High-affinity cross-reactive Anti-PSMA antibodies

Abstract

The application relates to the diagnosis and treatment of diseases, including cancer. The invention provides, and involves the use of, fully human antibody molecules that bind prostate-specific membrane antigen (PSMA) from humans, cynomolgus monkeys and guinea pigs with high affinity. The antibody molecules may be conjugated to a cytotoxic moiety.

Description

[0001] HIGH-AFFINITY CROSS-REACTIVE ANTI-PSMA ANTIBODIES Field of the Invention

[0002] The present invention relates to the diagnosis and treatment of diseases, including cancer. The invention provides and involves the use of antibody and antibody fragment molecules that bind prostate-specific membrane antigen (PSMA) from humans, cynomolgus monkeys, and guinea pigs with high affinity. The antibody and antibody fragment molecules may be conjugated to a radioisotope or a cytotoxic moiety.

[0003] Background

[0004] Prostate cancer is the second most common cancer in men. With an estimated 375,304 deaths in 2020 worldwide, prostate cancer is the fifth leading cause of death from cancer in men and represents 6.8% of total cancer death in males (Sung, CA Cancer J Clin, 71 , 209-249 (2021)).

[0005] Treatment of prostate cancer with androgen deprivation therapy (ADT) such as luteinising hormone-releasing hormone (LHRH) analogues or orchidectomy is usually initially effective at controlling metastatic diseases. However, patients inevitably progress from an androgen sensitive to a castration-resistant phenotype which is associated with 90% of overall mortality (Scher, PLoS One, 10, 0139440 (2015)).

[0006] Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein associated with tumour grade and development and is over-expressed in cancerous prostate cells. PSMA represents an ideal target for the development of targeted drug conjugates due to its low expression in normal organs.

[0007] A numberof antibodies which bind human PSMA have been described in the art, including WO2019191728, WO2023086336, WO2023026235, WO2022238522, W02020025564, WO2016145139, W02003064606, Tagawa et al., Cancer, (2010); 116(4 Suppl): 1075-1083. doi:10.1002 / cncr.24795, Wolf et al., The Prostate (2010), 70, 562-569, Smith-Jones at al., Cancer Research (2000), 60, 5237-5243.

[0008] However, to our knowledge no high-affinity anti-PSMA antibody which cross-reacts with human, cynomolgus monkey and guinea pig PSMA has been reported. The present invention has been devised in light of the above considerations.

[0009] Summary of the Invention

[0010] The present inventors have isolated high-affinity fully human antibody molecules which bind prostatespecific membrane antigen (PSMA), in human, cynomolgus monkey (macaca fascicularis) and guinea pig (cavia porcellus).

[0011] Cross-reactivity with cynomolgus monkey and guinea pig PSMA opens new avenues for evaluating the efficacy and tolerability of anti-PSMA antibody molecules intended for further clinical use.In a first aspect, the present invention thus provides an antibody molecule that binds human, cynomolgus monkey and guinea pig PSMA. The sequence of human PSMA is shown in SEQ ID NO: 1 , the sequence of cynomolgus monkey PSMA is shown in SEQ ID NO: 2, and the sequence of guinea pig PSMA is shown in SEQ ID NO: 3.

[0012] The antibody molecule preferably comprises the HCDR1, HCDR2, and HCDR3 sequences of the “CC105RR” antibody shown in SEQ ID NOs 4, 5 and 6, respectively, and / or the LCDR1, LCDR2 and LCDR3 sequences of the “CC105RR” antibody shown in SEQ ID NOs 7, 8 and 9, respectively. An antibody which comprises these 6 CDR sequences has been shown to be capable of binding the extracellular domain of human, cynomolgus monkey and guinea pig PSMA.

[0013] In some preferred embodiments, the antibody molecule may comprise the VH domain sequence or the VL domain sequence, but preferably the VH domain sequence and the VL domain sequence, of the CC105RR antibody molecule shown in SEQ ID NOs 10 and 11, respectively.

[0014] In a second aspect, the present invention provides an antibody molecule that binds human PSMA, wherein the antibody molecule comprises a VH domain comprising a set of complementarity determining regions HCDR1 , HCDR2 and HCDR3, and a VL domain comprising a set of complementarity determining regions LCDR1 , LCDR2 and LCDR3, wherein the HCDR1 , HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs 4, 5 and 6, respectively, and the LCDR1 , LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs 7, 8 and 9, respectively.

[0015] An antibody molecule, as referred to herein, may be in any suitable format. Many antibody molecule formats are known in the art and include both complete antibody molecule molecules, such as IgG, as well as antibody fragments, such as a single chain Fv (scFv), small immunoproteins (SIP), diabodies, or single-chain diabodies. The term “antibody molecule” as used herein encompasses both complete antibody molecule molecules and fragments of antibody molecules, in particular antigen-binding fragments. In a preferred embodiment, the antibody molecule consists of or comprises an scFv, a small immunoprotein, a diabody, a single-chain diabody, or a (complete) IgG molecule, such as an lgG1 or lgG4 molecule.

[0016] PSMA has been shown to be useful as a marker for prostate cancer. The antibody molecule of the invention may thus be employed in the imaging, detection, and diagnosis of diseases and disorders characterized, or associated with, the expression of PSMA. In this context, the antibody molecule may be used as is and later detected using e.g. a secondary antibody molecule, or it may be conjugated to a detectable label. An antibody molecule of the present invention may thus be used as is, i.e. in unconjugated form, or it may be conjugated to a therapeutic or diagnostic agent to provide a conjugate. Preferably, it is used in the form of a conjugate. The choice of agent conjugated to the antibody molecule will depend on the intended application of the conjugate. For example, where the conjugate is intendedfor the treatment of a disease or disorder, the conjugate may comprise an antibody molecule of the invention and a pro-inflammatory agent, a cytotoxic molecule, a radioisotope, a photosensitizer, an enzyme, a hormone, or an immunosuppressive agent. When the conjugate is intended for use in imaging, detecting, or diagnosing a disease or disorder, the conjugate may comprise an antibody molecule of the invention and a detectable label, such as a radioisotope, e.g. a non-therapeutic radioisotope. Depending on the agent conjugated to the antibody molecule, the conjugate may be or may comprise a single-chain protein. Where the conjugate is a single-chain protein, the entire protein can be expressed as a single polypeptide or fusion protein. In this case, the agent may be conjugated to the antibody molecule by means of a peptide linker. Fusion proteins have the advantage of being easier to produce and purify since they consist of a single species. This facilitates production of clinical-grade material. Alternatively, the agent may be conjugated to the antibody molecule by means of a cleavable linker.

[0017] The invention also provides isolated nucleic acids encoding the antibody molecules and conjugates of the invention. The skilled person would have no difficulty in preparing such nucleic acids using methods well-known in the art. An isolated nucleic acid may be used to express the antibody molecule or conjugate of the invention, for example by expression in a bacterial, yeast, insect or mammalian host cell. A preferred host cell is a mammalian cell. The nucleic acid will generally be provided in the form of a recombinant expression vector for expression. Host cells in vitro comprising such nucleic acids and expression vectors are part of the present invention, as is their use for expressing the antibody molecules and conjugates of the invention, which may subsequently be purified from cell culture and optionally further formulated into a pharmaceutical composition.

[0018] An antibody molecule or conjugate of the invention may be provided for example in a pharmaceutical composition and may be employed for medical use as described herein, either alone or in combination with one or more further therapeutic agents, such as a Janus kinase (JAK) inhibitor or an immunomodulatory agent, such as an anti-PD-1 antibody. Alternatively, the antibody molecule or conjugate of the invention may be provided in a diagnostic composition and may be employed for diagnostic use as described herein.

[0019] The present invention thus also relates to an antibody molecule or conjugate of the invention for use in a method for treatment of the human or animal body by therapy. For example, an antibody molecule or conjugate of the invention may be for use in a method of treating cancer, and / or treating a PSMA-associated disease in a patient, the method comprising administering a therapeutically effective amount of an antibody molecule or conjugate of the invention to the patient. The use of an antibody molecule or conjugate of the invention for the manufacture of a medicament for the treatment of cancer, and / or treating a PSMA-associated disease, is also contemplated.

[0020] A further aspect of the invention relates to an antibody molecule or conjugate of the invention for use in a method of imaging, detecting, or diagnosing cancer, and / or a PSMA-associated disease in a patient. Theinvention also relates to a method of imaging, detecting, or diagnosing cancer and / or a PSMA-associated disease in a patient comprising administering an antibody molecule or conjugate of the invention to the patient. The method may be an in vitro or an in vivo method. Also encompassed within the scope of the invention is the use of an antibody molecule or conjugate of the invention for the manufacture of a diagnostic product for imaging, detecting, or diagnosing cancer, and / or a PSMA-associated disease in a patient.

[0021] A patient, as referred to herein, is preferably a human patient. Alternatively, the patient may be an animal patient.

[0022] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0023] Summary of the Figures

[0024] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0025] Figure 1 shows the characterization of anti-PSMA scFv (CC105RR). Figure 1A shows the results of SDS-PAGE analysis of scFv (CC105RR). The scFv had the expected size of 25 kDa under non-reducing (NR) and reducing (R) conditions, respectively. Figure 1B shows the results of size exclusion chromatogram of scFv (CC105RR). The monomeric form of the scFv was eluted from the column at 12.5 mL (Superdex 75i column - GE Healthcare).

[0026] Figure 2 shows the characterization of anti-PSMA antibody CC105RR in lgG4 format. Figure 2A shows the results of SDS-PAGE analysis of CC105RR lgG4. The lgG4 had the expected size of 150 kDa under non-reducing conditions and 25 and 50 kDa under reducing conditions. Figure 2B shows a size exclusion chromatogram of CC105RR lgG4. The lgG4 was eluted from the column at 11.88 mL.

[0027] Figure 3 shows the characterization of anti-PSMA antibody CC105RR in SIP format. Figure 3A shows the results of SDS-PAGE analysis of CC105RR SIP. The protein had the expected size of 75 kDa under non-reducing conditions and 37 kDa under reducing conditions. Figure 3B shows a size exclusion chromatogram for CC105RR SIP. The CC105RR SIP was eluted from the column at 14 mL.

[0028] Figure 4 shows the quality control analysis of CC105RR-CSMut lgG1 (A) and CC105RR-GlyPro-MMAE (B). 4-12% SDS-PAGE gel was run in 1x MOPS buffer under reducing (R) and non-reducing (NR) conditions. L= ladder. Size exclusion chromatography (SEC) profiles on a Superdex column 200i. The main peak corresponds to the eluted proteins (around 11.7 mL). Figures 4C and 4D show the mass spectrometry quality control analysis of CC105RR-GlyPro-MMAE conjugation reaction: HPLC Chromatogram and HRMS deconvoluted spectra of CC105RR-CSMut lgG1 (C) and CC105RR-GlyPro-MMAE (D). Figure 4E shows a schematic illustration of the antibody-drug conjugate CC105RR-GlyPro- MMAE.

[0029] Figure 5 shows the results of BIAcore analysis using bivalent SIP (CC105RR) on a chip coated with human PSMA antigen at 500 nM concentration of the SIP.

[0030] Figure 6 shows the results of an ELISA evaluating binding of CC105RR to human, mouse, guinea pig and cynomolgus monkey PSMA. Anti-PSMA antibody CC105RR in SIP format was tested at a concentration of 5 pg / mL. PSMA antigens were coated onto the wells at a concentration of 100 nM. Anti-PSMA SIP antibody CC105RR showed binding to guinea pig and cynomolgus PSMA. The irrelevant antilysozyme “KSF” antibody in SIP format was used as a negative control, whereas an anti-His antibody was used as a positive control for antigen coating.

[0031] Figure 7 shows the results of a specificity ELISA evaluating binding of CC105RR to different proteins. Anti-PSMA antibody CC105RR in lgG4 format was tested at a concentration of 5 pg / mL. All protein antigens tested were coated onto the wells at a concentration of 100 nM. Anti-PSMA lgG4 antibody CC105RR showed binding only to PSMA. The KSF antibody in lgG4 format was used as a negative control, whereas an anti-His antibody was used as a positive control for antigen coating.

[0032] Figure 8 shows the results of flow cytometry analysis evaluating the binding of CC105RR in lgG4 format to HT1080.hPSMA cell line at varying concentrations of CC105RR. Antibody CC105RR in lgG4 format could bind to the PSMA-expressing cell lines with high specificity. Antibody KSF in lgG4 format was used as a negative control.

[0033] Figure 9 shows the results of ex-vivo immunofluorescence staining of HT1080.hPSMA tumor bearing mouse organs using CC105RR lgG4 (Figure 9A) and the irrelevant antibody KSF in lgG4 as negative control (Figure 9B).

[0034] Figure 10 shows the results of the in vivo therapy study of CC105RR-GlyPro-MMAE. Male BALB / c nu / nu mice bearing subcutaneous HT1080.hPSMA tumors were treated with CC105RR-GlyPro-MMAE and Cys-GlyPro-MMAE. Compounds were systemically administered three times (black arrows) by IV injection on alternate days at 125 nmol / kg (CC105RR-GlyPro-MMAE) or at 250 nmol / kg (Cys-GlyPro-MMAE SMDCs), doses that correspond to 250 nmol / kg of total administered MMAE. Cys-GlyPro-MMAE served as a negative control. Figure 10A shows the tumor volumes overtime, presented using single mouse plots (n= 5 animals / group). Figure 10B shows the body weight changes for each treatment group. Percentages represent body weight loss relative to the average body weight measured before IV administration. CR: complete tumor remission.Detailed Description of the Invention

[0035] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0036] Antibody molecule

[0037] The present invention provides antibody molecules that bind human, cynomolgus monkey, and guinea pig PSMA. Methods for determining binding an antigen, such as human, cynomolgus monkey, and guinea pig PSMA are known in the art and include for example ELISA, surface plasmon resonance, and flow cytometry.

[0038] The antibody molecule preferably binds PSMA specifically. The term “specific” may refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner, here PSMA. The term “specific” is also applicable where the antibody molecule is specific for a particular epitope, such as an epitope on PSMA, that is carried by a number of antigens, in which case the antibody molecule will be able to bind to the various antigens carrying the epitope.

[0039] In some embodiments, the antibody or antigen binding fragment may exhibit a binding affinity dissociation constant KD for human PSMA of about 100 nM or less. For example, the antibody or antigen binding fragment thereof may exhibit a binding affinity dissociation constant KD for human PSMA of from about 100 nM to about 10 pM; or from about 20nM to about 500pM. For example, the antibody molecule, in SIP format, may preferably bind human PSMA with an affinity (KD) of 550pM, or with a higher affinity.

[0040] The antibody molecule may further bind to cynomolgus monkey or guinea pig PSMA with the same affinity (KD) as an anti-PSMA antibody, and may be expressed in scFv format, consisting of the sequence shown in SEQ ID NO: 12 or a variant thereof, in SIP format, consisting of the sequence shown in SEQ ID NO: 16 or a variant thereof, or in IgG format, consisting of the sequences shown in SEQ ID NO: 14 or SEQ ID NO: 28 or SEQ ID NO: 29, and SEQ ID NO: 15 or a variant thereof, or with an affinity that is higher. The binding affinity of an antibody molecule to a cognate antigen, such as human, cynomolgus monkey or guinea pig PSMA can be determined by surface plasmon resonance (SPR), such as Biacore, e.g. as detailed in the examples.

[0041] The antibody molecule is preferably monoclonal. The antibody molecule may be human or humanized, but preferably is a human antibody molecule.

[0042] The antibody molecule may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides, and / or serum components.The antibody molecule may be natural or partly or wholly synthetically produced. For example, the antibody molecule may be a recombinant antibody molecule.

[0043] The antibody molecule may be an immunoglobulin, or an antigen-binding fragment thereof. For example, the antibody molecule may be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an lgG1, lgG2, lgG3 or lgG4 molecule, more preferably an lgG1 or lgG4 molecule, or an antigen-binding fragment thereof.

[0044] The antigen-binding site of an antibody molecule of the invention, such as an immunoglobulin or antigenbinding fragment thereof, binds PSMA. The antigen-binding site may comprise three CDRs, such as the three light chain variable domain (VL) CDRs or three heavy chain variable domain (VH) CDRs, but preferably comprises six CDRs, three VL CDRs and three VH CDRs. The three VH domain CDRs of the antigen-binding site may be located within an immunoglobulin VH domain and the three VL domain CDRs may be located within an immunoglobulin VL domain. The antibody molecule may comprise one or two antigen-binding sites for PSMA. Where the antibody molecule comprises two antigen-binding sites these are preferably identical. The antibody molecule thus may comprise one VH and one VL domain but preferably comprises two VH and two VL domains, i.e. two VHA / L domain pairs, as is the case in naturally-occurring immunoglobulin molecules, scFvs, diabodies and single-chain diabodies, for example.

[0045] The antigen-binding site of the antibody molecule preferably comprises the three VL domain CDRs and / or the three VH domain CDRs of antibody CC105RR. The VH and VL domain sequences of this antibody are shown in SEQ ID NOs 10 and 11, respectively, and the sequences of the CDRs of the CC105RR antibody may be readily determined from these VH and VL domain sequences by the skilled person using routine techniques. The CDR sequences may, for example, be determined according to Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991). In a preferred embodiment, the antigen-binding site of the antibody molecule comprises the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs 4, 5 and 6, respectively, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID Nos 7, 8 and 9, respectively.

[0046] In a further preferred embodiment, the antigen-binding site may comprise the VH domain (SEQ ID NO: 10) and / or VL domain (SEQ ID NO: 11) of antibody CC105RR, but preferably comprises the VH domain and VL domain of antibody CC105RR.

[0047] The antibody molecule may also comprise a variant of a CDR, VH domain, VL domain, heavy chain or light chain sequence, as described herein. A variant may for example comprise a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the CDR, VH domain, VL domain, heavy chain or light chain sequence described herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening. In a preferred embodiment, an antibody moleculecomprising one or more such variant sequences retain one or more of the functional characteristics of the parent antibody molecule, such as binding specificity and / or binding affinity for human, cynomolgus monkey and / or guinea pig PSMA. The parent antibody molecule is an antibody molecule which does not comprise the amino acid substitution(s), deletion(s), and / or insertion(s) which has (have) been incorporated into the variant antibody molecule.

[0048] The antibody molecule may comprise a VH domain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the VH domain of antibody CC105RR (SEQ ID NO: 10).

[0049] The antibody molecule may comprise a VL domain with at least 70%, more preferably one of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the VL domain of antibody CC105RR (SEQ ID NO: 11).

[0050] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC105RR in lgG4 format (SEQ ID NO: 14).

[0051] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC105RR-CSMut in lgG1 format (SEQ ID NO: 28).

[0052] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC105RR in lgG1 format (SEQ ID NO: 29).

[0053] The antibody molecule may comprise a light chain what has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the light chain of antibody CC105RR in lgG4 or lgG1 format (SEQ ID NO: 15). Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol.147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 253389-3402) may be used.

[0054] Variants of the CDRs, VH domain, VL domain, heavy chain or light chain sequences or other sequences disclosed herein comprising one or more, e.g. less than 20 alterations, less than 15 alterations, less than 10 alterations or less than 5 alterations, 4, 3, 2 or 1 , amino acid alterations (addition, deletion, substitution and / or insertion of an amino acid residue) may also be employed in antibody molecules or conjugates according to the invention. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening. Alterations may be made in one or more framework regions and / or one or more CDRs. In particular, alterations may be made in HCDR1 , HCDR2 and / or HCDR3, or in one or more framework regions of the heavy or light chain of the antibody molecule.

[0055] In one example, the heavy chain of an antibody molecule of the invention may comprise a C-terminal lysine residue as shown e.g. in SEQ ID NO: 14, 28 and 29 or said lysine residue may be deleted.

[0056] As noted above, the antibody molecule may be a whole antibody or a fragment thereof, in particular an antigen-binding fragment thereof.

[0057] Antigen-binding fragments of immunoglobulins include (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al. (1989) Nature 341 , 544-546; McCafferty et al., (1990) Nature, 348, 552-554; Holt et al. (2003) Trends in Biotechnology 21 , 484-490), which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al. (1988) Science, 242, 423-426; Huston et al. (1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fvdimers (WO1993 / 011161), (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (W02013 / 014149; WO94 / 13804; Holliger et al.

[0058] (1993a), Proc. Natl. Acad. Sci. USA 906444-6448) and (x) “single-chain diabodies” wherein two sets of VH and VL domains are connected together in sequence on the same polypeptide chain (Konterman & Muller, 1999). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245). Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. (1996), Cancer Res.,

[0059] 56(13):3055-61 ). Other examples of binding fragments are Fab’, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736). A SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform lgE-S2 (SS2-CH4; Batista et al., (1996), J. Exp. Med., 184: 2197-205) forming a homo-dimeric mini-immunoglobulin antibody molecule.

[0060] Preferably, the antibody molecule comprises or consists of a single-chain Fv (scFv), a small immunoprotein, a diabody, a single-chain diabody, or a (whole) IgG molecule, such as an lgG1 or lgG4 molecule.

[0061] Where the antibody molecule is an scFv, the VH and VL domains of the antibody are preferably linked by a 14 to 20 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Suitable linker sequences are known in the art and include the linker sequence shown in SEQ ID NO: 13.

[0062] In a preferred embodiment, the antibody molecule of the invention in scFv format comprises or consists of the sequence shown in SEQ ID NO: 12 or a variant thereof.

[0063] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen-binding site: antigen-binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804; Holligerand Winter, 1997; Holliger et al., 1993).

[0064] In a diabody or single-chain diabody, a heavy chain variable domain (VH) is connected to a light chain variable domain (VL) on the same polypeptide chain. The VH and VL domains are connected by a peptide linker that is too short to allow pairing between the two domains. This forces pairing with the complementary VH and VL domains of another chain. Where the antibody molecule is a diabody or single-chain diabody, the VH and VL domains are preferably linked by a 5 to 12 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 5, 6, 7, 8, 9, 10, 11 , or 12 amino acids in length. Preferably, the amino acid linker is 5 amino acids in length. Suitable linker sequences are known in the art and include the linker sequence shown in SEQ ID NO: 27.

[0065] In another preferred embodiment, the antibody molecule of the invention in diabody format comprises or consists of the sequence shown in SEQ ID NO: 24 or a variant thereof. In a single-chain diabody, two sets of VH and VL domains are connected together in sequence on the same polypeptide chain. For example, the two sets of VH and VL domains may be assembled in a single-chain sequence as follows: (VH-VL)--(VH-VL), where the brackets indicate a set. The two sets of VH and VL domains are connected as a single-chain by a long or ‘flexible’ peptide linker. This type of peptide linker sequence is long enoughto allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set. Generally, a long or ‘flexible’ linker is 15 to 20 amino acids.

[0066] In a further preferred embodiment, the antibody molecule of the invention in single-chain diabody format comprises or consists of the sequence shown in SEQ ID NO: 25 or in SEQ ID NO: 26, or a variant thereof.

[0067] Where the antibody is a small immunoprotein (SIP) e.g. as described in (Li etal., (1997), Protein Engineering, 10: 731-736), the VL domain of the scFv antibody is preferably linked to the CH4 domain of human IgE (Batista et al., (1996), J. Exp. Med., 184: 2197-205) via a 2 to 20 amino acid linker, more preferably a 2 to 10 amino acid linker.

[0068] In a yet further preferred embodiment, the antibody molecule of the invention in SIP format has the sequence shown in SEQ ID NO: 16 ora variant thereof.

[0069] Conjugate

[0070] Conjugates of the invention comprise an antibody molecule of the invention and a therapeutic or diagnostic agent. The therapeutic agent may be a pro-inflammatory agent, a cytotoxic molecule, a radioisotope, a photosensitizer, an enzyme, a hormone, or an immunosuppressive agent. Preferably, the therapeutic agent is a cytotoxic molecule, a radioisotope, or a pro-inflammatory agent. Cytotoxic molecules may include DNA-damaging and microtubule-disrupting agents. In some preferred embodiments, the therapeutic agent conjugated to the antibody molecule may be Calicheamicin, Duocarmycin, Doxorubicin (DNA-damaging), or Auristatin, Maytansinoids (microtubule-disrupting). Most preferably, the therapeutic agent conjugated to the antibody molecule of the invention is monomethyl auristatin E (MMAE).

[0071] Pro-inflammatory cytokines which may be conjugated to an antibody molecule of the invention include interleukin-2 (IL2) shown in SEQ ID NO: 18, interleukin-12 (IL-12) shown in SEQ ID NO: 22, and tumour necrosis factor (TNF), such as TNFa, shown in SEQ ID NO: 20.

[0072] The sequences of the remaining cytokines, as well as variants thereof which may be employed in the present invention, are known in the art.

[0073] A therapeutic agent may be conjugated to the N-terminus or C-terminus of the antibody molecule or both. Where a therapeutic agent is conjugated to both the N-terminus and the C-terminus of the antibody molecule, the therapeutic agents may be the same or different but preferably are different. Where the therapeutic agent is conjugated to the N-terminus of the antibody molecule, the C-terminus may be “free”, i.e. not conjugated to another moiety. Similarly, where the therapeutic agent is conjugated to the C-terminus of the antibody molecule, the N-terminus may be “free”, i.e. not conjugated to another moiety.In a preferred embodiment, the antibody molecule, preferably in lgG1 format, is conjugated to a cytotoxic molecule. In a preferred embodiment, the cysteine residues of the heavy chain in positions 219, 225, and 228 are mutated into serine residues (SEQ ID NO: 28). In a preferred embodiment, the C-terminus cysteine of the light chain (SEQ ID NO: 15) is conjugated to MC-GlyPro-PABC-MMAE to generate an antibody-drug-conjugate (CC105RR-GlyPro-MMAE).

[0074] In a preferred embodiment, the antibody molecule, preferably in single-chain diabody format, is conjugated to interleukin 12 (IL12). In a preferred embodiment, the antibody molecule is conjugated at its N-terminus to IL12. Suitable conjugate sequences include SEQ ID NO: 23 and variants thereof.

[0075] In a yet further preferred embodiment, the antibody molecule, preferably in scFv format, is conjugated, preferably at its C-terminus, to tumor necrosis factor alpha (TNFa). Suitable conjugate sequences include SEQ ID NO: 21 and variants thereof.

[0076] In another preferred embodiment, the antibody molecule, preferably in scFv format, is conjugated, preferably at its C-terminus to interleukin 2 (IL2). Suitable conjugate sequences include SEQ ID NO: 19 and variants thereof.

[0077] A diagnostic agent conjugated to the antibody molecule of the invention may be a detectable label, such as a radioisotope, e.g. a non-therapeutic radioisotope.

[0078] Radioisotopes which may be conjugated to an antibody molecule of the invention include isotopes such as94mTc,99mTc,186Re,188Re,203Pb,67Ga,68Ga,47Sc,111ln,97Ru,62Cu,64Cu,86Y,88Y, "Y,121Sn,161Tb,153Sm,166Ho,105Rh,177Lu,123l,124l,125l,131l,18F,211At and225Ac. Preferably, positron emitters, such as18F and124l, or gamma emitters, such as99mTc,111ln and123l, are used for diagnostic applications (e.g. for PET), while beta-emitters, such as131l, "Y and177Lu, are preferably used for therapeutic applications. Alpha-emitters, such as211At and225Ac may also be used for therapy. In one example, the antibody molecule may be conjugated to177Lu,131l, or"Y.

[0079] The antibody molecule may be conjugated with the therapeutic agent by means of a peptide bond or linker as described herein. Other means for conjugation include chemical conjugation, especially crosslinking using a bifunctional reagent (e.g. employing DOUBLE-REAGENTS™ Cross-linking Reagents Selection Guide, Pierce).

[0080] Linkers

[0081] The antibody molecule, e.g. scFv or IgG, and the therapeutic or diagnostic agent or molecule may be connected to each other directly, for example through any suitable chemical bond, but preferably are connected via a peptide linker. The chemical bond may be, for example, a covalent or ionic bond.

[0082] Examples of covalent bonds include peptide bonds (amide bonds) and disulfide bonds.Where the therapeutic or diagnostic agent is connected to the antibody molecule via a peptide linker, the peptide linker may be a short (2-30, preferably 10-20) residue stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. In one embodiment, the linker may be a cleavable linker. In a preferred embodiment, the cleavable linker may include a Gly-Pro motif. In a further preferred embodiment, the linker may also include a self-immolative spacer such as para-aminobenzyl carbamate (PABC).

[0083] Where the antibody molecule and therapeutic or diagnostic agent are connected via a peptide bond or peptide linker, the conjugate may be produced (secreted) as a single-chain polypeptide, such as a fusion protein.

[0084] Methods of treatment

[0085] As explained above, the presence of PSMA and cells expressing PSMA on their surface has been shown to be associated with a number of diseases and disorders, including cancer.

[0086] An antibody molecule or conjugate of the invention may therefore be for use as a medicament.

[0087] In particular, the antibody molecule or conjugate may be for use in a method of treatment (which may include prophylactic treatment) of the human or animal body.

[0088] Also provided is a method of treating a disease or disorder in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the antibody molecule or conjugate.

[0089] Further provided is the use of the antibody molecule or conjugate in the manufacture of a medicament for use in the treatment of a disease or disorder in a patient.

[0090] The patient may be a human patient or may be an animal patient.

[0091] Treatment may be any treatment or therapy in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the disease or disorder, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the disease or disorder, cure or remission (whether partial or total) of the disease or disorder, preventing, ameliorating, delaying, abating or arresting one or more symptoms and / or signs of the disease or disorder or prolonging survival of an individual or patient beyond that expected in the absence of treatment.

[0092] Treatment as a prophylactic measure (i.e. prophylaxis) is also included. For example, an individual susceptible to or at risk of the occurrence or re-occurrence of a disease or disorder may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of the disease or disorder in the individual.A method of treatment as described may comprise administering at least one further treatment to the individual in addition to the antibody molecule or conjugate. The antibody molecule or conjugate may thus be administered to an individual alone or in combination with one or more other treatments for the disease or disorder in question. Where the antibody molecule or conjugate is administered to the individual in combination with another treatment, the additional treatment may be administered to the individual concurrently with, sequentially to, or separately from the administration of the antibody molecule or conjugate. Where the additional treatment is administered concurrently with the antibody molecule or conjugate, the antibody molecule or conjugate and additional treatment may be administered to the patient as a combined preparation. For example, the additional therapy may be a known therapy or therapeutic agent for the disease or disorder to be treated.

[0093] In a preferred embodiment, the antibody or conjugate of the invention is administered to a patient in combination with a therapeutic agent, such as chemotherapy, radiation therapy, an immunomodulatory agent, and an immunoconjugate, such as an immunocytokine.

[0094] In another preferred embodiment, the therapeutic agent is selected from the group consisting of: chemotherapy, radiation therapy, immunomodulatory agent, an immunoconjugate, such as an immunocytokine.

[0095] In a further preferred embodiment, the antibody or conjugate of the invention is administered to a patient in combination with an immunomodulatory agent, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, or anti-TIM-3 antibody. Most preferably, the immunomodulatory agent is an anti-PD-1 antibody. Anti-PD-1, anti-PD-L1, anti-LAG-3, anti-TIGIT, and anti-TIM-3 antibodies are known in the art and are available to the skilled person. A number of anti-PD-1 and anti-PDL-1 antibodies are licensed for the treatment of cancer in human patients and can be employed in treatment of cancer in a patient in combination with an antibody or conjugate of the invention.

[0096] The disease or disorder to be treated may be a disease or disorder characterized by the expression or overexpression of PSMA on the surface of cells, such as cancer cells.

[0097] The disease or disorder to be treated using an antibody molecule or conjugate of the invention may be any disease or disorder characterized by, or associated with, the expression of PSMA

[0098] The disease or disorder to be treated may be cancer, preferably prostate cancer.

[0099] The disease or disorder to be treated using an antibody molecule or conjugate of the invention may be any disease or disorder characterized by, or associated with, the expression of PSMA. As explained above, expression of PSMA is limited in adult human tissues and is therefore expected to represent a disease-specific target for therapy in diseases or disorders characterized by expression of PSMA. The disease to be treated may be cancer, wherein the cancer cells express PSMA.The disease to be treated using an antibody molecule or conjugate of the invention may be cancer, as well as other tumours and neoplastic conditions.

[0100] The malignancy characterized by PSMA overexpression may be prostate cancer.

[0101] In some embodiments, the prostate cancer is metastatic prostate cancer, hormone sensitive prostate cancer (HSPC) or castrate resistant prostate cancer (CRPC). In some embodiments, the metastatic prostate cancer may be metastatic hormone sensitive prostate cancer (mHSPC) or metastatic castration resistant prostate cancer (mCRPC). Metastatic prostate cancer refers to prostate cancer which has spread or metastasized to another part of the body.

[0102] Hormone sensitive prostate cancer (HSPC) refers to prostate cancer whose growth is inhibited by a decrease in androgen levels or by inhibiting androgen action.

[0103] Castration resistant prostate cancer (CRPC) refers to prostate cancer which continues to grow even when androgen levels in the body are extremely low or undetectable.

[0104] Metastatic hormone sensitive prostate cancer (mHSPC) refers to prostate cancer which has spread or metastasised to another part of the body, and whose growth is inhibited by a decrease in androgen levels or by inhibiting androgen action.

[0105] Metastatic castration resistant prostate cancer (mCRPC) refers to prostate cancer which has spread or metastasized to another part of the body, and which continues to grow even when androgen levels in the body are extremely low or undetectable.

[0106] Methods of detection or diagnosis

[0107] The antibody molecules and conjugates of the invention are expected to be suitable for detecting PSMA in vivo and in vitro, and thus find application in the imaging, detection and diagnosis of disease characterized by, or associated with, expression of PSMA.

[0108] The present invention therefore also relates to the use of an antibody molecule or conjugate of the invention for detecting PSMA, e.g. cells, expressing PSMA on their cell surface, either in vitro or in vivo. The conjugate preferably comprises a detectable label to aid detection. The preparation of suitable conjugates is described elsewhere herein. Alternatively, binding of the antibody molecule to PSMA may be detected using a secondary antibody or other detection reagent. Where the antibody molecule is conjugated to a radioisotope, binding of the antibody molecule to PSMA in the patient may be detected using scintigraphy.Also provided is an in vitro method for detecting PSMA, the method comprising incubating the antibody molecule or conjugate with a sample obtained from an individual, e.g. a human patient, and detecting binding of the antibody molecule or conjugate to the sample, e.g. cells (such as fibroblasts) present in the sample, wherein binding of the antibody molecule or conjugate to the sample indicates the presence of PSMA. Methods for determining binding of an antibody molecule or antigen to a sample are known in the art and include, for example, ELISAs, flow cytometry, and immunostaining of tissue samples.

[0109] Further provided is the antibody molecule or conjugate for use in a method of detecting PSMA in vivo, the method comprising administering the antibody molecule or conjugate to an individual, e.g. a human patient, wherein localization of the antibody molecule or conjugate at a site in the individual, indicates expression of PSMA at said site.

[0110] As PSMA is rarely expressed in adult human tissues but is known to be expressed on tumour cells, the antibody molecules and conjugates of the invention are also expected to find application in the detection of diseases and disorders characterized by expression of PSMA. Thus, the present invention also provides an antibody molecule or conjugate of the invention for use a detection agent, diagnostic, or imaging agent.

[0111] Thus, the present invention also provides the antibody molecule or conjugate for use in a method of imaging, detecting, or diagnosing a disease or disorder in a patient.

[0112] Also provided is a method of imaging, detecting, or diagnosing a disease or disorder in a patient comprising administering an antibody molecule or conjugate of the invention to the patient.

[0113] Further provided is the use of an antibody molecule or conjugate of the invention in the manufacture of a diagnostic product for use in the detection or diagnosis of a disease or disorder.

[0114] The disease or disorder is preferably characterised by expression of PSMA, such as the presence of PSMA-expressing cells, and may be a disease or disorder as described herein, such as cancer.

[0115] Pharmaceutical compositions

[0116] Whilst an antibody molecule or conjugate may be administered alone, antibody molecules and conjugates will typically be administered in the form of a pharmaceutical composition. Thus, a further aspect of the present invention relates to a pharmaceutical composition comprising at least one antibody molecule or conjugate of the invention and at least one other component, such as a pharmaceutically acceptable excipient. A method comprising formulating an antibody molecule or conjugate into a pharmaceutical composition is also provided.

[0117] Pharmaceutical compositions may comprise, in addition to the antibody molecule or conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below. For parenteral, for example subcutaneous or intravenous administration, e.g. by injection, the pharmaceutical composition comprising the antibody molecule or conjugate may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be employed as required, including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0118] In some embodiments, the antibody molecules or conjugates may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antibody molecules or conjugates may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[0119] Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to an individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease or disorder being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule or conjugate, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody molecules are well known in the art (Ledermann et al., 1991 ; Bagshawe et al., 1991). Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for an antibody molecule being administered, may be used. Appropriate doses for conjugates are also known or can bedetermined. For example, a therapeutically effective amount or suitable dose of an antibody molecule or conjugate can be determined by comparing in vitro activity and in vivo activity in an animal model, such as a guinea pig, or a cynomolgus monkey. Methods for extrapolation of effective dosages in guinea pigs and cynomolgus monkey, as well as other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the antibody molecule or conjugate.

[0120] Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the antibody molecule or conjugate, the route of administration and the nature of the condition being treated.

[0121] Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g. about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are described above.

[0122] A pharmaceutical composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0123] Kits

[0124] Another aspect of the invention provides a therapeutic kit for use in the treatment of a disease or disorder comprising an antibody molecule or conjugate as described herein. The components of a kit are preferably sterile and in sealed vials or other containers.

[0125] A kit may further comprise instructions for use of the components in a method described herein. The components of the kit may be comprised or packaged in a container, for example a bag, box, jar, tin or blister pack.

[0126] Nucleic acids, vectors, host cells and methods of production

[0127] Provided is an isolated nucleic acid molecule encoding an antibody molecule or conjugate of the invention. Nucleic acid molecules may comprise DNA and / or RNA and may be partially or wholly synthetic.

[0128] An isolated nucleic acid molecule may be used to express an antibody molecule or conjugate of the invention. The nucleic acid will generally be provided in the form of an expression vector. Another aspect of the invention thus provides an expression vector comprising a nucleic acid as described above.

[0129] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, markergenes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate in the context.

[0130] A nucleic acid molecule or expression vector as described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid or vectors into host cells are well established in the art and any suitable technique may be employed. A range of host cells suitable for the production of recombinant antibody molecules and conjugates are known in the art, and include bacterial, yeast, insect or mammalian host cells. A preferred host cell is a mammalian cell, such as a CHO, CHO-S, NSO, or HEK cell, for example a HEK293 cell.

[0131] Another aspect of the invention provides a method of producing an antibody molecule, or conjugate, comprising expressing a nucleic acid encoding the antibody molecule, or conjugate, in a host cell and optionally isolating and / or purifying the antibody molecule, or conjugate, thus produced. Methods for culturing host cells are well-known in the art. The method may further comprise isolating and / or purifying the antibody molecule or conjugate. Techniques for the purification of recombinant antibody molecules, or conjugates, are well-known in the art and include, for example HPLC, FPLC, or affinity chromatography, e.g. using Protein A or Protein L. In some embodiments, purification may be performed using an affinity tag on antibody molecule. The method may also comprise formulating the antibody molecule, or conjugate, into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described herein.

[0132] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0133] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention shown above are illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0134] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0135] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0136] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0137] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of’.

[0138] Examples

[0139] EXAMPLE 1 - Cloning of Prostate Specific Membrane Antigen (PSMA) including characterization, phage display selection against antigen, and isolation of CC105RR antibody in scFv format

[0140] 1.1 Expression procedure

[0141] A human PSMA recombinant fragment containing a N-terminal His6 tag followed by an Avi-tag was expressed using transient gene expression (TGE) in CHO-S cells. For 1 mL of production 4 x 106CHO-S cells in suspension were centrifuged and resuspended in 1 mL of a suitable medium. 0.9 pg of plasmid DNA followed by 2.5 pg polyethylene imine (PEI; 1 mg / mL solution in water at pH 7.0) per million cells were then added to the cells and gently mixed. The transfected culture was incubated in a shaker incubator at 31 °C for 6 days. The protein fragment was purified from the cell culture medium by using nickel affinity chromatography and then dialyzed into HEPES buffer (50 mM HEPES.1 mM CaCI2, 0.5 mM MgCI2, and 100 mM NaCI, pH 7.5) and stored at -80°C.

[0142] 1.2 Antigen characterization

[0143] The human PSMA recombinant protein was analyzed by SDS-PAGE and by size exclusion chromatography using a Superdex200 increase 10 / 300 GL column on an KTA FPLC.

[0144] 1.3 Antigen biotinylationThe purified human PSMA recombinant fragment was site-specifically biotinylated with Bir-A and biotin. PD-10 desalting columns with sephadex G-25 M resin were used to replace the protein’s buffer with BirA buffer (100 mM Tris-HCI, 200 mM NaCI and 5 mM MgCI2 in Milli-Q water, pH 7.5) for site-specific biotinylation of the Avi-tagged recombinant PSMA. To perform the biotinylation reaction, a complete™, EDTA-free Protease Inhibitor Cocktail tablet was added to the recombinant protein and combined with 788 pl of Biotin (5 mM dissolved in DMSO), 245 pl of ATP disodium salt hydrate (0.5 M in 1 M Tris-HCI pH 9.5) and 150 pg of BirA-maltose-binding protein. The biotinylation reaction tube was covered with aluminum foil and shaken in a Sample Mixer at RT overnight. The next day, biotinylated human PSMA was purified by size exclusion chromatography on a Superdex200 increase column in HEPES buffer. Collected fractions were pooled, snap frozen in liquid nitrogen, and stored at -80°C.

[0145] 1.4 Phage display selection

[0146] The biotinylated human PSMA was used to perform biopanning with Dynabeads. Briefly, the biotinylated human PSMA (final concentration 120 pmol) was incubated with 800 pL of a pre-blocked phage display library for 30 minutes. After several washes with HEPES buffer (100 mM NaCI, 50 mM HEPES, pH 7.4), selected phages were eluted by reducing the disulfide bonds in the biotin linker with triethylamine.

[0147] Isolated phages were then amplified in E. coli strain TG-1 and precipitated from the supernatant with polyethylene glycol.

[0148] After two rounds of biopanning, clones were screened by ELISA. Avidin-coated ELISA plates were incubated with biotinylated human PSMA. The supernatants of selected induced monoclonal clones of the E. coli TG-1 cultures expressing scFv antibody fragments were added to the ELISA plates and bound scFvs were detected using the anti-c-myc antibody 9E10 followed by use of an anti-mouse IgG -horseradish peroxidase (HRP) conjugate.

[0149] 1.5 In-vitro characterization of antibody CC105RR in scFv format

[0150] The antibody clone that resulted in the highest ELISA signal, CC105RR, was produced in E. coli strain TG-1. A TG-1 culture was grown at 37°C in 2xTY / 100 pg / ml ampicillin. At ODeoo = 0.5, 1mM isopropyl-thio-galactopyranoside (IPTG) was added to induce expression of the scFv; the culture was incubated on a bacterial incubator shaking at 175 rpm at 30°C overnight. The culture was then centrifuged, and the supernatant purified from the cell culture medium by protein-A affinity chromatography and then dialyzed against PBS and stored in PBS at -80°C. The CC105RR scFv was then characterized by size exclusion chromatography using a Superdex 75 increase 10 / 300 GL column on an KTA FPLC. SDS-PAGE analysis was also performed with 4-12% Bis-Tris gel under reducing and non-reducing conditions (Figure 1).

[0151] 1.6 Cloning and expression of antibody CC105RR in scFv formatThe CC105RR scFv was cloned into a vector for mammalian expression. The primers were designed to add Nhel and Hindlll restriction sites: “Leader Seq DP47” > and “Hindi 11 Stop Myc” <.

[0152] The resulting fragment was PCR amplified with “Nhel Leader fwd” > and “Hindlll Stop Myc” < to add the restriction site for Nhel. The PCR product was digested with Nhel and Hindlll and ligated into a vector digested with the same enzymes.

[0153] CC105RR scFvwas then expressed using transient gene expression (TGE) in CHO-S cells (as described above). The CC105RR scFvwas purified from the cell culture medium by protein A affinity chromatography and then dialyzed against PBS and stored in PBS at -80°C. The amino acid sequence of CC105RR scFv is shown in SEQ ID NO: 12.

[0154] 1.7 Results

[0155] A new anti-PSMA antibody termed “CC105RR” in scFv format was isolated using phage display and characterized using SDS-PAGE (Figure 1A) and SEC (Figure 1B) analysis.

[0156] These results showed that CC105RR scFv had the expected molecular weight under reducing and nonreducing conditions and was eluted from the SEC columns at 12.5 mL. The antibody molecule showed excellent purity, as evidenced by the single peak observed by SEC.

[0157] EXAMPLE 2: Cloning, expression and in vitro characterization of the CC105RR antibody in lqG4 format

[0158] 2.1 Cloning of the CC105RR antibody into lgG4 format

[0159] Cloning of the CC105RR antibody in lgG4 format commenced by cloning the light chain. Primers were designed to insert Spel and BsiWI restriction sites:

[0160] “Leader DPK22 fwd”> and “DPK22 BsiWI” <

[0161] The resulting fragment was PCR amplified to add the leader sequence with the following primers: “Spel Leader fwd” > and “DPK22 BsiWI” <.

[0162] The resulting fragment was digested with Spel and BsiWI and ligated into a suitable vector previously digested with the same restriction enzymes.

[0163] The cloning procedure was continued with the cloning of the CC105RR heavy chain.

[0164] Primers were designed to insert Hindlll and Xhol restriction sites:

[0165] Hind II I Leader fwd” > and “DP47 Xhol” <The final PCR product was digested with Hindlll and Xhol and ligated into a suitable vector with the light chain as insert previously digested with the same restriction enzymes. The amino acid sequence of the CC105RR antibody in lgG4 format is shown in SEQ ID NOs 14 and 15.

[0166] The same cloning strategy was used to prepare the anti-hen egg lysozyme antibody “KSF” in lgG4 format (used herein as negative control).

[0167] 2.2 Characterization of the CC105RR antibody in lgG4 format

[0168] Antibody CC105RR in lgG4 format was expressed using transient gene expression (TGE) in CHO-S cells and purified by protein-A affinity chromatography, dialyzed, and stored in PBS (as described above).

[0169] Proteins were characterized by SDS-PAGE (Figure 2A) and size-exclusion chromatography using a Superdex200 increase 10 / 300 GL column on an KTA FPLC as also described above (Figure 2B).

[0170] 2.3 Results

[0171] The results show that the CC105RR antibody in lgG4 format had the expected molecular weight of 150 kDa under non-reducing conditions and 25 and 50 kDa under reducing conditions and showed excellent purity, as evidenced by SEC. Remarkably, the production yield for CC105RR antibody in lgG4 format was 50 mg / liter.

[0172] EXAMPLE 3: Cloning, expression and in vitro characterization of the CC105RR antibody in SIP format

[0173] 3.1 Cloning, expression and in vitro characterization of the CC105RR antibody in SIP format

[0174] The CC105RR antibody was cloned into SIP (small immunoprotein) format and the protein was expressed in CHO cells using pcDNA3.1 as the expression vector. The gene encoding the CC105RR antibody was PCR amplified with:

[0175] “Nhel Leader fwd” > (CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC)

[0176] and “DPK22_CH4_ba” <

[0177] A gene containing CH4 domain was PCR amplified with: “CH4_fwd” > and “Not_STOP_CH4_ba” <

[0178] The resulting PCR fragments were PCR assembled and cloned into the mammalian expression vector pcDNA3.1(+) using a Nhel / Notl restriction site, as described previously.

[0179] The SIP protein was expressed using transient gene expression in CHO cells as described previously and purified from the cell culture medium to homogeneity by Protein A chromatography. The CC105RRantibody in SIP format was analyzed by size-exclusion chromatography using a Superdex 200 increase 10 / 300 GL column on an AKTA FPLC. SDS-PAGE was performed using a 4-12% Bis-Tris gel under reducing and non-reducing conditions.

[0180] The amino acid sequence of CC105RR in SIP format is shown in SEQ ID NO: 16.

[0181] The same cloning strategy was used to prepare antibody KSF in SIP format.

[0182] 3.2 Characterization of the CC105RR antibody in SIP format

[0183] Antibody CC105RR in SIP format was expressed using transient gene expression (TGE) in CHO cells and purified by protein-A affinity chromatography, dialyzed and stored in PBS (as described above).

[0184] Proteins were characterized by SDS-PAGE (Figure 3A) and size-exclusion chromatography using a Superdex 200 increase 10 / 300 GL column on an KTA FPLC as also described above (Figure 3B).

[0185] 3.3 Results

[0186] The results show that the CC105RR antibody in SIP format had the expected molecular weight of 75 kDa under non-reducing conditions and 37 kDa under reducing conditions and showed excellent purity, as evidenced by SEC. Remarkably, the production yield for CC105RR antibody in SIP format was 6.5 mg / liter.

[0187] EXAMPLE 4: Cloning, expression of the CC105RR-CSMut lqG1, preparation of CC105RR-GlyPro-MMAE and their in vitro characterization

[0188] 4.1 Protein Production and Purification

[0189] The CC105RR-CSMut lgG1 antibody is the precursor of CC105RR-GlyPro-MMAE antibody-drug conjugate. The cysteine residues at positions 219, 225, and 228 of the heavy chain CC105RR antibody in lgG1 format were mutated into serine residues. For each light chain, only one single reactive cysteine residue (C215) was left at their C-terminus (SEQ ID NO: 15). The CC105RR-CSMut antibody in IgG 1 format (SEQ ID NO: 28) was cloned into the mammalian expression vector pCDNA(3.1)+ using strategies elsewhere described (Cazzamalli, J Am Chem Soc, 140, 1617-1621 (2018)). The recombinant protein was produced in CHO cells via polyethyleneimine (PEI)-mediated transient gene expression (TGE) and purified by Protein-A affinity chromatography (Roche). Buffer A (100 mM NaCI, 0.5 mM EDTA, 0.1% Tween 20 in PBS) and Buffer B (500 mM NaCI, 0.5 mM EDTA in PBS) were used during the purification process as washing steps. Antibody products were eluted using 10-15 ml 0.1 M glycine at pH = 3, fractions containing protein were collected, pooled, and dialyzed against phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. Purified proteins were characterized by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column on an AKTA Pure FPLC system (GE Healthcare).Protein integrity and purity were further assessed by SDS-PAGE using a 4-12% gradient gel in 1x MOPS buffer under reducing and non-reducing conditions (Figure 4A).

[0190] 4.2 Antibody Drug Conjugate Preparation

[0191] First, the two reactive cysteines of the CC105RR-CSMut lgG1 antibody (one in each light chain) were reduced with 20 equivalents of TCEP-HCI in PBS (pH = 7.4) at 37°C with overnight shaking at 550rpm. The next day, 40 equivalents of purified MC-GlyPro-PABC-MMAE were added to the reduced protein (5% v / v of DMSO). The mixture was stirred at 550 rpm at RT for 2 hours, and the final conjugated product (CC105RR-GlyPro-MMAE) was purified on a PD-10 column. Collected fractions containing protein were pooled and dialyzed against phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. The purified conjugated antibody was characterized by size-exclusion chromatography (SEC) using a Superdex200 Increase 10 / 300 GL column on an AKTA Pure FPLC system (GE Healthcare). Protein integrity and purity were further assessed by SDS-PAGE using a 4-12% gradient gel in 1x MOPS buffer under reducing and non-reducing conditions (Figure 4B). A schematic illustration of the antibody-drug conjugate is reported in Figure 4E.

[0192] 4.3 Mass Spectrometry Sample Preparation for Analytical Runs

[0193] Samples were diluted to about 0.1 mg / ml, and LC-MS was performed on a Waters Xevo G2XS Qtof instrument (ESI-ToF-MS) coupled to a Waters Acquity UPLC H-Class System using a 2.1 x 50 mm Acquity BEH300 C4 1.7 pm column (Waters). 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) were used as mobile phase at a flow rate of 0.4 ml / min. Gradient was programmed as follows: after 1.5 min isocratic with 95% solvent A, stepwise change from 95% solvent A to 95% solvent B in 4.5 min (10% increase every 0.5 min), back to 95% solvent A in 0.5 min, linearly to 95% solvent B and back to 95% solvent A in 2.25 min (last step repeated twice) (Figures 4C and 4D).

[0194] 4.4 Results

[0195] All cysteine residues in the hinge region of the CC105RR lgG1 heavy chain were mutated into serine to enable site-specific conjugation at the C-terminal cysteine residues present in the light chain using a thiolreactive maleimide-Glycine-Proline-MMAE linker-payload module (GlyPro-MMAE). Substitution of the CC105RR-CSMut lgG1 with a cysteine residue yielded a conjugate devoid of PSMA binding, which served as a negative control (Cys-GlyPro-MMAE). The identity and purity of the CC105RR-GlyPro-MMAE after production were assessed by SDS-PAGE, size-exclusion chromatography (SEC), and mass spectrometry (MS) (Figures 4B and 4D). CC105RR-GlyPro-MMAE antibody-drug conjugate exhibited a drug-to-antibody ratio (DAR) of 2.

[0196] EXAMPLE 5: Characterization of CC105RR in SIP format by Surface Plasmon Resonance5.1 Biacore analysis

[0197] Affinity measurements of the CC105RR in SIP format were performed by Surface Plasmon Resonance using BIAcore X100 instrument. A biotinylated human PSMA-coated SA chip was used and a final coating of 1500 RU was achieved, and samples were injected at a concentration of 500 nM.

[0198] No regeneration of the SA chip nor CM5 chip was performed, and the antibody fragments were left to detach from the antigen. KD measurements were assessed using the BIAevaluation 3.2 software.

[0199] 5.2 Results

[0200] The affinity (Kd) of the “CC105RR” antibody SIP (Figure 5) format against human PSMA as measured by BIAcore was calculated as 550 pM. Remarkably, the Kon was calculated as 2.23 x 105and the Koff was calculated as 1.22 x 10-4.

[0201] EXAMPLE 6: Cross-reactivity of CC105RR for human, cynomolqus monkey and guinea pig PSMA

[0202] 6.1 Cloning and expression of mouse, cynomolgous monkey, and guinea pig PSMA

[0203] The genes for the murine PSMA (mPSMA), for cynomolgous monkey PSMA (cmPSMA) and guinea pig PSMA (gpPSMA) were purchased in pcDNA 3.1 (+) vector.

[0204] All PSMA antigens were expressed using transient gene expression in CHO cells as described above and purified from the cell culture medium by using nickel affinity chromatography and then dialyzed into HEPES buffer (100 mM NaCI, 50 mM HEPES, pH 7.4) and stored at -80°C.

[0205] 6.2 ELISA analysis

[0206] Cross-reactivity of the anti-PSMA antibody CC105RR with mPSMA, cmPSMA and gpPSMA carrying a N-terminal His6 tag, was tested using ELISA. 100 nM of each mPSMA, cmPSMA and gpPSMA were coated on Maxisorp™ plates overnight at 4°C. The anti-hPSMA antibody CC105RR or the (negative control) KSF antibody, both in SIP format were incubated in the wells for 1 h at room temperature at a concentration of 5 pg / mL. For detection, Protein A-HRP (1 pg / ml in 2% MPBS, 50 pl / well) were given to the resulting complex for 1 h. In the final step, all wells were washed with 0.1% Tween in PBS (3x, 200 pl / well), PBS (3x, 200 pl / well), TMB (60 pl / well) was added, and the reaction was stopped with 1 M H2SO4 (30 pl / well). The absorbance (450 nm-620 nm) was measured using a microplate reader.

[0207] 6.3 Results

[0208] The anti-hPSMA CC105RR antibody was able to recognize and bind to cmPSMA and gpPSMA but not mPSMA (Figure 6). The negative control KSF antibody did not show binding to any antigen. An antihistidine tag antibody-HRP conjugate was used to confirm that the coating of the wells with mPSMA, cmPSMA or gpPSMA was performed correctly.The cross-reactivity of the CC105RR antibody with cynomolgous monkey and guinea pig PSMA in addition to human PSMA, allows the activity and tolerability of this antibody to be tested in many different animals that are expected to be more predictive of efficacy in human patients than mouse models, notably in models of cancer.

[0209] EXAMPLE 7: Specificity of CC105RR for PSMA

[0210] 7.1 ELISA Analysis

[0211] Specificity of the anti-PSMA antibody CC105RRto PSMA was tested using ELISA. 100 nM of different proteins were coated on Maxisorp™ plates overnight at 4°C. The anti-PSMA antibody CC105RR or the (negative control) KSF antibody, both in lgG4 format were incubated in the wells for 1 h at room temperature at a concentration of 5 pg / mL. For detection, anti-human-HRP (1 pg / ml in 2% MPBS, 50 pl / well) were given to the resulting complex for 1 h. In the final step, all wells were washed with 0.1% Tween in PBS (3x, 200 pl / well), PBS (3x, 200 pl / well), TMB (60 pl / well) was added, and the reaction was stopped with 1 M H2SO4 (30 pl / well). The absorbance (450 nm-620 nm) was measured using a microplate reader.

[0212] 7.2 Results

[0213] The anti-hPSMA CC105RR antibody was able to bind only to PSMA and not to other unrelated proteins such as CLL-1 , human FAP, human CAIX, etc. (Figure 7). The negative control KSF antibody did not show binding to any antigen. An anti-histidine tag antibody-HRP conjugate was used to confirm that the coating of the wells with all proteins was performed correctly.

[0214] EXAMPLE 8: Flow cytometry of CC105RR

[0215] 8.1 Flow Cytometry analysis

[0216] Binding of the CC105RR antibody in lgG4 format to cells expressing human PSMA was tested using a cell line artificially transduced to express human PSMA (hPSMA). The cell line used was the human fibrosarcoma cell line HT-1080.

[0217] Specifically, HT-1080-hPSMA cells were detached from cell culture plates using Accutase, counted and suspended to a final concentration of 1 x 106cells / mL in FACS buffer (0.5% BSA, 2mM EDTA in PBS). Cells were incubated with either CC105RR or KSF (negative isotype control) antibodies in lgG4 format and binding detected using a goat anti-human AlexaFluor488 antibody. Cells were analyzed on a CytoFLEX cytometer. The raw data were processed using the FlowJo 10.4 software.

[0218] 8.2 ResultsFlow cytometry analysis using HT-1080.hPSMA-confirmed the ability of the CC105RR antibody in lgG4 format to bind cells expressing human PSMA (Figure 8). The KSF lgG4 antibody (negative control) did not show any binding as expected.

[0219] EXAMPLE 9: Ex-vivo biodistribution analysis of the CC105RR antibody in tumor-bearing mice

[0220] 9.1 Ex-vivo experiment

[0221] 5x106HT1080.hPSMA cells were implanted subcutaneously in the flank of eight-week-old male BALB / c nude mice. For ex-vivo analysis, when tumors reached a size of 150-250 mm3, mice were injected with 200 pg CC105RR lgG4 and sacrificed 24 hours after injection. Organs were excised and embedded in cryo-embedding medium and cryostat section (10 pm). For detection, the antibody goat anti-human Alexa Fluor488 was used. Vasculature was visualized through staining with rat anti-mouse CD31 and donkey anti-rat Alexa Fluor594 antibodies.

[0222] 9.2 Results

[0223] The ability of the CC105RR antibody to efficiently target tumors was confirmed by ex-vivo analysis of binding of CC105RR (lgG4 format) to tumors expressing PSMA in tumor-bearing mice (Figure 9A). No binding was seen with the negative control KSF(lgG4) as expected (Figure 9B).

[0224] EXAMPLE 10: therapy studies with CC105RR-GlyPro-MMAE conjugate

[0225] 10.1 Experimental Animals

[0226] A total of 15 male BALB / c nu / nu mice (6-8 weeks old) were obtained from Janvier Labs (Le Genest-Saint-lsle, France). Animals were housed under specific pathogen-free conditions with controlled temperature (18-26°C), relative humidity (40-60%), and a 12 h light / dark cycle, in accordance with GVSOLAS and FELASA guidelines. Mice were kept in groups of up to five per cage and reallocated when necessary to avoid single housing. Experimental groups were assigned based on tumor volume, determined by caliper measurements and calculated as (length [mm] x width [mm]2) / 2. Blinding was not applied in this study.

[0227] 10.2 Synthesis of the negative control Cys-GlyPro-MMAE

[0228]

[0229] MC-GlyPro-PABC-MMAE (4.9 mg, 4.0 pmol, 1.0 equiv.) and Cysteine (0.6 mg, 4.8 pmol, 1.2 equiv.) were loaded into an Eppendorf tube and dissolved in a 1 :1 mixture of PBS and DMF (0.4 mL). DIPEA (3.5 pL, 20 pmol, 5.0 equiv.) was added and the reaction was incubated in a shaker incubator at 37 °C for 1 h.

[0230] The crude mixture was directly purified by RP-HPLC (gradient of 90:10 to 0:100 in 18 min water / ACN + 0.1% TFA) to afford Cys-GlyPro-MMAE as a white solid (4.0 mg, 3.0 pmol, 75% yield), m / z calculated for C67H103N10G16S: [M+H]+ 1335.73, detected: 1335.6.

[0231] 10.3 Therapy experiments

[0232] 5 x 106HT108O.hPSMA tumor cells were subcutaneously implanted into the right flank of 6- to 8-week-old male BALB / c nu / nu mice and allowed to grow to an average volume of 100 mm3. Mice were randomly assigned to therapy groups of 5 animals, and treatment started by injecting a solution of compounds or vehicle (PBS containing 2% of DMSO) intravenously (lateral tail vein) three times on alternate days at 125 nmol / kg (CC105RR-GlyPro-MMAE) or at 250 nmol / kg (Cys-GlyPro-MMAE), doses that correspond to equimolar doses of MMAE (250 nmol / kg). Cys-GlyPro-MMAE was injected as solutions in sterile PBS containing 2% DMSO. CC105RR-GlyPro-MMAE was injected as solutions in 1x PBS buffer. Animals were weighed and tumor sizes measured daily with an electronic caliper (Figure 10). The tumor volume was calculated according to the formula (long side) x (short side) x (short side) x 0.5. Animals were sacrificed when the termination criteria were reached. Prism 9 software (GraphPad Software) was used for data analysis (regular twoway ANOVA followed by Tukey’s test).

[0233] 10.4 Results

[0234] The in vivo efficacy of CC105RR-GlyPro-MMAE conjugate was evaluated in BALB / c nude mice bearing HT1080.hPSMA tumors (Figure 10A). CC105RR-GlyPro-MMAE and Cys-GlyPro-MMAE were given at equimolar doses of the cytotoxic payload, corresponding to 250 nmol / kg of MMAE. The CC105RR-GlyPro-MMAE conjugate induced complete responses in all treated animals bearing HT1080.hPSMA tumors (n = 5 mice / group), whereas no in vivo anti-tumor effect was observed in mice treated with the negative control Cys-GlyPro-MMAE. All treatments were well tolerated, with minimal changes in body weight observed (Figure 10B).Sequence Listing

[0235] SEQ ID NO: 1 - Amino acid sequence of the human Prostate Specific Membrane Antigen (hPSMA) -KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYD VLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFF KLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQR GNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKV PYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVV HEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVD CTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRA RYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVL RKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAF IDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETL SEVA

[0236] SEQ ID NO: 2 - Amino acid sequence of the cynomoloqus monkey Prostate Specific Membrane Antigen (cmPSMA) - KSSSEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELTHYD VLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFF KLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPADYFAPGVKSYPDGWNLPGGGVQR GNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSASPDSSWRGSLKV PYNVGPGFTGNFSTQKVKMHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVV HEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVD CTPLMYSLVYNLTKELESPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRA RYTKNWETNKFSSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSVVLPFDCRDYAVVL RKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAF IDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAAAETLS EVA

[0237] SEQ ID NO: 3 - Amino acid sequence of the guinea pig Prostate Specific Membrane Antigen (gpPSMA) KSSNEATNIAPQHGMKKAFLNGLKAENIKEFLYNFTRVPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAH YDVLLSYPSKTHPNYISVINEDGSEVFNTSLFEPPPPGYENVSDIVPPFSAFSPQGVPEGDLVYVNYARIE DFFKLEQDMKINCSGKIVIARYGKIFRGNKVKNAQLAGAKGVILYSDPADYFAPGVASYPDGWNLPGGGV QRGNILNLNGAGDPLTPGYPANEYAYRHGITEAVGLPHIPVHPIGYYDAQRLLEHMGGSAPPDSSWKGS LQVPYNVGPGFTGNFSTQKVRMHIHSSSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGA AVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEYGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTL RVDCTPLMYSLVYNLTKELQSPDEGFEGKSLYESWSEKSPSHELSGVPRISKLGSGNDFEVFFQRLGIAS GRARYTKNWETNKFSNYPLYHSVYETYELVEKFYDPTFKYHLTVAQVRGGMVFELANSVVLPFNCQDYAVVLKKYADKIYNISMKYPQEMKTYSVSFDSLFSAINNFTEIASKFNKRLQSLDKSNPILLRIMNDQLMFLER AFIDPLGLPDRPFYRHIIYAPSSHNKYAGESFPGIYDALFDIKSKTDPSKAWAEVKRQISIAAFTVQAAAGTL REVA

[0238] SEQ ID NO: 4 - Amino acid sequence of CC105RR CDR1 VH

[0239] GFTFSPFSMS

[0240] SEQ ID NO: 5 - Amino acid sequence of CC105RR CDR2 VH

[0241] AISDTGGTTYYADSVKG

[0242] SEQ ID NO: 6 - Amino acid sequence of CC105RR CDR3 VH

[0243] SRWVFDY

[0244] SEQ ID NO: 7 - Amino acid sequence of CC105RR CDR1 VL

[0245] RASQSVSSSYLA

[0246] SEQ ID NO: 8 - Amino acid sequence of CC105RR CDR2 VL

[0247] GASSRAT

[0248] SEQ ID NO: 9 - Amino acid sequence of CC105RR CDR3 VL

[0249] QQSMGLPAT

[0250] SEQ ID NO: 10 - Amino acid sequence of the CC105RR VH domain EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSS

[0251] SEQ ID NO: 11 - Amino acid sequence of the CC105RR VL domain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK

[0252] SEQ ID NO: 12 - Amino acid sequence of the CC105RR antibody molecule in scFv format

[0253] The linker sequence is underlined.

[0254] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK

[0255] SEQ ID NO: 13 - Amino acid sequence of the linker between VH and VL in CC105RR scFvGGGGSGGGGSGGGG

[0256] SEQ ID NO: 14 - Amino acid sequence of the CC105RR heavy chain in lqG4 format EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0257] SEQ ID NO: 15 - Amino acid sequence of the CC105RR light chain in lqG4 or lqG1 format EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

[0258] SEQ ID NO: 16 - Amino acid sequence of the CC105RR antibody molecule in SIP format

[0259] The linker sequences are underlined.

[0260] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIKSGGSGGPRAAPEVYAFATPEWPGSRDKRTLACLIQ NFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQT VQRAVSVNPESSRRGGC

[0261] SEQ ID NO: 17 - Amino acid sequence of the linker between scFvand ES2 CH4 domain in the SIP format SGGSGG

[0262] SEQ ID NO: 18 - Amino acid sequence of human interleukin 2 (IL-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0263] SEQ ID NO: 19 - Amino acid sequence of the CC105RR (scFv)-IL2 conjugate

[0264] The linker sequences are underlined.

[0265] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLD LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIV LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0266] SEQ ID NO: 20 - Amino acid sequence of the soluble form of the extracellular domain of human Tumor Necrosis Factor a (TNF) VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPD YLDFAESGQVYFGIIAL

[0267] SEQ ID NO: 21 - Amino acid sequence of the CC105RR (scFv)-TNF conjugate

[0268] The linker sequences are underlined.

[0269] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHWANP QAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTK VNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0270] SEQ ID NO: 22 - Amino acid sequence of human interleukin 12 (IL-12)

[0271] The linker sequence is underlined IWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCH KGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRG SSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIR DIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVIC RKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLL RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKT SFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEP DFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0272] SEQ ID NO: 23 - Amino acid sequence of the CC105RR (scDb)-IL12 conjugate IWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCH KGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIR DIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVIC RKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLL RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKT SFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEP DFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGSADGGSSAGGSDAGEVQLLESGGGLVQPGGSLRLSC AASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAKSRWVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK SSSSGSSSSGSSSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISD TGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGSGG EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK

[0273] SEQ ID NO: 24 - Amino acid sequence of the CC105RR antibody in diabody format (Db)

[0274] The linker sequence is underlined.

[0275] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPG ERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQSMGLPATFGQGTKVEIK

[0276] SEQ ID NO: 25 - Amino acid sequence of the CC105RR antibody in single-chain diabody format (scDb) The linker sequences are underlined.

[0277] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPG ERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQSMGLPATFGQGTKVEIKDIGGGSGGGGSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSP FSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSR WVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK

[0278] SEQ ID NO: 26 - Amino acid sequence of the CC105RR antibody in single-chain diabody format (scDb) The linker sequences are underlined.

[0279] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPG ERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQSMGLPATFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSP FSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSMGLPATFGQGTKVEIK

[0280] SEQ ID NO: 27 - Amino acid sequence of the linker between VH and VL in the CC105RR Db and CC105RR scDb

[0281] GGSGG

[0282] SEQ ID NO: 28 - Amino acid sequence of CC105RR -CSMut lqG1 heavy chain

[0283] The wild-type cysteine residues of the heavy chain in positions 219, 225, and 228 were mutated into serine residues (underlined) EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0284] SEQ ID NO: 29 - Amino acid sequence of CC105RR lqG1 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSPFSMSWVRQAPGKGLEWVSAISDTGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRWVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

Claims1. A fully human antibody molecule that specially binds to human, cynomolgus monkey and guinea pig prostate-specific membrane antigen (PSMA).

2. An antibody molecule according to claim 1 , wherein the antibody molecule comprises a VH domain comprising a set of complementarity determining regions HCDR1 , HCDR2 and HCDR3, and a VL domain comprising a set of complementarity determining regions LCDR1 , LCDR2 and LCDR3, wherein:the HCDR1 , HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs 4, 5 and 6, respectively, andthe LCDR1 , LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs 7, 8 and 9, respectively.

3. An antibody molecule according to claim 2, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO: 10 and / or the VL domain comprises the amino acid sequence shown in SEQ ID NO: 11.

4. An antibody molecule according to any one of claims 1 to 3, wherein the antibody molecule comprises or consists of: a single chain Fv (scFv), a small immunoprotein (SIP), a diabody (Db), a singlechain diabody (scDb), or an IgG molecule, optionally an IgG 1 or lgG4 molecule.

5. An antibody molecule according to claim 4, wherein:the antibody molecule is in scFv format and comprises the amino acid sequence set forth in SEQ ID NO: 12;the antibody molecule is in diabody format and comprises the amino acid sequence set forth in SEQ ID NO: 24;the antibody molecule is in single-chain diabody format and comprises the amino acid sequence set forth in SEQ ID NOs: 25 or 26;the antibody molecule is in lgG4 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 14, and the light chain amino acid sequence set forth in SEQ ID NO: 15;the antibody molecule is in lgG1 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 28, and the light chain amino acid sequence set forth in SEQ ID NO: 15;the antibody molecule is in lgG1 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 29, and the light chain amino acid sequence set forth in SEQ ID NO: 15; orthe antibody molecule is in small immunoprotein (SIP) format and comprises the amino acid sequence set forth in SEQ ID NO: 16.

6. A conjugate comprising an antibody molecule according to any one of claims 1 to 5 and a pro-inflammatory agent, a biocidal molecule, a cytotoxic molecule, or a radioisotope, optionally wherein the pro-inflammatory agent, biocidal molecule, cytotoxic molecule, or radioisotope is conjugated to the N-terminus or C-terminus of the antibody molecule.

7. A conjugate according to claim 6, wherein the pro-inflammatory agent is a cytokine, optionally wherein the cytokine is interleukin 2 (IL2), interleukin 12 (IL12), or tumor necrosis factor alpha (TNFa) or a variant thereof.

8. A conjugate according to claim 7, wherein(i) the antibody molecule is an scFv and is conjugated at its C-terminus to interleukin 2 (IL2), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 19;(ii) the antibody molecule is an scFv and is conjugated at its C-terminus to tumor necrosis factor alpha (TNFa), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 21,(iii) wherein the antibody molecule is a single-chain diabody and is conjugated at its N-terminus to interleukin 12 (IL12), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 23.

9. A conjugate according to claim 6, wherein the cytotoxic molecule is MMAE, optionally wherein the cytotoxic molecule is conjugated to the C-terminus of the light chain of the antibody molecule.

10. A conjugate according to claim 9, wherein the antibody molecule is an IgG antibody, wherein the conjugate optionally comprises the amino set forth in SEQ ID NOs: 28 and 15 and wherein the cytotoxic molecule is optionally conjugated through a MC-GlyPro-PBAC linker.

11. The antibody molecule or conjugate according to any one of claims 1 to 10 for use in a method of treating a disease or disorder characterised by expression of PSMA, optionally cancer, in a patient.

12. The antibody molecule or conjugate for use according to claim 11 , wherein the method further comprises administering a second therapeutic agent to the patient, optionally wherein the second therapeutic agent is an immunomodulatory agent, such as an anti-PD-1 antibody.

13. A nucleic acid molecule or expression vector encoding an antibody molecule or conjugate according to any one of claims 1 to 12.

14. A host cell comprising the nucleic acid or vector of claim 13.

15. A method of producing an antibody molecule or conjugate according to any one of claims 1 to 10, the method comprising culturing the host cell of claim 14 under conditions forexpression of the antibody molecule or conjugate, and optionally further isolating and / or purifying the antibody molecule or conjugate following expression.

16. A pharmaceutical composition comprising an antibody molecule or conjugate according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

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