High-affinity human Anti-ACP3 antibodies for diagnostic and therapeutic use

Abstract

The application relates to the diagnosis and treatment of diseases, including cancer. The invention provides, and involves the use of, fully human antibody molecules and antibody fragment molecules that bind prostatic acid phosphatase (ACP3) from humans and cynomolgus monkeys with high affinity. The antibody molecules may be conjugated to a cytotoxic moiety.

Description

[0001] HIGH-AFFINITY HUMAN ANTI-ACP3 ANTIBODIES FOR DIAGNOSTIC AND THERAPEUTIC USE

[0002] Field of the Invention

[0003] The present invention relates to the diagnosis and treatment of diseases, including cancer. The invention provides and involves the use of fully human antibody and antibody fragment molecules that bind prostatic acid phosphatase (ACP3) from humans and cynomolgus with high affinity. The antibody and antibody fragment molecules may be conjugated to a radioisotope or a cytotoxic moiety.

[0004] Background

[0005] Prostate cancer is the second most common cancer in men. With an estimated 375,304 deaths in 2020 worldwide, prostate cancer is the fifth leading cause of death from cancer in men and represents 6.8% of total cancer death in males (Sung 2021 CA Cancer J Clin 71(3):209-249).

[0006] Treatment of prostate cancer with androgen deprivation therapy (ADT) such as luteinising hormone-releasing hormone (LHRH) analogues or orchidectomy is usually initially effective at controlling metastatic diseases. However, patients inevitably progress from an androgen sensitive to a castration-resistant phenotype which is associated with 90% of overall mortality (Scher et al 2015 PL

[0007]

[0008] 0)).

[0009] Prostatic Acid Phosphatase antigen (ACP3) is a tyrosine phosphatase overexpressed in cancerous prostate cells. ACP3 represents an ideal target for the development of targeted drug conjugates due to its low expression in healthy organs.

[0010] Some low-affinity monoclonal antibodies that bind human ACP3 have been described in the art (see for example Lillehoj et al., Molecular Immunology (1982), 19, 1199-1202; Leroy et al. Cancer (1989), 64, 1-5; Ostrowski J Clin Biochem Nutr (2000), 28, 233-244; Chamberlain et al., Clin Chem (1995), 41 , 1495-1499; and W097 / 05901).

[0011] To our knowledge, only three anti-ACP3 monoclonal antibodies binding human ACP3 with high affinity have been described in the art, namely a rabbit monoclonal antibody marketed by abcam.com under catalog number 108984 trade name [EPR4066], and two mouse monoclonal antibodies (Hoyhtya et al. Clin Chem (1987), 33, 103-107). However, there is a need for antibodies binding human ACP3 with a higher affinity than these described antibodies. There is also a need for antibodies that cross-react with cynomolgus monkey (macaca fascicularis) ACP3 and are suitable for translational research.

[0012] Furthermore, anti-ACP3 monoclonal antibodies available on the market typically lack quality for staining tumor sections with sufficient precision. There is a need for antibodies that give higher sensitivity in immunofluorescence assays.The present invention has been devised in the light of the above considerations.

[0013] Summary of the Invention

[0014] The present inventors have isolated high-affinity fully human antibody molecules that bind prostatic acid phosphatase (ACP3) in humans and cynomolgus monkeys. Cross-reactivity with cynomolgus monkey ACP3 opens new avenues for evaluating the efficacy and tolerability of anti-ACP3 antibody molecules intended for further clinical use.

[0015] The isolated antibody molecules may also display improved binding to tumour tissue compared to existing anti-ACP3 monoclonal antibodies and may therefore find greater utility in vivo and in vitro applications. In a first aspect, the present invention thus provides an antibody molecule that binds human and cynomolgus monkey ACP3. The amino acid sequence of human ACP3 is shown in SEQ ID NO: 1 , the amino acid sequence of cynomolgus monkey ACP3 is shown in SEQ ID NO: 2.

[0016] The antibody may for example specifically bind to human ACP3 with a dissociation constant (KD) for ACP3 of less than 50pM.

[0017] In some embodiments, the antibody molecule may bind to an epitope within residues 281-299 and 313-331 of human ACP3 (SEQ ID NO:1).

[0018] The antibody molecule preferably comprises the HCDR1 , HCDR2, and HCDR3 sequences of the “CC106QQ” antibody shown in SEQ ID NOs 3, 4 and 5, respectively, and / or the LCDR1 , LCDR2 and LCDR3 sequences of the “CC106QQ” antibody shown in SEQ ID NOs 6, 7 and 8, respectively. An antibody which comprises these 6 CDR sequences has been shown to be capable of binding the extracellular domain of human and cynomolgus monkey ACP3.

[0019] In some preferred embodiments, the antibody molecule may comprise the VH domain sequence or the VL domain sequence, but preferably the VH domain sequence and the VL domain sequence, of the CC106QQ antibody molecule shown in SEQ ID NOs 9 and 10, respectively.

[0020] An antibody molecule, as referred to herein, may be in any suitable format. Many antibody molecule formats are known in the art and include both complete antibody molecule molecules, such as IgG, as well as antibody fragments, such as a single chain Fv (scFv), small immunoproteins (SIP), diabodies (Db), or single-chain diabodies (scDb). The term “antibody molecule” as used herein encompasses both complete antibody molecule molecules and fragments of antibody molecules, in particular antigen-binding fragments. In a preferred embodiment, the antibody molecule consists of or comprises an scFv, a smallimmunoprotein, a diabody, a single-chain diabody, or a (complete) IgG molecule, such as an lgG1 or lgG4 molecule.

[0021] ACP3 has been shown to be useful as a marker for prostate cancer. The antibody molecule of the invention may thus be employed in the imaging, detection, and diagnosis of diseases and disorders characterized, or associated with, the expression of ACP3. In this context, the antibody molecule may be used as it is and later detected using e.g. a secondary antibody molecule, or it may be conjugated to a detectable label. An antibody molecule of the present invention may thus be used as it is, i.e. in unconjugated form, or it may be conjugated to a therapeutic or diagnostic agent to provide a conjugate. Preferably, it is used in the form of a conjugate. The choice of agent conjugated to the antibody molecule will depend on the intended application of the conjugate. For example, where the conjugate is intended for the treatment of a disease or disorder, the conjugate may comprise an antibody molecule of the invention and a pro-inflammatory agent, a cytotoxic molecule, a radioisotope, a photosensitizer, an enzyme, a hormone, or an immunosuppressive agent. When the conjugate is intended for use in imaging, detecting, or diagnosing a disease or disorder, the conjugate may comprise an antibody molecule of the invention and a detectable label, such as a radioisotope, e.g. a non-therapeutic radioisotope. Depending on the agent conjugated to the antibody molecule, the conjugate may be or may comprise a single-chain protein. Where the conjugate is a single-chain protein, the entire protein can be expressed as a single polypeptide or fusion protein. In this case, the agent may be conjugated to the antibody molecule by means of a peptide linker. Fusion proteins have the advantage of being easier to produce and purify since they consist of a single species. This facilitates production of clinical-grade material. Alternatively, the agent may be conjugated to the antibody molecule by means of a cleavable linker.

[0022] The invention also provides isolated nucleic acids encoding the antibody molecules and conjugates of the invention. The skilled person would have no difficulty in preparing such nucleic acids using methods well-known in the art. An isolated nucleic acid may be used to express the antibody molecule or conjugate of the invention, for example by expression in a bacterial, yeast, insect, or mammalian host cell. A preferred host cell is a mammalian host cell. The nucleic acid will generally be provided in the form of a recombinant expression vector for expression. Host cells in vitro comprising such nucleic acids and expression vectors are part of the present invention, as is their use for expressing the antibody molecules and conjugates of the invention, which may subsequently be purified from cell culture and optionally further formulated into a pharmaceutical composition.

[0023] An antibody molecule or conjugate of the invention may be provided for example in a pharmaceutical composition and may be employed for medical use as described herein, either alone or in combination with one or more further therapeutic agents, such as an anti-PD-1 antibody. Alternatively, the antibody molecule or conjugate of the invention may be provided in a diagnostic composition and may be employed for diagnostic use as described herein.The present invention also relates to an antibody molecule or conjugate of the invention for use in a method for treatment of the human or animal body by therapy. For example, an antibody molecule or conjugate of the invention may be for use in a method of treating cancer, and / or treating an ACP3-associated disease in a patient, the method comprising administering a therapeutically effective amount of an antibody molecule or conjugate of the invention to the patient. The use of an antibody molecule or conjugate of the invention for the manufacture of a medicament for the treatment of cancer, and / or treating an ACP3-associated disease, is also contemplated.

[0024] A further aspect of the invention relates to an antibody molecule or conjugate of the invention for use in a method of imaging, detecting, or diagnosing cancer, and / or an ACP3-associated disease in a patient. The invention also relates to a method of imaging, detecting, or diagnosing cancer and / or an ACP3-associated disease in a patient comprising administering an antibody molecule or conjugate of the invention to the patient. The method may be an in vitro or an in vivo method. Also encompassed within the scope of the invention is the use of an antibody molecule or conjugate of the invention for the manufacture of a diagnostic product for imaging, detecting, or diagnosing cancer, and / or an ACP3-associated disease in a patient.

[0025] A patient, as referred to herein, is preferably a human patient. Alternatively, the patient may be a nonhuman animal patient.

[0026] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0027] Summary of the Figures

[0028] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0029] Figure 1 shows the characterization of anti-ACP3 scFv (CC106QQ). Figure 1A shows the results of SDS-PAGE analysis of scFv (CC106QQ). The scFv had the expected size of 25 kDa under non-reducing (NR) and reducing (R) conditions, respectively. Figure 1B shows the results of size exclusion chromatogram of scFv (CC106QQ). The monomeric form of the scFv was eluted from the column at 12.5 mL (Superdex 75i column - GE Healthcare).

[0030] Figure 2 shows the characterization of anti-ACP3 antibody CC106QQ in lgG4 format. Figure 2A shows the results of SDS-PAGE analysis of CC106QQ lgG4. The lgG4 had the expected size of 150 kDa under non-reducing conditions and 25 kDa and 50kDa under reducing conditions. Figure 2B shows a size exclusion chromatogram of CC106QQ lgG4. The lgG4 was eluted from the column at 11.8 mL (Superdex 200i column - GE Healthcare).Figure 3 shows the characterization of anti-ACP3 antibody CC106QQ in SIP format. Figure 3A shows the results of SDS-PAGE analysis of CC106QQ SIP. The protein had the expected size of 77 kDa under non-reducing conditions and 38 kDa under reducing conditions. Figure 3B shows a size exclusion chromatogram for CC106QQ SIP. The CC106QQ SIP was eluted from the column at 13.9 mL (Superdex 200i column - GE Healthcare).

[0031] Figure 4 shows the characterization of the IgG 1 anti-ACP3 antibody-drug-conjugate (CC106QQ-GlyPro-MMAE). Figure 4A shows the results of SDS-PAGE analysis of CC106QQ-CSMut and CC106QQ-GlyPro-MMAE. The heavy and light chains had the expected size of 50 kDa and 25 kDa, respectively, under non-reducing (NR) and reducing (R) conditions. Figure 4B shows the results of the size exclusion chromatogram of CC106QQ-GlyPro-MMAE. The antibody-drug-conjugate showed excellent purity (>98% purity), as evidenced by the single peak and eluted at around 10.7 mL on a Superdex 200i column.

[0032] Figure 4C shows the mass spectrometry analysis confirmed the successful conjugation of the CC106QQ-CSMut antibody (top chromatogram and deconvoluted spectrum) to MC-GlyPro-PABC-MMAE, as evidenced by the shift in molecular weight of the light chain of CC106QQ-GlyPro-PABC-MMAE (bottom chromatogram and deconvoluted spectrum). Figure 4D shows the C-terminus portion of the CC106QQ-GlyPro-PABC-MMAE antibody-drug-conjugate.

[0033] Figure 5 shows the results of BIAcore analysis using the anti-ACP3 lgG4 (CC106QQ) at a concentration of 10 nM on a chip coated with human ACP3 antigen.

[0034] Figure 6 shows the results of an ELISA evaluating binding of CC106QQ to human and cynomolgus monkey ACP3. Anti-ACP3 antibody CC106QQ in lgG4 format was tested at a concentration of 5 pg / mL. ACP3 antigens were coated onto the wells at a concentration of 100 nM. Anti-ACP3 lgG4 antibody CC106QQ showed binding to cynomolgus ACP3.

[0035] Figure 7 shows the results of a specificity ELISA evaluating binding of CC106QQ to different proteins. Anti-ACP3 antibody CC106QQ in lgG4 format was tested at a concentration of 5 pg / mL. All protein antigens tested were coated onto the wells at a concentration of 100 nM. Anti-ACP3 lgG4 antibody CC106QQ showed binding only to ACP3.

[0036] Figure 8 shows the results of flow cytometry analysis evaluating the binding of CC106QQ in lgG4 format to HT1080.hACP3 cell line at varying concentrations of CC106QQ. Antibody CC106QQ in lgG4 format could bind to the ACP3-expressing cell lines with high specificity. Antibody KSF in lgG4 format was used as a negative control.

[0037] Figure 9 shows the results of an ex-vivo immunofluorescence staining on organs and tumors of HT1080.hACP3 tumor-bearing mouse injected with CC106QQ lgG4 (Figure 9A). An irrelevant antibody, KSF, binding to hen-egg lysozyme, in lgG4 was used as a negative control (Figure 9B).Figure 10 shows the results of epitope mapping by PepSPOT Technology™ (Figure 10A) and a tridimensional representation of human ACP3, in dimeric form, with highlighted CC106QQ binding epitope (Figure 10B).

[0038] Figure 11 shows the results of a titration ELISA experiment in which CC106QQ was compared to the prior art antibody EPR4066 and showed a superior EC50.

[0039] Figure 12 shows the results of the in vivo therapy study of CC106QQ-GlyPro-MMAE. Male BALB / c nu / nu mice bearing subcutaneous HT1080. hACP3 tumor were treated with CC106QQ-GlyPro-MMAE and Cys-GlyPro-MMAE. Compounds were systemically administered three times (black arrows) by IV injection on alternate days at 125 nmol / kg (ADC) or at 250 nmol / kg (Cys-GlyPro-MMAE), doses that correspond to 250 nmol / kg of total administered MMAE. Cys-GlyPro-MMAE and saline served as a negative control. Figure 12A shows the tumor volumes overtime presented using single mouse plots (n = 5 animals / group). Figure 12B shows the body weight changes for each treatment group. CR: complete tumor remission.

[0040] Figure 13 shows the results of a comparative immunofluorescence study. The staining intensity of ACP3 (green signal) given by the CC106QQ antibody is remarkably more intense and crisper than the staining intensity of three other commercially available anti-ACP3 antibodies “EPR4066” (Abeam), “STJ197186” (St Johns Labs), and “BSM 60820” (Santa Cruz Biotech). The blue staining (DAPI) corresponds to nuclei. The KSF anti-lysozyme antibody serves as a negative control.

[0041] Figure 14 shows the results of a mass spectrometry-based biodistribution analysis of the CC106QQ antibody and the negative control KSF in mice bearing ACP3-positive tumors. The accumulation is measured 24 hours after injection and expressed as a percentage of the injected dose per gram of tissue. Compared to the negative control, CC106QQ demonstrates preferential tumor accumulation with %ID / g values above 15% and tumor-to-organ ratios above 4 in all organs except the lungs, which have a tumor-to-organ ratio of 2. In contrast, the negative control shows an average tumor-to-organ ratio of 1.

[0042] Detailed Description of the Invention

[0043] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0044] Antibody molecule

[0045] The present invention provides antibody molecules that bind human and cynomolgus monkey ACP3. Methods for determining binding an antigen, such as human and cynomolgus monkey ACP3 are known in the art and include for example ELISA, surface plasmon resonance, and flow cytometry.The antibody molecule preferably is fully human and preferably binds ACP3 specifically. The term “specific” may refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner, here ACP3. The term “specific” is also applicable where the antibody molecule is specific for a particular epitope, such as an epitope on ACP3, that is carried by a number of antigens, in which case the antibody molecule will be able to bind to the various antigens carrying the epitope.

[0046] In some embodiments, an antibody molecule described herein may bind to an epitope within amino acid residues 281-299 and 313-331 of human ACP3 (SEQ ID NO:1),

[0047] In some embodiments, an antibody molecule, such as an antibody or antigen binding fragment, may exhibit a binding affinity dissociation constant KD for human ACP3 of about 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, 50 pM or less, 20 pM or less, 15 pM or less or 10 pM or less. For example, the antibody or antigen binding fragment thereof may exhibit a binding affinity dissociation constant KD for human ACP3 of from about 100 nM to about 1 pM; or from about 20nM to about 5pM. In some embodiments, the antibody, in IgG format, may preferably bind human ACP3 with an affinity (KD) of 10.1pM, or with a higher affinity.

[0048] The antibody molecule may further bind to cynomolgus monkey ACP3 with the same affinity (KD) as an anti-ACP3 antibody, in scFv format, consisting of the sequence shown in SEQ ID NO: 11 , in SIP format, consisting of the sequence shown in SEQ ID NO: 15, or in IgG format, consisting of the sequence shown in SEQ ID NOs: 13, 14, in SEQ ID NOs: 23, 14, or in SEQ ID NOs: 24, 14 or with an affinity that is higher. The binding affinity of an antibody molecule to a cognate antigen, such as human or cynomolgus monkey ACP3 can be determined by surface plasmon resonance (SPR), such as Biacore, e.g. as detailed in the examples.

[0049] The antibody molecule is preferably monoclonal. The antibody molecule may be human or humanized, but preferably is a human antibody molecule.

[0050] The antibody molecule may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides, and / or serum components.

[0051] The antibody molecule may be natural or partly or wholly synthetically produced. For example, the antibody molecule may be a recombinant antibody molecule.

[0052] The antibody molecule may be an immunoglobulin, or an antigen-binding fragment thereof. For example, the antibody molecule may be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an IgG 1 , lgG2, lgG3 or lgG4 molecule, more preferably an IgG 1 or lgG4 molecule, or an antigen-binding fragment thereof.The antigen-binding site of an antibody molecule of the invention, such as an immunoglobulin or antigenbinding fragment thereof, binds ACP3. The antigen-binding site may comprise three CDRs, such as the three light chain variable domain (VL) CDRs or three heavy chain variable domain (VH) CDRs, but preferably comprises six CDRs, three VL CDRs and three VH CDRs. The three VH domain CDRs of the antigen-binding site may be located within an immunoglobulin VH domain and the three VL domain CDRs may be located within an immunoglobulin VL domain. The antibody molecule may comprise one or two antigen-binding sites for ACP3. Where the antibody molecule comprises two antigen-binding sites these are preferably identical. The antibody molecule thus may comprise one VH and one VL domain but preferably comprises two VH and two VL domains, i.e. two VHA / L domain pairs, as is the case in naturally-occurring immunoglobulin molecules, scFvs, diabodies and single-chain diabodies, for example.

[0053] The antigen-binding site of the antibody molecule preferably comprises the three VL domain CDRs and / or the three VH domain CDRs of antibody CC106QQ. The VH and VL domain sequences of this antibody are shown in SEQ ID NOs 9 and 10, respectively, and the sequences of the CDRs of the CC106QQ antibody may be readily determined from these VH and VL domain sequences by the skilled person using routine techniques. The CDR sequences may, for example, be determined according to Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991). In a preferred embodiment, the antigen-binding site of the antibody molecule comprises the HCDR1 , HCDR2, and HCDR3 sequences shown in SEQ ID NOs 3, 4 and 5, respectively, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID Nos 6, 7 and 8, respectively.

[0054] In a further preferred embodiment, the antigen-binding site may comprise the VH domain (SEQ ID NO: 9) and / or VL domain (SEQ ID NO: 10) of antibody CC106QQ but preferably comprises the VH domain and VL domain of antibody CC106QQ.

[0055] The antibody molecule may also comprise a variant of a CDR, VH domain, VL domain, heavy chain or light chain sequence described herein. A variant may for example comprise a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the CDR, VH domain, VL domain, heavy chain or light chain sequence described herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening. In a preferred embodiment, an antibody molecule comprising one or more such variant sequences retain one or more of the functional characteristics of the parent antibody molecule, such as binding specificity and / or binding affinity for human and / or cynomolgus monkey ACP3. The parent antibody molecule is an antibody molecule which does not comprise the amino acid substitution(s), deletion(s), and / or insertion(s) which has (have) been incorporated into the variant antibody molecule.The antibody molecule may comprise a VH domain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the VH domain of antibody CC106QQ (SEQ ID NO: 9).

[0056] The antibody molecule may comprise a VL domain with at least 70%, more preferably one of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the VL domain of antibody CC106QQ (SEQ ID NO: 10).

[0057] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC106QQ in lgG4 format (SEQ ID NO: 13).

[0058] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC106QQ in lgG1 format with C219S, C225S, and C228S mutations (SEQ ID NO: 23).

[0059] The antibody molecule may comprise a heavy chain which has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the heavy chain of antibody CC106QQ in lgG1 format (SEQ ID NO: 24).

[0060] The antibody molecule may comprise a light chain what has at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, sequence identity to the light chain of antibody CC106QQ in lgG4 or lgG1 format (SEQ ID NO: 14).

[0061] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used. Variants of the CDRs, VH domain, VL domain, heavy chain or light chain sequences or other sequences disclosed herein comprising one or more, e.g. less than 20 alterations, less than 15 alterations, less than10 alterations or less than 5 alterations, 4, 3, 2 or 1 , amino acid alterations (addition, deletion, substitution and / or insertion of an amino acid residue) may also be employed in antibody molecules or conjugates according to the invention. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening. Alterations may be made in one or more framework regions and / or one or more CDRs. In particular, alterations may be made in HCDR1 , HCDR2 and / or HCDR3, or in one or more framework regions of the heavy or light chain of the antibody molecule.

[0062] In one example, the heavy chain of an antibody molecule of the invention may comprise a C-terminal lysine residue as shown e.g. in SEQ ID NOs: 13, 23 and 24, or said lysine residue may be deleted.

[0063] As noted above, the antibody molecule may be a whole antibody or a fragment thereof, in particular an antigen-binding fragment thereof.

[0064] Antigen-binding fragments of immunglobulins include (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al. (1989) Nature 341 , 544-546; McCafferty et al., (1990) Nature, 348, 552-554; Holt et al. (2003) Trends in Biotechnology 21, 484-490), which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al. (1988) Science, 242, 423-426; Huston et al. (1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fv dimers (WO1993 / 011161), (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (W02013 / 014149; WO94 / 13804; Holliger et al.

[0065] (1993a), Proc. Natl. Acad. Sci. USA 906444-6448) and (x) “single-chain diabodies” wherein two sets of VH and VL domains are connected together in sequence on the same polypeptide chain (Konterman & Muller, 1999). Fv, scFv ordiabody molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245). Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. (1996), Cancer Res.,

[0066] 56(13):3055-61 ). Other examples of binding fragments are Fab’, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0067] A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736). A SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform lgE-S2 (ES2-CH4; Batista et al., (1996), J. Exp. Med., 184: 2197-205) forming a homo-dimeric mini-immunoglobulin antibody molecule as shown in SEQ ID NO:15 or a variant thereof.Preferably, the antibody molecule comprises or consists of a single-chain Fv (scFv), a small immunoprotein (SIP), a diabody, a single-chain diabody, or a (whole) IgG molecule, such as an IgG 1 or lgG4 molecule.

[0068] Where the antibody molecule is a scFv, the VH and VL domains of the antibody are preferably linked by a 14 to 20 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Suitable linker sequences are known in the art and include the linker sequence shown in SEQ ID NO:12.

[0069] In a preferred embodiment, the antibody molecule of the invention in scFv format comprises or consists of the sequence shown in SEQ ID NO: 11 or a variant thereof.

[0070] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen-binding site: antigen-binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804; Holliger and Winter, 1997; Holliger et al., 1993).

[0071] In a diabody or single-chain diabody, a heavy chain variable domain (VH) is connected to a light chain variable domain (VL) on the same polypeptide chain. The VH and VL domains are connected by a peptide linker that is too short to allow pairing between the two domains. This forces pairing with the complementary VH and VL domains of another chain.

[0072] Where the antibody molecule is a diabody or single-chain diabody, the VH and VL domains are preferably linked by a 5 to 12 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 5, 6, 7, 8, 9, 10, 11 , or 12 amino acids in length. Preferably, the amino acid linker is 5 amino acids in length.

[0073] In a single-chain diabody, two sets of VH and VL domains are connected together in sequence on the same polypeptide chain. For example, the two sets of VH and VL domains may be assembled in a singlechain sequence as follows: (VH-VL)-(VH-VL), where the brackets indicate a set. The two sets of VH and VL domains are connected as a single-chain by a long or ‘flexible’ peptide linker. This type of peptide linker sequence is long enough to allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set. Generally, a long or ‘flexible’ linker is 15 to 20 amino acids.

[0074] In a preferred embodiment, the antibody molecule of the invention in Db format comprises or consists of the sequence shown in SEQ ID NO: 25 or a variant thereof.In another preferred embodiment, the antibody molecule of the invention in scDb format comprises or consists of the sequence shown in SEQ ID NO: 26 or a variant thereof.

[0075] Where the antibody is a small immunoprotein (SIP) e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736), the VL domain of the scFv antibody is preferably linked to the CH4 domain of human IgE (Batista et al., (1996), J. Exp. Med., 184: 2197-205) via a 2 to 20 amino acid linker, more preferably a 2 to 10 amino acid linker.

[0076] In a further preferred embodiment, the antibody molecule of the invention in SIP format has the sequence shown in SEQ ID NO: 15 or a variant thereof.

[0077] Conjugates of the invention comprise an antibody molecule of the invention and a therapeutic or diagnostic agent. The therapeutic agent may be a pro-inflammatory agent, a cytotoxic molecule, a radioisotope, a photosensitizer, an enzyme, a hormone, or an immunosuppressive agent. Preferably, the therapeutic agent is a cytotoxic molecule, a radioisotope, or a pro-inflammatory agent. Cytotoxic molecules may include DNA-damaging and microtubule-disrupting agents. In some preferred embodiments, the therapeutic agent conjugated to the antibody molecule may be Calicheamicin, Duocarmycin, Doxorubicin (DNA-damaging), or Auristatin, Maytansinoids (microtubule-disrupting). Most preferably, the therapeutic agent conjugated to the antibody molecule of the invention is monomethyl auristatin E (MMAE).

[0078] Pro-inflammatory cytokines which may be conjugated to an antibody molecule of the invention include interleukin-2 (IL2) shown in SEQ ID NO:17 or a variant thereof, interleukin-12 (IL12) shown in SEQ ID NO:21 or a variant thereof, and tumour necrosis factor (TNF) or a variant thereof, such as TNFa, shown in SEQ ID NO: 19 or a variant thereof.

[0079] The sequences of the remaining cytokines, as well as variants thereof which may be employed in the present invention, are known in the art.

[0080] A therapeutic agent may be conjugated to the N-terminus or C-terminus of the antibody molecule or both. Where a therapeutic agent is conjugated to both the N-terminus and the C-terminus of the antibody molecule, the therapeutic agents may be the same or different but preferably are different. Where the therapeutic agent is conjugated to the N-terminus of the antibody molecule, the C-terminus may be “free”, i.e. not conjugated to another moiety. Similarly, where the therapeutic agent is conjugated to the C-terminus of the antibody molecule, the N-terminus may be “free”, i.e. not conjugated to another moiety. In a preferred embodiment, the antibody molecule, preferably in IgG 1 format, is conjugated to a cytotoxic molecule. In a preferred embodiment, the cysteine residues of the heavy chain in positions 219, 225, and 228 are mutated into serine residues (SEQ ID NO: 23). In a preferred embodiment, the C-terminuscysteine of the light chain (SEQ ID NO: 14) is conjugated to MC-GlyPro-PABC-MMAE to generate an antibody-drug-conjugate (CC106QQ-GlyPro-MMAE).

[0081] In a preferred embodiment, the antibody molecule, preferably in single-chain diabody format, is conjugated to interleukin 12 (IL12). In a preferred embodiment, the antibody molecule is conjugated at its N-terminus to IL12. Suitable conjugate sequences include SEQ ID NO: 22 and variants thereof.

[0082] In a yet further preferred embodiment, the antibody molecule, preferably in scFv format, is conjugated, preferably at its C-terminus, to tumor necrosis factor alpha (TNFa). Suitable conjugate sequences include SEQ ID NO: 20 and variants thereof.

[0083] In another preferred embodiment, the antibody molecule, preferably in scFv format, is conjugated, preferably at its C-terminus to interleukin 2 (IL2). Suitable conjugate sequences include SEQ ID NO: 18 and variants thereof.

[0084] A diagnostic agent conjugated to the antibody molecule of the invention may be a detectable label, such as a radioisotope, e.g. a non-therapeutic radioisotope.

[0085] Radioisotopes which may be conjugated to an antibody molecule of the invention include isotopes such as94mTc,99mTc,186Re,188Re,203Pb,67Ga,68Ga,47Sc,111l n,97Ru,62Cu,64Cu,86Y,88Y, "Y,121Sn,161Tb,153Sm,166Ho,105Rh,177Lu,123l,124l,125l,131l,18F,211At and225Ac. Preferably, positron emitters, such as18F and124l, or gamma emitters, such as99mTc,111ln and123l, are used for diagnostic applications (e.g. for PET), while beta-emitters, such as131l, "Y and177Lu, are preferably used for therapeutic applications. Alpha-emitters, such as211At and225Ac may also be used for therapy. In one example, the antibody molecule may be conjugated to177Lu,131l, or "Y.

[0086] The antibody molecule may be conjugated with the therapeutic agent by means of a peptide bond or linker as described herein. Other means for conjugation include chemical conjugation, especially crosslinking using a bifunctional reagent (e.g. employing DOUBLE-REAGENTS™ Cross-linking Reagents Selection Guide, Pierce).

[0087] Linkers

[0088] The antibody molecule, e.g. scFv or IgG, and the therapeutic or diagnostic agent or molecule may be connected to each other directly, for example through any suitable chemical bond, but preferably are connected via a peptide linker. The chemical bond may be, for example, a covalent or ionic bond.

[0089] Examples of covalent bonds include peptide bonds (amide bonds) and disulfide bonds.

[0090] Where the therapeutic or diagnostic agent is connected to the antibody molecule via a peptide linker, the peptide linker may be a short (2-30, preferably 10-20) residue stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. In oneembodiment, the linker may be a cleavable linker. In a preferred embodiment, the cleavable linker may include a Gly-Pro motif. In a further preferred embodiment, the linker may also include a self-immolative spacer, such as para-aminobenzyl carbamate (PABC).

[0091] Where the antibody molecule and therapeutic or diagnostic agent are connected via a peptide bond or peptide linker, the conjugate may be produced (secreted) as a single-chain polypeptide, such as a fusion protein.

[0092] Methods of treatment

[0093] As explained above, the presence of ACP3 and cells expressing ACP3 on their surface has been shown to be associated with a number of diseases and disorders, including cancer.

[0094] An antibody molecule or conjugate of the invention may therefore be for use as a medicament.

[0095] In particular, the antibody molecule or conjugate may be for use in a method of treatment (which may include prophylactic treatment) of the human or animal body.

[0096] Also provided is a method of treating a disease or disorder in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the antibody molecule or conjugate.

[0097] Further provided is the use of the antibody molecule or conjugate in the manufacture of a medicament for use in the treatment of a disease or disorder in a patient.

[0098] The patient may be a human patient or may be an animal patient.

[0099] Treatment may be any treatment or therapy in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the disease or disorder, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the disease or disorder, cure or remission (whether partial or total) of the disease or disorder, preventing, ameliorating, delaying, abating or arresting one or more symptoms and / or signs of the disease or disorder or prolonging survival of an individual or patient beyond that expected in the absence of treatment.

[0100] Treatment as a prophylactic measure (i.e. prophylaxis) is also included. For example, an individual susceptible to or at risk of the occurrence or re-occurrence of a disease or disorder may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of the disease or disorder in the individual.

[0101] A method of treatment as described may comprise administering at least one further treatment to the individual in addition to the antibody molecule or conjugate. The antibody molecule or conjugate may thus be administered to an individual alone or in combination with one or more other treatments for thedisease or disorder in question. Where the antibody molecule or conjugate is administered to the individual in combination with another treatment, the additional treatment may be administered to the individual concurrently with, sequentially to, or separately from the administration of the antibody molecule or conjugate. Where the additional treatment is administered concurrently with the antibody molecule or conjugate, the antibody molecule or conjugate and additional treatment may be administered to the patient as a combined preparation. For example, the additional therapy may be a known therapy or therapeutic agent for the disease or disorder to be treated.

[0102] In a preferred embodiment, the antibody or conjugate of the invention is administered to a patient in combination with a therapeutic agent, such as chemotherapy, radiation therapy, an immunomodulatory agent, and an immunoconjugate, such as an immunocytokine.

[0103] In another preferred embodiment, the therapeutic agent is selected from the group consisting of: chemotherapy, radiation therapy, an immunomodulatory agent, and an immunoconjugate, such as an immunocytokine.

[0104] In a further preferred embodiment, the antibody or conjugate of the invention is administered to a patient in combination with an immunomodulatory agent, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, or anti-TIM-3 antibody. Most preferably, the immunomodulatory agent is an anti-PD-1 antibody. Anti-PD-1, anti-PD-L1, anti-LAG-3, anti-TIGIT, and anti-TIM-3 antibodies are known in the art and are available to the skilled person. A number of anti-PD-1 and anti-PDL-1 antibodies are licensed for the treatment of cancer in human patients and can be employed in treatment of cancer in a patient in combination with an antibody or conjugate of the invention.

[0105] The disease or disorder to be treated may be a disease or disorder characterized by the expression or overexpression of ACP3 on the surface of cells, such as cancer cells. For example, the disease or disorder may be cancer that is characterized by the expression or overexpression of ACP3 (i.e. ACP3 positive cancer).

[0106] The disease or disorder to be treated using an antibody molecule or conjugate of the invention may be any disease or disorder characterized by, or associated with, the expression of ACP3.

[0107] The disease or disorder to be treated may be cancer, preferably prostate cancer.

[0108] The disease or disorder to be treated using an antibody molecule or conjugate of the invention may be any disease or disorder characterized by, or associated with, the expression of ACP3. As explained above, expression of ACP3 is limited in adult human tissues and is therefore expected to represent a disease-specific target for therapy in diseases or disorders characterized by expression of ACP3. The disease to be treated may be cancer, wherein the cancer cells express ACP3The disease to be treated using an antibody molecule or conjugate of the invention may be cancer, as well as other tumours and neoplastic conditions.

[0109] The cancer or malignancy characterized by ACP3 overexpression may be prostate cancer.

[0110] In some embodiments, a disease associated with ACP3 expression that is suitable for treatment as described, is preferably selected from pancreatitis, Paget's disease, sickle-cell disease, lysosomal storage diseases, and Gaucher's disease, more preferably, the disease is selected from prostate adenocarcinoma, ductal prostate cancer, ductal adenocarcinoma, clear cell adenocarcinoma, acinar adenocarcinoma, urothelial cancer, neuroendocrine prostate cancer, small cell prostate cancer or multiple myeloma.

[0111] Methods of detection or diagnosis

[0112] The antibody molecules and conjugates of the invention are suitable for detecting ACP3 in vivo and in vitro, and thus find application in the imaging, detection and diagnosis of disease characterized by, or associated with, expression of ACP3.

[0113] The present invention therefore also relates to the use of an antibody molecule or conjugate of the invention for detecting ACP3, e.g. cells, expressing ACP3 on their cell surface, either in vitro or in vivo. The conjugate preferably comprises a detectable label to aid detection. The preparation of suitable conjugates is described elsewhere herein. Alternatively, binding of the antibody molecule to ACP3 may be detected using a secondary antibody or other detection reagent. Where the antibody molecule is conjugated to a radioisotope, binding of the antibody molecule to ACP3 in the patient may be detected using scintigraphy.

[0114] Also provided is an in vitro method for detecting ACP3, the method comprising incubating the antibody molecule or conjugate with a sample obtained from an individual, e.g. a human patient, and detecting binding of the antibody molecule or conjugate to the sample, e.g. cells (such as fibroblasts) present in the sample, wherein binding of the antibody molecule or conjugate to the sample indicates the presence of ACP3. Methods for determining binding of an antibody molecule or antigen to a sample are known in the art and include, for example, ELISAs, flow cytometry, and immunofluorescence or immunostaining of tissue samples.

[0115] Further provided is the antibody molecule or conjugate for use in a method of detecting ACP3 in vivo, the method comprising administering the antibody molecule or conjugate to an individual, e.g. a human patient. Localization of the antibody molecule or conjugate at a site in the individual indicates expression of ACP3 at said site. The method may further comprise determining the localization of the antibody molecule or conjugate at sites in the individual.As ACP3 is rarely expressed in adult human tissues but is known to be expressed on tumour cells, the antibody molecules and conjugates of the invention are also expected to find application in the detection of diseases and disorders characterized by the expression of ACP3. Thus, the present invention also provides an antibody molecule or conjugate of the invention for use as a detection agent, diagnostic, or imaging agent.

[0116] Thus, the present invention also provides the antibody molecule or conjugate for use in a method of imaging, detecting, or diagnosing a disease or disorder in a patient.

[0117] Also provided is a method of imaging, detecting, or diagnosing a disease or disorder in a patient comprising administering an antibody molecule or conjugate of the invention to the patient.

[0118] Further provided is the use of an antibody molecule or conjugate of the invention in the manufacture of a diagnostic product for use in the detection or diagnosis of a disease or disorder.

[0119] The disease or disorder is preferably characterized by the expression of ACP3, such as the presence of ACP3-expressing cells, and may be a disease or disorder as described herein, such as cancer.

[0120] Pharmaceutical compositions

[0121] Whilst an antibody molecule or conjugate may be administered alone, antibody molecules and conjugates will typically be administered in the form of a pharmaceutical composition. Thus, a further aspect of the present invention relates to a pharmaceutical composition comprising at least one antibody molecule or conjugate of the invention and at least one other component, such as a pharmaceutically acceptable excipient. A method comprising formulating an antibody molecule or conjugate into a pharmaceutical composition is also provided.

[0122] Pharmaceutical compositions may comprise, in addition to the antibody molecule or conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below.

[0123] For parenteral, for example subcutaneous or intravenous administration, e.g. by injection, the pharmaceutical composition comprising the antibody molecule or conjugate may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be employed as required, including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0124] In some embodiments, the antibody molecules or conjugates may be provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules or conjugates may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[0125] Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to an individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease or disorder being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule or conjugate, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody molecules are well known in the art (Ledermann et al., 1991 ; Bagshawe et al., 1991). Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for an antibody molecule being administered, may be used. Appropriate doses for conjugates are also known or can be determined. For example, a therapeutically effective amount or suitable dose of an antibody molecule or conjugate can be determined by comparing in vitro activity and in vivo activity in an animal model, such as a cynomolgus monkey. Methods for extrapolation of effective dosages in cynomolgus monkeys, as well as other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the antibody molecule or conjugate.

[0126] Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the antibody molecule or conjugate, the route of administration and the nature of the condition being treated.Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g. about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are described above.

[0127] A pharmaceutical composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0128] Kits

[0129] Another aspect of the invention provides a therapeutic kit for use in the treatment of a disease or disorder comprising an antibody molecule or conjugate as described herein. The components of a kit are preferably sterile and in sealed vials or other containers.

[0130] A kit may further comprise instructions for use of the components in a method described herein. The components of the kit may be comprised or packaged in a container, for example a bag, box, jar, tin or blister pack.

[0131] Nucleic acids, vectors, host cells and methods of production

[0132] Provided is an isolated nucleic acid molecule encoding an antibody molecule or conjugate of the invention. Nucleic acid molecules may comprise DNA and / or RNA and may be partially or wholly synthetic.

[0133] An isolated nucleic acid molecule may be used to express an antibody molecule or conjugate of the invention. The nucleic acid will generally be provided in the form of an expression vector. Another aspect of the invention thus provides an expression vector comprising a nucleic acid as described above.

[0134] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate in the context.

[0135] A nucleic acid molecule or expression vector as described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid or vectors into host cells are well established in the art and any suitable technique may be employed. A range of host cells suitable for the production of recombinant antibody molecules and conjugates are known in the art, and include bacterial, yeast, insect or mammalian host cells. A preferred host cell is a mammalian cell, such as a CHO, NSO, or HEK cell, for example a HEK293 cell.Another aspect of the invention provides a method of producing an antibody molecule, or conjugate, comprising expressing a nucleic acid encoding the antibody molecule, or conjugate, in a host cell and optionally isolating and / or purifying the antibody molecule, or conjugate, thus produced. Methods for culturing host cells are well-known in the art. The method may further comprise isolating and / or purifying the antibody molecule or conjugate. Techniques for the purification of recombinant antibody molecules, or conjugates, are well-known in the art and include, for example HPLC, FPLC, or affinity chromatography, e.g. using Protein A or Protein L. In some embodiments, purification may be performed using an affinity tag on an antibody molecule. The method may also comprise formulating the antibody molecule, or conjugate, into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described herein.

[0136] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof.

[0137] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention shown above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0138] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0139] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0140] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0141] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0142] Examples

[0143] EXAMPLE 1 - Cloninq of ACP3 including characterization, phaqe display selection against antigen, and isolation of CC106QQ antibody in scFv format

[0144] 1.1 Expression procedure

[0145] A human ACP3 recombinant fragment containing a C-terminal His6 tag followed by an Avi-tag was expressed using transient gene expression (TGE) in CHO-S cells. For 1 mL of production 4 x 106CHO-S cells in suspension were centrifuged and resuspended in 1 mL of a suitable medium. 0.9 pg of plasmid DNA followed by 2.5 pg polyethylene imine (PEI; 1 mg / mL solution in water at pH 7.0) per million cells were then added to the cells and gently mixed. The transfected culture was incubated in a shaker incubator at 31 °C for 6 days. The protein fragment was purified from the cell culture medium by using nickel affinity chromatography and then dialyzed into 1x PBS buffer (pH 7.4) and stored at -80°C.

[0146] 1.2 Antigen characterization

[0147] The human ACP3 recombinant protein was analyzed by SDS-PAGE and by size exclusion chromatography using a Superdex 200 increase 10 / 300 GL column on an AKTA FPLC.

[0148] 1.3 Antigen biotinylation

[0149] The purified human ACP3 recombinant fragment was site-specifically biotinylated with Bir-A and biotin. PD-10 desalting columns with Sephadex G-25 M resin were used to replace the protein’s buffer with BirA buffer (100 mM Tris-HCI, 200 mM NaCI and 5 mM MgCI2 in Milli-Q water, pH 7.5) for site-specific biotinylation of the Avi-tagged recombinant ACP3. To perform the biotinylation reaction, a complete™, EDTA-free Protease Inhibitor Cocktail tablet was added to the recombinant protein and combined with 788 pl of Biotin (5 mM dissolved in DMSO), 245 pl of ATP disodium salt hydrate (0.5 M in 1 M Tris-HCI pH 9.5) and 150 pg of BirA-maltose-binding protein. The biotinylation reaction tube was covered with aluminum foil and shaken in a Sample Mixer at RT overnight. The next day, biotinylated human ACP3 was purified by size exclusion chromatography on a Superdex 200 increase column in PBS buffer.

[0150] Collected fractions were pooled, snap frozen in liquid nitrogen, and stored at -80°C.

[0151] 1.4 Phage display selection

[0152] The biotinylated human ACP3 was used to perform biopanning with Dynabeads. Briefly, the biotinylated human ACP3 (final concentration 120 pmol) was incubated with 800 pL of a pre-blocked phage display library for 30 minutes. After several washes with 1x PBS buffer (pH 7.4), selected phages were eluted byreducing the disulfide bonds in the biotin linker with triethylamine. Isolated phages were then amplified in E. coli strain TG-1 and precipitated from the supernatant with polyethylene glycol.

[0153] After two rounds of biopanning, clones were screened by ELISA. Avidin-coated ELISA plates were incubated with biotinylated human ACP3. The supernatants of selected induced monoclonal clones of the E. co / / TG-1 cultures expressing scFv antibody fragments were added to the ELISA plates and bound scFvs were detected using the anti-c-myc antibody 9E10 followed by an anti-mouse IgG -horseradish peroxidase (HRP) conjugate.

[0154] Clones were also screened by flow cytometry. HT1080.hACP3 cells were incubated with the supernatants of selected induced monoclonal clones of the E. co / / TG-1 cultures expressing scFv antibody fragments. Bound scFvs were detected using ProteinA-AlexaFluo488 antibody conjugate.

[0155] 1.5 In-vitro characterization of antibody CC106QQ in scFv format

[0156] The antibody clone that resulted in the highest ELISA and flow cytometry signals, CC106QQ, was produced in E. coli strain TG-1. A TG-1 culture was grown at 37°C in 2xTY / 100 pg / ml ampicillin. At ODeoo = 0.5, 1mM isopropyl-thio-galactopyranoside (IPTG) was added to induce expression of the scFv; the culture was incubated on a bacterial incubator shaking at 175 rpm at 30°C overnight. The culture was then centrifuged, and the supernatant purified from the cell culture medium by protein-A affinity chromatography and then dialyzed against PBS and stored in PBS at -80°C. The CC106QQ scFv was then characterized by size exclusion chromatography using a Superdex 75 increase 10 / 300 GL column on an AKTA FPLC. SDS-PAGE analysis was also performed with 4-12% Bis-Tris gel under reducing and non-reducing conditions (Figure 1).

[0157] 1.6 Cloning and expression of antibody CC106QQ in scFv format

[0158] The CC106QQ scFv was cloned into a vector for mammalian expression. The primers were designed to add Nhel and Hindi II restriction sites: “Leader Seq DP47” > and “Hindlll Stop Myc” <.

[0159] The resulting fragment was PCR amplified with “Nhel Leader fwd” > and “Hindlll Stop Myc” < to add the restriction site for Nhel. The PCR product was digested with Nhel and Hindlll and ligated into a vector previously digested with the same enzymes.

[0160] CC106QQ scFv was then expressed using transient gene expression (TGE) in CHO-S cells (as described above). The CC106QQ scFv was purified from the cell culture medium by protein A affinity chromatography, then dialyzed against PBS and stored at -80°C. The amino acid sequence of CC106QQ scFv is shown in SEQ ID NO: 11.

[0161] 1.7 ResultsA new anti-ACP3 antibody termed “CC106QQ” in scFv format was isolated using phage display and characterized using SDS-PAGE (Figure 1A) and SEC (Figure 1B) analysis.

[0162] These results showed that CC106QQ scFv had the expected molecular weight under reducing and nonreducing conditions and was eluted from the SEC column at 12.5 mL. The antibody molecule showed excellent purity, as evidenced by the major peak observed by SEC.

[0163] EXAMPLE 2: Cloning, expression and in vitro characterization of the CC106QQ antibody in lgG4 format

[0164] 2.1 Cloning of the CC106QQ antibody into lgG4 format

[0165] Cloning of the CC106QQ antibody in lgG4 format commenced by cloning the light chain. Primers were designed to insert Spel and BsiWI restriction sites:

[0166] “Leader DPK22 fwd”> and “DPK22 BsiWI” <

[0167] The resulting fragment was PCR amplified to add the leader sequence with the following primers: “Spel Leader fwd” > and “DPK22 BsiWI” <.

[0168] The resulting fragment was digested with Spel and BsiWI and ligated into a suitable vector previously digested with the same restriction enzymes.

[0169] The cloning procedure was continued with the cloning of the CC106QQ heavy chain.

[0170] Primers were designed to insert Hindi II and Xhol restriction sites:

[0171] “Hindi 11 Leader fwd” > and “DP47 Xhol” <

[0172] The final PCR product was digested with Hindi II and Xhol and ligated into a suitable vector with the light chain inserted and previously digested with the same restriction enzymes. The amino acid sequence of the CC106QQ antibody in lgG4 format is shown in SEQ ID NOs 13 and 14.

[0173] The same cloning strategy was used to prepare the anti-hen egg lysozyme antibody “KSF” in lgG4 format (used herein as negative control).

[0174] 2.2 Characterization of the CC106QQ antibody in lgG4 format

[0175] Antibody CC106QQ in lgG4 format was expressed using transient gene expression (TGE) in CHO-S cells and purified by protein-A affinity chromatography, dialyzed in PBS, and stored at -80°C (as described above).Proteins were characterized by SDS-PAGE (Figure 2A) and size-exclusion chromatography using a Superdex 200 increase 10 / 300 GL column on an AKTA FPLC, as also described above (Figure 2B).

[0176] EXAMPLE 3: Cloning, expression and in vitro characterization of the CC106QQ antibody in SIP format

[0177] 3.1 Cloning, expression and in vitro characterization of the CC106QQ antibody in SIP format

[0178] The CC106QQ antibody was cloned into SIP (small immunoprotein) format, and the protein was expressed in CHO cells using pcDNA3.1(+) as the expression vector. The gene encoding the CC106QQ antibody was PCR amplified with:

[0179] “Nhel Leader fwd” > (CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC)

[0180] and “DPK22_CH4_ba” <

[0181] A gene containing CH4 domain was PCR amplified with: “CH4_fwd” > and “Not_STOP_CH4_ba” <

[0182] The resulting PCR fragments were PCR assembled and cloned into the mammalian expression vector pcDNA3.1(+) using a Nhel / Notl restriction site.

[0183] The SIP protein was expressed using transient gene expression in CHO cells as described previously and purified from the cell culture medium to homogeneity by Protein A chromatography. The CC106QQ antibody in SIP format was analyzed by size-exclusion chromatography using a Superdex 200 increase 10 / 300 GL column on an KTA FPLC. SDS-PAGE was performed using a 4-12% Bis-Tris gel under reducing and non-reducing conditions.

[0184] The amino acid sequence of CC106QQ in SIP format is shown in SEQ ID NO: 15.

[0185] The same cloning strategy was used to prepare antibody KSF in SIP format.

[0186] 3.2 Characterization of the CC106QQ antibody in SIP format

[0187] Antibody CC106QQ in SIP format was expressed using transient gene expression (TGE) in CHO-S cells and purified by protein A affinity chromatography, dialyzed in PBS and stored at -80°C (as described above).

[0188] Proteins were characterized by SDS-PAGE (Figure 3A) and SEC using a Superdex 200 increase 10 / 300 GL column on an AKTA FPLC as also described above (Figure 3B).

[0189] EXAMPLE 4: Cloning, expression ofthe CC106QQ-CSMut lgG1 , preparation of CC106QQ-GlyPro-MMAE and their in vitro characterization4.1 Protein Production and Purification

[0190] CC106QQ-CSMut is the precursor of CC106QQ-GlyPro-PBAC-MMAE antibody-drug conjugate. The cysteine residues at positions 219, 225, and 228 of the heavy chain CC106QQ antibody in lgG1 format were mutated into serine residues. Only one single reactive cysteine residue was left at the C-terminus of the light chain. The recombinant antibody was produced in CHO cells via polyethyleneimine (PEI)-mediated transient gene expression (TGE) and purified by Protein-A affinity chromatography. Buffer A (100 mM NaCI, 0.5 mM EDTA, 0.1% Tween 20 in PBS) and Buffer B (500 mM NaCI, 0.5 mM EDTA in PBS) were used during the purification process as washing steps. Antibody product was eluted using IQ-15 ml 0.1 M glycine at pH = 3, fractions containing protein were collected, pooled, and dialyzed against phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. Protein integrity and purity were assessed by SDS-PAGE using a 4-12% gradient gel in 1 x MOPS buffer under reducing and non-reducing conditions (Figure 4A). Purified protein was characterized by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column on an AKTA Pure FPLC system.

[0191] 4.2 Antibody-Drug Conjugate Preparation

[0192] First, the two reactive cysteines of CC106QQ-CSMut (one in each light chain) were reduced with 20 equivalents of TCEP-HCI in PBS (pH = 7.4) at 37°C with overnight shaking at 550rpm. The next day, 40 equivalents of purified MC-GlyPro-PABC-MMAE were added to the reduced protein (5% v / v of DMSO). The mixture was stirred at 550 rpm at RT for 2 hours, and the final conjugated product was purified on a PD-10 column. Collected fractions containing protein were pooled and dialyzed against phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. Protein integrity and purity were assessed by SDS-PAGE using a 4-12% gradient gel in 1 x MOPS buffer under reducing and non-reducing conditions (Figure 4A).

[0193] Purified protein was characterized by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column on an KTA Pure FPLC system (Figure 4B). A schematic illustration of the antibody-drug conjugate is reported in Figure 4D.

[0194] 4.3 Mass Spectrometry Sample Preparation for Analytical Runs

[0195] Samples were diluted to about 0.1 mg / ml, and LC-MS was performed on a Waters Xevo G2XS Qtof instrument (ESI-ToF-MS) coupled to a Waters Acquity UPLC H-Class System using a 2.1 x 50 mm Acquity BEH300 C4 1.7 pm column (Waters). 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) were used as mobile phase at a flow rate of 0.4 ml / min. Gradient was programmed as follows: after 1.5 min isocratic with 95% solvent A, stepwise change from 95% solvent A to 95% solvent B in 4.5 min (10% increase every 0.5 min), back to 95% solvent A in 0.5 min, linearly to 95% solvent B and back to 95% solvent A in 2.25 min (last step repeated twice) (Figure 4C).

[0196] EXAMPLE 5: Characterization of CC106QQ by Surface Plasmon Resonance5.1 Biacore analysis

[0197] Affinity measurement of the CC106QQ in lgG4 format was performed by Surface Plasmon Resonance using BIAcore X100 instrument. A human ACP3-coated CM5 chip (in Acetate pH 5.0) was used and a final coating of 250 RU was achieved. The antibody CC106QQ was injected at a concentration of 10 nM. KD measurement was assessed using the BIAevaluation 3.2 software.

[0198] 5.2 Results

[0199] The affinity (KD) of the “CC106QQ” antibody in lgG4 (Figure 5) format against human ACP3 as measured by BIAcore was calculated as 10.1 pM.

[0200] EXAMPLE 6: Cross-reactivity of CC106QQ for human and cynomolgus monkey ACP3

[0201] 6.1 Cloning and expression of cynomolgus monkey ACP3

[0202] The gene for the cynomolgus monkey ACP3 (cmACP3) was purchased in pcDNA 3.1 (+) vector.

[0203] ACP3 antigen was expressed using transient gene expression in CHO cells as described above, purified from the cell culture medium by using nickel affinity chromatography, then dialyzed into 1x PBS buffer (pH 7.4) and stored at -80°C.

[0204] 6.2 ELISA analysis

[0205] Cross-reactivity of the anti-ACP3 antibody CC106QQ with cmACP3 and hACP3 carrying a C-terminal His6 tag, was tested using ELISA. 100 nM of each cmACP3 and hACP3 were coated on Maxisorp™ plates overnight at 4°C. The anti-ACP3 antibody CC106QQ or the (negative control) KSF antibody, both in lgG4 format were incubated in the wells for 1 h at room temperature at a concentration of 5 pg / mL. For detection, anti-human IgG-(Fc-specific) HRP (1 pg / ml in 2% MPBS, 50 pl / well) were given to the resulting complex for 1 h. In the final step, all wells were washed with 0.1% Tween in PBS (3x, 200 pl / well), PBS (3x, 200 pl / well), POD (60 pl / well) was added, and the reaction was stopped with 1 M H2SO4 (30 pl / well). The absorbance (450 nm-620 nm) was measured using a microplate reader.

[0206] 6.3 Results

[0207] The anti-ACP3 CC106QQ antibody was able to recognize and bind to cmACP3 and hACP3 (Figure 6).

[0208] The negative control KSF antibody did not show binding to any of the antigens tested. An anti-histidine tag antibody-HRP conjugate was used to confirm that the coating of the wells with cmACP3 or hACP3 was performed correctly.

[0209] The cross-reactivity of the CC106QQ antibody with cynomolgus monkey ACP3 in addition to human ACP3, allows the activity and tolerability of this antibody to be tested in a different animal. This isexpected to be more predictive of efficacy in human patients than mouse models, notably in models of cancer.

[0210] EXAMPLE 7: Specificity of CC106QQ for ACP3

[0211] 7.1 ELISA Analysis

[0212] Specificity of the anti-ACP3 antibody CC106QQ to ACP3 was tested using ELISA. 100 nM of different proteins were coated on Maxisorp™ plates overnight at 4°C. The anti-ACP3 antibody CC106QQ or the (negative control) KSF antibody, both in lgG4 format were incubated in the wells for 1 h at room temperature at a concentration of 5 pg / mL. For detection, anti-human-lgG-(Fc specific) HRP (1 pg / ml in 2% MPBS, 50 pl / well) were given to the resulting complex for 1 h. In the final step, all wells were washed with 0.1% Tween in PBS (3x, 200 pl / well), PBS (3x, 200 pl / well), POD (60 pl / well) was added and the reaction was stopped with 1 M H2SO4 (30 pl / well). The absorbance (450 nm-620 nm) was measured using a microplate reader.

[0213] 7.2 Results

[0214] The anti-ACP3 CC106QQ antibody was able to bind only to ACP3 and not to other unrelated proteins such as CLL-1 , human FAP, human CAIX, etc. (Figure 7). The negative control KSF antibody did not show binding to any antigen. An anti-histidine tag antibody HRP conjugate was used to confirm that the coating of the wells with all proteins was performed correctly.

[0215] EXAMPLE 8: Flow cytometry of CC106QQ

[0216] 8.1 Flow Cytometry analysis

[0217] Binding of the CC106QQ antibody in lgG4 format to cells expressing human ACP3 was tested using a cell line artificially transduced to express human ACP3 (hACP3). The cell line used was the human fibrosarcoma cell line HT-1080.

[0218] Specifically, HT-1080.hACP3 cells were detached from cell culture plates using Accutase, counted and suspended to a final concentration of 1 x 106cells / mL in FACS buffer (0.5% BSA, 2mM EDTA in PBS). Cells were incubated with either CC106QQ or KSF (negative isotype control) antibodies in lgG4 format and binding detected using a goat anti-human AlexaFluor488 antibody. Cells were analyzed on a CytoFLEX cytometer. The raw data were processed using the FlowJo 10.4 software.

[0219] 8.2 Results

[0220] Flow cytometry analysis using HT-1080.hACP3 confirmed the ability of the CC106QQ antibody in lgG4 format to bind cells expressing human ACP3 (Figure 8). The KSF lgG4 antibody (negative control) did not show any binding as expected.EXAMPLE 9: Ex-vivo biodistribution analysis of the CC106QQ antibody in tumor-bearinq mice

[0221] 9.1 Ex-vivo experiment

[0222] 5x106HT1080.hACP3 cells were implanted subcutaneously in the flank of eight-week-old male BALB / c nude mice. For ex-vivo analysis, when tumors reached a size of 150-250 mm3, mice were injected with 200pg CC106QQ lgG4 and sacrificed 24 hours after injection. Organs were excised and embedded in a cryo-embedding medium and cryostat sectioned (10pm). For detection, the antibody goat anti-human IgG Alexa Fluor488 was used.

[0223] 9.2 Results

[0224] The ability of the CC106QQ antibody to efficiently target tumors was confirmed through ex-vivo analysis of the binding of CC106QQ (lgG4 format) to tumors expressing ACP3 (Figure 9A). No binding was seen with the negative control KSF(lgG4), as expected (Figure 9B).

[0225] EXAMPLE 10:

[0226]

[0227] of CC106QQ anti

[0228]

[0229] 10.1 Epitope mapping

[0230] Overlapping peptides (15 amino acid-long) covering the extracellular domain sequence of human ACP3 were purchased from JPT (SPOT synthesis). The peptides were covalently bound to a cellulose membrane on 86 different spots. Before the first use, the membrane was rinsed with 30 ml methanol for 5 minutes to avoid precipitation of hydrophobic peptides during the following washing procedure. Then, the membrane was washed three times for 3 minutes with 50 mL of TBS-T buffer (50 mM TRIS; 137 mM NaCI; 2.7 mM KOI; 0.05% Tween 20; pH 8.0). To reduce the background signal, the membrane was blocked in 5% Milk-PBS at 4 °C overnight while gently shaking. The membrane was incubated with 5 pg / ml of CC106QQ lgG4 in 5% milk-PBS for 3 hours at room temperature with gentle shaking, followed by a secondary anti-human Fc specific conjugated to HRP. After the staining procedure, the membrane was washed three times in TBS-T for five minutes. In a dark room, the membrane was incubated with 1 ml of ECL start Western blotting detection reagent and developed according to the manufacturer’s instructions. The potential binding region of CC106QQ was aligned on the published crystal structure (PDB: 1 CVI), to highlight the potential binding epitope of CC106QQ.

[0231] 10.2. Results

[0232] The results of the epitope mapping showed that the CC106QQ antibody binds to AA residues 281-299 and 313-331 of human ACP3 (SEQ ID NO:1), as highlighted by the black spots on the membrane (Figure 10A) and by the arrows on the homodimeric tridimensional structure (Figure 10B).

[0233] EXAMPLE 11 : Titration ELISA of CC106QQ for ACP3

[0234] 11.1 ELISA AnalysisVarious concentrations of the anti-ACP3 antibody CC106QQ were tested against ACP3 and compared to a commercially available anti-ACP3 antibody EPR4066 from Abeam. 100 nM of hACP3 was coated on a Maxisorp™ plate overnight at 4°C. The anti-ACP3 antibody CC106QQ or the commercially available antibody EPR4066, both in lgG4 format, were incubated in the wells for 1 h at room temperature, starting from a concentration of 7.5 pg / mL and performing 1 :4 serial dilutions. For detection, Protein A-HRP (1 pg / ml in 2% MPBS, 50 pl / well) was given to the resulting complex for 1 h. In the final step, all wells were washed with 0.1% Tween in PBS (3x, 200 pl / well), PBS (3x, 200 pl / well), POD (60 pl / well) was added and the reaction was stopped with 1 M H2SO4 (30 pl / well). The absorbance (450 nm-620 nm) was measured using a microplate reader.

[0235] 11.2 Results

[0236] Both CC106QQ and EPR4066 antibodies were able to bind to ACP3 at various concentrations (Figure 11). CC106QQ showed a superior binding activity with a measured EC50 of 0.5993 nM as compared to an EC50 of 3.716 nM measured for EPR4066.

[0237] EXAMPLE 12: Therapy studies with CC106QQ-GlyPro-MMAE conjugate

[0238] 12.1 Experimental animals and tumor Implantation

[0239] A total of 15 male BALB / c nu / nu mice (6-8 weeks old) were obtained from Janvier Labs (Le Genest-Saint-lsle, France). Animals were housed under specific pathogen-free conditions with controlled temperature (18-26 °C), relative humidity (40-60%), and a 12 h light / dark cycle, in accordance with GV-SOLAS and FELASA guidelines. Mice were kept in groups of up to five per cage and reallocated when necessary to avoid single housing. Experimental groups were assigned based on tumor volume, determined by calliper measurements and calculated as (length [mm] x width [mm]2) / 2. Blinding was not applied in this study.

[0240] The human fibrosarcoma HT1080 cell lines stably expressing ACP3 were used for in vivo therapies. For tumor establishment, 5 x 106cells were subcutaneously implanted into the right flank of 6- to 8-week-old male BALB / c nu / nu mice.

[0241] 72.2 Synthesis of the negative control Cys-GlyPro-MMAE

[0242]

[0243] MC-GlyPro-PABC-MMAE (4.9 mg, 4.0 pmol, 1.0 equiv.) and Cysteine (0.6 mg, 4.8 pmol, 1.2 equiv.) were loaded into an Eppendorf tube and dissolved in a 1 :1 mixture of PBS and DMF (0.4 mL). DIPEA (3.5 pL, 20 pmol, 5.0 equiv.) was added and the reaction was incubated in a shaker incubator at 37 °C for 1 h. The crude mixture was directly purified by RP-HPLC (gradient of 90:10 to 0:100 in 18 min water / ACN + 0.1% TFA) to afford Cys-GlyPro-MMAE as a white solid (4.0 mg, 3.0 pmol, 75% yield), m / z calculated for C67H103N10G16S: [M+H]+ 1335.73, detected: 1335.6.

[0244] 12.3 Therapy experiments

[0245] HT1080.hACP3 tumor cells were implanted into male BALB / c nu / nu mice as described above and allowed to grow to an average volume of 100 mm3. Mice were randomly assigned to therapy groups of 5 animals, and treatment started by injecting a solution of compounds (CC106QQ-GlyPro-MMAE or Cys-GlyPro-MMAE) or vehicle (PBS containing 2% of DMSO) intravenously (lateral tail vein) at equimolar doses of MMAE (250 nmol / kg), three times (black arrows) on alternate days. Cys-GlyPro-MMAE was injected as solution in sterile PBS containing 2% DMSO. CC106QQ-GlyPro-MMAE conjugate was injected as solutions in 1x PBS buffer. Animals were weighed and tumor sizes measured daily with an electronic caliper. The tumor volume was calculated according to the formula (long side) x (short side) x (short side) x 0.5. Animals were sacrificed when the termination criteria were reached. Prism 9 software (GraphPad Software) was used for data analysis (regular two-way ANOVA followed by Tukey’s test).

[0246] 12.4 Results

[0247] The in vivo efficacy of the CC106QQ-GlyPro-MMAE conjugate was evaluated in nude mice bearing HT1080.hACP3 tumors (Figure 12). All conjugates were given at equimolar doses of the cytotoxic payload, corresponding to 250 nmol / kg of MMAE.

[0248] The CC106QQ-GlyPro-MMAE led to complete tumor remissions against ACP3-positive lesions. No in vivo anti-tumor effect was observed in mice treated with the negative control Cys-GlyPro-MMAE and the saline control (Figure 12A). All treatments were well tolerated, with minimal changes in body weight observed throughout the entire study (Figure 12B).EXAMPLE 13: Immunofluorescence of CC106QQ for ACP3

[0249] 13.1 Preparation of HT1080.hACP3 tumors cryosections

[0250] Eight-week-old male immunodeficient BALB / c nude mice were obtained from Janvier. Five million HT1080.hACP3 cells, expressing human ACP3, were injected subcutaneously into the right flank. Once tumors reached a volume of approximately 150 mm3(volume = length x width2 / 2), mice were sacrificed, and tumors were excised and embedded in a cryo-embedding medium. Tumor cryosections (10 pm) were prepared using a cryostat. Sections were fixed in ice-cold acetone. Slides were mounted with a fluorescence mounting medium (Dako Agilent) and analyzed using a Leica DMI6000B microscope (Leica Microsystems).

[0251] 13.2 Staining with anti-ACP3 antibodies

[0252] The cryosections were stained with four different anti-ACP3 antibodies in IgG format (CC106QQ, EPR4066, STJ197186, and BSM 60820). KSF IgG, an antibody that binds to hen-egg lysozyme, an irrelevant target, was used as a negative isotype control. All antibodies were used at an equimolar concentration of 5 pg / mL. For the detection of CC106QQ and KSF antibodies, a goat anti-human IgG Alexa Fluor 488 secondary antibody (Invitrogen™, A11013) was used. For detection of the EPR4066 antibody (Abeam) and of BSM 60820 antibody (Santa Cruz Biotech), a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen™, A11034) was used. For the detection of the STJ197186 antibody (St Johns Labs), a goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Invitrogen A11029) was used. Cell nuclei were visualized using DAPI (Invitrogen™, D1306). All secondary antibodies were diluted 1:200.

[0253] 13.3 Results

[0254] The immunofluorescence analysis of the HT1080.hACP3 tumor cryosections confirmed that anti-ACP3 antibodies CC106QQ, EPR4066, STJ197186, and BSM 60820 recognize the antigen. However, the staining intensity and ACP3 expression patterns were much stronger with CC106QQ as compared to EPR4066, STJ197186, and BSM 60820. No signal was observed with the negative control antibody (KSF) (Figure 13).

[0255] EXAMPLE 14: Mass spectrometry biodistribution analysis of CC106QQ in tumor-bearing mice.

[0256] 14.1 Production of Stable-lsotope-Labeled (SIL) antibodies

[0257] CHO cells expressing the CC106QQ antibody were grown in Power-CHO serum-free medium supplemented with 4 mM L-glutamine in a shaking incubator at 37 °C. For SIL protein production, the medium was changed to CHO-TF for SI LAC (Sartorius) supplemented with stable-isotopically labelled arginine and lysine (i.e., [13C6, 15N4]L-arginine; [13C6, 15N2]L-lysine, Silantes), according to themanufacturer's instructions, and incubated at 31 °C. Antibodies were purified by affinity chromatography using Protein A Sepharose. SIL CC106QQ antibody was used as a common Internal Standard for both CC106QQ and the negative-control anti-lysozyme KSF antibody.

[0258] 14.2 Animal preparation

[0259] Tumor-bearing mice were prepared for biodistribution analysis as described in Example 13.1. Tumors grew to a volume of 150-200 mm3before injection of 200 pg of both antibodies in lgG4 format into the lateral tail vein. Organs of euthanized mice were split in half, with a portion placed in a cryo-embedding medium for immunofluorescence analysis and the other half snap-frozen in liquid nitrogen and stored at -80 °C for LC-MS analysis.

[0260] 14.3 Sample preparation for LC-MS analysis

[0261] Tissues (20 mg) were resuspended in 800 pL of lysis buffer (0.25% sodium deoxycholate, 1 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% Igepal, protease inhibitor in PBS pH 7.4. 1 pmol of SIL Internal Standard was added. Tissues were then homogenized in a TissueLyser II for 30 minutes at 30 Hz. After homogenization, lysates were centrifuged at 21’000 g for 30 min. Supernatants were incubated with BSA-blocked protein A magnetic beads for 2 hours under rotation at 4 °C. Beads were recovered and washed with lysis buffer A (150 mM NaCI, 20 mM Tris-HCI, pH 7.5), and lysis buffer B (400 mM NaCI, 20 mM Tris-HCI, pH 7.5). Cystines were reduced and carbamidomethylated with Tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA), respectively. Finally, proteins were digested on beads overnight with trypsin (Sequencing grade) at 37 °C. Tryptic peptides were purified on Cis Spin columns (Thermo Fisher) according to the manufacturer’s instructions. Eluates were dried at room temperature with a vacuum centrifuge. Dried samples were finally resuspended in 100 pL of a solution containing 3% ACN and 0.1% FA before LC-MS analysis.

[0262] 14.4 LC-MS analysis

[0263] 1 pL of each sample was injected into the LC-MS system. Chromatographic separation was carried out on an DNV PepMap Neo RSLC column (75 pm x 15 cm, particle size 2 pm, pore size 100 A, Thermo Fisher) with a gradient program from 100% A (H2O, 0.1% FA), 0% B (ACN 0.1% FA) to 65% A, 35% B in 60 minutes on an Easy nanoLC 1000 (Thermo Fisher) at a flow rate of 300 nL / min. The LC system was coupled to a Q-Exactive mass spectrometer via a Nano Flex ion source. Ionization was carried out with a spray voltage of 2 kV, a capillary temperature of 250 °C, and a Lens RF level of 60 S. The mass spectrometer was operating in a data-dependent top 10 acquisition mode with the following parameters: MS1 scan range: 374.5-1425.5 m / z, HCD NCE: 27, Dynamic exclusion: 10 sec. MS / MS spectra were processed and analyzed using Proteome Discoverer (PD, Thermo Fisher, version 2.5). Database searches were performed against the antibody reference sequences using Sequest.

[0264] Carbamidomethylation of cysteines was set as a fixed modification, oxidation of methionine, [13Ce15N2]lysine, and [13Ce15N4] arginine were set as variable modifications, and trypsin was set as cleavage specificity, allowing a maximum of two missed cleavages. Data filtering was performed using Percolator with a 1% FDR. PD results were imported to Skyline (MacCoss Lab Software, version 22.2.0.527) and manually inspected. Peak areas of signature peptides were extracted, and their ratios were calculated.

[0265] 14.5 Results

[0266] The average ratios (analyte / IS) for each sample were corrected for matrix effects using a single external calibration point and normalized to the total weight of the tissue analyzed to obtain the percentage of injected dose per gram (% ID / g) which shows a preferential accumulation of CC106QQ on the tumor as compared to healthy organs. The negative control antibody KSF did not show any preferential accumulation on the tumor (Fig 14).Sequence Listing

[0267] SEQ ID NO: 1 - Amino acid sequence of the human Prostatic Acid Phosphatase Antiqen (hACP3) KELKFVTLVFRHGDRSPIDTFPTDPIKESSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQV YIRSTDVDRTLMSAMTNLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELESETLK SEEFQKRLHPYKDFIATLGKLSGLHGQDLFGIWSKVYDPLYCESVHNFTLPSWATEDTMTKLRELSELSLL SLYGIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMYSAHDTTVSGLQMALDVYNGLLPPYASCHL TELYFEKGEYFVEMYYRNETQHEPYPLMLPGCSPSCPLERFAELVGPVIPQDWSTECMTTNSHQGTEDS TD

[0268]

[0269] KELKFVTLVFRHGDRSPIDTFPTDPIKESSWPQGFGQLTQLGMEQHYELGEYIRKRYRTFLNESYKHEQV YIQSTDVDRTLMSAMTNLAALFPPEGVSIWNPNLLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELGSETLT SEEFQKRLHPYKDFIATLGKLSGFHDKDLFGIWSKIYDPLYCESVHNFTLPSWATEDAMTKLRELSELSLL SLYGIHKQKEKSRLQGGVLVNEILNHMKRATQMPSYKKLIMYSTHDTTVSGLQVALDVFNGLLPPYAACH LMELYFEKGEYFVEMYYRNETQHEPYLLTLPGCSSSCPLERFAELVGPVIPQDWSTECMTTSSHQGTED STD

[0270] SEQ ID NO: 3 - Amino acid sequence of CC106QQ CDR1 VH

[0271] GFTFSSYAMS

[0272] SEQ ID NO: 4- Amino acid :

[0273]

[0274] of CC106QQ CDR2 VH

[0275] AVSGSGGSTYYADSVKG

[0276] SEQ ID NO: 5 - Amino acid

[0277]

[0278] of CC106QQ CDR3 VH

[0279] YMPWFDY

[0280] SEQ ID NO: 6 - Amino acid

[0281]

[0282] of CC106QQ CDR1 VL

[0283] RASQSVSSSYLA

[0284] SEQ ID NO: 7 - Amino acid

[0285]

[0286] of CC106QQ CDR2 VL

[0287] GASSRAT

[0288] SEQ ID NO: 8 - Amino acid

[0289]

[0290] of CC106QQ CDR3 VL

[0291] QQPGLRPST

[0292] SEQ ID NO: 9 - Amino acid sequence of the CC106QQ VH domainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSS

[0293] SEQ ID NO: 10 - Amino acid sequence of the CC106QQ VL domain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIK

[0294] SEQ ID NO: 11 - Amino acid sequence of the CC106QQ antibody molecule in scFv format

[0295] The linker sequence is underlined.

[0296] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIK

[0297] SEQ ID NO: 12 - Amino acid sequence of the linker between VH and VL in CC106QQ scFv GGGGSGGGGSGGGG

[0298] SEQ ID NO: 13 - Amino acid sequence of the CC106QQ heavy chain in lqG4 format EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0299] SEQ ID NO: 14 - Amino acid sequence of the CC106QQ liqht chain in lqG4 and IqG 1 formats EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

[0300] SEQ ID NO: 15 - Amino acid sequence of the CC106QQ antibody molecule in SIP format

[0301] The linker sequences are underlined.

[0302] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIKSGGSGGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQT VQRAVSVNPESSRRGGC

[0303] SEQ ID NO: 16 - Amino acid sequence of the linker between scFv and eS2 CH4 domain in the SIP format SGGSGG

[0304] SEQ ID NO: 17 -Amino acid sequence of human interleukin 2 (IL-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0305] SEQ ID NO: 18 - Amino acid sequence of the CC106QQ-IL2 conjugate

[0306] The linker sequences are underlined.

[0307] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSP GERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQPGLRPSTFGQGTKVEIKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNY KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFCQSIISTLT

[0308] SEQ ID NO: 19 - Amino acid sequence of the soluble form of the extracellular domain of human Tumor Necrosis Factor a (TNF) VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPD YLDFAESGQVYFGIIAL

[0309] SEQ ID NO: 20 - Amino acid sequence of the CC106QQ-TNF conjugate

[0310] The linker sequences are underlined.

[0311] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLT QSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHVVANP QAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTK VNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALSEQ ID NO: 21 - Amino acid sequence of human interleukin 12 (IL-12)

[0312] The linker sequence is underlined IWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCH KGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRG SSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIR DIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVIC RKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLL RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKT SFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEP DFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0313] SEQ ID NO: 22 - Amino acid sequence of the CC106QQ-IL12 conjugate

[0314] The linker sequences are underlined.

[0315] IWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCH KGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRG SSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIR DIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVIC RKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLL RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKT SFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEP DFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGSADGGSSAGGSDAGEVQLLESGGGLVQPGGSLRLSC AASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKYMPWFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEI KSSSSGSSSSGSSSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAV SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGS GGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIK

[0316] SEQ ID NO: 23 - Amino acid sequence of CC106QQ-CSMut lqG1 heavy chain

[0317] The wild-type cysteine residues of the heavy chain in positions 219, 225, and 228 were mutated into serine residues (underlined) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0318] SEQ ID NO: 24 - Amino acid sequence of CC106QQ IqG 1 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0319] SEQ ID NO: 25 - Amino acid sequence of the CC106QQ in diabody format

[0320] The linker sequence is underlined.

[0321] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSP GERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQPGLRPSTFGQGTKVEIK

[0322] SEQ ID NO: 26 - Amino acid sequence of the CC106QQ in sinqle-chain diabody (scDb) format The linker sequences are underlined.

[0323] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYMPWFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSP GERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQPGLRPSTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSVWRQAPGKGLEVWSAVSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKY MPWFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQPGLRPSTFGQGTKVEIK

Claims

Claims1. An antibody molecule that specifically binds to human ACP3,wherein the antibody molecule comprises a VH domain comprising a set of complementarity determining regions HCDR1 , HCDR2 and HCDR3, and a VL domain comprising a set of complementarity determining regions LCDR1 , LCDR2 and LCDR3, wherein:the HCDR1 , HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs 3, 4 and 5, respectively, andthe LCDR1 , LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs 6, 7 and 8, respectively.

2. An antibody molecule according to claim 1 that specifically binds to human ACP3 with a dissociation constant (Kd) for ACP3 of less than 50pM.

3. An antibody molecule according to claim 1 or claim 2, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO: 9 and / or the VL domain comprises the amino acid sequence shown in SEQ ID NO: 10.

4. An antibody molecule according to any one of claims 1 to 3, wherein the antibody molecule comprises or consists of: a single chain Fv (scFv), a small immunoprotein (SIP), a diabody (Db), a singlechain diabody (scDb), or an IgG molecule, optionally an lgG1 or lgG4 molecule.

5. An antibody molecule according to claim 4, wherein:the antibody molecule is in scFv format and comprises the amino acid sequence set forth in SEQ ID NO: 11 ;the antibody molecule is in lgG4 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 13, and the light chain amino acid sequence set forth in SEQ ID NO: 14;the antibody molecule is in IgG 1 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 23, and the light chain amino acid sequence set forth in SEQ ID NO: 14;the antibody molecule is in IgG 1 format and comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 24 and the light chain amino acid sequence set forth in SEQ ID NO: 14;the antibody molecule is in Db format and comprises the amino acid sequence set forth in SEQ ID NO: 25;the antibody molecule is in scDb format and comprises the amino acid sequence set forth in SEQ ID NO: 26; orthe antibody molecule is in small immunoprotein (SIP) format and comprises the amino acid sequence set forth in SEQ ID NO: 15.

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

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

8. A conjugate according to claim 7, wherein(i) the antibody molecule is an scFv and is conjugated at its C-terminus to interleukin 2 (IL2), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 18;(ii) the antibody molecule is an scFv and is conjugated at its C-terminus to tumor necrosis factor alpha (TNFa), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 20,(iii) wherein the antibody molecule is a single-chain diabody and is conjugated at its N-terminus to interleukin 12 (IL12), and wherein the conjugate optionally comprises the amino acid sequence set forth in SEQ ID NO: 22.

9. A conjugate according to claim 6, wherein the cytotoxic molecule MMAE is conjugated to the C-terminus of SEQ ID NO: 14 through a MC-GlyPro-PBAC linker.

10. The antibody molecule according to any one of claims 1 to 5 or conjugate according to any one of claims 6 to 9 for use in a method of treating cancer characterised by expression of ACP3 in a patient.

11. The antibody molecule or conjugate for use according to claim 10, wherein the method further comprises administering a second therapeutic agent to the patient, optionally wherein the second therapeutic agent is an immunomodulatory agent, such as an anti-PD-1 antibody.

12. A nucleic acid molecule or expression vector encoding an antibody molecule according to any one of claims 1 to 5 or conjugate according to any one of claims 6 to 9.

13. A host cell comprising the nucleic acid or vector of claim 12.

14. A method of producing an antibody molecule according to any one of claims 1 to 5 or conjugate according to any one of claims 6 to 9, the method comprising culturing the host cell of claim 13 under conditions for expression of the antibody molecule or conjugate, and optionally further isolating and / or purifying the antibody molecule or conjugate following expression.

15. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 5 or conjugate according to any one of claims 6 to 9 and a pharmaceutically acceptable excipient.

16. A conjugate comprising an antibody molecule according to any one of claims 1 to 5 and a diagnostic agent, optionally wherein the diagnostic agent is a detectable label, such as a radioisotope.

17. The antibody molecule according to any one of claims 1 to 5 or conjugate according to claim 16 for use in a method of imaging, detecting or diagnosing of a disease characterised by expression of ACP3 in a patient.

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