' indicates a single or double bond.

[1020]

[0079] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises one of the following structures:

[1021]

[1022]

[0080] The antibody-drug conjugate of paragraph

[0064] , wherein the linker comprises 5 one of the following structures:

[1023]

[1024]

[0081] The antibody-drug conjugate of paragraph

[0058] , wherein the linker consists of 5 the following structure:

[1025]

[1026] where ' indicates a single or double bond.

[1027]

[0082] The antibody-drug conjugate of paragraph

[0058] , wherein the linker consists of the following structure:

[1028]

[1029]

[0083] The antibody-drug conjugate of paragraph

[0058] , wherein the linker consists of the following structure:

[1030]

[1031]

[0084] The antibody-drug conjugate of paragraph

[0058] , wherein the linker consists of a bis-succinimidocaproyl group, a valine-citrullinyl group, and a p- aminobenzyloxycarbonyl group.

[1032]

[0085] The antibody-drug conjugate of paragraph

[0058] , wherein the linker consists of a bis-maleimidocaproyl group, a valine-citrullinyl group, and a p- aminobenzyloxycarbonyl group.

[1033]

[0086] The antibody-drug conjugate of any one of paragraphs

[0081] -

[0085] , wherein the payload is MMAE.

[1034]

[0087] An antibody-drug conjugate (ADC) according to formula (I):

[1035] (T)p-Ab'-(L-(Payload)m)n

[1036] (I), a pharmaceutically acceptable salt thereof,

[1037] having

[1038] a controlled drug-to-antibody ratio (DAR) of (m x n); and a site-specific conjugation of L and optional T to Ab' at one or two of a pair of interchain cysteine residues of Ab' defined as the conjugated cysteine residues;

[1039] wherein in formula (I):

[1040] Ab' is a PSMA binding protein according to any one of paragraphs [l]-

[0039] and comprising two heavy chains and two light chains, each heavy chain and each light chain comprising one or more interchain cysteine residues, such that each of said conjugated cysteines is bound to either -L-(Payload)mor optional T fragment, and the remaining interchain cysteine residues form interchain disulfide bridges;

[1041] n is 1 or 2;

[1042] m is an integer larger than 0;p is 0 or 1;

[1043] T, if present, is a terminal group covalently bound to Sulfur atom of one of said pair of interchain cysteine residues of Ab' that is not covalently bound to L;

[1044] each L is independently a linear or branched di-, tri-, tetra-, penta-, hexa- or hepta-valent linker moiety covalently binding Payload to said Ab', wherein said L is attached to Ab' through Sulfur atoms of one or two of said pair of interchain cysteine residues and binds said Ab' with Payload;

[1045] Payload is a molecule selected from the group consisting of therapeutic agents, imaging agents, diagnostic agents, and combinations thereof.

[1046]

[0088] The antibody-drug conjugate of paragraph

[0087] , wherein each Payload is independently selected from the group consisting of cytotoxic agents, immunostimulatory agents, targeted protein degradation agents, immunocytokines, radiopharmaceutical agents, and combinations thereof.

[1047]

[0089] The antibody-drug conjugate of paragraph

[0087] , wherein each Payload is independently a cytotoxic agent, optionally selected from the group consisting of: dolastatins, maytansines, maytansinoids, antracyclins, calicheamicins, etoposides, taxanes, duocarymycins, benzodiazepines, benzodiazepine containing drugs, vinca alkaloids, amanitins, exatecan derivatives, exotoxins, vincristine derivatives, and combinations thereof.

[1048]

[0090] The antibody-drug conjugate of paragraph

[0087] , wherein each Payload independently comprises a dolastatin, optionally selected from the group consisting of: MMAE, MMAF, MMAD, auristatin E, and combinations thereof.

[1049]

[0091] The antibody-drug conjugate of paragraph

[0087] , wherein each Payload comprises MMAE.

[1050]

[0092] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0091] , wherein each L independently comprises a cleavable linker, a non-cleavable linker, and / or a self- immolative spacer, optionally wherein the linker comprises a cleavable linker that is a peptide linker.

[1051]

[0093] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0092] , wherein each L independently comprises a linker component selected from the group consisting of: valine-citrulline, alanine-phenylalanine, glycine-valine-citrulline, glycine-glycine- glycine, alanine-alanine- asparagine, 6-maleimidocaproyl, maleimidopropanoyl (MP), arylpropiolonitrile, alanine-phenylalanine (Ala-Phe), p-a mi nobenzyloxycarbonyl (PABC), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC), N-Succinimidyl (4-iodo- acetyl)aminobenzoate (SIAB), and combinations thereof.

[1052]

[0094] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0093] , wherein each L independently comprises a thio-reactive spacer, optionally a succinimidyl group, maleimidyl group or an arylpropiolonitrile group.

[1053]

[0095] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0093] , wherein each L independently comprises a succinimidyl group, a maleimidyl group, a caproyl group, a va li ne-citrul line group, and / or a -aminobenzyloxycarbonyl group.

[1054]

[0096] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0095] , wherein m is 1, 2, 3, 4, 5, or 6.

[1055]

[0097] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0096] , wherein the site-specific conjugation is at one or two of the pair of N-proximal inter(heavy chains) cysteine residues that are located nearest to the N-terminal end of said heavy chains.

[1056]

[0098] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0096] , wherein the PSMA binding protein is an IgGl, optionally human IgGl, and wherein the sitespecific conjugation is at one or two of Cysteine-239 of the two heavy chains according to Kabat numbering.

[1057]

[0099] The antibody-drug conjugate of any one of paragraphs

[0087] -

[0096] , wherein the PSMA binding protein is an IgGl, optionally human IgGl, and wherein the sitespecific conjugation is at Cysteine-239 of both of the two heavy chains according to Kabat numbering.

[1058]

[0100] The antibody-drug conjugate of paragraph

[0099] , wherein n is 2.

[1059]

[0101] The antibody-drug conjugate of paragraph

[0100] , wherein p is 0.

[1060]

[0102] The antibody-drug conjugate of paragraph

[0100] or

[0101] , wherein each L independently comprises a thio-reactive spacer, optionally a succinimidyl group, a maleimide group or an arylpropiolonitrile group.

[1061]

[0103] The antibody-drug conjugate of any one of paragraphs

[0100] -

[0102] , wherein each L independently comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein each L independently comprises a cleavable linker that is a peptide linker.

[1062]

[0104] The antibody-drug conjugate of any one of paragraphs

[0100] -

[0103] , wherein each L independently comprises a succinimidyl group, a maleimidyl group, a caproyl group, a va li ne-citrul li nyl group, and / or a -a mi nobenzyloxycarbonyl group.

[1063]

[0105] The antibody-drug conjugate of paragraph

[0099] , wherein n is 1.

[0106] The antibody-drug conjugate of paragraph

[0105] , wherein p is 0.

[1064]

[0107] The antibody-drug conjugate of paragraph

[0106] , wherein L is a tridentate linker.

[1065]

[0108] The antibody-drug conjugate of paragraph

[0106] , wherein L comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein L comprises a cleavable linker that is a peptide linker.

[1066]

[0109] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises a bis-succinimidyl group, a caproyl group, a va line-citrulli nyl group, and / or a -aminobenzyloxycarbonyl group.

[1067]

[0110] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises a bis-maleimidyl group, a caproyl group, a va line-citrulli nyl group, and / or a -aminobenzyloxycarbonyl group.

[1068]

[0111] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1069]

[1070] wherein

[1071]

[1072] ' indicates a single or double bond, and wherein W is substituted C6-14arylene, C5-14heteroarylene, C1-30alkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, or any combination thereof.

[1073]

[0112] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1074]

[1075] wherein W is substituted C6-14arylene, C5-14heteroarylene, C1-30alkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, or any combination thereof.

[1076]

[0113] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1077]

[1078] wherein W is substituted C6-14arylene, C5-14heteroarylene, C1-30alkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, or any combination thereof.

[1079]

[0114] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1080]

[1081] wherein:

[1082] ' indicates a single or double bond,

[1083] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[1084] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5-14heteroaryl;

[1085] R1is substituted or unsubstituted Ci-iOalkylene, C1-10heteroalkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, C1-10alkenylene, C1-10alkynylene, C6-14arylene,

[1086] C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[1087] a is 0 or 1;

[1088] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[1089] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[1090] R10, R11, and R12are each independently selected from H, C1-10alkyl, C3-10cycloalkyl, C3-10heterocycloalkyl, C1-10alkenyl, C1-10alkynyl, C6-14aryl, or C5-14heteroaryl;

[1091] R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, Ci-ioheteroalkylene, C3-i0cycloalkylene, heterocycloalkylene, C1-10alkenylene, C6-14arylene, C1-14heteroarylene, or any combination thereof;

[1092] Q is N or CR15;R15is selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, C1-10alkenyl, or Ci-ioalkynyl;

[1093] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[1094]

[0115] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1095]

[1096] wherein:

[1097] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[1098] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-i0heterocycloalkyl, Ci-iOalkenyl, Ci-ioalkynyl, C6-i4aryl, C5- C5-14heteroaryl;

[1099] R1is substituted or unsubstituted C1-10alkylene, C1-10heteroalkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, C1-10alkenylene, C1-10alkynylene, C6-14arylene,

[1100] C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[1101] a is 0 or 1;

[1102] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[1103] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[1104] R10, R11, and R12are each independently selected from H, C1-10alkyl, C3-10cycloalkyl, C3-10heterocycloalkyl, C1-10alkenyl, C1-10alkynyl, C6-14aryl, or C5-14heteroaryl;

[1105] R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, C1-10heteroalkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, C1-10alkenylene, C6-14arylene, C1-14heteroarylene, or any combination thereof;Q is N or CR15;

[1106] R15is selected from H, Ci-iOalkylene, C3-i0cycloalkylene, C3-i0heterocycloalkylene, Ci-ioalkenylene, or Ci-iOalkynylene;

[1107] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[1108]

[0116] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises the following structure:

[1109] Z— N

[1110] O 1

[1111] O |

[1112] y— N

[1113]

[1114] wherein:

[1115] Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;

[1116] R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5- C5-14heteroaryl;

[1117] R1is substituted or unsubstituted Ci-iOalkylene, Ci-ioheteroalkylene, C3- locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-14arylene,

[1118] C5-14heteroarylene, poly(ethylene glycol) chain, or any combination thereof;

[1119] a is 0 or 1;

[1120] b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[1121] X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;

[1122] R10, R11, and R12are each independently selected from H, C1-10alkyl, C3-i0cycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-14aryl, or C5-14heteroaryl;R13and R14are each independently selected from substituted or unsubstituted Ci-ioalkylene, C3-iocycloalkylene, C3-ioheterocycloalkylene, C1-10alkenylene, C6-14arylene, or C5-14heteroarylene or any combination thereof;

[1123] Q is N or CR15;

[1124] R15is selected from H, Ci-iOalkyl, C3-i0cycloalkyl, C3-i0heterocycloalkyl, C1-10alkenyl, or Ci-ioalkynyl;

[1125] x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

[1126]

[0117] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1127]

[1128] where

[1129]

[1130] ' indicates a single or double bond.

[1131]

[0118] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1132]

[1133]

[0119] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1134] and

[1135]

[1136]

[0120] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1137]

[1138] 5

[0121] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1139]

[1140]

[1141]

[0122] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1142]

[1143]

[0123] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1144]

[1145] where

[1146]

[1147] ' indicates a single or double bond.

[1148]

[0124] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L comprises one of the following structures:

[1149]

[1150]

[0125] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0108] , wherein L 5 comprises one of the following structures:

[1151]

[1152]

[0126] The antibody-drug conjugate of paragraph

[0106] , wherein L consists of the 5 following structure:

[1153]

[1154] where ' indicates a single or double bond.

[1155]

[0127] The antibody-drug conjugate of paragraph

[0106] , wherein L consists of the following structure:

[1156]

[1157]

[0128] The antibody-drug conjugate of paragraph

[0106] , wherein L consists of the following structure:

[1158]

[1159]

[0129] The antibody-drug conjugate of paragraph

[0106] , wherein L consists of a bis-succinimidocaproyl group, a va line-citrul line group, and a p-a mi nobenzyloxycarbonyl group.

[1160]

[0130] The antibody-drug conjugate of paragraph

[0106] , wherein L consists of a bis- maleimidocaproyl group, a valine-citrulline group, and a -a mi nobenzyloxycarbonyl group.

[1161]

[0131] The antibody-drug conjugate of any one of paragraphs

[0106] -

[0130] , wherein the Payload is MMAE.

[1162]

[0132] An antibody-drug conjugate (ADC) according to formula (I-DO):

[1163]

[1164] (I-DO), or a pharmaceutically acceptable salt thereof,

[1165] wherein in formula (I-DO):

[1166] indicates a single or double bond;Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1167] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1168]

[0133] An antibody-drug conjugate (ADC) according to formula (I-Dl):

[1169]

[1170] HO (I-Dl), or a pharmaceutically acceptable salt thereof,

[1171] wherein in formula (I-Dl):

[1172] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1173] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1174]

[0134] An antibody-drug conjugate (ADC) according to formula (I-D2):

[1175]

[1176] HO (I-D2), or a pharmaceutically acceptable salt thereof,

[1177] wherein in formula (I-D2):

[1178] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1179] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1180]

[0135] An antibody-drug conjugate (ADC) according to formula (I-EO):

[1181]

[1182] (I-EO), or a pharmaceutically acceptable salt thereof,

[1183] wherein in formula (I-EO):

[1184] indicates a single or double bond;Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and wherein the heavy chain comprises human IgGl Constant heavy chain (CH) region; and

[1185] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1186]

[0136] An antibody-drug conjugate (ADC) according to formula (I-El):

[1187]

[1188] (I-El), or a pharmaceutically acceptable salt thereof,

[1189] wherein in formula (I-El):

[1190] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and wherein the heavy chain comprises human IgGl Constant heavy chain (CH) region; and

[1191] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1192]

[0137] An antibody-drug conjugate (ADC) according to formula (I-E2):

[1193]

[1194] (I-E2), or a pharmaceutically acceptable salt thereof,

[1195] wherein in formula (I-E2):

[1196] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and wherein the heavy chain comprises human IgGl Constant heavy chain (CH) region; and

[1197] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1198]

[0138] An antibody-drug conjugate (ADC) according to formula (I-FO):

[1199]

[1200] (I-FO), or a pharmaceutically acceptable salt thereof,

[1201] wherein in formula (I-FO):

[1202] indicates a single or double bond;

[1203] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1204] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1205]

[0139] An antibody-drug conjugate (ADC) according to formula (I-Fl):

[1206]

[1207] (I-Fl), or a pharmaceutically acceptable salt thereof,

[1208] wherein in formula (I-Fl):

[1209] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1210] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1211]

[0140] An antibody-drug conjugate (ADC) according to formula (I-F2):

[1212]

[1213] (I-F2), or a pharmaceutically acceptable salt thereof,wherein in formula (I-F2):

[1214] Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; and

[1215] each S represents a sulfur atom of a Cysteine residue of Ab'.

[1216]

[0141] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0140] , wherein the heavy chain comprises a VH region comprising a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 7, and the light chain comprises a VL region comprising a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 8.

[1217]

[0142] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0140] , wherein the heavy chain comprises a VH region comprising the sequence of SEQ ID NO: 7, and the light chain comprises a VL region comprising the sequence of SEQ ID NO: 8.

[1218]

[0143] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0140] , wherein the heavy chain comprises a VH region according to the sequence of SEQ ID NO: 7, and the light chain comprises a VL region according to the sequence of SEQ ID NO: 8.

[1219]

[0144] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0142] , wherein Ab' comprises a Constant heavy chain (CH) region comprising the sequence SEQ ID NO: 9 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or a Constant light chain (CL) region comprising the sequence of SEQ ID NO: 10 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[1220]

[0145] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0142] , wherein Ab' comprises a CH region comprising the sequence of SEQ ID NO: 9, and a CL region comprising the sequence of SEQ ID NO: 10.

[1221]

[0146] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0142] , wherein Ab' comprises a CH region according to the sequence of SEQ ID NO: 9, and a CL region according to the sequence of SEQ ID NO: 10.

[0147] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0146] , wherein the heavy chain comprises the sequence SEQ ID NO: 11 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and the light chain comprises the sequence SEQ ID NO: 12 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[1222]

[0148] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0146] , wherein the heavy chain is according to the sequence SEQ ID NO: 11, and the light chain is according to the sequence of SEQ ID NO: 12.

[1223]

[0149] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0148] , wherein the two S represent a sulfur atom of a Cysteine residue of the heavy chain and the light chain, respectively.

[1224]

[0150] The antibody-drug conjugate of any one of paragraphs

[0132] -

[0148] , wherein each S represents a sulfur atom of a Cysteine residue of one of the two heavy chains, respectively.

[1225]

[0151] The antibody-drug conjugate of paragraph

[0150] , wherein each S represents a sulfur atom of Cysteine-239 of one of the two heavy chains, respectively, according to Kabat numbering.

[1226]

[0152] The antibody-drug conjugate of paragraph

[0150] , wherein:

[1227] (1) the CH regions of Ab' each comprise the sequence of SEQ ID NO: 9, and each S represents a sulfur atom of Cysteine at position 109 of SEQ ID NO: 9; or (2) the heavy chains of Ab' each comprise the sequence of SEQ ID NO: 11, and each S represents a sulfur atom of Cysteine at position 231 of SEQ ID NO: 11.

[1228]

[0153] The PSMA binding protein of any one of paragraphs [l]-

[0039] , the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] , or the antibody-drug conjugate of any one of paragraphs

[0052] -

[0152] for use in treatment, prevention and / or diagnosis of a disease or condition.

[1229]

[0154] The PSMA binding protein of any one of paragraphs [l]-

[0039] , the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] , or the antibody-drug conjugate of any one of paragraphs

[0052] -

[0152] for use of treatment, prevention and / or diagnosis of cancer, preferably selected from the group consisting of: prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas,gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

[1230]

[0155] The PSMA binding protein of any one of paragraphs [l]-

[0039] , the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] , or the antibody-drug conjugate of any one of paragraphs

[0052] -

[0152] for use of treatment, prevention and / or diagnosis of castration-resistant prostate cancer or metastatic castration-resistant prostate cancer.

[1231]

[0156] A pharmaceutical composition comprising a PSMA binding protein of any one of paragraphs [l]-

[0039] , and a pharmaceutically acceptable carrier or excipient.

[1232]

[0157] A pharmaceutical composition comprising the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] , and a pharmaceutically acceptable carrier or excipient.

[1233]

[0158] A pharmaceutical composition comprising the antibody-drug conjugate of any one of paragraphs

[0052] -

[0152] , and a pharmaceutically acceptable carrier.

[1234]

[0159] The pharmaceutical composition of any one of paragraphs

[0156] -[l 58] for use in treatment, prevention and / or diagnosis of a disease or condition.

[1235]

[0160] The pharmaceutical composition of any one of paragraphs

[0156] -[l 58] for use in treatment, prevention and / or diagnosis of cancer, preferably selected from the group consisting of: prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

[1236]

[0161] The pharmaceutical composition of any one of paragraphs

[0156] -[l 58] for use in treatment, prevention and / or diagnosis of castration-resistant prostate cancer or metastatic castration-resistant prostate cancer.

[1237]

[0162] A method of treating a disease or condition comprising administering to a subject in need thereof the PSMA binding protein of any one of paragraphs [l]-

[0039] or the antibody-drug conjugate of any one of paragraphs

[0052] -[l 52].

[1238]

[0163] A method of treating a disease or condition comprising administering to a subject in need thereof the one or more nucleic acids according to any one of paragraphs

[0040] -

[0045] or the pair of nucleic acids of paragraph

[0046] or

[0047] .

[0164] A method of treating a disease or condition comprising administering to a subject in need thereof the pharmaceutical composition of any one of paragraphs

[0156] -

[0158] .

[1239]

[0165] The method of any one of paragraphs

[0162] -

[0164] , wherein the disease or condition comprises a cancer, preferably selected from the group consisting of: prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

[1240]

[0166] The method of any one of paragraphs

[0162] -

[0164] , wherein the disease or condition comprises castration-resistant prostate cancer or metastatic castrationresistant prostate cancer.

[1241]

[0167] A conjugation complex of protein with a divalent metal atom M, represented by formula (II):

[1242] [Ab'-M]

[1243] (II),

[1244] or a salt thereof,

[1245] wherein Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6;

[1246] wherein M is a divalent transition metal;

[1247] wherein M bridges together sulfur atoms of two cysteine residues of Ab', so as to form the following group:

[1248] -S-M-S- within the coordination complex.

[1249]

[0168] A conjugation complex of protein with a divalent metal atom M, represented by:

[1250] [Ab'Red-M],

[1251] or a salt thereof,

[1252] wherein Ab'Red is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, andthe light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6;

[1253] wherein each of the heavy chains and light chains comprises one or more interchain cysteines that are reduced in the form of thiol -SH groups, but would form interchain disulfide bridges when oxidized;

[1254] wherein M is a divalent transition metal;

[1255] wherein M bridges together sulfur atoms of two cysteine residues of Ab', so as to form the following group:

[1256] -S-M-S- within the coordination complex.

[1257]

[0169] The conjugation complex of paragraph

[0167] or

[0168] , wherein M bridges together sulfur atoms of two interchain cysteine residues of Ab', wherein the two interchain Cysteine residues would otherwise form interchain disulfide bridges when Ab' is oxidized and not complexed with M.

[1258]

[0170] The conjugation complex of paragraph

[0169] , wherein the two interchain cysteine residues are on two heavy chains of Ab'.

[1259]

[0171] The conjugation complex of paragraph

[0169] , wherein the two interchain cysteine residues are on a heavy chain and a light chain of Ab', respectively.

[1260]

[0172] The conjugation complex of any one of paragraphs

[0167] -

[0172] , wherein the heavy chain comprises a VH region comprising the sequence of SEQ ID NO: 7 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and the light chain comprises a VL region comprising the sequence of SEQ ID NO: 8 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[1261]

[0173] The conjugation complex of any one of paragraphs

[0167] -

[0172] , wherein the heavy chain comprises a VH region comprising the sequence of SEQ ID NO: 7, and the light chain comprises a VL region comprising the sequence of SEQ ID NO: 8.

[1262]

[0174] The conjugation complex of any one of paragraphs

[0167] -

[0172] , wherein the heavy chain comprises a VH region according to the sequence of SEQ ID NO: 7, and the light chain comprises a VL region according to the sequence of SEQ ID NO: 8.

[1263]

[0175] The conjugation complex of any one of paragraphs

[0167] -

[0174] , wherein Ab' comprises a Constant heavy chain (CH) region comprising the sequence of SEQ ID NO: 9 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or a Constant light chain (CL) region comprising the sequence of SEQ ID NO: 10 or a sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[1264]

[0176] The conjugation complex of any one of paragraphs

[0167] -

[0174] , wherein Ab' comprises a CH region comprising the sequence of SEQ ID NO: 9, and a CL region comprising the sequence of SEQ ID NO: 10.

[1265]

[0177] The conjugation complex of any one of paragraphs

[0167] -

[0174] , wherein Ab' comprises a CH region according to the sequence of SEQ ID NO: 9, and a CL region according to the sequence of SEQ ID NO: 10.

[1266]

[0178] The conjugation complex of any one of paragraphs

[0167] -

[0177] , wherein the heavy chain comprises the sequence SEQ ID NO: 11 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and the light chain comprises the sequence SEQ ID NO: 12 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[1267]

[0179] The conjugation complex of any one of paragraphs

[0167] -

[0177] , wherein the heavy chain is according to the sequence SEQ ID NO: 11, and the light chain is according to the sequence of SEQ ID NO: 12.

[1268]

[0180] The conjugation complex of any one of paragraphs

[0167] -

[0179] , wherein the two S represent a sulfur atom of a Cysteine residue of the heavy chain and the light chain, respectively.

[1269]

[0181] The conjugation complex of any one of paragraphs

[0169] -

[0179] , each S represents a sulfur atom of Cysteine-239 of one of the two heavy chains, respectively, according to Kabat numbering.

[1270]

[0182] A method of preparing an antibody-drug conjugate according to any one of paragraphs

[0052] -

[0152] , the method comprising:

[1271] reducing the PSMA binding protein of any one of paragraphs

[0023] -

[0032] with a reducing agent to form a reduced version of the PSMA binding protein in which Sulfur atoms of all interchain cysteine residues are reduced and in the form of thiol -SH groups;

[1272] reacting the reduced version of the PSMA binding protein with a salt of metal M so as to form a metal coordination complex that comprises the PSMA binding protein complexed with M,

[1273] wherein M bridges together sulfur atoms of two of the reduced interchain cysteine residues of Ab' to form the following group:-S-M-S- within the coordination complex; and

[1274] incubating the metal coordination complex with an oxidizing agent, a metal complexing agent and a conjugation agent either sequentially or simultaneously to form the antibody-drug conjugate, wherein the conjugation agent comprises the linker and the payload of the antibody-drug conjugate, and a thiol-reactive group.

[1275]

[0183] A method of preparing an antibody-drug conjugate according to any one of paragraphs

[0132] -

[0152] , the method comprising:

[1276] reducing the PSMA binding protein according to any one of paragraphs

[0132] -

[0152] with a reducing agent to form a reduced version of the PSMA binding protein in which all Sulfur atoms of interchain cysteine residues are reduced and in the form of thiol -SH groups;

[1277] reacting the reduced version of the PSMA binding protein with a salt of metal M so as to form a metal coordination complex that comprises the PSMA binding protein complexed with M,

[1278] wherein M bridges together sulfur atoms of two of the reduced interchain cysteine residues of Ab', so as to form the following group:

[1279] -S-M-S- within the coordination complex; and

[1280] incubating the metal coordination complex with an oxidizing agent, a metal complexing agent and a conjugation agent either sequentially or simultaneously to form the antibody-drug conjugate, wherein the conjugation agent comprises the linker and the payload of the antibody-drug conjugate, and a thiol-reactive group.

[1281]

[0184] A method of preparing the conjugation complex of any one of paragraphs

[0168] -

[0181] , the method comprising:

[1282] reacting the PSMA binding protein according to any one of paragraphs

[0023] -

[0032] with a reducing agent so as to form a reduced version of the PSMA binding protein in which Sulfur atoms of all interchain cysteine residues are reduced and in the form of thiol -SH groups, and

[1283] reacting the reduced version of the PSMA binding protein with a salt of metal M so as to form a metal coordination complex that comprises the PSMA binding protein complexed with M,

[1284] wherein M bridges together sulfur atoms of two of the reduced interchain cysteine residues of Ab', so as to form the following group:

[1285] -S-M-S- within the coordination complex.

[0185] A method of preparing the conjugation complex according to any one of paragraphs

[0167] and

[0169] -

[0181] , the method comprising:

[1286] reacting the conjugation complex according to any one of paragraphs

[0168] -

[0181] with an oxidizing agent such that the reduced interchain cysteine residues, except the two interchain cysteine residues forming bond with a transition metal M, are reoxidized to form interchain disulfide bridges.

[1287]

[0186] The method of paragraph

[0182] ,

[0183] , or

[0185] , wherein the oxidizing agent is dehydroascorbic acid (DHAA), optionally the metal coordination complex is reacted with the oxidizing agent prior to the metal complexing agent and the conjugation agent.

[1288]

[0187] The method of any one of paragraphs

[0182] ,

[0183] , or

[0186] , wherein the metal complexing agent is selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA).

[1289]

[0188] The method of any one of paragraphs

[0182] -

[0184] ,

[0186] , or

[0187] , wherein 0.5 to 2.5, preferably between 1 and 2 equivalents of the salt of metal M is added to react with the reduced version of the PSMA binding protein.

[1290]

[0189] The method of any one of paragraphs

[0182] -

[0188] , wherein the metal M is selected from the group consisting of: Zn, Co, Cd, Hg, and any combinations thereof.EXAMPLES

[1291] Example 1: Binding kinetics measurement by BLI- XT42 and J591 are avidly reacting to human PSMA in Biolayer interferometry

[1292] One antibody according to some embodiments of the present disclosure is an IgGl having for heavy chain the amino acid sequence SEQ ID NO: 11 and for light chain the amino acid sequence SEQ ID NO: 12. This antibody is named hereinafter " XT42" in the following examples. Table 3 and 4 below summarize the sequences of XT42 described in this example.

[1293] Table 3. CDR sequences of XT42 according to different numbering schemes SEQ Name of the sequence Sequence

[1294] ID No

[1295] 1 CDR1-H of XT42 (Kabat) NYHMN

[1296] 2 CDR2-H of XT42 (Kabat) DISGSSRYIHYADFVKG

[1297] 3 CDR3-H of XT42 (Kabat) SSGGYYYGYGMDV

[1298] 4 CDR1-L of XT42 (Kabat) AGTSSDVGGYHYVS

[1299] 5 CDR2-L of XT42 (Kabat) DDSDRPS

[1300] 6 CDR3-L of XT42 (Kabat) SSGTYYSTRV

[1301] 19 CDR1-H of XT42 (Chothia) GFTFSNY

[1302] 20 CDR2-H of XT42 (Chothia) SGSSRY

[1303] 21 CDR3-H of XT42 (Chothia) SGGYYYGYGMD

[1304] 22 CDR1-L of XT42 (Chothia) TSSDVGGYHY

[1305] CDR2-L of XT42 (Chothia) DDS

[1306] 24 CDR3-L of XT42 (Chothia) GTYYSTR

[1307] 25 CDR1-H of XT42 (AbM) GFTFSNYHMN

[1308] 26 CDR2-H of XT42 (AbM) DISGSSRYIH

[1309] 27 CDR3-H of XT42 (AbM) SSGGYYYGYGM

[1310] 28 CDR1-L of XT42 (AbM) AGTSSDVGGYHYVS

[1311] 29 CDR2-L of XT42 (AbM) DDSDRPS

[1312] 30 CDR3-L of XT42 (AbM) SSGTYYSTRV

[1313] 31 CDR1-H of XT42 (Contact) SNYHMN

[1314] 32 CDR2-H of XT42 (Contact) WISDISGSSRYIH

[1315] 33 CDR3-H of XT42 (Contact) VRSSGGYYYGYGMD

[1316] 34 CDR1-L of XT42 (Contact) VGGYHYVSWY

[1317] 35 CDR2-L of XT42 (Contact) LMIYDDSDRP

[1318]

[1319] SEQ Name of the sequence Sequence

[1320] ID No

[1321] 36 CDR3-L of XT42 (Contact) SSGTYYSTR

[1322] 37 CDR1-H of XT42 (IMGT) GFTFSNYH

[1323] 38 CDR2-H of XT42 (IMGT) ISGSSRYI

[1324] 39 CDR3-H of XT42 (IMGT) VRSSGGYYYGYGMDV

[1325] 40 CDR1-L of XT42 (IMGT) SSDVGGYHY

[1326] CDR2-L of XT42 (IMGT) DDS

[1327] 42 CDR3-L of XT42 (IMGT) SSGTYYSTRV

[1328]

[1329] Table 4. Sequences of XT42

[1330] SEQ Name of Type Sequence

[1331] ID the

[1332] No sequence

[1333] 7 VH of XT42 amino EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYHMNWVRQAPGKGLEWISDISGSSRYIHYADFVKGRFTISRDNAKNSLYLQMNSLRA sequence EDTAVYYCVRSSGGYYYGYGMDVWGRGTLVTVSS

[1334] 8 VL of XT42 amino QSVLTQPASVSGSPGQSITICAGTSSSDVGGYHYVSWYQQHPGKAPKLMIYDDSDRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSGTYYSTRVFGGGTKLEIIK

[1335] 9 CH of XT42 amino ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG acid ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK sequence PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW E SNGQPENNYKTTPPVLD SDGS FFLY SKLTVDKSRWQQGNVFS C SVMHEALHNHYTQKSLSLSPGK

[1336] 10 CL of XT42 amino RTVAAPSVFIFPPSDEQLKSGSTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1337] 11 Heavy amino EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYHMNWVRQAPGKG chain of acid LEWISDISGSSRYIHYADFVKGRFTISRDNAKNSLYLQMNSLRA

[1338]

[1339] SEQ Name of Type Sequence

[1340] ID the

[1341] No sequence

[1342] XT42 sequence EDTAVYYCVRSSGGYYYGYGMDVWGRGTLVTVSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

[1343] 12 Light Chain amino QSVLTQPASVSGS PGQS I TI SCAGTS SDVGGYHYVSWYQQHPGK of XT42 acid APKLMIYDDSDRPSGVSNRFSGSKSGNTASLTISGLQAEDEADY sequence YCSSGTYYSTRVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1344] 13 VH of XT42 nucleic gaagtgcagctggtggagagcggaggaagcctggttaagcccgg acid aggaagcctcaggctgagctgtgctgctagtggatttaccttta sequence gtaactaccacatgaactgggtgaggcaggctcctggcaaagga ctggagtggatcagcgacattagcggcagtagcagatacatcca ctacgctgattttgtgaagggcaggttcaccatcagccgggaca acgctaagaacagtctgtatctgcagatgaacagcctgagggcc gaggacaccgctgtttactactgcgtgagaagctctggagggta ctactatggctacggcatggacgtttggggcaggggaaccttgg tgaccgtgagttcc

[1345] 14 VL of XT42 nucleic cagtctgtcctcacccagccagcatccgtctccggttctccagg acid acagtccatcaccatctcctgcgccggaacctcctccgatgtcg sequence gaggataccactacgtctcctggtaccagcagcaccccggaaaa gcacccaaactgatgatctacgacgacagcgaccgcccctccgg tgtttctaacaggttctccggctccaagtcaggcaacaccgcct ctctcaccatctccggtctgcaagctgaagatgaagctgactac tactgctcctccggcacctactactccaccagggtctttggcgg cggcacaaagctggagatcaag

[1346]

[1347] SEQ Name of Type Sequence

[1348] ID the

[1349] No sequence

[1350] 15 CH of XT42 nucleic gctagcaccaagggcccatcggtcttccccctggcaccctcctc acid caagagcacctctgggggcacagcggccctgggctgcctggtca sequence aggactacttccccgaaccggtgacggtgtcgtggaactcaggc gccctgaccagcggcgtgcacaccttcccggctgtcctacagtc ctcaggactctactccctcagcagcgtggtgaccgtgccctcca gcagcttgggcacccagacctacatctgcaacgtgaatcacaag cccagcaacaccaaggtggacaagaaagttgagcccaaatcttg tgacaaaactcacacatgcccaccgtgcccagcacctgaactcc tggggggaccgtcagtcttcctcttccccccaaaacccaaggac accctcatgatctcccggacccccgaggtcacatgcgtggtggt ggacgtgagccacgaagaccctgaggtcaagttcaactggtacg tggacggcgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgt cctgcaccaggactggctgaatggcaaggagtacaagtgcaagg tctccaacaaagccctcccagcccccatcgagaaaaccatctcc aaagccaaagggcagccccgagaaccacaggtgtacaccctgcc cccatcccgggacgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg gagagcaatgggcagccggagaacaactacaagaccacgcctcc cgtgctggactccgacggctccttcttcctctacagcaagctca ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgc tccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggtaaa

[1351] 16 CL of XT42 nucleic cgtacggtggctgcaccatctgtcttcatcttcccgccatctga acid tgagcagttgaaatctggaactgcctctgttgtgtgcctgctga sequence ataacttctatcccagagaggccaaagtacagtggaaggtggat aacgccctccaatcgggtaactcccaggagagtgtcacagagca ggacagcaaggacagcacctacagcctcagcagcaccctgacgc tgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagttcgcccgtcacaaagagcttcaa caggggagagtgt

[1352]

[1353] SEQ Name of Type Sequence

[1354] ID the

[1355] No sequence

[1356] 17 Heavy nucleic gaagtgcagctggtggagagcggaggaagcctggttaagcccgg Chain of acid aggaagcctcaggctgagctgtgctgctagtggatttaccttta XT42 sequence gtaactaccacatgaactgggtgaggcaggctcctggcaaagga ctggagtggatcagcgacattagcggcagtagcagatacatcca ctacgctgattttgtgaagggcaggttcaccatcagccgggaca acgctaagaacagtctgtatctgcagatgaacagcctgagggcc gaggacaccgctgtttactactgcgtgagaagctctggagggta ctactatggctacggcatggacgtttggggcaggggaaccttgg tgaccgtgagttccgctagcaccaagggcccatcggtcttcccc ctggcaccctcctccaagagcacctctgggggcacagcggccct gggctgcctggtcaaggactacttccccgaaccggtgacggtgt cgtggaactcaggcgccctgaccagcggcgtgcacaccttcccg gctgtcctacagtcctcaggactctactccctcagcagcgtggt gaccgtgccctccagcagcttgggcacccagacctacatctgca acgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgtgccc agcacctgaactcctggggggaccgtcagtcttcctcttccccc caaaacccaaggacaccctcatgatctcccggacccccgaggtc acatgcgtggtggtggacgtgagccacgaagaccctgaggtcaa gttcaactggtacgtggacggcgtggaggtgcataatgccaaga caaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggacgagctgaccaagaacca ggtcagcctgacctgcctggtcaaaggcttctatcccagcgaca tcgccgtggagtgggagagcaatgggcagccggagaacaactac aagaccacgcctcccgtgctggactccgacggctccttcttcct ctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccac tacacgcagaagagcctctccctgtctccgggtaaa

[1357]

[1358] SEQ Name of Type Sequence

[1359] ID the

[1360] No sequence

[1361] 18 Light Chain nucleic cagtctgtcctcacccagccagcatccgtctccggttctccagg of XT42 acid acagtccatcaccatctcctgcgccggaacctcctccgatgtcg sequence gaggataccactacgtctcctggtaccagcagcaccccggaaaa gcacccaaactgatgatctacgacgacagcgaccgcccctccgg tgtttctaacaggttctccggctccaagtcaggcaacaccgcct ctctcaccatctccggtctgcaagctgaagatgaagctgactac tactgctcctccggcacctactactccaccagggtctttggcgg cggcacaaagctggagatcaagcgtacggtggctgcaccatctg tcttcatcttcccgccatctgatgagcagttgaaatctggaact gcctctgttgtgtgcctgctgaataacttctatcccagagaggc caaagtacagtggaaggtggataacgccctccaatcgggtaact cccaggagagtgtcacagagcaggacagcaaggacagcacctac agcctcagcagcaccctgacgctgagcaaagcagactacgagaa acacaaagtctacgcctgcgaagtcacccatcagggcctgagtt cgcccgtcacaaagagcttcaacaggggagagtgt

[1362]

[1363] Sequences of SEQ ID NOs: 13-18 encode the amino acids sequences SEQ ID NOs: 7-12 respectively.

[1364] The binding kinetics of IgG XT42 and J591 (positive control, having the same sequences as the publicly available sequences of Rosopatamab, a humanized anti-PSMA monoclonal antibody) to the human PSMA (#PSA-H82Qb, ACROBiosystems) were assessed using Bio-Layer Interferometry (BLI).

[1365] Nickel prepared NTA (NTA: nitrilotriacetic acid) biosensors were hydrated in assay buffer (PBS2x 1%BSA 0.06%Tween, pH 7.4) for 10 minutes prior to use. The biosensors were then loaded with hPSMA-His via its histidine tag at a concentration of 8 pg / mL loading until BLI signal reached 0.4nm. IgGs (XT42 and J591) were prepared at various concentrations ranging from 100 nM to 6.25 nM in PBS2x 1% BSA 0.06%Tween for pH 7.4. The biosensors loaded with hPSMA were immersed in the IgG solutions, and real-time binding interactions were measured using the Octet BLI RH16 system. The association phase (kon) was measured for 120 seconds, during which the biosensors were exposed to the analyte solution. The dissociation phase (koff) was measured for an additional 120 seconds after transferring the biosensors to wells containing assay buffer without analyte. The signal, recorded innanometers (nm), reflected the amount of IgG analyte bound to the sensor surface over time. Kinetics values are the average of 3 and 5 experiments for XT42 and J591, respectively. The raw data were processed using the software provided with the Octet system.

[1366] The results obtained are represented in Figure 1. The analysis was performed for each tested antibody, and Figure 1 represents the resulting kinetic curves were compared between the test IgG (XT42) and the control (J591). Results showed that the XT42 antibody is binding with high affinity to hPSMA.

[1367] Example 2: Cross reactivity evaluation by BLI - XT42 cross reacts between human and cynomolgus PSMA but not to rodent surrogate

[1368] The binding kinetics of IgG XT42 (as per the invention) to the human PSMA (#PSA-H82Qb, ACROBiosystems) rat PSMA (#PSA-R5245, ACROBiosystems), cynomolgus PSMA (PSA-C5247, ACROBiosystems), and mouse (PSMA PSA-M5245, ACROBiosystems), and were assessed using Bio-Layer Interferometry (BLI).

[1369] Anti-Human IgG Fc Capture (AHC) biosensors were prepared and hydrated in assay buffer (PBS2x 1%BSA 0.06%Tween, pH 7.4) for 10 minutes prior to use. The biosensors were then loaded with XT42 IgG at a concentration of 4 pg / mL loading until BLI signal reached 0.4nm. PSMA (human, mouse, rat or cynomolgus) were prepared at 200 nM. The biosensors loaded with XT42 were immersed in the IgG solutions, and real-time binding interactions were measured using the Octet BLI Red96e system (Sartorius). The association phase (kon) was measured for 120 seconds, during which the biosensors were exposed to the analyte solution. The dissociation phase (koff) was measured for an additional 120 seconds after transferring the biosensors to wells containing assay buffer without analyte. The signal, recorded in nanometers (nm), reflected the amount of PSMA analyte bound to the sensor surface over time. The raw data were processed using the software provided with the Octet system.

[1370] The results obtained are represented in Figure 2. The analysis was performed for each tested PSMA. Results showed that XT42 is able to bind the human and the cynomolgus PSMA, whereas no binding was seen on the rodent PSMA.

[1371] Example 3: Epitope binning of XT42 against J591 on human PSMA- BLI epitope binning of XT42 against J591 on human PSMA demonstrates that the two antibodies recognize distinct, non-overlapping epitopes on PSMA.

[1372] The epitope binning experiment was performed using biolayer interferometry (BLI) to investigate whether XT42 and J591 compete for the same or different epitopes on human PSMA. The assay was carried out on the Octet RH16 (Sartorius) using an NTA biosensor coatedwith human PSMA, which allowed for real-time measurement of binding interactions and competition between the antibodies and their target.

[1373] Human histidine tagged PSMA (2 pg / mL) was loaded via its His tag onto the nickel loaded-NTA biosensor in a running buffer of PBS2X with 1% BSA 0.05% Tween-20 (Figure 3, I) then followed by a baseline step (Figure 3, II). After immobilization of PSMA, an initial saturating antibody (AB1S) (either J591 or XT42) was placed in contact to the biosensor to fully occupy the available binding sites on the PSMA antigen (Figure 3, III). The saturation process was monitored by the response unit (RU) until a stable signal was achieved.

[1374] Following the loading of AB1S, the biosensor was washed to remove any unbound antibodies. Next, a second antibody (AB2C - either J591 or XT42), was introduced to assess its ability to bind to hPSMA after saturation by AB1S (Figure 3, IV). The final step was a dissociation where biosensor was back into buffer (Figure 3, V) The level of binding of AB2C was measured in response units (RU) and compared to the control conditions.

[1375] Three experimental conditions were used to assess the binding competition:

[1376] - No competition control: In this condition, no AB1S was pre-loaded onto the biosensor, allowing AB2C to bind freely to PSMA. This served as the baseline for AB2C binding without any interference.

[1377] - Complete competition: In this condition, the same antibody was used for both AB1S and AB2C, ensuring that they would compete for the same epitope. As expected, no additional binding of AB2C was observed after the saturation of PSMA by AB1S.

[1378] - Partial competition: In this condition, different antibodies were used for AB1S and AB2C (J591 and XT42, or vice versa) to test whether the two antibodies recognized nonoverlapping epitopes on PSMA. If AB2C was able to bind after AB1S loading, it indicated that the antibodies targeted different epitopes.

[1379] The results obtained are represented in Figure 3. The results showed that XT42 and J591 exhibited no significant competition, as both antibodies were able to bind to PSMA even after the other was pre-loaded. This lack of competition indicates that XT42 and J591 recognize distinct, non-overlapping epitopes on PSMA.

[1380] Example 4: Binding to PSMA positive cells by Flow Cytometry - XT42 binding to PSMA positive cells by Flow Cytometry demonstrates its capacity to recognize PSMA at the surface of target cancer cells.

[1381] XT42 (as per the invention), and an irrelevant antibody 13R4 were tested on human PSMA-expressing LnCap clone FGC (#CRL-1740, ATCC) using flow cytometry to assess theeffects of dose on antibody binding. IgG antibodies were prepared at concentrations ranging from 200 nM to 6.1 pM.

[1382] The LnCap cells were cultured and suspended at a density of 1 x 106cells / mL in PBS 1% FBS buffer at pH 7.4. The antibodies were incubated with the cells at the aforementioned concentrations for 1 hour at 4°C to allow binding. After incubation, cells were washed with cold buffer, and the detection of bound antibodies was performed using an anti-human Fc-647 (CSA 3836 Cohesion Biosciences) secondary antibody. Flow cytometry analysis was conducted on a CytoFlex instrument (Beckmann Coulter) and FlowJo software.

[1383] Data collection focused on the geometric mean fluorescence intensity (Geomean) of the LnCap cells, representing the degree of antibody binding to the PSMA.

[1384] The results obtained are represented in Figure 4. Binding curves were plotted and the XT42 EC50 of 0.374nM was determined by fitting the binding data using nonlinear regression Sigmoidal, (three parameters).

[1385] Binding affinity of the antibody XT42 according to some embodiments of the present disclosure shows a notable sensitivity with a 0.374 nM EC50 on LnCap cells compared to irrelevant antibody which showed no binding to the cells.

[1386] Example 5: Internalization of XT42 antibody in human PSMA-positive LnCap cells - XT42 rapidly internalizes in PSMA-positive LnCap cells demonstrating its developability in ADC format

[1387] XT42 antibody internalization in LnCap cell line was measured with Incucyte® FabFluor-pH red antibody labelling reagent (Sartorius, Gottingen, Germany) according to the manufacturer's instructions. In details, LnCap clone FGC (#CRL-1740, ATCC) were seeded at 10 000 cells / well in a 96-well flat bottom culture plate. Cells were treated with XT42, 13R4 (irrelevant antibody) or J 591 to evaluate antibody internalization. Antibodies were individually labelled with human FabFluor-pH red reagent at a molar ratio of 1:3 in medium and incubated for 15 min at 37°C protected from light to allow conjugation. The labelled antibodies were added to the LnCap cells at 4 pg / mL final concentration. Cell images (magnification lOx) were analysed for fluorescence area and intensity every 45 min for up to 24 h using Incucyte® S3 ZOOM live-cell monitoring system. Data are presented as red fluorescence area per well. The results obtained are represented in Figure 5 and highlight the internalization rates of the tested antibodies.

[1388] The results obtained are represented in Figure 5. Despite different avidity kinetics, XT42 and J591 displayed a comparable internalization rates in LnCap, while 13R4 irrelevant antibody showed no internalization.Example 6: Preparation and characterization of the conjugates

[1389] General procedure A for the preparation of conventional DAR4 conjugates

[1390] To a solution of Protein (100 pL, 40 pM in PBS pH 7.4) were added EDTA (0.5 M in water, 0.5 pL, pH 8.0) and TCEP’HCI (10 mM in water, 2.4 pL, 2.25 equiv.). The mixture was incubated at 37 °C for 2 hours, followed by cooling to 0°C. To the resulting mixture was added the solution of Payload (10 mM in DMSO, 1.2 pL, 3 equiv.) and DMSO if required (max 10%). The mixture was incubated until completion of the conjugation reaction (e.g. 1.5 hours at 4°C for maleimide-based payloads, 16h at 20°C for less reactive payloads). The resulting conjugate was purified using Bio-Spin® P-30Gel Column (Bio-Rad) equilibrated with PBS (IX, pH 7.4).

[1391] General procedure B for the preparation of conventional DAR8 conjugates

[1392] To a solution of Protein (100 pL, 40 pM in PBS pH 7.4) were added EDTA (0.5 M in water, 0.5 pL, pH 8.0) and TCEP’HCI (10 mM in water, 2.4 pL, 6 equiv.). The mixture was incubated at 37 °C for 2 hours, followed by cooling to 0°C. To the resulting mixture was added DMSO and the solution of Payload (10 mM in DMSO, 1.2 pL, 3 equiv.) to reach a final DMSO content of 10%. The mixture was incubated until completion of the conjugation reaction (e.g.

[1393] 1.5 hours at 4°C for maleimide-based payloads). The resulting conjugate was purified using Bio-Spin® P-30Gel Column (Bio-Rad) equilibrated with PBS (IX, pH 7.4).

[1394] General procedure C for the preparation of conventional Lys conjugates

[1395] To a solution of Protein (100 pL, 40 pM in PBS pH 7.4) was added the solution of Payload (NHS ester or isothiocyanate reagent at 10 mM in DMSO, equiv. depending on the desired level of modification but without exceeding 10% DMSO). The mixture was incubated until completion of the conjugation reaction (e.g. 2 hours at rt or overnight at 4°C). Next, a quenching buffer (e.g., 50 mM Tris or 1 M glycine, pH 7.5) was added to neutralize unreacted NHS esters. Incubate for 15 minutes at room temperature. The resulting conjugate was purified using Bio-Spin® P-30Gel Column (Bio-Rad) equilibrated with PBS (IX, pH 7.4). General procedure D for the preparation of GeminiMab conjugates (DARI, DAR2)

[1396] To a solution of Protein (100 pL, 40 pM in PBS pH 7.4) were added TCEP’HCI (10 mM in water, 2.4 pL, 6 equiv.). The mixture was incubated at 37 °C for 2 hours, followed by cooling to 20°C. Subsequently, a solution of Zn(OAc)z (4 mM in water, 1.4 pL, 1.4 equiv.) was added to the mixture, followed by the addition of dehydroascorbic acid (DHAA, 10 mM in DMSO, 6 pL, 15 equiv.). The resulting mixture was incubated at 20°C for 1 hour, then purified using Bio-Spin® P-30Gel Column (Bio-Rad) equilibrated with PBS (IX, pH 7.4) and cooled to 4°C. To the resulting mixture was added the solution of thiol -reactive payload (10 mM inDMSO, 1.2 pL, 3 equiv.) followed by EDTA (0.5 M in water, 0.5 pL, pH 8.0). The mixture was incubated until completion of the conjugation reaction (e.g. 1.5 hours at 4°C for maleimide-based payloads, 16h at 20°C for less reactive payloads). The resulting conjugate was purified using Bio-Spin® P-30Gel Column (Bio-Rad) equilibrated with PBS (IX, pH 7.4).

[1397] General procedure E for reverse-phase high performance liquid chromatography - mass spectrometry (RP-HPLC MS) characterization of the conjugates

[1398] RP-HPLC MS analyses were conducted using a ThermoFisher Ultimate 3000 HPLC system (Thermo Fisher Scientific, Germering, Germany), coupled to an Exactive™ Plus Extended Mass Range (EMR, Orbitrap analyzer, Thermo Fisher Scientific, Bremen, Germany). This setup was equipped with an HESI-II probe source operating in positive ion mode. Instrument control and data acquisition were handled using the Thermo Xcalibur™ mass spectrometry data system. For the analysis, an Agilent PLRP-S, 1000 A, 2.1 x 50 mm, 5 pm column (P / N: PL1912-1502) was utilized and maintained at 40 °C. Mobile phase A was 0.1% formic acid in DI water, mobile phase B was 0.1% formic acid in acetonitrile. The separation process involved a flow rate of 0.600 mL / min and employed the following gradient: 0-4 min, isocratic 20% B; 4-5 min, 20% to 40% B; 5-10 min, 40% to 70% B; 10-11 min, 70% to 90% B. Full MS spectra were acquired in positive polarity with a scan range of 800-4,000 m / z. The resolution was set at 70,000, with an AGC target of 1 x 106ions. Sheath gas was set to 35 AU and auxiliary gas was set to 10 AU. The spray voltage was 2.2 kV, the capillary temperature was 250 °C. Prior to analysis, all protein samples were deglycosylated using Endo S enzyme (New England Biolabs, P / N: P0741S) following manufacturer's instructions. For each analysis, 1 pg of the protein conjugate was injected. All MS data of the conjugates were deconvoluted and analyzed using UniDec software (version 5.2.1, Marty et al. Anal. Chem. 2015. DOI: 10.1021 / acs.analchem.5b00140).

[1399] General procedure F for peptide mapping

[1400] 50 pg of ADC were solubilized 80 pL of 50 mM NH4HCO3, 0.1% RapiGest (Waters) at pH 7.8. IdeS digestion was performed by adding 50 units of FabRICATOR (Genovis) at 37°C for 30 min. Disulfide reduction was performed by incubating with 5 mM TCEP for 30 min at 57°C. Alkylation of cysteines was performed in 10 mM IAM in the dark at 20°C for 40 min. Trypsin (Promega, V5111) was prepared by suspending 20 pg in 100 pL of H2O. Digestion was performed by adding 5 pL of trypsin solution, which corresponds to a 1:50 enzyme: substrate ratio. Samples were incubated overnight at 20°C. The reaction was stopped by adding 1 pL of TFA. RapiGest was eliminated by incubation at 37°C for 30 min and centrifugation at 13,000 g for 5 min.NanoLC-MS / MS analysis was performed using a nanoACQUITY Ultra-Performance-LC (Waters Corporation, Milford, USA) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The nanoLC system was composed of ACQUITY UPLC® CSH130 C18 column (250 mm x 75 pm with a 1.7 pm particle size, Waters Corporation, Milford, USA) and a Symmetry C18 precolumn (20 mm x 180 pm with a 5 pm particle size, Waters Corporation, Milford, USA). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Sample was loaded into the enrichment column during 3 min at 5 pL / min with 99% of solvent A and 1% of solvent B. Elution of the peptides was performed at a flow rate of 350 nL / min with a 2-44% linear gradient of solvent B in 38 minutes, followed by a 44-64% linear gradient in 30 minutes.

[1401] The Q-Exactive Plus was operated in data-dependent acquisition mode by automatically switching between full MS and consecutive MS / MS acquisitions. Full-scan MS spectra were collected from 300 - 1,800 m / z at a resolution of 70,000 at 200 m / z with an automatic gain control target fixed at 3 x 106ions and a maximum injection time of 50 ms. The top 10 precursor ions with an intensity exceeding 5 x 104ions and charge states > 2 were selected from each MS spectrum for fragmentation by higher-energy collisional dissociation. Spectra were collected at a resolution of 17,500 at 200 m / z with a fixed first mass of 100 m / z, an automatic gain control target fixed at 1 x 105ions and a maximum injection time of 100 ms. Dynamic exclusion time was set to 3 s.

[1402] Peptides containing drug linker were identified by using Byos® Desktop Software (Protein Metrics) or searched manually in raw data.

[1403] Compound 1

[1404]

[1405] Compound 1Compound 1 (also named in the following examples J 591 -ADC) was prepared following General procedure A, using J591 as protein and MC-VC-MMAE (CAS 646502-53-6) as thiolreactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1406] Expected mass of the major species 150075 Da; observed mass of the major species 150078 Da.

[1407] Compound 2

[1408]

[1409] Compound 2 was prepared following General procedure A, using XT42 as protein and MC-VC-MMAE (CAS 646502-53-6) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1410] Expected mass of the major species 151448 Da; observed mass of the major species 151443 Da.

[1411] Compound 3

[1412]

[1413] Compound 3 was prepared following General procedure A, using XT42 as protein and APN-MC-VC-MMAE (CAS 2252392-31-5) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1414] Expected mass of the major species 152492 Da; observed mass of the major species 152500 Da.

[1415] Compound 4

[1416] XT42

[1417]

[1418] Compound 4

[1419] Compound 4 was prepared following General procedure A, using XT42 as protein and MC-MMAF (CAS 863971-19-1) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1420] Expected mass of the major species 149884 Da; observed mass of the major species 149888 Da.Compound 5

[1421] OH

[1422] MC-EVC-MMAF

[1423] XT42

[1424]

[1425] Compound 5

[1426] Compound 5 was prepared following General procedure A, using XT42 as protein and MC-EVC-MMAF as thiol -reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1427] Expected mass of the major species 152020 Da; observed mass of the major species 152013 Da.

[1428]

[1429] Compound 6

[1430] Compound 6 was prepared following General procedure D, using XT42 as protein and MC-VC-MMAE (CAS 646502-53-6) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1431] Expected mass of the major species 148816 Da; observed mass of the major species 148813 Da.

[1432] Compound 7

[1433]

[1434] Compound 7

[1435] Compound 7 was prepared following General procedure D, using XT42 as protein and APN-MC-VC-MMAE (CAS 2252392-31-5) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1436] Expected mass of the major species 149338 Da; observed mass of the major species 149337 Da.Compound 8

[1437]

[1438] Compound 8

[1439] Compound 8 was prepared following General procedure D, using XT42 as protein and MC-MMAF (CAS 863971-19-1) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1440] Expected mass of the major species 148034 Da; observed mass of the major species 148029 Da.Compound 9

[1441]

[1442] Compound 9 (also named in the following examples XT42-ADC) was prepared following General procedure D, using XT42 as protein and Bis-Mal-MC-VC-MMAE (CAS 1620837-70-8) as thiol -reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1443] Expected mass of the major species 147598 Da; observed mass of the major species 147595 Da.Compound 9 was characterized by peptide mapping following General Procedure F. The analysis confirmed that the predominant conjugation site for the payload is at C239 (according to Kabat numbering) on the heavy chain of the XT42 antibody.

[1444] Compound 10

[1445] HO

[1446]

[1447] HO Compound 10 was prepared following General procedure D, using XT42 as protein and BisAlphaMal-VC-MMAE as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1448] Expected mass of the major species 147656 Da; observed mass of the major species 147653 Da.Compound 11

[1449]

[1450] Compound 11 was prepared following General procedure D, using XT42 as protein and BisBetaMal-VC-MMAE as thiol -reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1451] Expected mass of the major species 147684 Da; observed mass of the major species 147690 Da.

[1452]

[1453] Compound 12

[1454] Compound 12 was prepared following General procedure A, using XT42 as protein and Deruxtecan (CAS 1599440-13-7) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1455] Expected mass of the major species 150316 Da; observed mass of the major species 150319 Da.

[1456]

[1457] Compound 13 was prepared following General procedure B, using XT42 as protein and Deruxtecan (CAS 1599440-13-7) as thiol-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1458] Expected mass of the major species 154448 Da; observed mass of the major species 154447 Da.Compound 14

[1459]

[1460] Compound 14

[1461] Compound 14 was prepared following General procedure A, using XT42 as protein and BetaMal-Dota (CAS 1006711-90-5) as thiol -reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1462] Expected mass of the major species 147238 Da; observed mass of the major species 147237 Da.

[1463] Compound 15

[1464]

[1465] Compound 15

[1466] Compound 15 was prepared following General procedure C, using XT42 as protein and SMCC-DM1 (CAS 1228105-51-8) as lysine-reactive payload. The product was characterized by RP-HPLC MS following General procedure E.

[1467] Expected mass of the major species 149253 Da; observed mass of the major species 149259 Da.

[1468] Example 7: In Vitro Potency and Selectivity of Anti-PSMA ADCs

[1469] The in vitro potency and specificity of anti-PSMA ADCs were evaluated using the PSMA- positive C4-2 cell line, representative of metastatic castration-resistant prostate cancer (mCRPC), and the PSMA-negative PC-3 cell line as a control. Cells were maintained in a high-glucose DMEM medium supplemented with 10% fetal calf serum, penicillin-streptomycin, and L-glutamine.

[1470] To assess anti-proliferative activity, concentrations of all ADCs were normalized by the amount of the conjugated payload and cells were incubated with corresponding ADCs for 96 hours at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA), and absorbance was measured at 515 nm using a microplate reader. Untreated cells were used as a negative control, with viability set at 100%. Potency (IC50) values were calculated using GraphPad Prism software.

[1471] The results demonstrate that anti-PSMA ADCs selectively inhibit the growth of PSMA-expressing C4-2 cells, while no cytotoxicity was observed in PSMA-negative PC-3 cells. The selective cytotoxic activity suggests that the ADC-mediated cell killing is target-dependent.

[1472] These findings indicate that the disclosed anti-PSMA ADCs provide targeted cytotoxicity against PSMA-expressing cells, minimizing off-target toxicity. The lack of activity in PSMA-negative PC-3 cells further confirms that the ADCs require target engagement for cytotoxic effects, highlighting their potential as a therapeutic approach for PSMA-positive cancers.

[1473] Table 5. In vitro cell-killing activity in LnCaP-C4.2 and PC-3 cell lines ADC IC50 (nM) in IC50 (nM)

[1474] LnCaP-C4.2 cells in PC-3 cells

[1475] Compound 1 0.32 No activity

[1476] Compound 3 0.38 No activity

[1477] Compound 7 0.31 No activity

[1478] Compound 8 0.63 No activity

[1479] Compound 9 0.16 No activity

[1480]

[1481] Example 8: Pharmacokinetic study in Sprague-Dawley rats

[1482] Pharmacokinetic (PK) evaluation of ADC stability and tolerability was performed in healthy female Sprague Dawley rats following a single intravenous (IV) administration. The study assessed the systemic clearance and stability of XT42-ADC compared to the unconjugated XT42 antibody. Six rats, 7-8 weeks old, were randomized based on body weight. Animals received a single IV injection via the caudal vein at a dose of 10 mg / kg with an injection volume of 5 mL / kg. Blood samples were collected at lh, 4h, 12h, 48h, 120h,168h, and 336h post-administration, with intra-cardiac collection performed as a terminal procedure.

[1483] Plasma was prepared by collecting blood into tubes containing K3 EDTA as an anticoagulant. The tubes were centrifuged at 2000 g for 10 minutes at 4°C to separate plasma. Aliquots of 100 pL and 50 pL were prepared and stored in polypropylene tubes at -80°C until analysis.

[1484] Plasma samples were analyzed using the Human IgG ELISA Antibody Pair Kit (Stemcell, Reference: 01994) according to the manufacturer's instructions.

[1485] PK analysis revealed that XT42-ADC (Compound 9) exhibited a systemic clearance profile similar to the unconjugated XT42 antibody, indicating that the conjugation process did not significantly alter antibody stability or half-life. The ADC was well tolerated, with no signs of adverse effects at the administered dose.

[1486] The results of the PK study are presented in Figure 6, showing the plasma concentration-time profiles of the tested compounds. The calculated PK parameters for XT42 and XT42-ADC, are summarized in Table 6. These findings demonstrate that the disclosed XT42-ADC maintains the pharmacokinetic profile of the XT42 antibody, supporting the therapeutic potential of both XT42 and XT42-ADC. The results support the suitability of XT42-ADC for targeted therapy, minimizing off-target toxicity while ensuring efficient delivery of the cytotoxic payload.

[1487] Table 6. Calculated PK parameters of XT42 and XT42-ADC (compound 9) Calculated PK Parameter Summary (10 mg / kg IV)

[1488] Parameter XT42 XT42-ADC

[1489] Dose (mg / kg) 10 10

[1490] AUC (0-t)* (ug.h / mL) 52947 45081

[1491] AUC (0-inf) (ug.h / mL) 88761 70628

[1492] DNAUC (0-inf) h*kg*ug / mL / mg) 8876 7063

[1493] T 1 / 2 (h) 269 234

[1494] MRT (h) 374 326

[1495] Clb (mL / min / kg) 0.00191 0.00244

[1496] Vss (L / kg) 0.04168 0.04758

[1497]

[1498] Cmax (ug / mL) 546 460

[1499] Example 9: In vivo efficacy study in LnCaP-C4.2 model

[1500] The anti-tumor efficacy of ADCs was evaluated in a xenograft model using male CB17 SCID mice bearing subcutaneous LnCaP-C4.2 tumors. The study aimed to compare the therapeutic effectiveness of XT42-ADC (compound 9) and J591-ADC (compound 1).To establish tumors, 7-week-old male CB17 SCID mice were subcutaneously injected with 1 x 107LnCaP-C4.2 cells suspended in 200 pL of RPMI 1640 medium without phenol red, containing 50% (v / v) Matrigel. Tumor cell implantation was performed 24 hours post-whole-body irradiation using a gamma source (1.44 Gy,60Co, BioMep, France). Tumor growth was monitored, and animals were randomized into three treatment groups (n=10 per group) when tumor volumes reached an average of 178 mm3. Randomization was performed based on individual tumor volumes, ensuring homogeneity between groups through analysis of variance (ANOVA).

[1501] XT42-ADC, J591-ADC, and vehicle (PBS) were administered via intravenous (IV) injection into the caudal vein at the time of randomization. ADCs were dosed to deliver 38 pg / kg of conjugated MMAE in an administration volume of 5 mL / kg. Tumor length and width were measured biweekly using calipers, and tumor volumes were calculated using the formula:

[1502] Tumor Volume = Length x Width x Width I 2

[1503] Body weights and tumor volumes were monitored over a 41-day period. The results, depicted in Figure 7, demonstrate a significantly stronger anti-tumor effect of XT42-ADC compared to J591-ADC. The study findings indicate that XT42-ADC provides superior tumor inhibition, supporting its potential therapeutic application.

[1504] Example 10: In vivo efficacy study in a patient-derived xenograft LuCap96CR model The anti-tumor efficacy of XT42-ADC was evaluated in a LuCaP96CR patient-derived xenograft (PDX) model using male CB17 SCID mice bearing subcutaneous human prostate tumors. The study aimed to compare the therapeutic effectiveness of XT42-ADC with the standard-of-care treatment enzalutamide.

[1505] Two weeks prior to tumor implantation, CB17 SCID mice were surgically castrated. On the day of implantation, tumors from the amplification phase were excised, and tumor fragments (30-50 mg) were implanted subcutaneously into the right flank of male CB17 SCID mice (one LuCaP96CR fragment per mouse). Following tumor induction, mice were provided with a high-fat diet to mitigate body weight loss associated with cachexia in the LuCaP96CR PDX model (intervention applied when body weight loss exceeded 10%).

[1506] Animals were randomized into treatment groups when the average tumor volume reached 120 mm3. On the day of randomization (D24 post-implantation), three groups of 10 mice each were assigned to the following treatments:

[1507] 1. Vehicle (PBS) - Single IV dose

[1508] 2. XT42-ADC - Single IV dose at 10 mg / kg3. Enzalutamide - Oral administration, (Q1D x 5) x 6

[1509] Body weights and tumor volumes were measured twice weekly for a period of 59 days post-implantation. Tumor volumes were calculated using the standard formula:

[1510] Tumor Volume = Length x Width x Width I 2

[1511] The results, depicted in Figure 8, demonstrate that XT42-ADC exhibited significantly higher anti-tumor activity compared to enzalutamide. XT42-ADC induced complete responses in 10 / 10 mice, whereas no complete responses (0 / 10) were observed in the enzalutamide-treated group.

[1512] Example 11: Tolerability study in cynomolgus monkeys

[1513] A tolerability study was conducted in naive cynomolgus monkeys aged 2.5 to 3 years at the time of dose initiation. Two monkeys received escalating intravenous (IV) doses of XT42 according to the following schedule: 4 mg / kg on Day 1, 12 mg / kg on Day 22, 18 mg / kg on Day 43, and 24 mg / kg on Day 64.

[1514] Mortality and moribundity observations were performed twice daily, while cage-side observations were conducted once daily. Detailed clinical observations were recorded once during the pretest phase, once weekly, and once prior to necropsy. Body weight was measured twice during the pretest phase, weekly throughout the study, and once prior to necropsy. Body temperature was assessed twice pretest and two hours after each dose administration. Clinical pathology evaluations included hematology, coagulation, and clinical chemistry assessments, performed 24 hours before and 48 hours after each dose, and once prior to necropsy. Urinalysis was conducted once pretest and 24 hours after each dose.

[1515] Based on the recorded observations, XT42-ADC was well tolerated, with no signs of severe toxicity at doses up to 24 mg / kg. This indicates a significantly lower toxicity compared to the state-of-the-art ARX517 ADC, for which the highest non-severely toxic dose (HNSTD) in cynomolgus monkeys was reported as 6 mg / kg (Mol Cancer Ther (2024) 23 (12): 1842-1853).

[1516] Example 12: In vivo dose-response study in the LnCaP-C4.2 model

[1517] The objective of this study was to determine the minimum efficacious dose (MED) of XT42-ADC in male CB17 SCID mice bearing subcutaneous LnCaP-C4.2 tumors.

[1518] The anti-tumor efficacy of XT42-ADC was assessed in a xenograft model using male CB17 SCID mice implanted with LnCaP-C4.2 tumors. The study compared the therapeutic effectiveness of XT42-ADC at three dose levels: 10 mg / kg, 5 mg / kg, and 2.5 mg / kg.

[1519] To establish tumors, 7-week-old male CB17 SCID mice were injected subcutaneously with 1 x 107LnCaP-C4.2 cells, suspended in 200 pL of RPMI 1640 medium (phenol red-free)containing 50% (v / v) Matrigel. Tumor implantation was performed 72 hours after whole-body irradiation with a gamma source (1.44 Gy,60Co, BioMep, France). Tumor growth was monitored, and animals were randomized into four treatment groups (n = 10 per group) when tumor volumes reached an average of 177 mm3. Randomization was based on individual tumor volumes, ensuring homogeneity between groups through analysis of variance (ANOVA).

[1520] The groups were assigned as follows: one group received a single intravenous (IV) dose of vehicle (PBS), while the remaining three groups received XT42-ADC as a single IV dose at 2.5 mg / kg, 5 mg / kg, or 10 mg / kg.

[1521] Tumor dimensions (length and width) were measured biweekly using calipers, and tumor volumes were calculated using the formula:

[1522] Tumor Volume = Length x Width x Width I 2

[1523] Body weight and tumor volume were monitored over 46 days post-implantation. The results, depicted in Figure 9, indicate that the minimum efficacious dose (MED) of XT42-ADC is 2.5 mg / kg. At this dose, tumor stasis was achieved with a single administration of XT42-ADC, demonstrating a significantly higher anti-tumor activity compared to the state-of-the-art ARX517 ADC, which required three administrations at 3 mg / kg to achieve a comparable level of efficacy in the same tumor model.

[1524] Example 13: In vitro potency of Compound 10

[1525] The objective of this study was to assess potency of Compound 10 in comparison with Compound 9, both of which were synthesized according to Example 6, against metastatic castration-resistant prostate cancer (mCRPC) in a cellular model.

[1526] The in vitro potency of Compound 10 and Compound 9 were evaluated using the PSMA-positive C4-2 cell line, representative of metastatic castration-resistant prostate cancer (mCRPC). Cells were maintained in GIBCO™ RPMI 1640 Medium, GLUTAMAX™ Supplement (Thermo Fisher Scientific), supplemented with 10% fetal calf serum and penicillinstreptomycin, at 37°C in a humidified atmosphere containing 5% CO2.

[1527] For anti-proliferative assays, cells were seeded at a density of 8,000 cells per well in 96-well plates and allowed to adhere for 24 hours. To assess anti-proliferative activity, cells were then treated with the corresponding ADCs and incubated for an additional 96 hours. Cell viability was determined using the ALAMARBLUE™ High Sensitivity (HS) Cell Viability Reagent (Invitrogen), following the manufacturer's protocol. Fluorescence was measured using a microplate reader with excitation at 531 / 25 nm and emission at 595 / 60 nm. Untreated cells served as the negative control, with viability set at 100%. Potency (IC50) values were calculated using GraphPad Prism version 8.0.1 (GraphPad Software, USA). As shown in Figure10, Compound 10 and Compound 9 exerted similar level of cytotoxic activity on the PSMA-positive C4-2 cell line. IC50 of Compound 10 in PSMA-positive C4-2 cell line was measured to be 2.10 nM, whereas Compound 9 was 2.17 nM.

[1528] While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A PSMA binding protein comprising:(a) (i) any one or a combination of CDRs selected from the group consisting of:CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 7, and CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 8; or (ii) a CDR variant of (i), wherein the variant has 1, 2, or 3 amino acid modifications; or(b) a VH region comprising a sequence at least 80% identical to SEQ ID NO: 7, and / or a VL region comprising a sequence at least 80% identical to SEQ ID NO: 8.

2. The PSMA binding protein of claim 1, wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR- H3 of sequence SEQ ID NO: 3; a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6, with up to five amino acid modifications across said six CDR sequences.

3. The PSMA binding protein of claim 2, wherein the PSMA binding protein comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, a CDR- H3 of sequence SEQ ID NO: 3, a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6.

4. The PSMA binding protein of any one of claim 1-3, wherein the VH region of the PSMA binding protein comprises the sequence of SEQ ID NO: 7 or a sequence having at least 90% identity thereto, and / or the VL region of the PSMA binding protein comprises the sequence of SEQ ID No: 8 or a sequence having at least 90% identity thereto.

5. The PSMA binding protein of any one of claim 1-4, wherein the PSMA binding protein binds to human PSMA, and does not bind to PSMA through the same epitope as Rosopatamab (J591).

6. The PSMA binding protein of any one of claim 1-5, wherein the PSMA binding protein comprises two heavy chains and two light chains.

7. The PSMA binding protein of any one of claim 1-6, wherein the PSMA binding protein comprises a Constant heavy chain (CH) region comprising the sequence SEQ ID NO: 9 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and / or a Constant light chain (CL) region comprising the sequenceof SEQ ID NO: 10 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

8. The PSMA binding protein of any one of claim 1-7, wherein the PSMA binding protein comprises a heavy chain comprising the sequence SEQ ID NO: 11 or a sequence having at least 90% identity thereto, and / or a light chain comprising the sequence of SEQ ID NO: 12 or a sequence having at least 90% identity thereto.

9. An antibody-drug conjugate comprising the PSMA binding protein according to any one of claims 1-8 and a payload, wherein the payload is conjugated to the PSMA binding protein.

10. The antibody-drug conjugate of claim 9, wherein the payload is selected from the group consisting of cytotoxic agents, immunostimulatory agents, targeted protein degradation agents, immunocytokines, radiopharmaceutical agents, and combinations thereof.

11. An antibody-drug conjugate (ADC) according to formula (I):(T)p-Ab'-(L-(Payload)m)n(I), a pharmaceutically acceptable salt thereof,havinga controlled drug-to-antibody ratio (DAR) of (m x n); anda site-specific conjugation of L and optional T to Ab' at one or two of a pair of interchain cysteine residues of Ab' defined as the conjugated cysteine residues;wherein in formula (I):Ab' is a PSMA binding protein according to any one of claims 1-11 and comprising two heavy chains and two light chains, each heavy chain and each light chain comprising one or more interchain cysteine residues, such that each of said conjugated cysteines is bound to either -L-(Payload)mor optional T fragment, and the remaining interchain cysteine residues form interchain disulfide bridges;n is 1 or 2;m is 1, 2, 3, 4, 5, or 6;p is 0 or 1;T, if present, is a terminal group covalently bound to Sulfur atom of one of said pair of interchain cysteine residues of Ab' that is not covalently bound to L;each L is independently a linear or branched di-, tri-, tetra-, penta-, hexa- or hepta-valent linker moiety covalently binding Payload to said Ab', wherein said L isattached to Ab' through Sulfur atoms of one or two of said pair of interchain cysteine residues and binds said Ab' with Payload;Payload is a molecule selected from the group consisting of therapeutic agents, imaging agents, diagnostic agents, and combinations thereof.

12. The antibody-drug conjugate of claim 11, wherein each Payload independently comprises a dolastatin selected from the group consisting of: MMAE, MMAF, MMAD, auristatin E, and combinations thereof, optionally each Payload comprises MMAE.

13. The antibody-drug conjugate of claim 11 or 12, wherein each L independently comprises a cleavable linker, a non-cleavable linker, and / or a self-immolative spacer, optionally wherein the linker comprises a cleavable linker that is a peptide linker.

14. The antibody-drug conjugate of any one of claims 11-13, wherein the site-specific conjugation is at one or two of the pair of N-proximal inter(heavy chains) cysteine residues that are located nearest to the N-terminal end of said heavy chains.

15. The antibody-drug conjugate of any one of claims 11-14, wherein the PSMA binding protein is a human IgGl, and wherein the site-specific conjugation is at Cysteine- 239 of both of the two heavy chains according to Kabat numbering.

16. The antibody-drug conjugate of claim 15, wherein n is 1 and p is 0, and wherein L is a tridentate linker.

17. The antibody-drug conjugate of claim 16, wherein L comprises the following structure:wherein' indicates a single or double bond, and wherein W is substituted C6-14arylene, C5-14heteroarylene, C1-30alkylene, C3-10cycloalkylene, C3-10heterocycloalkylene, or any combination thereof.

18. The antibody-drug conjugate of claim 16, wherein L comprises the following structure:wherein:' indicates a single or double bond,Z is O, S, NR2, C(=O)O, C(=O)NR3, C(=S)O, C(=S)NR4, C(=S)S, NR5C(=O)NR6, NR7C(=S)NR8, or OC(=O)NR9;R2, R3, R4, R5, R6, R7, R8, and R9are each independently selected from H, Ci-ioalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, Ci-iOalkynyl, C6-i4aryl, C5- C5-14heteroaryl;R1is substituted or unsubstituted Ci-iOalkylene, Cnoheteroalkylene, C3- locycloalkylene, C3-i0heterocycloalkylene, Ci-iOalkenylene, Ci-iOalkynylene, C6-14arylene, C5-i4heteroarylene, polyethylene glycol) chain, or any combination thereof;a is 0 or 1;b is 0, 1, 2, 3, 4, 5, 6, 7, or 8;X is —NR10—, — O—, — S—, — C(=O)—, — C(=S)—, — NR11C(=O)—, — NR12C(=S)—, — OC(=O)—, — OC(=S)—;R10, R11, and R12are each independently selected from H, C1-10alkyl, C3-i0cycloalkyl, C3-ioheterocycloalkyl, Ci-iOalkenyl, C1-10alkynyl, C6-i4aryl, or C5-i4heteroaryl;R13and R14are each independently selected from substituted or unsubstituted Ci- walkylene, Cnoheteroalkylene, C3-i0cycloalkylene, heterocycloalkylene, Ci- walkenylene, C6-i4arylene, Ci-i4heteroarylene, or any combination thereof;Q is N or CR15;R15is selected from H, Ci-iOalkyl, C3-iocycloalkyl, C3-ioheterocycloalkyl, C1-10alkenyl, or Ci-ioalkynyl;x and y are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and x+y>l.

19. The antibody-drug conjugate of claim 16, wherein L comprises one of the following structures:where' indicates a single or double bond.

20. An antibody-drug conjugate (ADC) according to formula (I-D2):HO (I-D2), or a pharmaceutically acceptable salt thereof,wherein in formula (I-D2):Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6; andeach S represents a sulfur atom of a Cysteine residue of Ab'.

21. The antibody-drug conjugate of claim 20, wherein the heavy chain comprises the sequence SEQ ID NO: 11 or a sequence having at least 90% identity thereto, and the light chain comprises the sequence SEQ ID NO: 12 or a sequence having at least 90% identity thereto.

22. The antibody-drug conjugate of claim 20 or 21, wherein each S represents a sulfur atom of Cysteine-239 of one of the two heavy chains, respectively, according to Kabat numbering.

23. One or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein according to any one of claim 1-8.

24. One or more nucleic acids comprising:(1) a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 13, and / or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 14; or(2) a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 17, and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identity to the sequence of SEQ ID NO: 18.

25. A vector comprising the one or more nucleic acids according to claim 23 or 24.

26. A host cell comprising one or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein according to any one of claims 1-8.

27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a PSMA binding protein of any one of claims 1-8, the one or more nucleic acids according to claim 23 or 24, or the antibody-drug conjugate of any one of paragraphs 9-22.

28. The pharmaceutical composition of claim 27 for use in treatment, prevention and / or diagnosis of a disease or condition.

29. The pharmaceutical composition of claim 27 for use in treatment, prevention and / or diagnosis of cancer, preferably selected from the group consisting of: prostate cancer, lung cancer, brain tumors, skin cancer, breast cancer, kidney cancer, aggressive sarcomas, gynecological cancers, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, testicular cancer, bladder cancer and thyroid cancer.

30. A method of making a PSMA binding protein of any one of claims 1-8, the method comprising culturing a host cell comprising one or more nucleic acids encoding polypeptides capable of forming the PSMA binding protein, or one or more nucleic acids according to claim 23 or 24, under conditions suitable for producing the PSMA binding protein.

31. A method of preparing an antibody-drug conjugate, the method comprising:reducing the PSMA binding protein of any one of claims 1-8 with a reducing agent to form a reduced version of the PSMA binding protein in which Sulfur atoms of all interchain cysteine residues are reduced and in the form of thiol -SH groups; reacting the reduced version of the PSMA binding protein with a salt of metal M so as to form a metal coordination complex that comprises the PSMA binding protein complexed with M,wherein M bridges together sulfur atoms of two of the reduced interchain cysteine residues of Ab' to form the following group:-S-M-S- within the coordination complex; andincubating the metal coordination complex with an oxidizing agent, a metal complexing agent and a conjugation agent either sequentially or simultaneously to form the antibody-drug conjugate, wherein the conjugation agent comprises the linker and the payload of the antibody-drug conjugate, and a thiol-reactive group.

32. A conjugation complex of protein with a divalent metal atom M, represented by formula (II):[Ab'-M](II),or a salt thereof,wherein Ab' is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6;wherein M is a divalent transition metal;wherein M bridges together sulfur atoms of two cysteine residues of Ab', so as to form the following group:-S-M-S- within the coordination complex.

33. A conjugation complex of protein with a divalent metal atom M, represented by:[Ab'Red-M],or a salt thereof,wherein Ab'Red is a PSMA binding protein comprising two heavy chains and two light chains, wherein the heavy chain comprises a CDR-H1 of sequence SEQ ID NO: 1, a CDR-H2 of sequence SEQ ID NO: 2, and a CDR-H3 of sequence SEQ ID NO: 3, and the light chain comprises a CDR-L1 of sequence SEQ ID NO: 4, a CDR-L2 of sequence SEQ ID NO: 5, and a CDR-L3 of sequence SEQ ID NO: 6;wherein each of the heavy chains and light chains comprises one or more interchain cysteines that are reduced in the form of thiol -SH groups, but would form interchain disulfide bridges when oxidized;wherein M is a divalent transition metal;wherein M bridges together sulfur atoms of two cysteine residues of Ab', so as to form the following group:-S-M-S- within the coordination complex.

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