PSMA binding molecule

Abstract

The present invention relates to PSMA-binding binding molecules, polypeptides and constructs comprising PSMA-specific single domain variable heavy immunoglobulin (VHH) domains. The present invention further relates to payload-comprising conjugates thereof.

Description

[0001] PSMA BINDING MOLECULE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to PSMA-binding binding molecules, polypeptides and constructs comprising PSMA-specific single domain variable heavy immunoglobulin (VHH) domains. The present invention further relates to payload-comprising conjugates thereof.

[0004] BACKGROUND TO THE INVENTION

[0005] Prostate-specific membrane antigen (PSMA) is a membrane protein commonly expressed in prostate tissue, comprising an internal portion, a transmembrane portion, and an external portion. PSMA has increased expression in cancer tissue, including in prostate cancer but also in other types of cancer. PSMA has therefore been proposed as a diagnostic and therapeutic target for such cancers, including via the use of antibodies targeting PSMA.

[0006] Antibodies, also known as immunoglobulins, are large, multi-domain, multi-subunit proteins that are used in immunotherapy to bind to (and inhibit) therapeutic targets. Single domain variable heavy immunoglobulins (comprising VHHs, also known as nanobodies or single-domain antibodies) are small antibody-based fragments comprising a single variable heavy (VH) immunoglobulin domain. A key difference between single domain antibodies and conventional antibodies is therefore the lack of constant heavy 1 (CH1), variable light (VL) and constant light (CL) domains. VHHs per se refer to the variable domain and typically do not include any constant domains (i.e. CH2 and CH3 domains).

[0007] In some approaches, therapeutic or diagnostic agents may be conjugated to an antibody (or fragment derived therefrom), resulting in e.g. antibody drug conjugates (ADCs). Such ADCs can comprise VHHs.

[0008] An early affinity study has been carried out to investigate the targeting of PSMA with VHH fragments (Zare et al., Int J Biol Markers 2014; 29(2):e169-e179), but the authors acknowledge that this preliminary work requires further validation for the strategy of using VHH fragments alone. Another study into VHHs targeting PSMA discloses inhibitory effects of VHH Humabody-based ADCs, with a conjugated DGN549 payload (Nessler et al., Cancer Res.

[0009] 2020; 80(6): 1268-1278). This study attributed its most promising in vivo results to a poorly / slowly internalising single VHH domain fused to an albumin-binding domain. A study has also been carried out on a VHH fragment conjugated to111In, which was unable to achieve more than 37% internalisation after 90 minutes at 37°C (Chatalic et al., J Nucl Med 2015; 56:1094-1099).Although various approaches have been investigated in the art, the present inventors have identified that there is still a need for novel, improved immunotherapeutic candidates targeting PSMA, e.g. with improved internalisation. For such candidates, there is also a need to provide improved antibody-based formats to optimise the delivery of conjugated payloads, whilst simultaneously obtaining desirable producibility characteristics. In addition, some preliminary VHH fragments targeting PSMA have been investigated to an extent, but it has not yet been considered whether a novel VHH-based format could be used to target PSMA, whilst also obtaining the benefits of Fc region functionality, and / or the benefits of further “adaptor” domains and moieties to increase functionality. Thus, there is a need in the art to provide new and advantageous anti-PSMA binding molecules and conjugates. However, the need to balance many desirable properties in this respect (e.g. target binding and affinity, internalisation, stability, producibility, payload optimisation, effector functions and efficacy) means that it is far from straightforward to develop suitable new candidates.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides novel, advantageous binding molecules, constructs and conjugates targeting PSMA. In particular, the present invention provides advantageous VHH domain candidates and the sequences thereof, which have many advantageous properties including consistently and surprisingly high internalisation rates, and excellent target affinities. The present invention further provides VHH-based conjugates having optimised payload delivery whilst also maintaining target binding, stability, producibility and effector functions (where present). Further, the present invention validates novel approaches of anti-PSMA VHH-based constructs comprising further immunoglobulin domains for improved functionality - either an Fc region or certain constant immunoglobulin domains with an adaptor domain / moiety (e.g. a further VHH domain targeting an ALFA tag).

[0012] Accordingly, in an aspect, the present invention provides a binding molecule comprising a single domain variable heavy immunoglobulin (VHH) domain that specifically binds to prostate-specific membrane antigen (PSMA). In some embodiments, the binding molecule is a polypeptide. In some embodiments, the binding molecule comprises SEQ ID NOs: 1 to 3, and / or SEQ ID NOs 4 to 6, and / or SEQ ID NO: 7 (candidate PSMA1). In some embodiments, the binding molecule comprises SEQ ID NOs: 8 to 10, and / or SEQ ID NOs 11 to 13, and / or SEQ ID NO: 14 (candidate PSMA2). In some embodiments, the binding molecule comprises SEQ ID NOs: 15 to 17, and / or SEQ ID NOs 18 to 20, and / or SEQ ID NO: 21 (candidate PSMA3).In some embodiments, the binding molecule of the present invention comprises a VHH domain and one or more immunoglobulin constant domains.

[0013] In some embodiments, the binding molecule of the present invention comprises a VHH domain and an additional binding moiety or a cognate of a binding moiety (e.g. a further VHH domain that specifically binds to an ALFA-tag (NbALFA)). In some embodiments, the binding molecule comprises one or more immunoglobulin constant domains and an additional binding moiety or cognate of a binding moiety. In some embodiments, the binding molecule comprises an anti-PSMA VHH, an Fc domain comprising a KiH mutation, and an NbALFA domain, optionally in that order (Figure 1, RD3). In some embodiments, the binding molecule comprises an anti-PSMA VHH, an Fc domain, and an NbALFA domain, optionally in that order (Figure 1, RD4). In some embodiments, the binding molecule comprises an anti-PSMA VHH, a CH3 domain optionally comprising a KiH mutation, and an NbALFA domain, optionally in that order (Figure 1, RD5). In some embodiments, the binding molecule comprises an anti-PSMA VHH, an NbALFA domain and an Fc domain optionally comprising a KiH mutation, optionally in that order (Figure 1, RD6). In some embodiments, the order in the binding molecule in question is N to C terminal, e.g. of the polypeptide.

[0014] In some embodiments, the binding molecule of the present invention further comprises a hinge region located between the VHH domain and the additional domain(s) or moiety, e.g. located between the VHH domain and the one or more immunoglobulin constant domains. In some embodiments, the hinge region is typically located between the VHH domain and the Fc region. In an embodiment, the hinge region is derived from lgG1, lgG2, lgG3 or lgG4 (e.g. it comprises or consists of a sequence derived from an lgG1 lgG2, lgG3 or lgG4 hinge sequence). In a highly preferred embodiment, the hinge region is derived from lgG1 or lgG3 (e.g. it comprises or consists of a sequence derived from an lgG1 or lgG3 hinge sequence, preferably having 80% identity to SEQ ID NO: 22 or SEQ ID NO: 23). In an embodiment, the hinge region is not derived from I gG2, an lgG2 hinge region or a variant thereof. Typically, the hinge region of the invention is used to link the (anti-PSMA) VHH domain of the binding molecule to an Fc region. Thus, typically, the binding molecule of the present invention comprises a VHH domain, a hinge region and an Fc region, e.g. wherein the hinge region links the VHH domain and the Fc region. As the hinge region may provide sites for conjugation to payloads, such embodiments of the present invention comprising the hinge region are particularly important for the binding molecule-payload conjugates (BPCs) of the present invention.

[0015] In an aspect, the present invention provides a construct comprising a first binding molecule according to the invention and a second binding molecule according to the invention.In an aspect, the present invention provides a binding molecule-payload conjugate (BPC) comprising a binding molecule or construct according to the invention, and one or more payload(s), optionally further comprising a linker connecting the binding molecule or conjugate to the payload(s). In some embodiments, the conjugate comprising a binding molecule according to the invention has a payload to binding molecule ratio of 2 or 4, preferably wherein the conjugate comprises a hinge region derived from lgG1 or lgG3 respectively. In some embodiments, the conjugate comprising the construct of the present invention (e.g. comprising a first and second binding molecule of the invention) has a payload to BPC ratio of 4 or 8, preferably wherein each of the first and second binding molecules in the construct comprises a hinge region derived from lgG1 or lgG3 respectively.

[0016] In an aspect, the present invention provides one or more nucleic acid sequence(s) capable of expressing the binding molecule or construct of the invention.

[0017] In an aspect, the present invention provides a vector comprising the one or more nucleic acid sequence(s) of the invention.

[0018] In an aspect, the present invention provides a cell comprising the binding molecule, construct, BPC, one or more nucleic acid sequence(s) or vector of the invention.

[0019] In an aspect, the present invention provides a method for making a cell according to the invention, comprising the step of introducing the one or more nucleic acid sequence(s) or vector of the invention into said cell.

[0020] In an aspect, the present invention provides a method for producing the binding molecule or construct according to the invention, wherein the method comprises the steps of:

[0021] (i) introducing the one or more nucleic acid sequence(s) or vector according to the invention into a cell; and

[0022] (ii) expressing the binding molecule or construct in the cell;

[0023] and optionally (iii) harvesting the binding molecule or construct thereof from the cell or cell culture supernatant of the cell.

[0024] In an aspect, the present invention provides a composition comprising the binding molecule, construct or BPC of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.In an aspect, the present invention provides an in vitro method comprising contacting a cell with the binding molecule, construct, BPC or composition of the invention.

[0025] In an aspect, the present invention provides the binding molecule, construct, BPC or composition of the invention for use in a method of therapy or a diagnostic method.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Overview of NbALFA (ProteinDocker) construct formats. Anti-PSMA VHHs are depicted in dark grey, immunoglobulin constant domains derived from the Fc part are depicted in medium grey. ALFA-binding VHHs (NbALFA) are highlighted in light grey. The format description including the valency is stated below each format illustration, while the format abbreviation as used herein is written above in bold letters. KiH: Knob-into-Hole.

[0028] Figure 2: Internalization of ProteinDocker constructs. Internalization of ProteinDocker constructs into target positive LNCaP cells was investigated using a FACS-based assay.

[0029] Figure 3: Overview of VHH-Fc construct formats. Anti-PSMA VHHs are depicted in dark grey, lgG1 Fc parts are depicted in light grey. Interchain disulfide bonds within the hinge regions are depicted as horizontal lines in medium grey.

[0030] Figure 4: Internalization of VHH-Fc fusion constructs. Internalization of VHH-Fcs into target positive LNCaP cells was investigated using a FACS-based assay.

[0031] Figure 5: Internalization of VHH-Fc fusion proteins using any high content imagingbased assay. 5A) shows the internalization of PSMA1 VHH-Fc lgG1, PSMA1 VHH-Fc lgG3, PSMA2 VHH-Fc lgG1, PSMA2 VHH-Fc lgG3, PSMA3 VHH-Fc lgG1, PSMA3 VHH-Fc lgG3 into CHO-FIpIn-human PSMA cells by high-content imaging. Percent internalization of 5nM anti-PSMA VHH-Fc was determined after 0, 1 or 4 hrs incubation at 37°C. At timepoint 0, anti-PSMA VHH-Fc binding to the cells was determined and plotted as mean total cellular Alexa Fluor 488 Intensity. 5B) shows the internalization of PSMA1 VHH-Fc lgG1, PSMA1 VHH-Fc lgG3, PSMA2 VHH-Fc lgG1, PSMA2 VHH-Fc lgG3, PSMA3 VHH-Fc lgG1, PSMA3 VHH-Fc lgG3 into LNCaP cells by high-content imaging. Percent internalization of 5nM anti-PSMA VHH-Fc was determined after 0, 1 or 4 hrs incubation at 37°C. At timepoint 0, anti-PSMA VHH-Fc binding to the cells was determined and plotted as mean total cellular Alexa Fluor 488 Intensity.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] VHH domains and binding moleculesIn an aspect, the present invention provides a binding molecule comprising a single domain variable heavy immunoglobulin (VHH) domain that specifically binds to prostatespecific membrane antigen (PSMA). Accordingly, in embodiments, PSMA is considered the antigen of the binding molecules of the present invention.

[0034] In embodiments, it will be understood herein that “specifically binds to” refers to the antibody-like binding of the VHH, e.g. via the VHH HCDRs 1-3, to its target: PSMA. Thus, in embodiments, it will be understood herein that the term “specifically” does not exclude the binding molecule from having other targets. In some embodiments, “specifically binds to” means that the VHH domain binds to an epitope on PSMA. In some embodiments, PSMA is human PSMA. In some embodiments, PSMA is defined according to the UniProt entry Q04609 (UniProtKB online database Release 2023_05).

[0035] In some embodiments, PSMA may comprise an amino acid sequence according to SEQ ID NO: 29 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have 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 SEQ ID NO: 29. In some embodiments, PSMA may consist of an amino acid sequence according to SEQ ID NO: 29.

[0036] SEQ ID NO: 29:

[0037] MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKH NMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHY DVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPE GDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILY SDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGI AEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFST QKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVV HEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYIN ADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFS GMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVE KFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQ EMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFID PLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYV AAFTVQAAAETLSEVA

[0038] Suitable assays and techniques for measuring / quantifying binding activity of the binding molecule according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM),bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art. For example, it will be understood that EC50 is a measure of the concentration of a binding molecule that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of a binding molecule to bind to a target.

[0039] It will be understood that in embodiments herein, the binding molecule of the present invention is essentially based on a VHH domain, but may comprise further domains e.g. further domains fused to the VHH domain. In some embodiments, the VHH domain specifically binds to PSMA. In some embodiments, the binding molecule does not comprise a VL domain. In some embodiments, the binding molecule does not comprise a CH1 domain. In some embodiments, the binding molecule comprises two VHH domains. In some embodiments, the binding molecule comprises two VHH domains that specifically bind to PSMA (which may optionally be the same). In some embodiments, the binding molecule comprises two VHH domains in tandem. In some embodiments, the binding molecule comprises three VHH domains. In some embodiments, the binding molecule comprises two VHH domains that specifically bind to PSMA (which may optionally be the same) and a third, different VHH domain that specifically binds to a different target (e.g. to an ALFA tag).

[0040] In some embodiments, the VHH domain may comprise one or more complementarity determining regions (CDRs). In some embodiments, the VHH domain may comprise one, two or three CDRs as defined with reference to SEQ ID NOs herein. In some embodiments, the VHH domain may comprise three CDRs as defined with reference to SEQ ID NOs herein. It will be understood that CDRs of the VHH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the VHH domain may be termed HCDR1, HCDR2 and HCDR3 respectively. In some embodiments, the VHH domain further comprises framework regions. In some embodiments, the framework regions are a scaffold (e.g. amino acid sequences) that support the CDRs such that the VHH domain specifically binds to PSMA via the CDRs.

[0041] The terms “complementarity determining region” or “CDR” refer to a highly variable loop in the variable region which interact with a cognate antigen and is largely responsible for determining the ability of a binding molecule (e.g. a VHH) to bind to an antigen and for determining binding affinity. The CDRs within a variable region are typically numbered from the amino to the carboxy terminus, with CDR1 being closest to the amino terminus of the variable region and CDR3 being closest to the carboxy terminus of the variable region. The CDRs may be determined according to the Kabat definition and / or the IMGT definition, both of which are well-known in the art.In some embodiments, the VHH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:

[0042] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (NYALS),

[0043] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (AITRAGDPINVADSVKG), and

[0044] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (WDYSDSDVEDY);

[0045] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0046] In some embodiments, the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:

[0047] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (GLTFSNYA),

[0048] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (ITRAGDPI), and

[0049] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (NAWDYSDSDVEDY);

[0050] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0051] In some embodiments, the VHH domain comprises the amino acid sequence according to SEQ ID NO: 7 (QLQLVESGGGLVQPGGSLRLSCAASGLTFSNYALSWNRQAPGKERELVAAITRAGDPINV ADSVKGRFTITRDNAKKTVYLQMNSLKPEDTAVYYCNAWDYSDSDVEDYWGQGTLVTVSS

[0052] ), or an amino acid sequence having at least 80% identity thereto. In some embodiments, the amino acid sequence has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or has 100% identity to SEQ ID NO: 7. In some embodiments, the VHH domain may comprise or consist of the sequence in question.

[0053] In some embodiments, the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 8 (DYGVG),

[0054] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 9 (AISWSGSNTYYSDSVKG), and

[0055] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 10 (RAAPEIPDDRATVDS);

[0056] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0057] In some embodiments, the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:

[0058] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 11 (GRTVWDYG),

[0059] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 12 (ISWSGSNT), and

[0060] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 13 (AARAAPEIPDDRATVDS);

[0061] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0062] In some embodiments, the VHH domain comprises the amino acid sequence according to SEQ ID NO: 14 (QVQLVESGGGLVQAGGSLRLSCAASGRTVWDYGVGWFRQAPGKVREFVAAISWSGSNT YYSDSVKGRFTISRDVAKNTVFLQMNSLKPEDTAIYYCAARAAPEIPDDRATVDSWGQGTL VTVSS), or an amino acid sequence having at least 80% identity thereto. In some embodiments, the amino acid sequence has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or has 100% identity to SEQ ID NO: 14. In some embodiments, the VHH domain may comprise or consist of the sequence in question.

[0063] In some embodiments, the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:

[0064] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 15 (TYDMA),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 16 (AISWSGVNTYYADSVKG), and

[0065] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 17 (G AAASG RYYDTG EG EYDY) ;

[0066] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0067] In some embodiments, the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:

[0068] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 18 (GRTFSTYD),

[0069] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 19 (ISWSGVNT), and

[0070] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 20 (AAG AAASG RYYDTG EG EYDY) ;

[0071] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0072] In some embodiments, the VHH domain comprises the amino acid sequence according to SEQ ID NO: 21 (QVQLVESGGGLVQAGDSLRLSCAASGRTFSTYDMAWFRQAPGKEREFVAAISWSGVNTY YADSVKGRFTISRENTKNAINLQMNNLKPEDTAVYYCAAGAAASGRYYDTGEGEYDYWGQ GTQVTVSS), or an amino acid sequence having at least 80% identity thereto. In some embodiments, the amino acid sequence has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or has 100% identity to SEQ ID NO: 21. In some embodiments, the VHH domain may comprise or consist of the sequence in question.

[0073] In some embodiments, one or more of the HCDRs may comprise one, two or three amino acid mutations.

[0074] In some embodiments, HCDR1 may comprise one, two or three amino acid mutations.

[0075] In some embodiments, HCDR2 may comprise one, two or three amino acid mutations.In some embodiments, HCDR3 may comprise one, two or three amino acid mutations.

[0076] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the binding molecule according to the invention from binding to PSMA. In other words, a binding molecule according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to PSMA. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to PSMA as the parent binding molecule. In some embodiments, the mutation suitably maintains the same internalisation rate as the parent binding molecule. The term “parent binding molecule” in this context refers to the binding molecule without the mutation in question.

[0077] It will be understood that a VHH domain having % identity to the SEQ ID NO of any VHH domain defined herein may have an equivalent function to the VHH domain having the sequence set forth in the SEQ ID NO defined herein and may suitably maintain the capacity to bind to PSMA, e.g. the same capacity as the VHH domain having the sequence set forth in the SEQ ID NO defined herein.

[0078] In some embodiments, the VHH domain of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequences of one, two or three, preferably all three, of the HCDRs of the corresponding candidate.

[0079] In some embodiments, the present invention relates to a binding molecule comprising a VHH domain that specifically binds to the same epitope on human PSMA as either:

[0080] a) a VHH domain comprising the CDRS of SEQ ID NOs 1 , 2 and 3; the CDRs of SEQ ID NOs 4, 5 or 6, and / or the VHH sequence of SEQ ID NO: 7;

[0081] b) a VHH domain comprising the CDRS of SEQ ID NOs 8, 9 and 10; the CDRs of SEQ ID NOs 11, 12 or 13, and / or the VHH sequence of SEQ ID NO: 14; or

[0082] c) a VHH domain comprising the CDRS of SEQ ID NOs 15, 16 and 17; the CDRs of SEQ ID NOs 18, 19 or 20, and / or the VHH sequence of SEQ ID NO: 21.

[0083] In some embodiments of this type, the binding molecule of the present invention may have at least, substantially the same or the same affinity for human PSMA as the VHH domain according to a), b) or c) respectively. In some embodiments of this type, the binding molecule of the present invention may have at least, substantially the same or the same rate ofinternalisation on cells expressing human PSMA as the VHH domain according to a), b) or c) respectively. This also applies to constructs of the invention and BPCs comprising binding molecules of these embodiments.

[0084] Further domains / regions of binding molecules

[0085] In some embodiments, the binding molecule comprises one or more additional immunoglobulin domains, e.g. further to one or more VHH domains as defined above. In some embodiments, the binding molecule comprises one or more immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 2 (CH2) domain. In some embodiments, the immunoglobulin constant domains consist of a CH3 domain. In some embodiments, the immunoglobulin constant domains comprise (or consist of) a CH2 and a CH3 domain. In some embodiments, the CH2 and CH3 domains are considered to be an Fc (fragment crystallisable) region. In some embodiments, the binding molecule comprises an Fc region. In some embodiments, the Fc region is a AK447 Fc region. In some embodiments, the Fc region is an Fc region containing K447. In some embodiments, the additional immunoglobulin domains comprise (or consist of) a VHH domain that specifically binds to a target other than PSMA, e.g. an ALFA tag.

[0086] In some embodiments, the binding molecule or construct of the present invention, e.g. via the Fc region, binds to one or more or all of the Fc receptors. In preferred embodiments, the Fc receptors comprise one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16), C1q and FcRn. In some embodiments, the binding molecule or construct comprises a hinge domain as defined herein (e.g. that is derived from IgG 1 or lgG3) and maintains Fc receptor binding. In some embodiments, the binding molecule or construct of the present invention, e.g. via the Fc region, binds to FcyRI. In some embodiments, the binding molecule comprises a hinge domain as defined herein (e.g. that is derived from lgG1 or lgG3) and maintains FcyRI binding. In some embodiments, the Fc receptor / FcyRI binding of the binding molecule or construct of the present invention is the same as or substantially the same as that of a full-length antibody comprising the same Fc region as the binding molecule or construct of the present invention.

[0087] It will be understood that the Fc region may interact with Fc receptors presented on the surface of a cell and / or may interact with proteins of the complement system. The Fc receptors may be Fc gamma receptors, e.g. FcyRI. The proteins of the complement system may include C1q. In some embodiments, the Fc region of the binding molecule according tothe invention may comprise a hinge region. In some embodiments, the Fc region of the binding molecule according to the invention may comprise a modified hinge region.

[0088] In some embodiments, the construct of the invention comprising an Fc region maintains the same Fc functionality as an antibody comprising the same Fc region. In some embodiments, Fc-mediated effector functions include antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0089] However, in other embodiments the Fc region of the construct is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. In some embodiments, the Fc region of the construct is modified to negate one or more Fc receptor functionalities. In some embodiments, the Fc region of the construct of the present invention is silenced in respect of one or more or all of FcyRI (CD64), FcyRI la (CD32A), FcyRllb (CD32B), FcyRI II (CD16) and C1q functionality.

[0090] In some such embodiments, the Fc region of the construct comprises a silencing modification selected from the STR mutation, the LALA mutation, the LALA delta-A mutation and the LALA-KA mutation as defined herein. In an embodiment, the Fc region of the construct comprises the STR Mutation: L234S, L235T, G236R by Ell numbering. The STR mutation is further described in WO2021234402A2, which is incorporated by reference herein. In an embodiment, the Fc region of the construct comprises the LALA Mutation: L234A, L235A by EU numbering. In an embodiment, the Fc region of the construct comprises the LALA delta-A mutation: L234A, L235A, A327G, A330S, P331S by EU numbering. In an embodiment, the Fc region of the construct comprises the LALA-KA mutation: L234A, L235A, K322A by EU numbering. In an embodiment the Fc region of the construct comprises the amino acid residue S at position 234, T at position 235 and R at position 236 by EU numbering. In an embodiment the Fc region of the construct comprises the amino acid residue A at position 234 and A at position 235 by EU numbering. In an embodiment the Fc region of the construct comprises the amino acid residue A at position 234, A at position 235, G at position 327, S at position 330 and S at position 331 by EU numbering. In an embodiment the Fc region of the construct comprises the amino acid residue A at position 234, A at position 235 and A at position 322 by EU numbering. For the avoidance of doubt, each of the positions of specific Fc silencing mutations described herein are with reference to EU residue numbering of the Fc region.

[0091] In some embodiments, the binding molecule comprises a hinge region between the VHH domain and the constant domains. This equally applies to the binding molecules in the constructs and BPCs of the present invention.In some embodiments, the hinge region is derived from lgG1 or lgG3.

[0092] In some embodiments, the hinge region is derived from lgG1.

[0093] In some embodiments, the hinge region is derived from lgG3.

[0094] In some embodiments, the hinge region is derived from an lgG1 hinge region or an lgG3 hinge region.

[0095] In some embodiments, the hinge region is derived from an lgG1 hinge region.

[0096] In some embodiments, the hinge region is derived from an lgG3 hinge region.

[0097] In some embodiments, the hinge region is an lgG1 hinge region or an lgG3 hinge region.

[0098] In some embodiments, the hinge region is an lgG1 hinge region.

[0099] In some embodiments, the hinge region is an lgG3 hinge region.

[0100] In some embodiments, the hinge region is suitable for the production of pure and / or non-aggregated constructs comprising binding molecules of the present invention.

[0101] In some embodiments, the hinge region does not result in aggregation when used to produce a construct comprising binding molecules of the present invention.

[0102] In some embodiments, the hinge region substantially results in the production of a single molecular species when used to produce a construct comprising binding molecules of the present invention.

[0103] In some embodiments, the hinge regions do not form intermolecular disulphide bonds. It will be understood that intermolecular disulphide bonds in this context are between molecules of the construct or BPC comprising the binding molecules of the present invention.

[0104] In some embodiments, the construct or BPC of the present invention comprises binding molecules which each comprise the same hinge region.

[0105] In some embodiments, the lack of aggregation is determined by size exclusion chromatography, although any suitable means are known to the skilled person and may be used.In some embodiments, the hinge region (e.g. the hinge region derived from lgG1) has at least 80% identity to SEQ ID NO: 22 (DKTHTCPPCP). In some embodiments, the hinge region has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity to SEQ ID NO: 22. In some embodiments, the hinge region may comprise or consist of the sequence in question.

[0106] In some embodiments, the hinge region (e.g. the hinge region derived from lgG3) has at least 80% identity to SEQ ID NO: 23 (PRCPEPKACDAPPPCPRCP). In some embodiments, the hinge region has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity to SEQ ID NO: 23. In some embodiments, the hinge region may comprise or consist of the sequence in question. In some embodiments, the hinge region may comprise a PTM modification.

[0107] In some embodiments, the hinge region (e.g. the hinge region derived from lgG3) has at least 80% identity to SEQ ID NO: 30 (PRCPEPKSCDTPPPCPRCP). In some embodiments, the hinge region has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity to SEQ ID NO: 30. In some embodiments, the hinge region may comprise or consist of the sequence in question.

[0108] It will be understood that in some embodiments herein, e.g. for the provision of a binding molecule suitable for forming a payload conjugate, the binding molecule comprises one or more sites suitable for conjugation to a payload. In embodiments, it is generally understood herein that the sites in question are comprised in the hinge region. In some embodiments, it is generally understood that the sites in the hinge region comprise cysteine residues. In some embodiments, e.g. when the construct of the present invention is partially reduced, the sites in the hinge region comprise free thiol moieties. Thus, in some embodiments herein the binding molecule comprises a VHH domain and a hinge region as defined herein. In some embodiments, the binding molecule comprises an Fc region capable of forming one or more disulphide bonds that are not for conjugation, and which do not count as a site for conjugation or unpaired cysteine etc. as defined herein.

[0109] In some embodiments, the binding molecule, e.g. a single polypeptide chain, comprises 1, 2, 3, 4, 5, 6 or more than 6 sites suitable for conjugation to payloads, preferably 2 to 4 sites suitable for conjugation to payloads, most preferably 2 or 4 sites suitable forconjugation to payloads. In some embodiments, there are 2 sites suitable for conjugation to payloads. In some embodiments, there are 4 sites suitable for conjugation to payloads.

[0110] In some specific embodiments of the invention, the sites suitable for conjugation to payloads are thiol moieties provided by thiol-moiety-containing residues, e.g. cysteine residues, present in the hinge region.

[0111] In some embodiments, the sites suitable for conjugation to payloads are suitable for reduction and then conjugation to a payload.

[0112] In some embodiments, the binding molecule further comprises an additional binding moiety or a cognate of a binding moiety. In some embodiments, the cognate is an antigen, an epitope or a polypeptide tag that is specifically recognised by an antigen-binding fragment of an antibody. In a preferred embodiment, the cognate is a polypeptide tag. In a preferred embodiment, the cognate is a polypeptide tag that is specifically recognised by an antigenbinding fragment of an antibody. In a particularly preferred embodiment, the cognate is an ALFA tag (as described in Gdtzke etal., Nat Commun, 2019, 10:4403). In some embodiments, the cognate is a polypeptide comprising or consisting of the sequence SRLEEELRRRLTE (SEQ ID NO: 26). In some embodiments, the binding moiety is an antigen-binding fragment that is capable of specifically binding to an ALFA tag. In some embodiments, the binding moiety is an antigen-binding fragment that is capable of specifically binding to a polypeptide comprising or consisting of the sequence SRLEEELRRRLTE (SEQ ID NO: 26). In some embodiments, the binding moiety is a VHH domain capable of specifically binding to the ALFA tag. In some embodiments, the binding moiety is VHH domain capable of specifically binding to a polypeptide comprising or consisting of the sequence SRLEEELRRRLTE (SEQ ID NO: 26). In some embodiments, the binding moiety is a nanobody which specifically binds to the ALFA tag. In some embodiments, the binding moiety is wild-type, e.g. the sequence of the binding moiety has not been substantially modified. In some embodiments, the binding moiety is humanised. In some embodiments, the binding moiety is a (humanised) nanobody comprising a sequence having at least 80%, 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having 100% identity to SEQ ID NO: 27. A suitable binding moiety is described in WQ2020 / 053239A1 (incorporated herein by reference).

[0113] Accordingly, in some embodiments, the binding molecule comprises an additional immunoglobulin domain comprising an anti-ALFA VHH (NbALFA) domain. In some embodiments, the additional immunoglobulin domains of the binding molecule comprise (or consist of) one, two or more humanised NbALFA domains. In some embodiments, theadditional immunoglobulin domains of the polypeptide comprise (or consist of) one, two or more wild-type NbALFA domains. In some embodiments, the binding molecule comprises an NbALFA domain and does not comprise any constant immunoglobulin domains. In some embodiments, the binding molecule comprises one or more additional constant immunoglobulin domains, e.g. as defined above, as well as an NbALFA domain. In some embodiments, the binding molecule comprises a CH3 domain and an NbALFA domain. In some embodiments, the binding molecule comprises a CH2 domain and an NbALFA domain. In some embodiments, the binding molecule comprises a CH2 domain, a CH3 domain and an NbALFA domain. In some embodiments, the binding molecule comprises an Fc region and an NbALFA domain.

[0114] In an embodiment, the Fc region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having 100% identity to SEQ ID NO: 28. In some embodiments of this type the Fc region is a AK447 Fc region.

[0115] In some embodiments, the binding molecules (or constructs) of the present invention do not comprise albumin-binding domain.

[0116] Constructs comprising binding molecules

[0117] In an aspect, the present invention provides a construct comprising a first binding molecule according to the invention and a second binding molecule according to the invention. It will be understood that within the scope of the invention the first binding molecule according to the invention and the second binding molecule according to the invention may be the same or different, such that each binding molecule may independently possess any combination of the features defined herein.

[0118] In some embodiments, only one of the binding molecules in the construct comprises an additional binding moiety or a cognate of a binding moiety as defined above. In some embodiments, both of the binding molecules in the construct comprise an additional binding moiety or a cognate of a binding moiety as defined above. In some embodiments, both of the binding molecules in the construct specifically bind to PSMA. In some embodiments, the construct is monoparatopic. In some embodiments, the construct is biparatopic. In some embodiments, the construct is monovalent. In some embodiments, the construct is bivalent. In some embodiments, the construct is multivalent.

[0119] Since the constructs of the present invention comprise a first and second binding molecule according to the present invention, it will generally be understood herein that theconstruct will comprise at least a first and second set - and hence at least double - of the features of the binding molecules of the present invention, e.g. the construct of the present invention will generally comprise double the number of sites suitable for conjugation to payloads as defined herein, compared to the binding molecule of the present invention. Thus, in some embodiments, where the polypeptide of the invention comprises e.g. 2 or 4 sites suitable for conjugation to payloads, the corresponding construct of the invention comprises 4 or 8 sites suitable for conjugation to payloads respectively.

[0120] In some embodiments, the construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 sites suitable for conjugation to payloads, preferably 4 to 8 sites suitable for conjugation to payloads.

[0121] In some embodiments, the first binding molecule and / or the second binding molecule independently comprise one or more modifications that enhance the formation of the construct. In some embodiments, the modifications comprise knobs-into-holes modifications.

[0122] In some embodiments, the first and second binding molecules comprise modifications that enhance the formation of a dimer, comprising one monomer of the first binding molecule and one monomer of the second binding molecule. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the modifications can be those modifications which are widely known in the art of antibody technology for the purpose of (hetero)dimerisation between antibody chains (e.g. “knobs-into-holes” (KiH) modifications). In a typical embodiment, the modifications are present in the CH domains, e.g. preferably the CH3 domains, of the first and second binding molecules.

[0123] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that enhances the formation of the dimeric construct comprising the first and second binding molecules. In some embodiments wherein the first and second binding molecules each comprise more than one constant immunoglobulin domain, e.g. a CH2 domain or further CH3 domain, one or more or all of the CH domains may comprise such modifications. Thus, in some embodiments herein, reference to a “CH” domain may be a reference to a CH3 or CH2 domain or to the Fc region. In preferred embodiments herein, reference to the “CH domain” may be a reference to a CH3 domain.

[0124] As used herein, the term “enhances the formation of the construct comprising the first and second binding molecules” includes promoting dimerisation of the first and second binding molecules. Thus, in some embodiments, the modifications promote dimerisation of the firstand second binding molecules. In some embodiments, the modifications promote heterodimerisation of the first and second binding molecules. By heterodimerisation it is meant that the first binding molecule is different to the second binding molecule, and the first binding molecule will form a dimer with the second binding molecule, but the first binding molecule will not form a dimer with the first binding molecule and the second binding molecule will not form a dimer with the second binding molecule. In some embodiments, the modifications promote homodimerisation of the first and second binding molecule. By homodimerisation it is meant that the first binding molecule is the same as the second binding molecule, and the first binding molecule will form a dimer with the second binding molecule.

[0125] Various means of promoting dimerisation of two domains (e.g. homo- or heterodimerisation) are known in the art. In some embodiments, the two domains may comprise a modification that enhances formation of dimer, e.g. a modification that increases the affinity of the two domains, promotes and / or enables the formation of one or more disulphide bonds, reduces steric hindrance to the dimerisation, promotes electrostatic and / or hydrophilic / hydrophobic interactions between the two domains, or any combination thereof.

[0126] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that enhances the affinity between the first and second binding molecules. In some embodiments, the modification enhances the affinity between the CH domains of the first and second binding molecules.

[0127] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that reduces steric hindrance to the formation of the construct comprising the first and second binding molecules. In some embodiments, the modification reduces steric hindrance between the CH domains of the first and second binding molecules. In some embodiments, one or more of the CH domains of the first binding molecule and one or more of the CH domains of the second binding molecule comprise a ‘knobs-into-holes’ modification. In some embodiments, one or more of the CH domains of the first binding molecule and one or more of the CH domains of the second binding molecule are associated via a ‘knobs-into-holes’ modification.

[0128] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that promotes electrostatic interactions between the first and second binding molecules. In some embodiments, the modification promotes electrostatic interactions between the CH domains of the first and second binding molecules. In some embodiments, the electrostaticinteractions that are promoted are favourable to the formation of a dimer (e.g. a heterodimer) comprising the first and second binding molecules.

[0129] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that promotes hydrophilic / hydrophobic interactions between the first and second binding molecules. In some embodiments, the modification promotes hydrophilic / hydrophobic interactions between the CH domains of the first and second binding molecules. In some embodiments, the hydrophilic / hydrophobic interactions that are promoted are favourable to the formation of a dimer comprising the first and second binding molecules.

[0130] In some embodiments, a CH domain of the first binding molecule forms a disulphide bond with a CH domain of the second binding molecule. In a preferred embodiment, a CH domain of the first binding molecule forms a disulphide bond with a CH domain of the second binding molecule, and in addition one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that enhances the affinity between the first and second binding molecules.

[0131] Any suitable technology for promoting the dimerisation of CH domains, i.e. of at least one of the CH domains of the first binding molecule and at least one of the CH domains of the second binding molecule, may be employed in the practice of the invention. Preferably, the modification that enhances the formation of a dimer between the CH domains is KiH technology (Ridgeway et al., Protein Engineering, Design and Selection, 1996, 9: 617-621 and Merchant et al., Nat Biotechnol, 1998, 16: 677-681). The KiH technology is based on an engineered pair of CH domains that heterodimerises (CH heterodimer) in which asymmetric hydrophobic mutations are introduced between the homodimeric CH domain. KiH involves introducing mutations that create a protuberance (“knob”) in the interface of the first CH domain and a corresponding cavity (“hole”) in the interface of the second CH domain, such that the protuberance can be positioned in the cavity to promote heterodimer assembly and hinder homodimer formation. KiH variants therefore thermodynamically favour the formation of heterodimers rather than homodimers.

[0132] By way of further example, the present invention may employ: the strand-exchange engineered domain (SEED) CH3 dimers (Davis et al., Protein Eng Des Sei., 2010, 23:195-202); electrostatic steering employing DD-KK variants with asymmetric electrostatic interactions (Gunasekaran etal., J Biol Chem., 2010 , 285: 19637-46): Azymetric technologies by Zymeworks (https: / / www.zvmeworks.com / technologies / azvmetric / ): Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT) (Skegro et al., J Biol Chem.,2017, 292: 9745-9759): Fast-lg and ART-lg (https: / / www.chugai-pharm.co.ip / english / profile / rd / technologies.html): DEKK dimerisation technology (https: / / merus.nl / technology / multiclonics-platform / and Nardis et al, J Biol Chem., 2017, 292: 14706-14717): HA-TF variants with asymmetric hydrophobic interactions (Moore etal., MAbs, 2011, 3: 546-57): computationally designed CH3 interfaces, such as the 7.8.60 design (Leaver-Fay et al., Structure, 2016, 24: 641-651): EW-RVT variants, which were designed to replace the conserved electrostatic interactions with asymmetric hydrophobic interactions and to add asymmetric long-range electrostatic interactions at the rim of the heterodimeric CH3 interface (Choi et al., Mol Cancer Then, 2013, 12: 2748-59): and the K370ECH3A-E357NCH3B mutations employed in the “A107” variant which replaced the homodimer-favouring electrostatic interactions with heterodimer-stabilizing hydrogen bonds. Herein, Ell numbering is used to denote the residues of an immunoglobulin domain (Edelman et al., Proc Natl Acad Sci U S A., 1969, 63: 78-85).

[0133] In some embodiments, one or more of the CH domains of the first binding molecule and one or more of the CH domains of the second binding molecule are associated via a KiH modification. In some embodiments, the CH domain of the first binding molecule and the CH domain of the second binding molecule, where present, each comprise a KiH modification. Any suitable KiH modifications known in the art may be employed in the practice of the present invention.

[0134] In another aspect, the present invention provides a construct comprising a first binding molecule according to the invention and a second binding molecule comprising a VHH domain. In some embodiments of this type, the second binding molecule does not specifically bind to PSMA. In some embodiments of this type, the second binding molecule further comprises one or more constant immunoglobulin domains (e.g. a CH3 domain or an Fc region) and / or a hinge region (e.g. an lgG1 -derived hinge region or an lgG3-derived hinge region) as is defined in respect of the binding molecules of the present invention herein. Other aspects and embodiments of the present invention, e.g. conjugates, modifications and methods herein, will generally be understood as equally applying to both binding molecules of the construct and the construct perse as defined in this aspect, in this paragraph. In some embodiments, the constructs of this aspect are biparatopic.

[0135] In some embodiments, the constructs of the present invention are VHH-Fc fusion constructs. “VHH-Fc fusion constructs” are defined herein as constructs comprising a first binding molecule and a second binding molecule, each of which comprises a VHH domain fused to an Fc region, preferably via a hinge region.Conjugates of binding molecules and constructs

[0136] In an aspect, the present invention provides a binding molecule-payload conjugate (BPC) comprising a binding molecule or construct according to the invention, and one or more payload(s). In some preferred embodiments, the BPC comprises a construct of the present invention. In some embodiments, the BPC further comprises a linker connecting the binding molecule or conjugate to the payload(s). In some embodiments, the payload is a therapeutic agent. In some embodiments, the payload is a diagnostic agent. In some embodiments, the payloads comprise both therapeutic and diagnostic agents. It is to be understood herein that the term “BPC” is analogous to the term “ADC”, as in “antibody-drug conjugate”, except that the present invention utilises binding molecules comprising VHH domains instead of full-length antibodies per se, which may be conjugated to any useful conjugate molecule(s), e.g. any therapeutic or diagnostic agents, more generally than a drug.

[0137] It will be understood that the term “payload” may be interchangeable with “cargo”. It will also be understood that the payload may be a drug.

[0138] ADCs are a class of targeted therapeutics that can improve the selectivity and the cytotoxic activity of cancer drugs. Upon binding of an ADC to a target antigen present on the surface of a cell, the ADC may become internalised and trafficked to intracellular compartments (e.g. a lysosome) where the payload is released from the ADC. When the payload is a drug, the release of the drug from the ADC may allow the drug to exert its effect on the cell. The payload may be released from the ADC by proteolysis of a cleavable linker (if present) or by degradation of the antibody of the ADC.

[0139] In some embodiments the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 unpaired cysteine residues. In some embodiments, the unpaired cysteine residues are bound to the payloads of the BPC.

[0140] In some embodiments, the payload is a chemotherapeutic entity, a radionuclide or a detection entity. In some embodiments, the payload is a drug. In some embodiments, the drug is a cytotoxic drug, a DNA-damaging agent, a tubulin inhibitor, a topoisomerase inhibitor, an immunomodulator or a STING agonist. In some embodiments, the topoisomerase inhibitor may be a topoisomerase I inhibitor.

[0141] In some embodiments, the payload is a nucleic acid, such as a therapeutic or diagnostic nucleic acid. In some embodiments, the nucleic acid is a payload nucleic acid as defined herein. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the nucleic acid is non-coding RNA (ncRNA). In some embodiments, the nucleic acid is an RNA interference (RNAi) nucleic acid. In some embodiments, the nucleic acid is microRNA (miRNA). In some embodiments, the nucleic acid is small inhibitory RNA (siRNA). In some embodiments, the nucleic acid is an oligonucleotide. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid itself comprises a conjugate, e.g. a chemical conjugate. In some embodiments, the nucleic acid comprises one or more modifications, such as one or more non-naturally occurring modifications.

[0142] In some embodiments, the payload is a drug capable of treating cancer. In some embodiments, the payload is a drug capable of treating cancer in humans. In some embodiments, the payload is a drug capable of treating a cancer with increased PSMA expression relative to the same non-cancer cell(s) (e.g. the wild-type). In some embodiments, the payload is a drug capable of treating prostate cancer. In some embodiments, the payload is a drug capable of treating prostate cancer in humans.

[0143] It will be understood that a cytotoxic drug, chemotherapeutic drug, or chemotherapeutic entity may refer to a drug or molecule that:

[0144] • is destructive to a cell;

[0145] • induces apoptosis in a cell;

[0146] • inhibits or prevents the function of a cell;

[0147] • inhibits or prevents a cell from proliferating; and / or

[0148] • reduces the viability of a cell.

[0149] It will be understood that the term “radionuclide” may refer to a radioimmunoconjugate which uses radiation for therapeutic and / or diagnostic purposes. For diagnosis purposes, the payload of the BPC may be a label compatible with imaging procedures, such as single photon emission computed tomography or positron emission tomography (PET). For therapeutic purposes, the payload of the BPC may be an emitter of radiation that may be used to irradiate a tumour. Such radionuclides for either of these purposes will be known in the art, and will depend on e.g. the intended use, target tissue type, and size and location of a tumour.

[0150] It will be understood that the term “detection entity” may refer to a payload that produces a detectable signal, such as a fluorescent signal released, for example by a fluorescent peptide or dye or label or fluorophore, which following excitation emits light at a detectable wavelength. Suitable detection entities will be known in the art, and will depend on e.g. the intended use and target tissue type, and size and location of a tumour.In some embodiments:

[0151] (i) the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 payloads, preferably 4 to 8 payloads, preferably 4 or 8 payloads; or

[0152] (ii) the BPC has a payload to BPC ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12, preferably of 4 to 8, preferably 4 or 8;

[0153] optionally wherein the payloads are different or are the same. In some embodiments, the payloads may be different. In some embodiments, the BPC comprises two or more than two different types of payload. In some embodiments, the payloads may be the same. In some embodiments, the payloads are drugs. Thus, in some embodiments, the payload to BPC ratio is a drug to BPC ratio. It will generally be understood herein that in embodiments the payload to BPC ratio refers to the ratio between the number of conjugated payloads to the BPC and the BPC itself, i.e. the whole BPC which is typically a single construct of the present invention and therefore has a value of “1” in this context. Thus, the payload to BPC ratio is effectively the same as the number of payload molecules that are conjugated to the BPC in question. It is to be understood herein that the payload to BPC ratio is also analogous to and may equally be referred to as “DAR”, as in “drug-antibody ratio”, as is typically used to define ADCs in the art, although for the purposes of the present invention it will be understood that the binding molecule comprises a VHH domain and is not necessarily a full-length antibody. In some embodiment, it will be understood that the payload to BPC ratio / DAR is usually calculated from the average number of payloads / drugs per BPC / construct.

[0154] In some embodiments, the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 unpaired cysteine residues conjugated to payloads, preferably 4 to 8 unpaired cysteine residues conjugated to payloads, most preferably 4 or 8 unpaired cysteine residues conjugated to payloads. In some embodiments, there are 4 unpaired cysteine residues conjugated to payloads. In some embodiments, there are 8 unpaired cysteine residues conjugated to payloads. In some residues, the unpaired cysteine residues are comprised in the hinge regions of the BPC. In some embodiments, an unpaired cysteine residue is defined as a reduced cysteine residue. In some embodiments, an unpaired cysteine residue is defined as a reduced cysteine residue which does not form a disulphide bond with another cysteine residue comprised in a polypeptide sequence, e.g. in another binding molecule such as in another binding molecule of the invention.

[0155] In some embodiments, the BPC may be directly attached to the one or more payload(s). In some embodiments, the BPC may be indirectly attached to the one or morepayload(s). In some embodiments, the attachment is from unpaired cysteine residues of the BPC to the payloads, e.g., for each unpaired cysteine residue, one unpaired cysteine residue to one payload. In some embodiments, the ADC may comprise a linker that connects (e.g. conjugates) the antibody or fragment thereof to the payload(s). In some embodiments, the one or more payload(s) may be covalently attached to the antibody or fragment thereof to form the ADC.

[0156] It will be understood that a linker may be used to connect (e.g. conjugate) the antibody or fragment thereof to the payload(s) to produce an ADC. Such linkers may be any appropriate linker known in the art, such as a linker that has chemically reactive groups at each end. These linkers can form a covalent attachment between two molecules, e.g. the antibody or fragment thereof and the drug or payload. The linker may form, for example, hydrazone, disulfide or amide bonds between the antibody or fragment thereof and the payload. In some embodiments, one end of the linker may be linked to the antibody and the other end of the linker may be linked to the payload. In some embodiments, the linker may be a flexible linker. In some embodiments, the linker may be a cleavable linker.

[0157] In some embodiments, the BPC comprises 4 payloads and each binding molecule in the BPC comprises a hinge region defined herein as being derived from lgG1. In some embodiments, the BPC has a payload to BPC ratio of 4 and each binding molecule in the BPC comprises a hinge region defined herein as being derived from lgG1. In some embodiments, the BPC comprises 8 payloads and each binding molecule in the BPC comprises a hinge region defined herein as being derived from lgG3. In some embodiments, the BPC has a payload to BPC ratio of 8 and each binding molecule in the BPC comprises a hinge region defined herein as being derived from lgG3.

[0158] In some embodiments, the conjugates referred to herein (BPCs) are not recombinantly fused to a payload.

[0159] Payload nucleic acids

[0160] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, forexample, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0161] The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0162] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes II, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0163] A modified purine (A or G) or pyrimidine (C, T, or II) base moiety is preferably modified by one or more alkyl groups, more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkylcytosine, 5-C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0164] Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, thesealtered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, 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%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0165] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0166] The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, 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%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0167] In some embodiments, the DNA is present in the form of a vector. In some embodiments, the vector is a DNA vector. In some embodiments, the vector comprises DNA encoding an amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity."RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0168] In some embodiments, the RNA is single stranded RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is generated by RNA in vitro transcription. In some embodiments, the RNA comprises a 5' cap structure. In some embodiments, the RNA does not comprise modified ribonucleotides. In some embodiments, the RNA comprises modified ribonucleotides. In some embodiments, the modified ribonucleotides comprise modified uridines. In some embodiments, the modified uridines comprise N1-methyl-pseudouridine.

[0169] In some embodiments, the RNA described herein is single-stranded RNA that may be translated into the respective protein upon entering cells, e.g., cells used in the assays described herein and cells of a recipient. In addition to wildtype or codon-optimized sequences encoding the amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity, e.g., a pharmaceutically active peptide or polypeptide such as antigen sequence, the RNA may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A)-tail). In one embodiment, the RNA contains all of these elements. In one embodiment, beta-S-ARCA(DI) (m27,2'-OGppSpG) or m27,3’-OGppp(m12’-O)ApG may be utilized as specific capping structure at the 5'-end of the RNA drug substances. As 5'-UTR sequence, the 5'-UTR sequence of the human alpha-globin mRNA, optionally with an optimized ‘Kozak sequence’ to increase translational efficiency may be used. As 3'-UTR sequence, a combination of two sequence elements (Fl element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) placed between the coding sequence and the poly(A)-tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used. These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017 / 060314, herein incorporated by reference). Alternatively, the 3‘-UTR may be two re-iterated 3'-UTRs of the human beta-globin mRNA. Furthermore, a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues may be used. This poly(A)-tail sequence was designed to enhance RNA stability and translational efficiency.The amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity, e.g., a pharmaceutically active peptide or polypeptide such as antigen sequence, may comprise amino acid sequences other than the amino acid sequence of a peptide or polypeptide having biological activity. Such other amino acid sequences may support the function or activity of the peptide or polypeptide having biological activity. In some embodiments, such other amino acid sequences comprise an amino acid sequence enhancing antigen processing and / or presentation. Alternatively, or additionally, such other amino acid sequences comprise an amino acid sequence which breaks immunological tolerance. Alternatively, or additionally, such other amino acid sequences comprise an amino acid sequence which produces bioluminescence. Such other amino acid sequences may be useful for determining the amount of the amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity or a fragment thereof in the assays described herein. In particular, such other amino acid sequences may be useful for quantification by LC-MS / MS analysis.

[0170] According to the present disclosure, the term "mRNA" means "messenger-RNA" and relates to a "transcript" which may be generated by using a DNA template and may encode a peptide or polypeptide. Typically, an mRNA comprises a 5'-UTR, a peptide / polypeptide coding region, and a 3'-UTR. In the context of the present disclosure, mRNA may be generated by in vitro transcription (IVT) from a DNA template. As set forth above, the in vitro transcription methodology is known to the skilled person, and a variety of in vitro transcription kits is commercially available.

[0171] mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0172] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0173] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide or polypeptide.

[0174] In some embodiments, the mRNA which preferably encodes a peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 ,000, at least 1 ,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, atleast 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0175] As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf. , e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified mRNA, correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0176] In some embodiments, mRNA is in vitro transcribed mRNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0177] In some embodiments of the present disclosure, the mRNA is "replicon mRNA" or simply a "replicon", in particular "self-replicating mRNA" or "self-amplifying mRNA". In certain embodiments, the replicon or self-replicating mRNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and inprotein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0178] In some embodiments of the present disclosure, the mRNA contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the mRNA, it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and / or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).In some embodiments, the mRNA comprises a 5'-cap structure. In some embodiments, the mRNA does not have uncapped 5'-triphosphates. In some embodiments, the mRNA may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the mRNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at position N7 (resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the p-phosphate (such as m27,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or -S-ARCA)), as described in PCT / EP2019 / 056502. Providing an mRNA with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated mRNA strand, or the mRNA may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0179] In some embodiments, the mRNA comprises a 5'-cap structure selected from the group consisting of m27,2'OG(5’)ppSp(5')G (in particular its D1 diastereomer), m27,3'OG(5')ppp(5')G, and m27,3'-OGppp(m12'-O)ApG.

[0180] In some embodiments, the mRNA comprises a capO, cap1, or cap2, preferably cap1 or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "cap1" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0181] The D1 diastereomer of beta-S-ARCA (P-S-ARCA) has the following structure:

[0182]

[0183] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(DI)" is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta- S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml / min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0184] The 5'-cap analog m27,3'-OGppp(m12'-O)ApG (also referred to as m27,3'OG(5')ppp(5')m2'-OApG) which is a building block of a cap1 has the following structure:

[0185]

[0186] An exemplary capO mRNA comprising P-S-ARCA and mRNA has the following structure:

[0187]

[0188] An exemplary capO mRNA comprising m27,3'OG(5')ppp(5')G and mRNA has the following structure:

[0189]

[0190] An exemplary cap1 mRNA comprising m27,3'-OGppp(m12'-O)ApG and mRNA has the following structure:

[0191]

[0192] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an mRNA molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the mRNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. mRNAs disclosed herein can have a poly-A tail attached to the free 3'-end of the mRNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0193] It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0194] The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as II nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0195] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0196] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides(dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an mRNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, II). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0197] In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.

[0198] In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.

[0199] In some embodiments, mRNA used in present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf. , e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal-globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0200] The mRNA may have modified ribonucleotides in order to increase its stability and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in some embodiments, uridine in the mRNA described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0201] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (qj), N1-methyl-pseudouridine (m1i ), 5-methyl-uridine (m5U), and combinations thereof.

[0202] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the mRNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (im5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4i ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3i ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ip), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (i m), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0203] An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "^P-modified", whereas the term "ml^P-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine). Such

[0204]

[0205] or ml^P- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine replacing completely uridine

[0206] The codons of the mRNA used in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and / or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the mRNA used in the present disclosure is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0207] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the mRNA described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in theoccurrence of tRNAs in cells. Thus, the sequence of mRNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

[0208] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the mRNA described herein is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the mRNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the mRNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine) / C (cytosine) content are more stable than sequences having an increased A (adenosine) / U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the mRNA, there are various possibilities for modification of the mRNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or II nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or II or contain a lower content of A and / or II nucleotides.

[0209] In various embodiments, the G / C content of the coding region of the mRNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild type RNA.

[0210] A combination of the above described modifications, i.e. , incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'-and / or3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (^P) or N(1)-methylpseudouridine (ml^P) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the mRNA used in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'-and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (^P) or N(1)-methylpseudouridine (ml^P) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.Internalisation properties

[0211] In some embodiments, the VHH domain that specifically binds to PSMA induces internalisation of the binding molecule, construct or BPC of the invention into a cell expressing PSMA.

[0212] In some embodiments, the VHH domain that specifically binds to PSMA induces a high rate of internalisation of the binding molecule, construct or BPC into a cell expressing PSMA. In some embodiments, a “high rate of internalisation” refers to an internalisation rate of 40% or higher. In some embodiments, a “high rate of internalisation” refers to a rate of internalisation that is higher than that of a reference anti-PSMA antibody or VHH.

[0213] In some embodiments, the rate of internalisation is determined by fluorescence activated cell sorting (FACS). In some embodiments, the rate of internalisation is determined by microscopy.

[0214] In some embodiments, the rate of internalisation is at least 20% at 1 hour, optionally wherein the rate of internalisation is at least 25% at 1 hour, at least 30% at 1 hour, at least 35% at 1 hour, at least 40% at 1 hour, at least 45% at 1 hour, at least 50% at 1 hour, at least 55% at 1 hour, at least 60% at 1 hour, at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, at least 80% at 1 hour, at least 81% at 1 hour, at least 82% at 1 hour, at least 83% at 1 hour, at least 84% at 1 hour, at least 85% at 1 hour, at least 86% at 1 hour, at least 87% at 1 hour, at least 88% at 1 hour, at least 89% at 1 hour, at least 90% at 1 hour, at least 91% at 1 hour, at least 92% at 1 hour, at least 93% at 1 hour, at least 94% at 1 hour, at least 95% at 1 hour, at least 96% at 1 hour, at least 97% at 1 hour, at least 98% at 1 hour, at least 99% at 1 hour, or at least 100% at 1 hour.

[0215] In some embodiments, typically the rate of internalisation is determined herein following incubation with PSMA-positive cells at 37°C for 1 hour. In some embodiments, typically the rate of internalisation is determined as the % of an appropriate dose (e.g. 5nM) of the binding molecule or construct of the presentation that has internalised into PSMA-positive cells, e.g. following incubation at 37°C for 1 hour.

[0216] In some embodiments, the rate of internalisation is in respect of cells expressing human PSMA. In some embodiments, the rate of internalisation is in respect of cells engineered to express human PSMA. In some embodiments, the rate of internalisation is in respect of cells over-expressing human PSMA. In some embodiments, the rate of internalisation is in respect of cells engineered to over-express human PSMA. In someembodiments, the rate of internalisation is in respect of CHO cells, e.g. CHO cells engineered to (over)express human PSMA. In some embodiments, the rate of internalisation is in respect of Flpln CHO cells (e.g. https: / / www.thermofisher.com / order / catalog / product / R75807) engineered to express human PSMA. Herein, in embodiments, “engineered” means that the cells have been recombinantly modified to express a human PSMA transgene, e.g. which has been introduced to the cells by transduction, transfection, viral vector, CRISPR / Cas systems, electroporation or other similar means to introduce a recombinant gene sequence, which are known to the skilled person.

[0217] In some embodiments, the rate of internalisation is in respect of cells that endogenously express human PSMA, which may be at lower expression levels and / or with more heterogeneity than cells that are engineered to (over)express human PSMA. In some embodiments, the rate of internalisation is in respect of cancer cells that (endogenously) express human PSMA. In some embodiments, the rate of internalisation is in respect of lymph node carcinoma of the prostate (LNCaP) cells (e.g. https: / / www.atcc.org / products / crl-1740) that (endogenously) express human PSMA. Herein, in embodiments, “endogenously” expressing PSMA means that the cell has not been engineered to overexpress PSMA.

[0218] In some embodiments, the rate of internalisation of the binding molecule of the present invention is at least about 80% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 1-3, 4-6 and / or 7 (PSMA1) as described herein, optionally wherein the binding molecule comprises a VHH domain that specifically binds to PSMA and a VHH domain that specifically binds to an ALFA tag, optionally wherein the binding molecule comprises one or more immunoglobulin constant domains. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0219] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is about 100% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 1-3, 4-6 and / or 7 (PSMA1) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from lgG1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0220] In some embodiments, the rate of internalisation of the construct of the present invention is about 100% when incubated with cells engineered to express human PSMA,wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 1-3, 4-6 and / or 7 (PSMA1) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from I gG 1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0221] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 1-3, 4-6 and / or 7 (PSMA1) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from I gG 1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0222] In some embodiments, the rate of internalisation of the construct of the present invention is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 1-3, 4-6 and / or 7 (PSMA1) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0223] In some embodiments, the rate of internalisation of the binding molecule of the present invention is at least about 20% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein the binding molecule comprises a VHH domain that specifically binds to PSMA and a VHH domain that specifically binds to an ALFA tag, optionally wherein the binding molecule comprises one or more immunoglobulin constant domains. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0224] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is at least about 90% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hingeregion derived from lgG1. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0225] In some embodiments, the rate of internalisation of the construct of the present invention is at least 90% when incubated with cells engineered to express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG1. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0226] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is about 100% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0227] In some embodiments, the rate of internalisation of the construct of the present invention is at least 100% when incubated with cells engineered to express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0228] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is at least 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from I gG 1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.In some embodiments, the rate of internalisation of the construct of the present invention is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 8-10, 11-13 and / or 14 (PSMA2) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0229] In some embodiments, the rate of internalisation of the binding molecule of the present invention is at least about 80% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein the binding molecule comprises a VHH domain that specifically binds to PSMA and a VHH domain that specifically binds to an ALFA tag, optionally wherein the binding molecule comprises one or more immunoglobulin constant domains. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0230] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is about 100% when incubated with cells engineered to express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from I gG 1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0231] In some embodiments, the rate of internalisation of the construct of the present invention is about 100% when incubated with cells engineered to express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from I gG 1 or lgG3. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0232] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domain of the binding molecule is defined with reference toSEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from lgG1. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0233] In some embodiments, the rate of internalisation of the construct of the present invention is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG1. In embodiments, the rate of internalisation is after 1 hour of incubation at 37°C.

[0234] In some embodiments, when the binding molecule of the present invention is comprised in a construct comprising a first and second binding molecule, the rate of internalisation is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domain of the binding molecule is defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein each binding molecule comprises a VHH domain and an Fc region, optionally further comprising a hinge region derived from lgG1. In embodiments, the rate of internalisation is after 4 hours of incubation at 37°C.

[0235] In some embodiments, the rate of internalisation of the construct of the present invention is at least about 50% when incubated with cells that endogenously express human PSMA, wherein the VHH domains in the construct of the present invention are defined with reference to SEQ ID NOs: 15-17, 18-20 and / or 21 (PSMA3) as described herein, optionally wherein the construct of the present invention is a VHH-Fc fusion construct, optionally comprising a hinge region derived from lgG1. In embodiments, the rate of internalisation is after 4 hours of incubation at 37°C.

[0236] In some embodiments, the binding molecule, construct or BPC has a rate of internalisation into a cell expressing PSMA that is greater than, equivalent to, substantially the same as, or the same as, that of a control. In some embodiments, the control is a positive control. In some embodiments, a positive control is a binding molecule, construct of BPC that has been identified as having a high level of internalisation into a cell expressing PSMA. In some embodiments, the binding molecule, construct or BPC has a rate of internalisation into a cell expressing PSMA that is within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of that of a positive control, wherein the %values are absolute amounts relating to the rate of internalisation. For example, in some such embodiments, for a construct having a rate of internalisation that is within 5% of that of a control with a rate of internalisation of 95%, the construct may have a rate of internalisation that is as low as 90%. In some embodiments, the binding molecule, construct or BPC has a rate of internalisation into a cell expressing PSMA that is within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of that of a positive control, wherein the % values are calculated as a % of the rate of internalisation of the control. For example, in some such embodiments, for a construct having a rate of internalisation that is within 5% of that of a control with a rate of internalisation of 80%, the construct may have a rate of internalisation that is as low as 72%.

[0237] In some embodiments, the (positive) control is a binding molecule, construct or BPC of PSMA1 , PSMA2 or PSMA3. In the context of being used as a control, PSMA1 is the binding molecule, construct or BPC defined as comprising the VHH domain of SEQ ID NO: 7. In the context of being used as a control, PSMA2 is the binding molecule, construct or BPC defined as comprising the VHH domain of SEQ ID NO: 14. In the context of being used as a control, PSMA3 is the binding molecule, construct or BPC defined as comprising the VHH domain of SEQ ID NO: 21.

[0238] In some embodiments, the control is a negative control. In some embodiments, the binding molecule, construct or BPC of the invention has an internalisation rate that is greater than that of a negative control. In an embodiment, the negative control is 111ln-JVZ-007-c-myc-his (Chatalic et al., J Nucl Med 2015; 56:1094-1099). In an embodiment, the negative control is 111ln-JVZ-007-cys (also Chatalic etal., J Nucl Med 2015; 56:1094-1099).

[0239] In some embodiments, the binding molecule, construct or BPC of the invention has an internalisation rate on LNCaP cells (endogenously expressing PSMA) that is greater than about 30% or about 37%. In some embodiments of this type, the binding molecule, construct or BPC of the invention has an internalisation rate of at least about 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 49% or 50% on LNCaP cells. In some embodiments of this type, the internalisation rate is following incubation for 90 minutes at 37°C.

[0240] As used herein, in embodiments, “incubation” means contacting the cell expressing PSMA with the binding molecule, construct or BPC in appropriate conditions for binding and internalisation, which the skilled person can determine. The incubation time is therefore the duration for which the cells are exposed to the binding molecule, construct or BPC. The incubation temperature is therefore the temperature of the cells during the incubation. It is to be understood that the incubation time, temperature and rate of internalisation are all to bedetermined within margins that are as accurate as can reasonably be determined by the skilled person.

[0241] Other properties

[0242] In some embodiments, the binding molecule, construct or BPC exhibits a high affinity (e.g. a low dissociation constant, KD) for human PSMA. In some embodiments, the affinity for human PSMA is substantially the same as, the same as or at least as high as that of one or more of the positive controls as defined above in respect of internalisation. In some embodiments, the affinity for human PSMA is in the nanomolar, sub-nanomolar or picomolar range. In some embodiments, the KD value for human PSMA is in the picomolar range, such as in the double digit picomolar range. In some embodiments, the KD value for human PSMA is about 10E'9M or less, preferably about 10E'10M or less. In some embodiments, the KD for human PSMA is less than about 10E'11M. In some embodiments, the binding molecule, construct or BPC has a slow off-rate (Koff). In some embodiments, the Koff is about 1E'4M or less. In some embodiments, the Koff is about 1.0E'7M or less.

[0243] In some embodiments, the binding molecule, construct or BPC comprises a polypeptide sequence (e.g. the V region or Fr1-Fr3) having at least 70% identity, such as from about 70% identity to about 80% identity inclusive, to a polypeptide encoded by the human germline IGHV3-23*04 V gene (e.g. the V region or Fr1-Fr3) (SEQ ID NO: 24).

[0244] In some embodiments, the binding molecule, construct or BPC comprises a polypeptide sequence (e.g. the V region or Fr1-Fr3) having at least 70% identity, such as from about 70% identity to about 80% identity inclusive, to a polypeptide encoded by the human germline or IGHV3-30*02 V gene (e.g. the V region or Fr1-Fr3) (SEQ ID NO: 25).

[0245] In some embodiments, the binding molecule, construct or BPC inhibits the enzymatic activity of human PSMA.

[0246] Nucleic acid sequence(s)

[0247] In an aspect, the present invention provides one or more nucleic acid sequence(s) capable of expressing the binding molecule or construct of the invention.

[0248] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. The nucleic acid sequence(s) may be RNA or DNA sequences, or a mixture of RNA and DNA sequences.In an embodiment, the nucleic acid sequence(s) are one or more DNA sequences, such as cDNA sequences. In an embodiment, the nucleic acid sequence is a DNA sequence, such as a cDNA sequence. In an embodiment, the nucleic acid sequence(s) are RNA sequences, such as mRNA sequences. In an embodiment, the nucleic acid sequence is an RNA sequence, such as an mRNA sequence.

[0249] The nucleic acid sequence(s) may be single-stranded or may be double-stranded. The nucleic acid sequence(s) may be, for example, genomic, recombinant, mRNA or cDNA. The nucleic acid sequence(s) may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance in vivo activity and / or stability.

[0250] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These variations in nucleic acid sequences are encompassed by the present invention. Therefore, multiple nucleic acid sequence(s) are envisaged, each of which may be different, but which still encode a binding molecule or construct according to the present invention. It is known in the art how to design and produce such nucleic acid sequences.

[0251] In some embodiments, the nucleic acid sequence(s) may be codon optimised for production in the host cell of choice. In some embodiments, the nucleic acid sequence(s) may be operably linked to further sequence(s) such as control sequence(s), e.g. promoter sequence(s), enhancer sequence(s), polyadenylation signal sequence(s) and / or other regulatory sequence(s), which control transcription and / or translation. The nucleic acid sequence(s) may be in the form of one or more expression cassettes. The nucleic acid sequences may be suitable for expression in prokaryotic cells or in eukaryotic cells, such as mammalian cells. Any promoter may be used, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0252] Vector

[0253] In an aspect, the present invention provides a vector comprising the one or more nucleic acid sequence(s) of the invention.

[0254] Accordingly, the vector may comprise a polynucleotide comprising a nucleic acid sequence or sequences encoding the binding molecule according to the invention or the construct according to the invention.The vector may be used to introduce nucleic acid sequence(s) according to the invention into a cell so that the cell expresses and / or produces the binding molecule according to the invention or the construct according to the invention.

[0255] As used herein, the term “vector” may be considered interchangeable with the term “expression vector” and “expression construct”. The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[0256] The vector according to the invention may be any agent capable of delivering nucleic acid sequence(s) according to the invention to a cell and / or expressing nucleic acid sequence(s) according to the invention in a cell. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0257] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector.

[0258] The vector may be capable of transfecting or transducing a cell.

[0259] Cells and related methods

[0260] In an aspect, the present invention provides a cell comprising the binding molecule, construct, BPC, one or more nucleic acid sequence(s) or vector of the invention.

[0261] The nucleic acid or vector may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo.

[0262] In an aspect, the present invention therefore provides a method for making a cell according to the invention, comprising the step of introducing the one or more nucleic acid sequence(s) or vector of the invention into said cell. In some embodiments, the nucleic acid sequence(s) or vector may be introduced as described herein.

[0263] In some embodiments, the cell may be capable of expressing the binding molecule or construct of the invention. In some embodiments, the cell may be capable of producing the binding molecule according to the invention or the construct according to the invention. In some embodiments, the cell may be capable of expressing and / or producing the binding molecule according to the invention or the construct according to the invention when the cell is cultured under suitable conditions.In an aspect, the present invention therefore provides a method for producing the binding molecule or construct according to the invention, wherein the method comprises the steps of:

[0264] (i) introducing the one or more nucleic acid sequence(s) or vector according to the invention into a cell; and

[0265] (ii) expressing the binding molecule or construct in the cell;

[0266] and optionally (iii) harvesting the binding molecule or construct thereof from the cell or cell culture supernatant of the cell.

[0267] In some embodiments of the methods according to the invention, the nucleic acid sequence(s) or vector may be introduced into the cell by transduction or transfection in vitro or ex vivo.

[0268] In some embodiments of the methods according to the invention, culturing the cell under suitable conditions may result in the cell expressing and / or producing the binding molecule or construct of the invention.

[0269] In some embodiments, the method for producing the binding molecule or the construct of the invention may further comprise step (iii) harvesting the binding molecule or construct of the invention from the cell or cell culture supernatant of the cell.

[0270] It will be understood that the binding molecule according to the invention or the construct according to the invention may be harvested from the cell. It will also be understood that the binding molecule according to the invention or the construct according to the invention may be harvested from supernatant of the cell, for example when the binding molecule or construct is released out of the cell into the cell culture medium that the cell is cultured in.

[0271] In some embodiments, the cell may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell may be a bacterial cell, a fungal cell, a yeast cell, a plant cell or an animal cell. In some embodiments, the cell may be a mammalian cell or an insect cell. In some embodiments, the cell may be a human cell.

[0272] Pharmaceutical compositions

[0273] In an aspect, the present invention provides a composition comprising the binding molecule, construct or BPC of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.In some embodiments, the pharmaceutical composition may comprise the binding molecule according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0274] In some embodiments, the pharmaceutical composition may comprise the construct according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0275] In some embodiments, the pharmaceutical composition may comprise the BPC according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0276] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more selected from this list consisting of: a pharmaceutically acceptable adjuvant, salt, active polypeptide, compound, component and active agent.

[0277] Pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition according to the invention may be produced using current good manufacturing practices (CGMP).

[0278] The pharmaceutical composition may be formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Pharmaceutical compositions can be formulated for administration by different routes, for example, for oral, parenteral, topical, inhalative, intravenous, intramuscular, rectal, sublingual, transdermal, subcutaneous, intratumoral application routes, according to their chemical and physical properties.

[0279] The pharmaceutical composition may be in the form of a tablet, a coated tablet, powder, granulate, a pellet, a capsule, an effervescent tablet or a transdermal therapeutic system. The pharmaceutical composition may be in the form of a liquid composition, selected from the group consisting of a solution, a syrup, an infusion, an extract, a solution for intravenous application, or a solution for infusion. The pharmaceutical composition may be in the form of a semisolid composition such as an emulsion, a suspension, a cream, a lotion, a gel, a globule, a buccal tablet or a suppository.

[0280] The term “carrier”, as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. A sterilesaline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0281] Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0282] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The composition of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0283] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

[0284] In some embodiments, the pharmaceutical composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0285] In some embodiments, the pharmaceutical composition may be in a form suitable for intravenous infusion. In some embodiments, the pharmaceutical composition may be administered intravenously.

[0286] In some embodiments, the pharmaceutical composition may comprise one or more vesicles, nanoparticles, lipid nanoparticle (LNPs), liposomes or polymeric mixtures.

[0287] KitsIn an aspect, the present invention also provides a kit comprising:

[0288] (i) the pharmaceutical composition according to the invention; and

[0289] (ii) a pharmaceutical composition comprising an adaptor molecule comprising a second binding component and one or more payload(s);

[0290] wherein the second binding component is capable of binding to a first binding component of the binding molecule, conjugate or BPC of the present invention. In some embodiments, the second binding component comprises a binding moiety or cognate, wherein the binding molecule, construct or BPC of the invention comprises the cognate thereof or binding moiety thereof respectively. In some embodiments, the second binding component comprises a VHH domain that specifically binds to an ALFA tag and the binding molecule, construct or BPC of the invention comprises an ALFA tag. In some embodiments, the second binding component comprises an ALFA tag and the binding molecule, conjugate or BPC of the present invention comprises a VHH domain that specifically binds to an ALFA tag.

[0291] In an aspect, the present invention also provides a kit comprising:

[0292] (i) the pharmaceutical composition according to the invention; and

[0293] (ii) a pharmaceutical composition comprising one or more payload(s);

[0294] wherein the one or more payload(s) is capable of binding to the binding molecule or construct of the pharmaceutical composition of (i).

[0295] In some embodiments, the kit may optionally comprise (iii) instructions for using the kit to target the one or more payload(s) to a cell expressing PSMA.

[0296] Methods and uses

[0297] In an aspect, the present invention provides an in vitro method comprising contacting a cell with the binding molecule, construct, BPC or composition of the invention.

[0298] In an aspect, the present invention provides a method of therapy or a diagnostic method comprising administering the binding molecule, construct, BPC or composition of the invention to a subject.

[0299] In an aspect, the present invention provides the binding molecule, construct, BPC or composition of the present invention for use in the manufacture of a medicament or diagnostic agent.In some embodiments, the method is a method of treating, preventing or diagnosing cancer.

[0300] In some embodiments, the cancer expresses PSMA, wherein the expression of PSMA in the cancer is increased compared to the expression of PSMA by the same non-cancerous tissue or cells.

[0301] In some embodiments, the cancer is selected from the list consisting of brain cancer, pancreatic cancer, cancer of the pituitary gland, cancer of the hypothalamus, prostate cancer, testicular cancer, skin cancer, renal cancer, cervical cancer, breast cancer, ovarian cancer, cancer of the fallopian tube, stomach cancer, lung cancer, thymus cancer, colorectal cancer, gastro-intestinal cancer neuroendocrine cancer, and rectal neuroendocrine cancer.

[0302] In some embodiments, the cancer is prostate cancer.

[0303] In some embodiments, the binding molecule according to the invention, the conjugate according to the invention, the BPC according to the invention, or the pharmaceutical composition according to the invention, may be administered to a subject systemically.

[0304] In some embodiments, the binding molecule according to the invention, the conjugate according to the invention, the BPC according to the invention, or the pharmaceutical composition according to the invention may be administered intravenously to a subject.

[0305] The term "disease" and "disorder" are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a cell, a tissue, an organ, or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a cell, a tissue, an organ, or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease.

[0306] A progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or "bettering" of the disease. The "worsening" of a disease is characterised by a decreasing ability of a cell, tissue, organ or individual / patient to fulfil its function efficiently, whereas the "bettering" of a disease is typically characterised by an increase in the ability of a cell, tissue, an organ or an individual / patient to fulfil its function efficiently.

[0307] The terms “treat”, “treatment” and “treating” refers to lessening, reducing or improving at least one symptom associated with an existing disease or condition and / or to slow down,reduce or block the progression of the disease or condition and / or to delay or prevent the onset of symptoms (such as further symptoms) of the disease or condition.

[0308] The terms “prevent”, “prevention” and “preventing” refers to preventing the onset of symptoms of a disease or condition, and as such encompasses prophylactic treatment.

[0309] The medical uses and methods of treatment described herein may be used in combination with additional treatments and / or medicaments.

[0310] In some embodiments, the binding molecule according to the invention, the conjugate according to the invention, the BPC according to the invention, or the pharmaceutical composition according to the invention may be used in a method for diagnosing a disease. In some embodiments, the disease may be any disease described herein.

[0311] It will be understood that binding of the binding molecule according to the invention, the conjugate according to the invention, the BPC according to the invention to a target cell expressing PSMA may be used to indicate the presence of a disease state, such as a disease associated with PSMA expression or increased PSMA expression (e.g. cancer or prostate cancer).

[0312] It will also be understood that the binding molecule according to the invention, the conjugate according to the invention may be conjugated to a detection entity, such as a detectable marker, to form a BPC as described herein. Examples of a detectable marker include a fluorescent marker such as a fluorophore, which emits light at a detectable wavelength following excitation of the fluorophore.

[0313] Without wishing to be bound by theory, when the BPC according to the invention comprises a detection entity as a payload (e.g. a fluorophore) and is administered to a subject or is exposed to one or more cells, the signal produced by the detection entity of the BPC may be used to detect the presence of PSMA and thus may be used to diagnose a disease associated with PSMA expression (e.g. cancer) in a subject.

[0314] Accordingly, in some embodiments, the binding molecule according to the invention, the antibody or fragment thereof according to the invention, the PSMA according to the invention, or the pharmaceutical composition according to the invention may be used to identify a subject that is likely to benefit from a treatment of a disease.

[0315] Without wishing to be bound by theory, once a disease associated with PSMA expression (e.g. cancer) has been identified, an BPC according to the invention comprising adrug (e.g. a cytotoxic drug) may be administered to the subject in order to treat the disease associated with PSMA expression (e.g. cancer).

[0316] Subjects

[0317] In some embodiments, the subject may be a mammal.

[0318] In some embodiments, the subject may be a human.

[0319] In some embodiments, the subject may be a non-human mammal, including for example, a dog, a cat, a horse, a cow, a sheep or a pig.

[0320] As used herein, the terms “patient” and “subject” may be used interchangeably.

[0321] In some embodiments, the subject may be a patient. In some embodiments, the subject may be a human patient.

[0322] In some embodiments, the subject may be a human male. In some embodiments, the subject may be a human female.

[0323] Identity

[0324] The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of amino acids (expressed in percent) of an amino acid sequence in a peptide or protein, which across the amino acid sequence, are identical to a reference sequence.

[0325] The percentage of identity is thus calculated by counting the number of aligned amino acids that are identical (a Match) between two sequences (in the amino acids sequence of the peptide or protein of the invention and in the reference sequence), dividing that number by the total number of amino acids in the aligned region and multiplying by 100.

[0326] Therefore, Percentage of Identity = (Matches divided by Length of the aligned region) multiplied by 100.

[0327] Insertions and deletions are not allowed in the calculation the percentage of identity of an amino acid sequence.

[0328] Any method of calculating percentage identity is permitted within the scope of the invention.

[0329] General termsThis disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[0330] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.

[0331] The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”.

[0332] The term “residues” is used to refer to amino acids in an amino acid sequence.

[0333] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0334] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0335] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0336] The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0337] As used herein, the term “variant” is synonymous with the term “mutant” and refers to an amino acid sequence or a nucleic acid sequence that differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a protein comprising an amino acid sequence or a polynucleotide comprising an amino acid sequence, which isidentical to the native protein or native polynucleotide (e.g. gene) respectively. The variant may have an equivalent function to the amino acid sequences or nucleic acid sequences described herein, but may include one or more amino acid or nucleic acid (respectively) substitutions, insertions or deletions. Amino acid substitutions, insertions and / or deletions may be considered as mutations. Nucleic acid substitutions, insertions and / or deletions may be considered as mutations.

[0338] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0339] Sequences

[0340] The VHH domain candidates used herein are PSMA1, PSMA2 and PSMA3.

[0341]

[0342]

[0343]

[0344] NUMBERED PARAGRAPHS

[0345] 1. A binding molecule comprising a single domain variable heavy immunoglobulin (VHH) domain that specifically binds to prostate-specific membrane antigen (PSMA).

[0346] 2. The binding molecule of paragraph 1 , wherein the VHH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:

[0347] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (NYALS),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (AITRAGDPINVADSVKG), and

[0348] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (WDYSDSDVEDY);

[0349] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0350] 3. The binding molecule of paragraph 1 or 2, wherein the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:

[0351] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (GLTFSNYA),

[0352] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (ITRAGDPI), and

[0353] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (NAWDYSDSDVEDY);

[0354] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0355] 4. The binding molecule of any one of paragraphs 1 to 3, wherein the VHH domain comprises the amino acid sequence according to SEQ ID NO: 7 (QLQLVESGGGLVQPGGSLRLSCAASGLTFSNYALSWNRQAPGKERELVAAITRAGDPINV ADSVKGRFTITRDNAKKTVYLQMNSLKPEDTAVYYCNAWDYSDSDVEDYWGQGTLVTVSS

[0356] ), or an amino acid sequence having at least 80% identity thereto.

[0357] 5. The binding molecule of paragraph 1, wherein the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:

[0358] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 8 (DYGVG),

[0359] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 9 (AISWSGSNTYYSDSVKG), and

[0360] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 10 (RAAPEIPDDRATVDS);optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0361] 6. The binding molecule of paragraph 1 or 5, wherein the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:

[0362] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 11 (GRTVWDYG),

[0363] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 12 (ISWSGSNT), and

[0364] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 13 (AARAAPEIPDDRATVDS);

[0365] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0366] 7. The binding molecule of paragraph 1, 5 or 6, wherein the VHH domain comprises the amino acid sequence according to SEQ ID NO: 14 (QVQLVESGGGLVQAGGSLRLSCAASGRTVWDYGVGWFRQAPGKVREFVAAISWSGSNT YYSDSVKGRFTISRDVAKNTVFLQMNSLKPEDTAIYYCAARAAPEIPDDRATVDSWGQGTL VTVSS), or an amino acid sequence having at least 80% identity thereto.

[0367] 8. The binding molecule of paragraph 1, wherein the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:

[0368] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 15 (TYDMA),

[0369] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 16 (AISWSGVNTYYADSVKG), and

[0370] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 17 (G AAASG RYYDTG EG EYDY) ;

[0371] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0372] 9. The binding molecule of paragraph 1 or 8, wherein the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 18 (GRTFSTYD),

[0373] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 19 (ISWSGVNT), and

[0374] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 20 (AAG AAASG RYYDTG EG EYDY) ;

[0375] optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0376] 10. The binding molecule of paragraph 1, 8 or 9, wherein the VHH domain comprises the amino acid sequence according to SEQ ID NO: 21 (QVQLVESGGGLVQAGDSLRLSCAASGRTFSTYDMAWFRQAPGKEREFVAAISWSGVNTY YADSVKGRFTISRENTKNAINLQMNNLKPEDTAVYYCAAGAAASGRYYDTGEGEYDYWGQ GTQVTVSS), or an amino acid sequence having at least 80% identity thereto.

[0377] 11. The binding molecule of any one of paragraphs 1 to 10, wherein the binding molecule comprises one or more immunoglobulin constant domains.

[0378] 12. The binding molecule of paragraph 11 , wherein the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain.

[0379] 13. The binding molecule of paragraph 12, wherein the immunoglobulin constant domains further comprise a constant heavy 2 (CH2) domain.

[0380] 14. The binding molecule of any one of paragraphs 11 to 13, wherein the binding molecule comprises a hinge region between the VHH domain and the constant domains, optionally wherein the hinge region is derived from lgG1 or lgG3, optionally wherein the hinge region has at least 80% identity to SEQ ID NO: 22 (DKTHTCPPCP) or SEQ ID NO: 23 (PRCPEPKACDAPPPCPRCP).

[0381] 15. The binding molecule of any one of paragraphs 11 to 14, wherein the binding molecule comprises 1, 2, 3, 4, 5, 6 or more than 6 sites suitable for conjugation to payloads, preferably 2 to 4 sites suitable for conjugation to payloads.

[0382] 16. The binding molecule of any one of paragraphs 1 to 15, wherein the binding molecule further comprises an additional binding moiety or a cognate of a binding moiety, optionallywherein the additional binding moiety is an antigen-binding fragment that specifically binds to an ALFA-tag, optionally wherein the cognate of a binding moiety is an ALFA tag.

[0383] 17. A construct comprising a first binding molecule according to any one of paragraphs 1 to 16 and a second binding molecule according to any one of paragraphs 1 to 16.

[0384] 18. The construct of paragraph 17, wherein the construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 sites suitable for conjugation to payloads, preferably 4 to 8 sites suitable for conjugation to payloads.

[0385] 19. The construct of paragraph 17 or 18, wherein the first binding molecule and / or the second binding molecule comprise one or more modifications that enhance the formation of the construct, optionally wherein the modifications comprise knobs-into-holes modifications.

[0386] 20. A binding molecule-payload conjugate (BPC) comprising a binding molecule or construct according to any one of paragraphs 1 to 19, and one or more payload(s), optionally further comprising a linker connecting the binding molecule or conjugate to the payload(s).

[0387] 21. The BPC according to paragraph 20, wherein the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 unpaired cysteine residues, preferably 4 to 8 unpaired cysteine residues.

[0388] 22. The BPC according to paragraph 21, wherein the unpaired cysteine residues are bound to the payloads of the BPC.

[0389] 23. The BPC according to any one of paragraphs 20 to 22, wherein the payload is a chemotherapeutic entity, a radionuclide or a detection entity.

[0390] 24. The BPC according to any one of paragraphs 20 to 23, wherein the payload is a drug, optionally wherein the drug is a cytotoxic drug, a DNA-damaging agent, a tubulin inhibitor, a topoisomerase I inhibitor, an immunomodulator, a STING agonist or a nucleic acid, optionally wherein the nucleic acid is an antisense oligonucleotide, siRNA or miRNA.

[0391] 25. The BPC according to any one of paragraphs 20 to 24, wherein the payload is a drug capable of treating cancer, optionally wherein the payload is a drug capable of treating prostate cancer in humans.

[0392] 26. The BPC according to any one of paragraphs 20 to 25, wherein

[0393] (i) the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 payloads, preferably 4 to 8 payloads; or(ii) the BPC has a payload to BPC ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12, preferably of 4 to 8;

[0394] optionally wherein the payloads are different or are the same.

[0395] 27. The binding molecule, construct or BPC of any one of paragraphs 1 to 26, wherein the VHH domain that specifically binds to PSMA induces internalisation of the binding molecule, construct or BPC into a cell expressing PSMA.

[0396] 28. The binding molecule, construct or BPC of any one of paragraphs 1 to 27, wherein the VHH domain that specifically binds to PSMA induces a high rate of internalisation of the binding molecule, construct or BPC into a cell expressing PSMA.

[0397] 29. The binding molecule, construct or BPC of paragraph 28, wherein the rate of internalisation is determined by fluorescence activated cell sorting (FACS) or microscopy.

[0398] 30. The binding molecule, construct or BPC of paragraph 28 or 29, wherein the rate of internalisation is at least 20% at 1 hour, optionally wherein the rate of internalisation is at least 25% at 1 hour, at least 30% at 1 hour, at least 35% at 1 hour, at least 40% at 1 hour, at least 45% at 1 hour, at least 50% at 1 hour, at least 55% at 1 hour, at least 60% at 1 hour, at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, at least 80% at 1 hour, at least 81% at 1 hour, at least 82% at 1 hour, at least 83% at 1 hour, at least 84% at 1 hour, at least 85% at 1 hour, at least 86% at 1 hour, at least 87% at 1 hour, at least 88% at 1 hour, at least 89% at 1 hour, at least 90% at 1 hour, at least 91% at 1 hour, at least 92% at 1 hour, at least 93% at 1 hour, at least 94% at 1 hour, at least 95% at 1 hour, at least 96% at 1 hour, at least 97% at 1 hour, at least 98% at 1 hour, at least 99% at 1 hour, or at least 100% at 1 hour.

[0399] 31. The binding molecule, construct or BPC of any one of paragraphs 28 to 30, wherein the rate of internalisation is following incubation with PSMA-positive cells at 37°C for 1 hour.

[0400] 32. The binding molecule, construct or BPC of any one of paragraphs 1 to 31 , wherein the binding molecule, construct or BPC has a rate of internalisation into a cell expressing PSMA that is greater than, equivalent to, substantially the same as, or the same as, that of a control.

[0401] 33. The binding molecule, construct or BPC of paragraph 32, wherein the control is a binding molecule, construct or BPC of PSMA1, PSMA2 or PSMA3.

[0402] 34. The binding molecule, construct or BPC of any one of paragraphs 1 to 33, wherein the binding molecule, construct or BPC comprises a polypeptide sequence having at least 70%identity to a polypeptide encoded by the human germline IGHV3-23*04 V gene (SEQ ID NO: 24).

[0403] 35. The binding molecule, construct or BPC of any one of paragraphs 1 to 33, wherein the binding molecule, construct or BPC comprises a polypeptide sequence having at least 70% identity to a polypeptide encoded by the human germline or IGHV3-30*02 V gene (SEQ ID NO: 25).

[0404] 36. One or more nucleic acid sequence(s) capable of expressing the binding molecule or construct of any one of paragraphs 1 to 19 or 27 to 35.

[0405] 37. The one or more nucleic acid sequence(s) of paragraph 36, wherein the one or more nucleic acid sequence(s) is an RNA sequence.

[0406] 38. A vector comprising the one or more nucleic acid sequence(s) of paragraph 36 or 37.

[0407] 39. A cell comprising the binding molecule, construct, BPC, one or more nucleic acid sequence(s) or vector of any one of paragraphs 1 to 38.

[0408] 40. The cell of paragraph 39, wherein the cell is capable of expressing the binding molecule or construct of any one of paragraphs 1 to 19 or 27 to 35.

[0409] 41. A method for making a cell according to paragraph 39 or 40, comprising the step of introducing the one or more nucleic acid sequence(s) or vector according to any one of paragraphs 36 to 38 into said cell.

[0410] 42. A method for producing the binding molecule or construct according to any of paragraphs 1 to 19 or 27 to 35, wherein the method comprises the steps of:

[0411] (i) introducing the one or more nucleic acid sequence(s) or vector according to any one of paragraphs 36 to 38 into a cell; and

[0412] (ii) expressing the binding molecule or construct in the cell;

[0413] and optionally (iii) harvesting the binding molecule or construct thereof from the cell or cell culture supernatant of the cell.

[0414] 43. A composition comprising the binding molecule, construct or BPC of any one of paragraphs 1 to 35, together with a pharmaceutically acceptable carrier, diluent or excipient.44. An in vitro method comprising contacting a cell with the binding molecule, construct, BPC or composition of any one of paragraphs 1 to 35 or 43.

[0415] 45. The binding molecule, construct, BPC or composition of any one of paragraphs 1 to 35 or 43 for use in a method of therapy or a diagnostic method.

[0416] 46. The binding molecule, construct, BPC or composition for use of paragraph 45, wherein the method is a method of treating, preventing or diagnosing cancer.

[0417] 47. The binding molecule, construct, BPC or composition for use for use of paragraph 46, wherein the cancer expresses PSMA, wherein the expression of PSMA in the cancer is increased compared to the expression of PSMA by the same non-cancerous tissue or cells.

[0418] 48. The binding molecule, construct, BPC or composition for use of paragraph 46 or 47, wherein the cancer is selected from the list consisting of brain cancer, pancreatic cancer, cancer of the pituitary gland, cancer of the hypothalamus, prostate cancer, testicular cancer, skin cancer, renal cancer, cervical cancer, breast cancer, ovarian cancer, cancer of the fallopian tube, stomach cancer, lung cancer, thymus cancer, colorectal cancer, gastrointestinal cancer neuroendocrine cancer, and rectal neuroendocrine cancer.

[0419] 49. The binding molecule, construct, BPC or composition for use of any one of paragraphs 46 to 48, wherein the cancer is prostate cancer.

[0420] 50. A kit comprising the binding molecule, construct, BPC or composition of any one of paragraphs 1 to 35 or 43, optionally further comprising instructions for using the kit to inhibit PSMA in vitro.

[0421] EXAMPLES

[0422] Materials and Methods

[0423] Expression of recombinant proteins

[0424] Human codon optimized protein sequences were generated by gene synthesis and cloned into pTWIST expression vectors (Twist Bioscience). For recombinant expression of the VHH-Fc fusions (constructs comprising an anti-PSMA VHH domain, an engineered hinge region and an Fc region) and ProteinDocker constructs (constructs comprising at least an anti-PSMA VHH domain and an NbALFA domain as well as potential lgG1-derived constant domains), Expi293 cells were transfected with ExpiFectamine (ThermoFisher Scientific) using the manufacturer’s protocol. Culture supernatants were harvested from Expi293 producer celllines after 4-5 days, centrifuged, and the produced recombinant proteins were used for further characterization and purification.

[0425] DLS and NanoDSF

[0426] Thermal stabilities (via NanoDSF) and size distribution (via DLS) were investigated using the Prometheus PANTA from NanoTemper. 10 pL of the fusion proteins were loaded into capillaries. After loading, the capillaries were mounted into the instrument and the DLS analysis was performed. To assess thermal stability, a heat ramp of 1°C / min from 25°C to 95°C was subsequently applied to all samples. During this process the intrinsic fluorescence of the proteins was measured at 350 nm and 330 nm. The ratio was plotted against the temperature and the first derivative was calculated. Minima and maxima corresponded to the TM values.

[0427] Analytical SEC

[0428] Aggregation analysis was performed via SEC utilizing an Agilent Infinity II HPLC and a Biozen 1.8 pm dSEC-2, 200 A LC column (300 x 4.6 mm). Flowrates were adjusted to 0.25 mL / min, resulting in approximately 255 bar pressure. As mobile phase 0.2 M potassium phosphate, 250 mM KCI, pH 6.2, 5% acetonitrile was used. Each run took 20 min excluding a 2-3 min wash step between each analysis. 10 pL of the fusion proteins were applied and detected by absorption at 280 nm.

[0429] Affinity to recombinant human PSMA for NbALFA-fusion proteins

[0430] For affinity measurements an Octet HTX device from Sartorius was used.

[0431] Commercially available SAX2.0 biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the biotinylated ALFA-tag peptide with a concentration of 83 ng / mL was loaded for 240 sec. Subsequently, a quenching step in a 100 pg / mL biocytin solution was performed for 120 sec, followed by a 120 sec baseline in KB.

[0432] The ProteinDocker was applied with a concentration of 10 nM to all biosensors for 300 sec or until a threshold of 0.7 nm in response was reached. Following a 60 sec baseline in KB, association was measured over 600 sec using seven different concentrations of PSMA starting at 50 nM in a 1:1 serial dilution series down to 0.78125 nM. As a reference, KB without the PSMA was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. Forinter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.

[0433] Affinity to CD64 for NbALFA-fusion proteins

[0434] For affinity measurements an Octet HTX device from Sartorius was used.

[0435] Commercially available SAX2.0 biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the biotinylated ALFA-tag peptide with a concentration of 83 ng / mL was loaded for 240 sec. Subsequently, a quenching step in a 100 pg / mL biocytin solution was performed for 120 sec, followed by a 120 sec baseline in KB.

[0436] The ProteinDocker was applied with a concentration of 10 nM to all biosensors for 300 sec or until a threshold of 0.7 nm in response was reached. Following a 60 sec baseline in KB, association was measured over 600 sec using seven different concentrations of recombinant human CD64 starting at 5 nM in a 1:1 serial dilution series down to 0.08 nM. As a reference, KB without CD64 was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.

[0437] Affinity to the ALFA tag

[0438] For affinity measurements an Octet HTX device from Sartorius was used.

[0439] Commercially available SAX2.0 biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the biotinylated ALFA-tag peptide was loaded for 240 sec. Subsequently, a quenching step in a 100 pg / mL biocytin solution was performed for 120 sec, followed by a 120 sec baseline in KB.

[0440] Association was measured over 900 sec using seven different concentrations of a ProteinDocker starting at 10 nM in a 1:1 serial dilution series down to 156.25 pM. As a reference, KB without the ProteinDocker was measured. The dissociation was acquired over 1500 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the second baseline. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.

[0441] Cell binding: EC50 determination

[0442] PSMA-positive cells were seeded in a 96-well microtiter plate (round button) and incubated in the presence of different concentrations of the VHH-Fc fusion for 30 min at 4°C. Afterwards, cells were washed twice with FACS-buffer (1x phosphate-buffer saline + 10 mL 0.5 M ethylenediaminetetraacetic acid + 10 mL Fetal Bovine Serum (FBS)) and once with 1x phosphate-buffer saline and subsequently incubated with an anti-human APC detection antibody (for the detection of VHH-Fc) or ALFA-AF647 (for detection of ProteinDocker) for 30 min at 4°C. After another washing step with FACS-buffer cells were fixated with BD Fixative and analysed with a flow cytometer (BD Bioscience. Mean fluorescence intensities (MFI) values were plotted against the concentration of the VHH-Fc or the ProteinDocker and the resulting data points were fitted using a non-linear fit, resulting in the EC50 values of the respective VHH-Fc or ProteinDocker.

[0443] Analysis of internalization via flow cytometry

[0444] PSMA positive cells were seeded in FACS tubes and incubated in the presence of 100 nM or 500 nM VHH-Fc fusion or ProteinDocker for 30 min at 4°C.

[0445] Afterwards, cells were washed twice with FACS-buffer and once with 1x phosphate-buffer saline and were subsequently resuspended in 1 mL growth medium. Cells were incubated for either 30 min, 1 h, 2h or 4h at either 37°C or on ice. Cells were washed twice with FACS buffer and once with PBS before an anti-human APC detection antibody (for the detection of VHH-Fc) or ALFA-AF647 (for detection of ProteinDocker) was applied for 30 min. After another washing step with FACS-buffer and PBS, cells were fixated with BD Fixative and analysed with a flow cytometer (BD Biosciences). The MFI values of the 4°C samples were compared with their respective 37°C counterpart to visualize the internalization-mediated difference in fluorescence intensity.

[0446] Affinity to recombinant human PSMA of VHH-Fc fusions

[0447] For affinity measurements an Octet HTX device from Sartorius was used.

[0448] Commercially available AHC biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the VHH-Fc proteins were loaded onto the sensors loaded for 300 sec or until a threshold of 0.9 nm in response was reached. Following a 120 sec baseline in KB, association to PSMA wasmeasured over 600 sec using seven different concentrations of the PSMA starting at 50 nM in a 1:1 serial dilution series down to 0.78125 nM. As a reference, KB without PSMA was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.

[0449] Affinity to CD64 of VHH-Fc fusions

[0450] For affinity measurements an Octet HTX device from Sartorius was used.

[0451] Commercially available Ni-NTA biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the His-tagged CD64 was loaded for 300 sec or until a threshold of 1 nm in response was reached. Following a 120 sec baseline in KB, association to the VHH-Fcs were measured over 600 sec using seven different concentrations of the VHH-Fcs starting at 50 nM in a 1:1 serial dilution series down to 0.78125 nM. As a reference, KB without the VHH-Fcs was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.

[0452] High-content imaging internalization assay

[0453] The anti-PSMA-VHH-Fc fusions of the invention were evaluated in their internalization rate and strength in overexpressing CHO-FIpIn-human PSMA and prostate adenocarcinoma LNCaP cells via high-content imaging.

[0454] Cells were seeded in 384 well plates overnight. Each of the anti-PSMA- VHH-Fc lgG1-hinge or lgG3 hinge constructs (see e.g. Figure 3a and 3b) was premixed at 5nM with 10nM Alexafluor 488 anti-human IgG-Fab fragment (Dianova) for 30 minutes at 4°C. The premix was then allowed to bind to Hoechst 33342 stained cells for 30 minutes at 4°C and afterwards washed off. The plates were cultivated at 37°C in humidified CO2 incubator for either 0, 1 or 4 hrs. Subsequently part of the replicate conditions were either treated with cell medium (nonquenched) or 100nM anti-AlexaFluor488-lgG quenching antibody for 30 minutes at4°C. After a washing step, plates were measured for Hoechst and Alexa Fluor 488 staining in the high-content imager Celllnsight CX5 (Thermo Fisher). The software module SpotDetector was deployed for cell masking and calculation of the mean Alexa Fluor 488 Intensity per well accounting for each single cell, mean Alexa Fluor 488 spot intensity.

[0455] Percent internalization was calculated from the ratio of mean Alexa Fluor 488 intensity from the quenched condition to the mean intensity of AlexaFluor 488 without quenching. In the quenched condition only internalized VHH-Fc fusions are detected which are increasing over time proportional to the rate of internalization.

[0456] Example 1 - Construction and characterization of anti-PSMAxNbALFA fusion proteins in different formats

[0457] The present inventors strived to investigate suitable antibody formats for PSMA-directed ALFA-tagged payload delivery. Therefore, the VHHs PSMA2 and PSMA1 were on a genetic level fused to certain immunoglobulin constant domains of a human lgG1 antibody and a humanized NbALFA VHH (SEQ ID NO: 27). These formats differ in molecular size, valency and general architecture (Figure 1), and are referred to as ProteinDocker herein. Consequently, different in vivo half-lives are expected from these molecules and different ALFA-payload molecules are suitable. E.g. the VHH-CH3 fusion format RD5 as illustrated in Figure 1 lacks the interaction site of the neonatal Fc receptor (FcRn) in its constant domains due to the deletion of the CH2 domain. Combined with its smaller molecular size, a shorter half-life compared to other formats is expected. The formats RD3 and RD5 differ in valencies to ALFA-tagged molecules. While RD3 binds only one ALFA-conjugated compound, RD4 can bind two ALFA tags simultaneously. This allows to usage of mono- or bis-ALFA conjugates, respectively, resulting in different mode of actions. The RD6 format exhibits similar size and the same valencies as the RD3 format, but differs in domain orientation. The resulting six proteins were produced in ExpiHEK cells and after purification tested for a variety of biophysical parameters using NanoDSF, DLS and analytical SEC (Table 1).

[0458] Table 1: Determination of biophysical properties of ProteinDocker fusion proteins.

[0459] Melting temperature (TM values), as well as onset temperatures (ON) are shown. Temperature-dependent turbidity inflection point (IP) and onsets values as well as temperature-dependent aggregation (cumulant radius ON) values are given under thermal stability. DLS measurements of the non-heated sample as well as purity in SEC analysis are depicted for all candidates.

[0460]

[0461]

[0462] Even though differences in TM values were observed based on the format and the respective anti-PSMA VHH, all proteins were ranked as thermostable, i.e. the apparent differences did not have a significant negative effect on the thermostability of any generated construct. A promising aggregation behaviour for all constructs was further confirmed by DLS and SEC.

[0463] Besides favourable biophysical properties, the functionality of these proteins is one of the most crucial parameters. Consequently, the inventors investigated the binding affinities to recombinant PSMA as well as FcyRI (CD64) and the ALFA tag (see Table 2 below).

[0464] Table 2: BLI-assisted affinity determination to PSMA, the ALFA tag and CD64 of ProteinDocker fusion proteins.

[0465]

[0466] The determination of the binding affinity to PSMA clearly showed that the different molecular designs do not influence the high affinity recognition of the antigen. The same effect wasobserved for interaction with the ALFA tag. Only the RD4 format showed a slower off-rate in ALFA binding, which is expected as the bivalent NbALFA mediated avidity effects by binding to immobilized ALFA peptides. Further, binding to FcyRI was not altered except for the RD5 format. Due to the deletion of the CH2 domain and the resulting lack of the CD64 binding epitope in the RD5 format, this was expected.

[0467] Next, the inventors wanted to elucidate the binding behaviour of the ProteinDocker on PSMA endogenously expressing LNCaP cells (Table 3). Once again it was demonstrated that, independently from the molecular format, strong bindings were observed with EC50 values in the single digit nanomolar range.

[0468] Table 3: EC50 values for LNCaP cell binding of the ProteinDocker.

[0469]

[0470] In order to deliver an ALFA-tagged payload efficiently to a PSMA-expressing target cell, the internalization properties of the ProteinDockers are key. Consequently, internalization into the PSMA-expressing prostate cancer cell line LNCaP was investigated using a FACS-based assay. It was found that all ProteinDockers internalized strong and fast (Figure 2). Within the first 30 min of the experiment, most (over 60%) of the anti-PSMA molecules were already internalized. After 4 hours, over 80% of the anti-PSMA molecules were internalized. These unexpectedly rapid and efficacious levels of internalization underline the highly advantageous suitability of ProteinDocker binders comprising the PSMA2 and PSMA1 VHHs for e.g. PSMA-related therapeutic applications.

[0471] Taken together, the inventors demonstrated herein the successful evaluation of the PSMA-binding molecules PSMA2 and PSMA1, fused with NbALFA, in a variety of designs. Besides high binding affinity and stability, strong cell binding and surprisingly fast / efficacious internalization was observed.

[0472] Example 2 - Hinge-engineered PSMA-binders for ADC applicationsBesides ALFA-tagged payload delivery, the inventors strived to investigate whether the anti-PSMA VHHs are suitable for direct conjugation and their usage as antibody drug conjugates. The antibody format chosen for that is referred to as VHH-Fc, describing the N-terminal fusion of the VHH to a (human IgG 1 ) Fc region. For providing such formats, various hinge sequences for linking the VHH targeting domain and Fc region were investigated.

[0473] In one format, a partial human lgG1 hinge (SEQ ID NO: 22) was included connecting the VHH with the Fc. In this partial hinge the Cys220 (Ell numbering) was absent, as this residue in conventional lgG1 molecules forms a disulfide bond with the light chain. As no light chains are used herein, and to avoid unwanted unpaired cysteines, the residues 216-220 (Ell numbering) were removed in these constructs. The remaining two interchain disulfide bonds allow for partial reduction and subsequent conjugation with thiol-reactive payload-linker molecules. Consequently, a construct / BPC with a drug to antibody ratio (DAR) of 4 can be achieved with this format.

[0474] The present inventors further explored the possibility of a second format allowing for higher DAR values, through further non-obvious modifications and design choices regarding the hinge region. In particular, the inventors exchanged the lgG1-hinge by a truncated and post translational modification (PTM)-modified lgG3 hinge region (Figure 3, right-hand schematic, SEQ ID NO: 23). This construct exhibits four interchain disulfides that, upon reduction, result in eight free cysteines. Consequently, a construct / BPC with a DAR of 8 can be achieved with this format.

[0475] Without wishing to be bound by theory, the first construct format comprising an lgG1-derived hinge should provide a DAR of 4, and the second construct format comprising an lgG3-derived hinge should provide a DAR of 8. However, it remained to be determined whether the formats in question would provide their respective DAR values while still maintaining the required and favourable properties of these formats as anti-PSMA binders. Thus, the present inventors constructed and produced VHH-Fc fusion proteins, exhibiting either the described lgG1- or lgG3-derived hinge, using the anti-PSMA VHHs PSMA1, PSMA2 and PSMA3. After production in ExpiHEK cells, the biophysical properties of the molecules were assessed using NanoDSF, DLS and analytical SEC (Table 4).

[0476] Table 4: Determination of biophysical properties of VHH-Fc fusion proteins. Melting temperature (TM values), as well as onset temperatures (ON) are shown. Temperaturedependent turbidity inflection point (IP) and onsets values as well as temperature-dependent aggregation (cumulant radius ON) values are given under thermal stability. DLSmeasurements of the non-heated sample as well as purity in SEC analysis are depicted for all candidates.

[0477]

[0478] As before, high stabilities of the VHH fusion proteins were observed, paired with excellent aggregation behaviours. After confirming the biophysical stability of the molecules, the present inventors investigated the binding properties of the VHH-Fcs to PSMA and FcyRI (Table 5).

[0479] Table 5: BLI-assisted affinity determination to PSMA and CD64 of VHH-Fc fusion proteins.

[0480]

[0481]

[0482] Surprisingly, the present inventors observed that the binding to CD64 was unaltered in BLI experiments when the lgG1-derived hinge was exchanged to the lgG3-derived hinge, even though the CD64 epitope of the CH2 domain is in close proximity to the altered hinge region. Further, very high binding affinities were observed in both formats for all three VHH candidates, underlining the advantageous suitability of the VHHs as well as of both I gG 1 and lgG3-derived formats for further ADC development.

[0483] However, to efficiently target cancer cells, efficient binding to PSMA-positive cells and subsequent internalization is key. Consequently, EC50 values of the VHH-Fc fusion proteins on PSMA-expressing LNCaP cells were determined (Table 6).

[0484] Table 6: EC50 values for LNCaP cell binding of the VHH-Fc fusions.

[0485]

[0486] Acceptable EC50 values were shown for all candidate molecules. With the exception of PSMA3-derived candidates, sub-nanomolar EC50 values were observed, with no differences between the hinge variants found.As a strong cell binding was verified, internalization studies were subsequently performed. Therefore, the above-mentioned FACS-based assay was conducted (Figure 4).

[0487] As for the ProteinDockers in Example 1, within 30 min of incubation time, the majority (over 60%) of the VHH-Fc fusions already internalized into LNCaP cells.

[0488] Additionally, an orthogonal internalization assay using high content imaging was conducted, further underlining the surprisingly fast internalization kinetics of all candidates. This was demonstrated with PSMA-overexpressing CHO cells (Figure 5a, Table 7) as well as with LNCaP cells endogenously expressing PSMA (Figure 5b, Table 8).

[0489] Table 7: Percent internalization of PSMA1 VHH-Fc lgG1, PSMA1 VHH-Fc lgG3, PSMA2 VHH-Fc lgG1, PSMA2 VHH-Fc lgG3, PSMA3 VHH-Fc lgG1, PSMA3 VHH-Fc lgG3 into CHO-FIpIn-human PSMA detected by high-content imaging. Percent internalization of 5nM anti-PSMA VHH-Fc was determined after 0, 1 or 4 hrs incubation at 37°C.

[0490]

[0491] As determined by high-content imaging of CHO cells engineered to express PSMA, internalisation was over 90% after 1 hour of incubation for each construct tested.

[0492] Table 8: Percent internalization of PSMA1 VHH-Fc lgG1, PSMA1 VHH-Fc lgG3, PSMA2 VHH-Fc lgG1, PSMA2 VHH-Fc lgG3, PSMA3 VHH-Fc lgG1, PSMA3 VHH-Fc lgG3 into LNCaP detected by high-content imaging. Percent internalization of 5nM anti-PSMA VHH-Fc was determined after 0, 1 or 4 hrs incubation at 37°C.

[0493]

[0494] As determined by high-content imaging of LNCaP cells endogenously expressing PSMA, internalisation was over 80% after 4 hours of incubation for each construct tested.

[0495] In summary, the present inventors demonstrated that the anti-PSMA VHH-Fc fusion proteins are producible molecules with excellent biophysical properties, high affinity and strong cell binding. Additionally, a surprisingly very fast internalization into PSMA expressing cells could be demonstrated using two orthogonal assays underlining the suitability of those molecules to be developed as anti-PSMA ADCs.

[0496] These properties, in particular the rapid internalisation of the anti-PSMA VHH candidates PSMA1, PSMA2 and PSMA3 across multiple formats, were unexpected and represent a clear improvement in suitability for PSMA-based medical applications.

[0497] It was also unexpected that the lgG1- and lgG3-based hinge regions DARs of 4 and 8 respectively were found to be suitable while still maintaining suitable and advantageous PSMA binder properties such as internalisation, affinity, producibility and receptor (e.g. CD64) binding.

Claims

CLAIMS1. A binding molecule comprising a single domain variable heavy immunoglobulin (VHH) domain that specifically binds to prostate-specific membrane antigen (PSMA).

2. The binding molecule of claim 1, wherein either:a) one or more of the following i)-iii):i) the VHH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (NYALS),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (AITRAGDPINVADSVKG), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (WDYSDSDVEDY);optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;ii) the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (GLTFSNYA),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (ITRAGDPI), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (NAWDYSDSDVEDY);optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;iii) the VHH domain comprises the amino acid sequence according to SEQ ID NO: 7 (QLQLVESGGGLVQPGGSLRLSCAASGLTFSNYALSWNRQAPGKERELVAAITRAG DPINVADSVKGRFTITRDNAKKTVYLQMNSLKPEDTAVYYCNAWDYSDSDVEDYW GQGTLVTVSS), or an amino acid sequence having at least 80% identity thereto;orb) one or more of the following i)-iii):i) the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 8 (DYGVG),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 9 (AISWSGSNTYYSDSVKG), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 10 (RAAPEIPDDRATVDS);optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;ii) the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 11 (GRTVWDYG),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 12 (ISWSGSNT), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 13 (AARAAPEIPDDRATVDS);optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;iii) the VHH domain comprises the amino acid sequence according to SEQ ID NO: 14 (QVQLVESGGGLVQAGGSLRLSCAASGRTVWDYGVGWFRQAPGKVREFVAAISW SGSNTYYSDSVKGRFTISRDVAKNTVFLQMNSLKPEDTAIYYCAARAAPEIPDDRAT VDSWGQGTLVTVSS), or an amino acid sequence having at least 80% identity thereto;orc) one or more of the following i)-iii):i) the VHH domain comprises HCDRs 1-3 as defined by the Kabat definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 15 (TYDMA),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 16 (AISWSGVNTYYADSVKG), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 17 (G AAASG RYYDTG EG EYDY) ;optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;ii) the VHH domain comprises HCDRs 1-3 as defined by the IMGT definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 18 (GRTFSTYD),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 19 (ISWSGVNT), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 20 (AAG AAASG RYYDTG EG EYDY) ;optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences; and / oriii) the VHH domain comprises the amino acid sequence according to SEQ ID NO: 21 (QVQLVESGGGLVQAGDSLRLSCAASGRTFSTYDMAWFRQAPGKEREFVAAISWS GVNTYYADSVKGRFTISRENTKNAINLQMNNLKPEDTAVYYCAAGAAASGRYYDTG EGEYDYWGQGTQVTVSS), or an amino acid sequence having at least 80% identity thereto.

3. The binding molecule of claim 1 or 2, wherein the binding molecule comprises one or more immunoglobulin constant domains; optionally the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain; optionally wherein the immunoglobulin constant domains further comprise a constant heavy 2 (CH2) domain; optionally wherein the binding molecule comprises a hinge region between the VHH domain and the constant domains, preferably wherein the hinge region is derived from I gG 1 or I gG3, optionally wherein the hinge region has at least 80% identity to SEQ ID NO: 22 (DKTHTCPPCP) or SEQ ID NO: 23 (PRCPEPKACDAPPPCPRCP).

4. The binding molecule of claim 3, wherein the binding molecule comprises 1, 2, 3, 4, 5, 6 or more than 6 sites suitable for conjugation to payloads, preferably 2 to 4 sites suitable for conjugation to payloads.

5. The binding molecule of any one of claims 1 to 4, wherein the binding molecule further comprises an additional binding moiety or a cognate of a binding moiety, optionally wherein the additional binding moiety is an antigen-binding fragment that specifically binds to an ALFA-tag, optionally wherein the cognate of a binding moiety is an ALFA tag.

6. A construct comprising a first binding molecule according to any one of claims 1 to 5 and a second binding molecule according to any one of claims 1 to 5;optionally wherein the construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 sites suitable for conjugation to payloads, preferably 4 to 8 sites suitable for conjugation to payloads;optionally wherein the first binding molecule and / or the second binding molecule comprise one or more modifications that enhance the formation of the construct, optionally wherein the modifications comprise knobs-into-holes modifications.

7. A binding molecule-payload conjugate (BPC) comprising a binding molecule or construct according to any one of claims 1 to 6, and one or more payload(s), optionally further comprising a linker connecting the binding molecule or conjugate to the payload(s); optionally wherein the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 unpaired cysteine residues, preferably 4 to 8 unpaired cysteine residues; optionally wherein the unpaired cysteine residues are bound to the payloads of the BPC; optionally wherein the payload is a chemotherapeutic entity, a radionuclide or a detection entity; optionally wherein the payload is a drug, optionally wherein the drug is a cytotoxic drug, a DNA-damaging agent, a tubulin inhibitor, a topoisomerase I inhibitor, an immunomodulator, a STING agonist or a nucleic acid, optionally an antisense oligonucleotide, miRNA or siRNA; optionally wherein the payload is a drug capable of treating cancer, optionally wherein the payload is a drug capable of treating prostate cancer in humans;further optionally wherein(i) the BPC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 payloads, preferably 4 to 8 payloads; or(ii) the BPC has a payload to BPC ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12, preferably of 4 to 8;optionally wherein the payloads are different or are the same.

8. The binding molecule, construct or BPC of any one of claims 1 to 7, wherein the VHH domain that specifically binds to PSMA induces internalisation of the binding molecule, construct or BPC into a cell expressing PSMA; optionally wherein the VHH domain that specifically binds to PSMA induces a high rate of internalisation of the binding molecule, construct or BPC into a cell expressing PSMA; optionally wherein the rate of internalisation is determined by fluorescence activated cell sorting (FACS) or microscopy; optionally wherein the rate of internalisation is at least 20% at 1 hour, at least 25% at 1 hour, at least 30% at 1 hour, at least 35% at 1 hour, at least 40% at 1 hour, at least 45% at 1 hour, at least 50% at 1 hour, at least 55% at 1 hour, at least 60% at 1 hour, at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, at least 80% at 1 hour, at least 81% at 1 hour, at least 82% at 1 hour, at least 83% at 1 hour, at least 84% at 1 hour, at least 85% at 1 hour, at least 86% at 1 hour, at least 87% at 1 hour, at least 88% at 1 hour, at least 89% at 1 hour, at least 90% at 1 hour, at least 91% at 1 hour, at least 92% at 1 hour, at least 93% at 1 hour, at least 94% at 1 hour, at least 95% at 1 hour, at least 96% at 1 hour, at least 97% at 1 hour, at least 98% at 1 hour, at least 99% at 1 hour, or at least 100% at 1 hour; optionally wherein the rate of internalisation is following incubation with PSMA-positive cells at 37°C for 1 hour.

9. The binding molecule, construct or BPC of any one of claims 1 to 8, wherein the binding molecule, construct or BPC has a rate of internalisation into a cell expressing PSMA that is greater than, equivalent to, substantially the same as, or the same as, that of a control; optionally wherein the control is a binding molecule, construct or BPC of PSMA1, PSMA2 or PSMA3.

10. The binding molecule, construct or BPC of any one of claims 1 to 9, wherein the binding molecule, construct or BPC comprises: a) a polypeptide sequence having at least 70% identity to a polypeptide encoded by the human germline IGHV3-23*04 V gene (SEQ ID NO: 24), or b) a polypeptide sequence having at least 70% identity to a polypeptide encoded by the human germline or IGHV3-30*02 V gene (SEQ ID NO: 25).

11. One or more nucleic acid sequence(s) capable of expressing the binding molecule or construct of any one of claims 1 to 6 or 8 to 10; optionally wherein the one or more nucleic acid sequence(s) is an RNA sequence; optionally wherein the one or more nucleic acid sequence(s) are comprised in a vector.

12. A cell comprising the binding molecule, construct, BPC, one or more nucleic acid sequence(s) or vector of any one of claims 1 to 11; optionally wherein the cell is capable of expressing the binding molecule or construct of any one of claims 1 to 6 or 8 to 10.

13. A composition comprising the binding molecule, construct or BPC of any one of claims 1 to 12, together with a pharmaceutically acceptable carrier, diluent or excipient.

14. An in vitro method comprising contacting a cell with the binding molecule, construct, BPC or composition of any one of claims 1 to 10 or 13.

15. The binding molecule, construct, BPC or composition of any one of claims 1 to 10 or 13 for use in a method of therapy or a diagnostic method; optionally wherein the method is a method of treating, preventing or diagnosing cancer; optionally wherein the cancer expresses PSMA, wherein the expression of PSMA in the cancer is increased compared to the expression of PSMA by the same non-cancerous tissue or cells; optionally wherein the cancer is selected from the list consisting of brain cancer, pancreatic cancer, cancer of the pituitary gland, cancer of the hypothalamus, prostate cancer, testicular cancer, skin cancer, renal cancer, cervical cancer, breast cancer, ovarian cancer, cancer of the fallopian tube, stomach cancer, lung cancer, thymus cancer, colorectal cancer, gastro-intestinal cancer neuroendocrine cancer, and rectal neuroendocrine cancer, preferably wherein the cancer is prostate cancer.

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