Antibody-drug conjugates and immunotoxins

Abstract

The present invention relates to conjugates, in particular antibody-drug conjugates and immunotoxins, having the formula I:
A-(L-D)p(I)
or a pharmaceutically acceptable salts or solvates thereof,
wherein:
A is an antibody that selectively binds FAP;
L is a linker;
D is a drug comprising a cytolysin or a Nigrin-b A-chain; and
p is 1 to 10, and to use of such conjugates in the therapeutic treatment of tumors. Methods of producing such conjugates and components for use in such methods are disclosed.

Claims

1. A conjugate having the formula I:
A-(L-D).sub.p(I) or a pharmaceutically acceptable salt or solvate thereof, wherein: A is an antibody that selectively binds FAP; L is a linker comprising a spacer; D is a drug comprising a cytolysin of formula IV: ##STR00031## wherein: R.sup.2 is H or C.sub.1-C.sub.4 alkyl; R.sup.6 is C.sub.1-C.sub.6 alkyl; R.sup.7 is C.sub.1-C.sub.6 alkyl, CH.sub.2OR.sup.19 or CH.sub.2OCOR.sup.20, wherein R.sup.19 is alkyl, R.sup.20 is C.sub.2-C.sub.6-alkenyl, phenyl, or CH.sub.2-phenyl; R.sup.9 is C.sub.1-C.sub.6 alkyl; R.sup.10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R.sup.11 has the following structure: R.sup.21 ##STR00032## wherein R.sup.21 is H, OH, halogen, NH.sub.2, alkyloxy, phenyl, alkyl amino or dialkyl amino; R.sup.16 is H or a C.sub.1-C.sub.6-alkyl group; n=2, 3 or 4; q is 0, 1, 2 or 3; and p is 1 to 10.

2. The conjugate of claim 1, wherein A comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: (i) CDRH1: SEQ ID NO: 7; (ii) CDRH2: SEQ ID NO: 8; (iii) CDRH3: SEQ ID NO: 9; (iv) CDRL1: SEQ ID NO: 10; (v) CDRL2: SEQ ID NO: 11; and (vi) CDRL3: SEQ ID NO: 12.

3. The conjugate of claim 1, wherein L comprises an attachment group for attachment to A and a protease cleavable portion.

4. The conjugate of claim 3, wherein L comprises maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

5. The conjugate of claim 1, wherein R.sup.2 is methyl, f=2, R.sup.6 is C.sub.4 alkyl, R.sup.7 is C.sub.3 alkyl, R.sup.9 is C.sub.3 alkyl, R.sup.10 is O-alkyl, R.sup.21 is OH, q is 1, and R.sup.16 is methyl.

6. The conjugate of claim 1, wherein n=3.

7. A method of treating a tumor in a mammalian subject, wherein said tumor and/or stroma surrounding said tumor expresses fibroblast activation protein alpha (FAP), said method comprising administering a therapeutically effective amount of a conjugate of claim 1 to a subject in need thereof.

8. The method of claim 7, wherein said conjugate is administered simultaneously, sequentially or separately with one or more other antitumor drugs.

9. The method of claim 8, wherein said one or more other antitumor drugs comprise a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

10. The method of claim 9, wherein said one or more other antitumor drugs comprise Gemcitabine, Abraxane, bevacizumab, itraconazole, or carboxyamidotriazole, an anti-PD-1 molecule or an anti-PD-L1 molecule.

11. The method of claim 10, wherein said anti-PD-1 molecule or anti-PD-L1 molecule comprises nivolumab or pembrolizumab.

12. The method of claim 7, wherein the FAP expressing tumor is a solid tumor.

13. The method of claim 12, wherein the solid tumor is an FAP expressing solid tumor of pancreatic cancer, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer or colon cancer.

14. A method of treating a fibroblast activation protein alpha (FAP) expressing inflammatory condition in a mammalian subject, comprising administering a therapeutically effective amount of a conjugate of claim 1 to a subject in need thereof.

15. The method of claim 14, wherein said FAP expressing inflammatory condition is FAP expressing rheumatoid arthritis.

16. A conjugate having the formula I:
A-(L-D).sub.p(I) or a pharmaceutically acceptable salt or solvate thereof, wherein: A is an antibody that selectively binds FAP; L is a linker; D is a drug having the structure: ##STR00033## wherein p is 1 to 10.

17. The conjugate of claim 16, wherein L comprises maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1C show characterization of humanized scFv hu33 and hu36. FIG. 1A) SDS-PAGE analysis of purified scFv fragments. Coomassie staining. R-reducing, NRnon reducing. FIG. 1B) Flow cytometry analysis of binding of hu36 (humanized) and mo36 (chimeric) to HT1080-huFAP cells. Bound antibodies were detected with an anti-His-tag antibody (n=2). FIG. 1C) ELISA of binding of hu36 scFv and mo36 scFv to immobilized recombinant human FAP (coated at 100 ng/ml). Bound antibodies were detected with an HRP-conjugated anti-Myc-tag antibody.

(2) FIGS. 2A and 2B show ELISA of anti-FAP mo36-IgG1 (circles) and hu36-IgG1 (squares) for binding to recombinant human FAP (rhFAP) or control protein (BSA) (triangles and inverted triangles, respectively) (FIG. 2A). 50 ng protein were coated per well. Bound antibodies were detected with HRP-conjugated anti-human IgG-Fc. Flow cytometry analysis of anti-FAP mo36-IgG1 (triangles and stars) and hu36-IgG1 (squares and circles) for binding to HT1080-FAP (FIG. 2B). Bound proteins were detected with a PE-labeled anti-hu IgG-Fc antibody.

(3) FIGS. 3A and 3B show flow cytometry analysis of binding of hu36-IgG1 to stably transfected HT1080 to express human FAP (HT1080-huFAP) (FIG. 3A) and mouse FAP (HT1080-moFAP) (FIG. 3B). Bound antibodies were detected with a PE-labeled anti-human Fc antibody.

(4) FIG. 4 shows confocal microscopy of HT1080-FAP cells, incubated with hu36-IgG1 for various times (0, 30 and 60 mins) and stained with FITC-labelled anti-IgG antibody, WGA-TRed (membrane staining), and DAPI (nucleus). The right-hand panels show a merged image of the three stains.

(5) FIG. 5 shows analysis of internalization of hu36-IgG1 by discrimination of cells (n=10-30) showing only membrane staining (PM; open bars), PM and intracellular staining (shaded bars), or only intracellular staining (filled bars). A clear time-dependent internalization is evidenced.

(6) FIG. 6 shows MALDI-Tof profile of recombinant nigrin-b A-chain. Observed mass (Da): 28546.55; Expected mass (Da): 28546.09; Mass deviation: 0.5; Mass Accuracy: 16 ppm.

(7) FIG. 7 shows ribosome inactivating protein (RIP) activity of recombinant Nigrin-b A-chain (recNgA) tested in rabbit reticulocyte cell-free lysates (RRL) versus native (WT) Nigrin-b. (3a, 3b, 6c, 9c) represent different formulations of recNgA.

(8) FIG. 8 shows cytotoxicity of recNgA tested on HT1080-FAP cell line through crystal violet viability assay (native Nigrindiamonds; recombinant Nigrin-b A-chainsquares).

(9) FIG. 9 shows RIP activity of anti-FAP hu36-IgG1-recNgbA immunotoxin conjugates (HSP131-001; crosses) in an RRL assay compared to native (WT) nigrin (triangles) and recombinant Nigrin-b A-chain (recNgA; squares).

(10) FIGS. 10A and 10B show cytotoxic activity of anti-FAP hu36-IgG1-recNgbA immunotoxin conjugates (HSP131-001; triangles), unconjugated (naked) anti-FAP hu36-IgG1 (squares) and recombinant Nigrin-b A-chain (recNgA; diamonds) on HT1080-WT cell line (FIG. 10A); and HT1080-FAP cell line (FIG. 10B). Fold-change in proliferation is plotted against antibody/immunotoxin concentration.

(11) FIG. 11 shows the general antibody conjugate structure for a cytolysin-conjugated antibody via a vcPABA linker. Attachment of the cytolysin may be via R.sub.1 or R.sub.4 (identified by arrows).

(12) FIG. 12 shows immunodetection of anti-FAP hu36 tumour sections of patient-derived xenograft (PDX) mice (pancreatic tumour). Specific Dose- and Time-dependent staining of stroma is observed in subcutaneous tumors from PDX mouse model for pancreas cancer (Panc185)Single dose (1 & 5 mg/kg) of anti-hu/moFAP hu36 IgG1 was administrated intraperitoneally in PDX mice Panc-185; immunodetection was performed with an anti-human IgG1 secondary antibody20 scale pictures are shown. Control-48 h: Mice administrated with Vehicle and tumors excised after 48 h.

(13) FIG. 13 shows animal weight monitored after treatment with anti-FAP:recNgA immunotoxin at different doses (2.5, 1, 0.5, 0.25, 0.1 mg/kg) administrated once a week. Significant weight loss and toxicity was observed in Group 1 and 2 (2.5 and 1 mg/kg, respectively), similarly to treatment with 5 mg/kg (not shown); 0.5 mg/kg was the highest tolerated dose when applied as single agent.

(14) FIGS. 14A and 14B show Relative Body weight (FIG. 14A) and Tumor volume (FIG. 14B) measured from patient-derived xenograft mice (PAXF 736) untreated (Vehicle; 10 ml/kg/day; once a week), treated with Gemcitabine (GEM; 150 mg/kg; once a week), or antiFAP:recNgA immunotoxin (OMTX505; 0.5/0.25 mg/kg; once a week), or both (OMTX505 (0.25 mg/kg):GEM(150 mg/kg)), for 4 weeks (treatment days 1, 8, 15, 22, 29).

(15) FIGS. 15A-15C show ELISA and FACS analysis of ADC471 binding to FAP target. ELISA detection of ADC-471 binding to huFAP fusion protein compared to naked anti-hu/mo FAP hu36 antibody; EC50 values are indicated for HPS124-3 ADC-471 molecule with DAR=3.48 (FIG. 15A); FACS analysis of binding on HT1080-huFAP, HT1080-wt and HEK293 cells of HPS131-143-1 (ADC-471; DAR 4), HPS131-124-1 (ADC-467; DAR 1.2) and HPS131-124-3 (ADC-471; DAR 3.48) ADCs (FIGS. 15B and 15C). EC.sub.50 values are indicated for this latter (FIG. 15B).

(16) FIG. 16 shows Time-lapse immunofluorescence analysis of internalization capacity of anti-FAP hu36:cytolysin ADC (ADC-471; HPS131-124-3) on living HT1080-FAP cells. Left panel: Incubation with naked anti-hu/moFAP hu36 (FITC-AB; green); Right panel: Incubation with ADC-471 (FITC-ADC; green). Time 0, 30, 60, 90 min (upper panels): HT1080-FAP cells. Time 30 min (lower panels): HT1080-wild type cells.

(17) FIGS. 17A and 17B show in vitro cytotoxic effect of anti-hu/moFAP hu36: cytolysin ADCs on HT1080-wt (FIG. 17A) and FAP(+) cells

(18) (FIG. 17B). Cell proliferation arrest was evidenced through crystal violet staining after 72 h incubation of each compound at a concentration range from 10.sup.6 to 10.sup.12M. Parental TAM334 cytolysin was used as positive control for unspecific cytotoxicity.

(19) FIGS. 18A and 18B show tumor growth inhibition effect of anti-hu/moFAP hu36:cytolysin ADC candidates. DC471 versus ADC551 (FIG. 18A); ADC471 and ADC553 (OMTX705-553) versus ADC558 (OMTX705-558) (FIG. 18B). Vehicle and GEM (Gemcitabine): negative and positive control groups.

DETAILED DESCRIPTION OF THE INVENTION

(20) In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

(21) FAP

(22) As used herein Fibroblast activation protein, fibroblast activating protein, FAP and FAP are used interchangeably. The FAP may be an FAP of any mammalian species. In some cases FAP is human FAP (also known as Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblast activation protein alpha or integral membrane serine protease), the amino acid sequence of which is disclosed at UniProt accession No. Q12884 (Version 140, dated 11 Dec. 2013) (SEQ ID NO: 15). In some cases, a molecule that binds FAP (e.g. an antibody molecule or a conjugate thereof) may bind to a region of the extracellular domain of FAP. The extracellular domain of human FAP comprises residues 26-760 of the full-length human FAP protein. In some cases FAP is murine FAP (also known as fibroblast activation protein alpha or integral membrane serine protease), the amino acid sequence of which is disclosed at UniProt accession No. P97321 (Version 117, dated 11 Dec. 2013) (SEQ ID NO: 16). The extracellular domain of murine FAP comprises residues 26-761 of the full-length murine FAP protein.

(23) Conjugate

(24) As used herein conjugate includes the resultant structure formed by linking molecules and specifically includes antibody-drug conjugates (ADCs) and immunotoxins (ITs).

(25) Selectively Binds

(26) The terms selectively binds and selective binding refer to binding of an antibody, or binding fragment thereof, to a predetermined molecule (e.g. an antigen) in a specific manner. For example, the antibody, or binding fragment thereof, may bind to FAP, e.g. an extracellular portion thereof, with an affinity of at least about 110.sup.7M.sup.1, and may bind to the predetermined molecule with an affinity that is at least two-fold greater (e.g. five-fold or ten-fold greater) than its affinity for binding to a molecule other than the predetermined molecule.

(27) Antibody Molecule

(28) As used herein with reference to all aspects of the invention, the term antibody or antibody molecule includes any immunoglobulin whether natural or partly or wholly synthetically produced. The term antibody or antibody molecule includes monoclonal antibodies (mAb) and polyclonal antibodies (including polyclonal antisera). Antibodies may be intact or fragments derived from full antibodies (see below). Antibodies may be human antibodies, humanised antibodies or antibodies of non-human origin. Monoclonal antibodies are homogeneous, highly specific antibody populations directed against a single antigenic site or determinant of the target molecule. Polyclonal antibodies include heterogeneous antibody populations that are directed against different antigenic determinants of the target molecule. The term antiserum or antisera refers to blood serum containing antibodies obtained from immunized animals.

(29) It has been shown that fragments of a whole antibody can perform the function of binding antigens. Thus reference to antibody herein, and with reference to the methods, arrays and kits of the invention, covers a full antibody and also covers any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) the Fab fragment consisting of V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) the Fd fragment consisting of the V.sub.H and C.sub.H1 domains; (iii) the Fv fragment consisting of the V.sub.L and V.sub.H domains of a single antibody; (iv) the dAb fragment which consists of a V.sub.H domain; (v) isolated CDR regions; (vi) F(ab).sub.2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a V.sub.H domain and a V.sub.L domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (WO 93/11161) and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; 58). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made.

(30) In relation to a an antibody molecule, the term selectively binds may be used herein to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

(31) In some cases in accordance with the present invention the antibody may be a fully human antibody.

(32) Cytotoxic Chemotherapeutic Agents

(33) In some cases in accordance with any aspect of the present invention, the conjugate of the invention may administered with, or for administration with, (whether simultaneously, sequentially or separately) one or more other antitumor drugs, including, but not limited to, a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

(34) Cytotoxic chemotherapeutic agents are well known in the art and include anti-cancer agents such as: Alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; 10 ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U) and streptozoein (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2-deoxycofonnycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorabicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin Q; enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and antbracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o, p-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen. A further preferred cytotoxic agent is Gemcitabine (Gemzar). A further preferred cytotoxic agent is Paclitaxel bound to human serum albumin (Abraxane).

(35) Anti-angiogenic agents are well known in the art and include anti-cancer agents such as bevacizumab, itraconazole, and carboxyamidotriazole.

(36) Immunotherapeutic agents are known in the art and include, for example, anti-programmed cell death protein 1 (PD-1) antibodies and anti-programmed death-ligand 1 (PD-L1) antibodies, including Nivolumab (MDX1106) and Pembrolizumab (MK-3475).

(37) Pharmaceutical Compositions

(38) The conjugates of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

(39) A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the conjugate, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the conjugate.

(40) In some embodiments, conjugates of the present invention may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised conjugates may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

(41) Conjugates of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the conjugate. Thus pharmaceutical compositions may comprise, in addition to the conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the conjugate. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

(42) For intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the 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, asparagines, 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 non-ionic surfactants, such as TWEEN, PLURONICS or polyethylene glycol (PEG).

(43) Subject

(44) The subject may be a human, a companion animal (e.g. a dog or cat), a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep). Preferably, the subject is a human. In some cases the subject may be a human diagnosed with or classified as being at risk of developing a cancer, e.g., an epithelial tumor. In certain cases the subject may be a laboratory animal, e.g., a mouse model of a cancer. In certain cases the subject may be a mammal (e.g. a human) that has been diagnosed with or classified as being at risk of developing an inflammatory condition, such as rheumatoid arthritis (RA). In particular, the subject may be a human having RA.

(45) Cancer

(46) The anti-FAP conjugates described herein find use in the treatment of a tumor in a mammalian subject. The tumor may be a solid tumor. In particular, the tumor may be a pancreatic cancer, breast cancer, melanoma, lung cancer, head & neck cancer, ovarian cancer, bladder cancer or colon cancer.

(47) Inflammatory Condition

(48) In some cases in accordance with the present invention, the anti-FAP antibody or the antibody drug conjugate may be for use in the treatment of an inflammatory condition. FAP expression has been reported in fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) patients (see, e.g., Bauer et al., Arthritis Res. Therp. (2006):8(6); R171). The present inventors believe that the anti-FAP antibodies described herein, and/or conjugates thereof described herein, are able to ameliorate RA and/or symptoms of RA.

(49) The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Example 1Production of Anti-FAP Antibodies

(50) Anti-FAP scFvs selected by phage display from an immunized FAP.sup./ knock-out mouse have been described previously (28). Two scFvs, MO36 and MO33, cross-reactive for human and murine FAP (28) were converted into full-length IgG for subsequent characterisation studies and for generation of immunotoxins and ADCs. These scFv (scFv33 and scFv36) were used to generate chimeric antibodies, fusing heavy and light chain constant domains to VH and VL, respectively. In addition, both were humanized by CDR grafting and tested for binding to FAP-expressing cells and recombinant FAP in comparison to the parental scFv. From this comparison, the best binder was used to generate full-length IgG. All scFvs were produced in E. coli and purified by IMAC, IgGs were produced in mammalian cells (CHO) using the Lonza GS expression vectors pEE6.4 and pEE14.4 developed for antibody production. Features of the scFvs are summarized in Table 1.

(51) TABLE-US-00001 TABLE 1 antibodies, specificities, subclass, and vectors used as starting material Vl Plasmid Format Species Antigen Clone Subclass Vector DNA # scFv mouse hu/mo FAP mo33 lambda pAB1 376 scFv mouse hu/mo FAP mo36 kappa pAB1 277 scFv humanized hu/mo FAP hu33 lambda pAB1 1214 scFv humanized hu/mo FAP hu36 kappa pAB1 1215

(52) All scFvs were bacterially produced in E. coli TG1 and purified from the periplasmic extracts of 1 L cultures by IMAC. Both humanized antibodies (scFv hu33 and hu36) were purified in soluble form with yields of approximately 0.6 mg/L culture. In SDS-PAGE the proteins migrated with the expected size of approximately 30 kDa (FIG. 1A). Purity was estimated to be >90%. In flow cytometry experiments using HT1080 cells expressing human FAP (stable transfectants), a similar binding was observed for scFv hu36 and mo36 scFv, which was also produced in bacteria (not shown). EC.sub.50 values were in the low nanomolar range. Some differences were observed at higher concentrations (FIG. 1B). scFv hu33 showed no binding or only marginal binding in these experiments. Further development therefore focused on hu36. Binding of hu36 scFv was also observed by ELISA with recombinant human FAP (extracellular region aa 26-760; R&D systems), although binding was somewhat weaker than that seen for mo36 scFv (FIG. 1C).

(53) Plasmids corresponding to full length IgG1 antibodies were generated and transfected into CHO cells for production of antibodies in Lonza's CHO expressing system with yields of approximately 1 mg/L of cell culture (lab scale). Antibodies were purified from cell culture supernatant by protein A chromatography. Purified proteins were characterized by SDS-PAGE and size exclusion chromatography. Bioactivity was analyzed by ELISA using recombinant FAP and detection of bound antibodies with HRP-conjugated anti-human IgG antibodies. Cell binding was analyzed by flow cytometry using HT1080-FAP cell line.

(54) Results:

(55) Plasmids generated (and sequenced):

(56) TABLE-US-00002 mo36 IgG1: pEE14.4 mo36-IgG1 OCMTX001p (chimeric anti-FAP IgG1) hu36 IgG1: pEE14.4 hu36-IgG1 OCMTX002p (humanized anti-FAP IgG1)

Example 2Characterisation of Anti-FAP Antibodies

(57) The amino acid sequences of humanized anti-FAP IgG1 hu36 (hu36-IgG1) heavy chain (HC) and light chain (LC), respectively are shown below:

(58) Anti-FAP hu36-IgG1-HC:

(59) TABLE-US-00003 (SEQIDNO:1) 0 GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK aa 449 MWofprocessedHC 49,069 TheoreticalpI 8.69 Potentialglycosylationsite(doubleunderlined):N297 MutationsleadingtoADCCandCDCdeficiencyareshowninbolditalics (seealsoWO99/58572) Signalsequenceisshownboxed VHdomainisunderlined;CDRH1-H3areshowninboldandcurved underlined.
Anti-FAP hu36-IgG1-LC:

(60) TABLE-US-00004 (SEQIDNO:2) NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC aa 218 MWofprocessedHC 23,919 theoreticalpI 7.77 signalsequenceisboxed VLdomainisunderlined;CDRL1-L3areshowninboldandcurved underlined. hu36-IgG1-HCwithoutsignalsequence: (SEQIDNO:3) GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS 0 YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK hu36-IgG1-LCwithoutsignalsequence: (SEQIDNO:4) NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC hu36-VH: (SEQIDNO:5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFKDRVTM TADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSS hu36-VL: (SEQIDNO:6) DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKR hu36-CDRH1: (SEQIDNO:7) ENIIH hu36-CDRH2: (SEQIDNO:8) WFHPGSGSIKYNEKFKD hu36-CDRH3: (SEQIDNO:9) HGGTGRGAMDY hu36-CDRL1: (SEQIDNO:10) RASKSVSTSAYSYMH hu36-CDRL2: (SEQIDNO:11) LASNLES hu36-CDRL3: (SEQIDNO:12) QHSRELPYT Parametersofthefullhu36-IgGareasfollows: Totallengthoffull-lengthIgG(aa):1,334 Calculatedmolecularmassoffull-lengthIgG:145,922 Calculatedextinctioncoefficientoffull-lengthIgG:209,420 Abs0.1%(=g/l) 1.435 theoreticalpI: 8.60 potentialglycosylationsite: N297

(61) Purified chimeric and human anti-FAP antibodies mo36 and hu36 were analyzed in ELISA for binding to recombinant FAP. Both anti-FAP antibodies showed specific and strong binding to recombinant FAP with similar EC.sub.50 values (around 5 nM) (FIG. 2A). Furthermore, both antibodies showed binding to HTP1080-FAP expressing human FAP on their cell surface (FIG. 2B). The humanized IgG gave stronger signals compared with the chimeric IgG, however, with similar EC.sub.50 values. The humanized hu36 anti-FAP antibody was able to cross-react to both human and murine FAP as shown by FACS analysis (FIGS. 3A and 3B). Hu36-IgG1 bound in a concentration-dependent manner to both cell lines with subnanomolar EC50 values (0.33 and 0.25 nM).

(62) For scale-up the antibody constructs were cloned in GS double vectors (pEE14.4). The DNA plasmids were transformed, amplified, and transiently transfected into CHOK1SV cells for expression evaluation at a volume of 200 ml. In a second step the antibodies were transiently expressed in 5-10 L large scale cultures. Clarified culture supernatant was purified using one-step Protein A chromatography. Product quality analysis through SE-HPLC, SDS-PAGE and LAL was carried out using purified material at a concentration of 1 mg/ml, alongside an in-house human antibody as a control sample.

(63) The purified protein samples were filtered through a 0.2 m filter and analysed by SE-HPLC chromatograms. The antibodies were purified to >98.8%. The endotoxin levels were <0.5 EU/mg.

(64) All purified proteins were analyzed by SDS-PAGE in reducing and non-reducing conditions (data not shown).

(65) Purified proteins hu36-IgG and mu36-IgG were characterized by SDS-PAGE and size exclusion chromatography. Bioactivity was analyzed by ELISA, using recombinant FAP and detection of bound antibodies with HRP-conjugated anti-human IgG antibodies. Cell binding was analyzed by flow cytometry, using HT1080-FAP cell line. Melting points were determined by dynamic light scattering using a zetasizer nano. Affinities were determined by QCM using an Attana A100. Internalization study was performed by indirect immunofluorescence confocal microscopy on permeabilized cells, detecting bound and internalized antibodies with a FITC-labeled secondary antibody.

(66) The full-length IgG1 purified antibodies were successfully produced at both lab scale and large scale, for the generation of immunoconjugates. A summary of antibody properties is shown in Table 2. The antibodies retained their specificity, as shown by ELISA and flow cytometry experiments. The antibodies bound FAP-expressing cells with subnanomolar EC.sub.50 values. Affinities, as determined by QCM, were comparable with that of parental antibodies. QCM measurements indicated the contribution of avidity effects to high-affinity binding. Thermal stability differed between the different IgGs (77-80 C.)

(67) Rapid internalisation was shown for hu36-IgG1 (humanized anti-FAP antibody) on HT1080-FAP cells (see FIGS. 4 and 5).

(68) TABLE-US-00005 TABLE 2 Summary of antibody properties antibody mo36-IgG1 hu36-IgG1 antigen hu and mo FAP hu and mo FAP isotype 1*/ 1*/ IgG type chimeric humanized plasmid OCMTX001p OCMTX002p purity (SEC) minor aggregates Tm (DLS) 77 C. 80 C. EC.sub.50 ELISA 3 nM (rhFAP) 3 nM (rhFAP) EC.sub.50 FACS 0.5 nM (HT1080- 0.3 nM (HT1080- huFAP) huFAP) 0.2 nM (HT1080- moFAP) binding to n.d. + primary tumor fibroblasts binding rhFAP: rhFAP: constants K.sub.D K.sub.D1 = 112 nM K.sub.D1 = 218 nM (QCM) K.sub.D2 = 0.6 nM K.sub.D2 = 0.4 nM internalization n.d. HT1080-FAP 30-60 min 1* = deficient for ADCC and CDC (see Amour et al., 1999; Richter et al., 2013).
Anti-FAP IgG1 In Vivo Binding.

(69) Anti-FAP IgG1 hu36 was administrated intraperitoneally to patient-derived xenograft mice for pancreas cancer at a single dose of 1 and 5 mg/kg. Tumors were excised after 12, 24, and 48 h administration, formalin-fixed and paraffin-embedded. Immunodetection of anti-FAP hu36 was performed with an anti-human IgG secondary antibody. FIG. 12 shows the specific dose- and time-dependent staining of stroma, only in tumor samples from treated mice.

Example 3Nigrin-b A-Chain

(70) In order to avoid side effects of free toxin that could be released in the bloodstream and to reduce potential immunogenicity of the RIP toxin, as extensively described with ricin, the enzymatic domain of Nigrin b, the A chain, was cloned and expressed in bacteria. The present inventors hypothesized that, if the A chain produced in bacteria was able to retain its activity, it would not be able to enter the cells, unless conjugated to a vehicle molecule, such as an antibody.

(71) Production

(72) Nigrin-b A-chain was synthetized taking into account codon optimization for bacterial expression and the synthetized gene was cloned in two different vectors, Nigrin_pET30b-3 and Nigrin_pET33b-1 (+/His tag) for expression in two different E. coli strains, E. coli BLR(DE3) and E. coli HMS174(DE3). Different culture media were used to check different expression conditions. Process purification was established using Capto Q chromatography and SP Sepharose High Performance. Purified recombinant Nigrin-b A-chain (recNgA) was formulated at 5 mg/ml in PBS 1 pH7.4, DTT 0.5 mM, glycerol 10%. Endotoxin levels were <1 EU/mg of Nigrin and the purity >99% in monomeric form.

(73) Eldman N-terminal sequencing revealed that N-terminal end of recNgA corresponded to the expected sequence.

(74) TABLE-US-00006 RecombinantNigrin-bA-chainaminoacidsequence: (SEQIDNO:13) MIDYPSVSFNLDGAKSATYRDFLSNLRKTVATGTYEVNGLPVLRRESEVQ VKSRFVLVPLTNYNGNTVTLAVDVTNLYVVAFSGNANSYFFKDATEVQKS NLFVGTKQNTLSFTGNYDNLETAANTRRESIELGPSPLDGAITSLYHGDS VARSLLVVIQMVSEAARFRYIEQEVRRSLQQATSFTPNALMLSMENNWSS MSLEIQQAGNNVSPFFGTVQLLNYDHTHRLVDNFEELYKITGIAILLFRC SSPSND

(75) The recombinant Nigrin-b A-chain has the following characteristics:

(76) Number of amino acids: 256

(77) Molecular weight: 28546.0

(78) Theoretical pI: 5.45

(79) The nucleotide sequence encoding recombinant Nigrin-b A-chain is as follows:

(80) TABLE-US-00007 (SEQIDNO:14) atagactatccctccgtctccttcaacttg gatggagccaagtcggctacatacagggac ttcctcagcaacctgcgaaaaacagtggca actggcacctatgaagtaaacggtttacca gtactgaggcgcgaaagtgaagtacaggtc aagagtcggttcgttctcgtccctctcacc aattacaatggaaacaccgtcacgttggca gtagatgtgaccaacctttacgtggtggct tttagtggaaatgcaaactcctactttttc aaggacgctacggaagttcaaaagagtaat ttattcgttggcaccaagcaaaatacgtta tccttcacgggtaattatgacaaccttgag actgcggcgaatactaggagggagtctatc gaactgggacccagtccgctagatggagcc attacaagtttgtatcatggtgatagcgta gcccgatctctccttgtggtaattcagatg gtctcggaagcggcaaggttcagatacatt gagcaagaagtgcgccgaagcctacagcag gctacaagcttcacaccaaatgctttgatg ctgagcatggagaacaactggtcgtctatg tccttggagatccagcaggcgggaaataat gtatcacccttctttgggaccgttcagctt ctaaattacgatcacactcaccgcctagtt gacaactttgaggaactctataagattacg gggatagcaattcttctcttccgttgctcc tcaccaagcaatgat
Materials Nigrin_pET30b-3 genetic construct. Escherichia coli (Migula) Castellani and Palmers BLR(DE3) Culture media: auto induced medium (AIM) Extraction culture buffer: Glycine/NaOH 10 mM, Leupeptine 1 g/ml, Pepstatine 1 g/ml, pH 9.5. Extraction supernatant buffer Tris-HCl 50 mM, NaCl 200 mM, MgCl.sub.2 2 mM, leupeptine 1 gml.sup.1, pepstatine 1 gml.sup.1, lysozyme 0.1 mgml.sup.1, pH8.0. Dialysis solution: Citric acid/NaOH 25 mM pH5.0. -Capto Q FPLC: Equilibration buffer A: Glycine/NaOH 50 mM pH9.5. Elution buffer B: Glycine/NaOH 50 mM pH9.5, NaCl 1 M. Pooled fractions from Capto Q step (+80 ml extraction). SP Sepharose HP FPLC: Equilibration buffer A: Citric acid 25 mM pH4.0.Elution buffer B: Citric acid 25 mM pH4.0, NaCl 1 M.
Methods

(81) E. coli BLR(DE3) holding expression Nigrin_pET30b-3 cultivated in 1 L format of Auto Inducible Medium (AIM) with 30 gml.sup.1 Kanamycin. Protein expression was triggered by lactose activation and glucose depletion after about 3-4 hours of growth. Then, the temperature was lowered to 20 C. for an overnight duration.

(82) For extraction, each cell pellet was initially resuspended in 80 ml of extraction buffer per liter of culture, and 3 cycles of 7 minutes disintegration at 1100-110 Bar were performed after 30 minutes of incubation at 8 C. under shaking. Then the extract underwent 60 minutes centrifugation at 15,900 g, 8 C. The supernatant was the purification's starting material.

(83) Capto Q FPLC: 160 ml of extracted product from 81 culture were loaded into 160 ml Capto Q and equilibrated using 4CV of equilibration buffer and washed with 15 CV of equilibration buffer. Elution was carried in three steps: 15 CV at 1.5 mS/cm (7.6% B); 20 CV at 23.8 mS/cm (18.9% B); 20 CV 100% B.

(84) Dialysis was performed at the following conditions: 650 ml of the product were dialyzed in 45Lbathsin in citric acid/NaOH 25 mM pH5.0, cut-off 6-8000 Da. Dialysis factor 3500, <24 h. After dialysis, a 30 minutes centrifugation at 20,500 g and 8 C. allowed to separate soluble from insoluble fractions. SDS-PAGE was performed on the total and soluble fractions both pre and post dialysis (10 l loaded on SDS-PAGE). The eluent was dialysed into PBS pH7.4 and filtered T=0.22 m using 220 cm.sup.2 EKV filters.

(85) SP Sepharose HP: 610 ml of dialyzed pool of Capto Q in Citric acid 25 mM pH5.0 were loaded into 240 ml SP Sepharose High Performance with 4 CV of equilibration buffer and washed with 15CV of equilibration buffer and eluted at 25 Cv gradient to 20% B; 4 CV step of 100% B.

(86) Pooled fractions from SP Sepharose HP step were dialysed in PBS pH7.4, DTT 0.5 mM (54 L baths, pooled fractions of 950 mL at 0.97 mg/ml). Cut off was 6-8000 Da, dialysis factor was 3130, time >24 h. Afterwards a 30 min centrifugation at 20,55 g and 8 C. allowed to separate soluble from insoluble fractions. 10% glycerol was added afterwards.

(87) Finally the eluent was dialysed into PBS pH7.4 (5 baths 3100) and filtered =0.2 m, then the recNg b A batch was snap frozen at 80 C. A SEC in Semi-Preparative 5200 Superdex was later carried out.

(88) Size exclusion chromatography and mass spectrometry analysis demonstrated monomeric and purification status of the obtained recombinant nigrin-b A-chain (recNgA) (FIG. 6).

(89) Stability studies were performed to evaluate pH and temperature effect on nigrin-b A-chain protein itself and its activity. recNgA is stable at pH ranging from 5 to 9, and in presence or not of glycerol (from 10 to 45%) (data not shown).

(90) Activity

(91) The ribosome-inactivating protein (RIP) activity of recombinant Nigrin-b A-chain was tested in rabbit reticulocyte cell-free lysates: IC.sub.50 value obtained was similar to native nigrin-b and within 2.5 to 25 pM range (see FIG. 7). Thus, the A chain from Nigrin-b, expressed as a recombinant protein in bacteria, maintains its enzymatic activity, supporting that glycosylation is not required for RIP activity of Nigrin-b A-chain.

(92) RecNgA retains its activity in rabbit reticulocyte cell-free lysates if stored frozen at (80 C.) and below 3 freeze-thaw cycles (not shown).

(93) The cytotoxic activity of recNgA was tested on cell cultures through crystal violet-based viability assay. recNgA, lacking the B chain to translocate within cells, presents a 100 to 1000 less toxic activity than native Nigrin-b, as shown in FIG. 8. Native nigrin b showed an IC.sub.50210.sup.8M (similar to previous published data; see 33), while recNgA showed an IC.sub.50210.sup.6M.

(94) Previously published studies showed that native Nigrin b presents higher RIP activity than Ricin in RRL assay, while it is much less toxic (30-10,000 time, approximately) in cells or in vivo (see IC.sub.50 and LD.sub.50 values in Table 3).

(95) Upon removing of B chain, Ricin A chain loses activity in both RRL assay and cytotoxicity assay. Unexpectedly, Nigrin b A chain, generated for the first time in this present invention, only loses activity in cell cytotoxicity assay, while it was even increased in RRL assay with respect to native Nigrin b. These data were suggesting that, in the case of Ricin, removing B chain was affecting not only binding and translocation of A chain, but also its RIP activity, while this was not the case for Nigrin b A chain that retains and even increases its RRL activity with respect to its native counterpart. As a result, Nigrin b A chain is 50 times more active than the Ricin A chain in RRL.

(96) Consequently, upon conjugation, Nigrin b A chain conjugates present higher cytotoxic activity (IC.sub.50 within pM range) than Ricin A chain conjugates (nM range) (data not shown).

(97) TABLE-US-00008 TABLE 3 In vitro and in vivo activity data for Ricin and Nigrin (native and A chain). Rabbit Lysate HeLa Cells IC.sub.50 Mouse LD.sub.50 IC.sub.50 (pM) (pM) (gkg.sup.1) Nigrin b 30 27,600.00 (20-2300 nM; 12,000.00 dpt cell line) Nigrin b 6.5 750,000.00 ND A chain (HT1080-FAP) 300,000.00 (HT1080) Ricin 100 0.67 3.00 Ricin A 300 260,000.00 (T ND chain cells) (Inventors' Own data - Nigrin b A chain; see also Ferreras J. M. et al., Toxins, 3: 420, 2011; Svinth M. et al., BBRC, 249: 637, 1998)

Example 4Conjugation of Nigrin-b A-Chain to Anti-FAP Antibodies

(98) For immunoconjugates containing RIPs to exhibit maximal cytotoxicity the RIP must be released from the targeting vehicle in fully active form, which requires avoiding steric hindrance (34)). The disulfide bond is the only type of linkage that fit this criterium (35, 36). This bond allows conjugation using reagents for the introduction of free sulfhydryl groups such as N-succynimidyl 3(2-pyridyl-dithiopropionate) (SPDP) and 4-succynimidyloxycarbonyl--methyl- (2-pyridyl-dithio)toluene (SMPT). Immunotoxins consisting of mAbs covalently bound to toxins by hindered disulfide linkers, often labeled as second generation immunotoxins, are stable, long lived and display potent cytotoxicity to target cells (37).

(99) SPDP has already been used in the making of immunotoxins (ITs) containing nigrin b (38, 39). Moreover SMPT protects the disulfide bond from attack by thiolate anions, improving in vivo stability of the linkage (40, 41).

(100) Material

(101) Recombinant nigrin b A chain in PBS, pH7.4, 10% glycerol, 0,5 mM DTT, 4.92 gl.sup.1, stored at 5 C. 5,5-dithio-bis-(2-nitrobenzoic acid) GE PD MiniTrap G-10 desalting columns. 0.2 m 28 mm sterile Minisart filters. Sciclone ALH 3000 workstation. Sarstedt Microtest Plate 96-Well Flat Bottom, ref n 82.1581.
Methods

(102) Dithiothreitol (DTT, Cleland's reagent) is a redox agent that will be used to free the thiol groups present in the protein sample. Once said groups have been freed and so are available for reacting 5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman reagent) will be added. Ellman reagent disulphide bridge will be cleaved and the 2 resulting thio-nitrobenzoate molecules (TNB) will attach to the protein at the thiol group sites. To titrate the TNBs absorbance values will be taken at =412 nm, a wavelength at which DTT is not absorbed, rendering the concentration of thiol groups. The proportion of these with the concentration of the protein taken from its absorbance at =280 will yield the number of free thiol groups per protein molecule.

(103) Direct thiol titration was performed as follows: 204 l recNg b A were dissolved in 796 l 20 mM phosphate 250 mM NaCl 1 mM EDTA pH 7.0 (assay buffer) (1.0033 gl.sup.1=final concentration). Ellman reagent was dissolved in phosphate 0.2 M at 3 gl.sup.1. For both buffers monobasic and dibasic sodium phosphate were added in a 1.61 to 1 mass proportion. PH was adjusted at room temperature and buffers were filtered. 100 ml Ellman buffer and 500 ml assay buffer were prepared. Ellman reagent was completely resuspended rather than weighed.

(104) The recNgA sample was incubated in the presence of 4.8 mM DTT at room temperature for 30 min. The recNgbA sample was then purified in the column and the first 10 ml of the eluent aliquoted (V=0.5 ml). The A.sub.280 of the aliquots was taken and the two most concentrated mixed. A.sub.280 was taken again. 10 l of 3 gl.sup.1 DTNB were added and A.sub.412 measured after 2 min (n=1), using Ellman diluted in assay buffer in the same concentration as a blank (n.sub.b=3). Readings belonged to the 0.1-3 AU linear range. Protein solutions were pipetted right beneath the meniscus after vortexing. 100 l were pipetted per well. The results of this study show that the thiol group belonging to recNgA's single cysteine residue is free and available for reaction, not being blocked by its tertiary structure. This will allow recNgbA to be conjugated using a linker that requires a hindered inter-chain disulfide bond.

(105) It is well established that immunoconjugates which contain ribosome-inactivating proteins exhibit maximal cytotoxicity only when the toxin molecule is released from the targeting vehicle in a fully active form. The separation of the RIP molecule from the carrier is required to avoid steric hindrance and to allow an effective translocation of the toxin into the cytoplasm (34)). At present, the disulfide bond is the only type of linkage which appears to fit these criteria (36).

(106) The coupling of two different protein macromolecules, that results in heterodimer formation, requires that each protein is modified prior to mixing them to react. In the case of the A chains of type 2 RIPs, the modification is limited to the reductive cleavage of the native cysteine residue that links the active (A) and the binding (B) chains of the molecule.

(107) For IgG molecules, this is not possible because cysteine residues are involved in maintaining the tertiary and/or quaternary structure of the protein, so that it is not possible to reduce them without loss of the specific protein functions. Moreover, presumably some of the cysteine residues are not sterically accessible, as it was demonstrated by the 10 thiols groups per immunoglobulin that had to be generated for an optimal conjugation to an activated RIP (42).

(108) For these reasons, in most IgG molecules, thiol groups are chemically inserted using hetero-bifunctional reagents, and several methods have been developed in order to generate hetero-conjugates avoiding or reducing to a minimum the formation of homopolymers. In most cases, the reagents used to introduce thiol groups react with amino groups, forming amide or amidine bonds. Amino groups are reactive, abundant and, in a limited way for most proteins, expendable. That is, a limited number of amino groups can be modified without diminishing the biological activity of the protein (36).

(109) The most commonly used reagents for the introduction of free sulphydryl groups are N-succynimidyl 3-(2-pyridyl-dithiopropionate) (SPDP) and 4-succynimidyloxycarbonyl--methyl--(2-pyridyl-dithio)toluene (SMPT), that introduce 2-pyridyl disulphide groups into the protein by reacting with amino groups to form neutral amides, and methyl 4-mercaptobutyrimidate (2-iminothiolane.Traut's reagent) that introduces mercaptobutyrimidoyl groups, reacting to form charged amidines, thus preserving the positive charge of the derivatized amino acid (36, 41).

(110) SPDP and SMPT introduce hindered disulphide bond, while 2-iminothiolane SH must be protected by reacting it with 5,5-dithiobis-2-nitrobenzoic acid (Ellman's reagent). The reaction with Ellman's reagent is also used for the quick measurement of protein sulphydryl groups (43, 44).

(111) SMPT has a methyl group and a benzene ring attached to the carbon atom adjacent to disulphide bond that protects it from attack by thiolate anions, thus improving the in vivo stability of the linkage (40, 41).

(112) Based on these data, IgG proteins can be modified with SMPT, which do not significantly affect the antigen binding property of the molecules in the following conditions, even if they change the charge of the protein in the reaction site.

(113) In the present study the inventors investigated conjugating humanized anti-FAP-IgGls with recNgA, using 2 different recNgA:mAb molar ratios of 2.5 and 3.5, after derivatization using an SMPT:mAb molar ratio of 6, following conjugation protocols (36). Purification was performed by Size Exclusion chromatography on Sephacryl 5200 (37).

(114) Under the described conditions, the immunotoxin is predominantly a mixture of antibody linked to one or two toxin molecules, with the presence of high molecular weight components (IgG linked to several RIP proteins), as well as free and polymeric RIPs (dimeric in the case of recNgA) and free antibody. Thus, a careful purification is thought to be desirable to obtain a pure product.

(115) Biochemical Characterization

(116) Anti-FAP hu36-IgG1-recNgA immunotoxin conjugates were produced and characterized as follows: Conjugate HPS131-001-1 Concentration 0.277 mg/ml Drug:antibody ratio (DAR): 1.8 PM: 182 kDa Purity: 87% (13% of free mAb)
In Vitro Activity Testing

(117) Activity testing on conjugates prepared as described above was performed though evaluation of RIP activity in rabbit reticulocyte cell-free lysate (RRL) assay (FIG. 9), and cytotoxic effect on cell cultures (FIGS. 10A and 10B).

(118) The RRL assay results show that the anti-FAP hu36-IgG1-recNgA conjugates (HPS131-001-1) presented similar IC50 values as native Nigrin-b or recNgA and were in the 3 M range, showing that antibody conjugation did not diminish the enzymatic activity of recNgA (see FIG. 9).

(119) The cell cytotoxicity results show that, on HT1080 wild-type cells, conjugated antibody HPS131-001-1 displays only slight toxicity (if any) and only at highest concentration, naked anti-FAP hu36-IgG1 does not have any effect, and recNgA shows cytotoxic effect only at 10.sup.6M and after 72 h incubation (see FIG. 10A).

(120) However, on FAP-expressing cells, HT1080-FAP, only HPS131-001-1 conjugated anti-FAP antibodies strongly reduce HT-1080-FAP cell viability in the picomolar concentration range, with IC.sub.50 values of 5 M (see FIG. 10B).

(121) These results show that: 1) anti-FAP:recNgA immunotoxins are highly active in vitro, being cytotoxic at picomolar range; 2) Activity is highly specific to FAP-expression, since no significant effect was observed in HT1080-WT; 3) Anti-FAP hu36-IgG1 specificity for its target is not affected by the conjugation to recNgA, neither is the enzymatic RIP activity of recNgA; 4) Activity is specific of the conjugated anti-FAP hu36-IgG1, since no effect was observed with the naked IgG1; 5) Anti-FAP:recNgA immunotoxins are internalized, since non conjugated recNgA (lacking membrane binding domain) shows almost no cytotoxic effect (IC.sub.50>1 M)(see FIG. 8).

(122) In summary, anti-FAP:recNgA immunotoxins have the ability in vitro to specifically recognize the target (FAP), to be internalized within the cytosol and release the recNgA effector moiety to actively inhibit ribosomes, resulting in cytotoxicity IC.sub.50 values within the picomolar range.

(123) In Vivo Evaluation of Anti-Tumoral Effect

(124) Immunotoxin anti-FAP:recNgA has been tested in vivo in both cell-derived and patient-derived xenograft mouse models for pancreas cancer. A dose range study was first performed to define the maximum tolerated dose in normal mice and each of these models: doses from 5 to 0.1 mg/kg were administrated intraperitoneally once a week during 3 weeks, and animal weight was monitored every 2 days to detect possible weight loss due to toxic effect of the immunotoxin. Results are presented in FIG. 13.

(125) High doses (>0.5 mg/kg) induced hepatotoxicity in normal mice, while no FAP-dependent toxicity was observed after pathological analysis of uterus and skeletal muscle, where low FAP expression has been described (Dolznig H., et al., Cancer Immun., 5:10, 2005; Roberts E. W., et al., J. Exp. Med., 210:1137, 2013), nor in heart and kidney. Doses lower than 0.5 mg/kg did not induce any detectable non-specific toxicity in cell line-derived orthotopic (FIG. 13) and patient-derived subcutaneous (FIG. 14A) xenograft murine models of pancreas cancer.

(126) In efficacy studies performed then at nontoxic doses from 0.5 to 0.1 mg/kg, anti-FAP:recNgA immunotoxin, applied as single agent or in combination with Gemcitabine (240 mg/kg), has shown no in vivo antitumoral efficacy in FAP () cell line derived orthotopic xenograft murine models (not shown), while high in vivo antitumoral efficacy was evidenced at a dose of 0.5 mg/kg in FAP (+) patient-derived subcutaneous xenograft murine models of pancreas cancer (FIG. 14B). When combined with Gemcitabine (150 mg/kg), it even showed 100% tumor growth inhibition and tumor regression.

Example 5Cytolysins and their Conjugation to Anti-FAP Antibodies

(127) Tubulysins are recently discovered natural compounds isolated from Myxobacteria, able to destabilize the tubulin skeleton, inducing apoptosis with a very high activity.

(128) Leading to a fast, irreversible and strong change in the cell morphology, tubulysins and their synthetic tetrapeptidic analogues, the cytolysins, are highly potent cell-killing agents (nM to pM activity). Tubulysin A inhibits tubulin polymerization in vitro with an IC.sub.50 of 0.75-1 M, thus blocking the formation of mitotic spindles and inducing cell cycle arrest in G2/M phase. Tubulysins compete strongly with vinblastine through binding on the vinblastine binding site of tubulin. Furthermore they are stable in lysosome enriched cell fractions (45-48).

(129) Amenable to conjugation, many different tubulysin/cytolysin derivatives are accessible by total synthesis in sufficient quantities for preclinical and clinical development; functional groups in their structure can be adapted to several different linker technologies.

(130) The cytolysins employed for conjugation studies were chosen from the general structure shown above (formula IV). These structures exhibit activity against different cancer cell lines (nM to pM range).

(131) Various linker systems can be used and attached to either R.sup.2 or R.sup.17 position of the molecule.

(132) The general outline of the cytolysin conjugates, including the vcPABA linker and anti-FAP antibody, is shown in FIG. 11 (in the structure depicted in FIG. 11, the attachment site of the cytolysin to the vcPABA linker is at position R.sub.1 or R.sub.4the R.sub.1 and R.sub.4 numbering system used in FIG. 11 differs from the R group numbering system used, e.g., in the claims; it is intended that R.sub.1 of FIG. 11 corresponds to R.sup.2 in the claims and that R.sub.4 of FIG. 11 corresponds to R.sup.17 of the claims).

(133) The vcPABA (valine-citrulline-PRBC) protease-cleavable linker has been previously used in the ADC molecule Brentuximab Vedotine, developed by Seattle Genetics and Takeda, and recently approved by the FDA and EMEA as Adcetris (2011, and November 2012, respectively). In this ADC the vcPABA has been coupled at its free NH2 to maleimide caproyl for thiol-based conjugation on mAb (cAC10 anti-CD30 antibody). On the other side, vcPABA has been conjugated through its COOH to the Auristatin cytotoxic drug from Seattle Genetics (MMAE). (see 49)

(134) The present inventors have used this linker (maleimide caproyl-vcPABA) to conjugate anti-FAP antibodies through thiol-based reaction with the maleimide caproyl, and on the other end, to the cytolysin cytotoxic molecules through its cyclic piperidine with vcPABA (R1 or R4 positions of the cytolysin shown in FIG. 11).

(135) Synthesis of Maleimido-val-cit-PABOCO-Tubulysin/Cytolysin-TAM461:

(136) ##STR00023##
TAM461 (Tubulysin/Cytolysin): 30.0 mg (0.041 mmol)
DMF: 3 mL
TAM465 (Linker): 35 mg (0.045 mmol)
HOBt: 1.4 mg
DIPEA: 10 L

(137) TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z=1371 [M+H].

(138) Synthesis of Maleimido-val-cit-PABOCO-Tubulysin/Cytolysin-TAM470:

(139) ##STR00024##
TAM470 (Tubulysin/Cytolysin): 0.07 mmol
DMF: 5 mL
TAM466 (Linker): 50 mg (0.065 mmol)
HOBt: 2.4 mg
DIPEA: 18 L

(140) TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h and then analysed with TLC, indicating completion of reaction. The reaction mixture was concentrated and the resulting oil was purified with column chromatography using 4-12% methanol: DCM to give 56 mg of TAM471 (yield: 62%). ESI-MS: 1384.6 [M+1].

(141) In vitro activity testing is performed. Functional activity will be evaluated through microtubule inhibition assay, while cytotoxic activity is determined through crystal violet viability assay.

(142) Generation of Cytolysin-Linker Derivatives

(143) Different cytolysin-linker derivatives were synthesized according to the general structure presented in FIG. 11, where vcPABA linker was added either in position R1 (TAM467, TAM551) or R4 (TAM471, TAM553, TAM558), alone or with ethylene-glycol spacer (EG; n=1 to 3), or substituted by ethylene glycol groups (n=3) (TAM552). The respective chemical structures are presented in Table 4.

(144) TABLE-US-00009 TABLE 4 Chemical structures of cytolysin -linker derivatives Mol. Product Code Wt. TAM467 1370.7 TAM551 1356.7 TAM471 1384.7 TAM552 1198.5 TAM553 1499.8 0 TAM558 1603.9 Microtubule inhibition inhibition activity and cytotoxic activity of each new derivative was evaluated through tubulin polymerization inhibition assay (TPI; Tubulin Polymerization assay kit; Cytoskeleton, Cat. #BK011P), and cell proliferation arrest on HT1080 cells (CPA; crystal violet). IC50 were calculated and results are presented in Table 5.

(145) TABLE-US-00010 TABLE 5 Microtubule inhibition activity and Cell Cytotoxicity activity of cytolysin-linker derivatives. IC.sub.50 (TPI IC.sub.50 (CPA Compound assay; M) assay; nM) TAM467 (Linker in R1) 150 230-420 TAM551 (Linker in R1) ND 90 TAM471 (Linker in R4; 14 17-42 vcPABA) TAM552 (Linker in R4; no 1.9 10 vcPABA; 3EG) TAM553 (Linker in R4; 6 98 vcPABA; 1EG) TAM558 (Linker in R4; 1.9 98 vcPABA; 3EG) TAM334 (parental cytolysin; 2 0.3-0.6 no linker) Tubulysin A ND 0.04-0.2 Tubulysin A + linker ND 5-20 MMAE (Seattle Genetics) ND 0.1-0.6 DM1-DM4 (Immunogen) ND 0.01-0.1 (ND: Not determined)

(146) In vitro activity of parental cytolysin TAM334 is within the same range of other payloads currently used for the generation of antibody-drug conjugates such as auristatins (MMAE) or maytansinoids (DM1-DM4). As expected and previously described for other compounds from the Tubulysin A family, upon addition of linker, cell cytotoxic activity of cytolysins was decreased with respect to the parental compound TAM334. In addition, TAM467 derivative was presenting significantly lowest activity in both assays. All the derivatives were used in conjugation to generate ADC molecules.

(147) Conjugation and Chemical Characterization of ADCs

(148) Each of the newly generated derivatives was conjugated to the anti-FAP hu36 following a non-site-specific conjugation method on cysteine residues. To this aim, one batch of antibody was reduced and reacted with each of the derivatives. Different TCEP ratios were tested to reach optimal DAR of 3-4, less than 10% of free antibody and drug. Optimal conjugation conditions were as follows: TCEP=2.5 and 3.57 Thiol levels Ellmann's. Conjugates were then purified on G25 Sephadex and analysed through Size Exclusion Chromatography (SEC) to determine their purity, as well as Hydrophobic Interaction Chromatography (HIC) and Polymeric liquid reversed-phase chromatography (PLRP) to determine DAR, content of free antibody and distribution profile of different ADC species (0-8 drugs/mAb). Content of free drug was evaluated by UV detection method at 280 nm. Results of chemical analysis (SEC, HIC and PRLP profiles) were determined for each ADC and for free antibody (data not shown). Biochemical characteristics of the ADCs is shown in Table 6.

(149) TABLE-US-00011 TABLE 6 Summary of chemical characteristics of the different ADC molecules HIC SEC mAb free purity Free Lot Drug Conc. mAb DAR 280 nm Drug Volume HPS157- TAM471 1.195 10.1% 3.38 92% 0% ~5.8 mL 039-001 mg/mL (6.931 mg) HPS157- TAM551 1.332 22.4% 3.08 74% 0% ~5.8 mL 039-002 mg/mL (7.726 mg) HPS157- TAM552 1.319 5.1% 3.84 97% 0% ~5.8 mL 039-003 mg/mL (7.650 mg) HPS157- TAM553 1.305 7.0% 4.10 84% 0% ~5.8 mL 039-004 mg/mL (7.569 mg) HPS157- TAM558 1.332 5.8% 3.92 93% 0% ~5.8 mL 039-005 mg/mL (7.726 mg)

(150) The various drugs produced different levels of aggregation. Specifically ADC HPS157-039-002 (TAM551) showed highest level of aggregation already at DAR=3.08, leaving 22.4% of unconjugated antibody. A preliminary conjugation with TAM467 also showed high level of aggregation: at DAR 3.27, SEC purity was already only 67% with 16% of free drug (data not shown). These data were suggesting that vcPABA linker in position R1 was apparently less than optimal for this type of cytolysin molecule under these conditions.

(151) Target Binding of Conjugates

(152) Anti-FAP hu36:TAM471 ADC binding to huFAP fusion protein was analysed by ELISA, and binding to HT1080-FAP cells by FACS (FIG. 15). For FACS analysis, compounds were incubated either at serial dilutions (FIG. 15B) or at one dilution (FIG. 15C; 10 nM) and detected with an anti-human IgG-PE (V chain specific).

(153) EC.sub.50 values obtained in both assays showed no significant difference with respect to naked anti-hu/moFAP hu36 antibody (FIGS. 15A & 15B). No binding was observed in FAP() cells such as HT1080-wt and HEK293 cells (FIG. 15C).

(154) FIG. 16 shows that ADC-471 (FIG. 16B) specifically binds and gets fully internalized after 90 min in HT1080-FAP cells, similarly to naked anti-FAP antibody (FIG. 16A). These results evidenced that conjugation did not affect target specificity and affinity, or internalization ability of the anti-FAP hu36 IgG1.

Example 6Evaluation of In Vitro Cytotoxic Activity and In Vivo Anti-Tumoral Effect

(155) Anti-FAP:cytolysin ADC candidates were evaluated in vitro through proliferation arrest assay (crystal violet staining). Results are presented in FIG. 17 and IC.sub.50 values in Table 7. Anti-tumoral effect of each ADC candidate was evaluated in a patient-derived xenograft (PDX) mouse model for pancreas cancer (PAXF-736). This model was previously selected for FAP expression level and stroma expansion. ADC compounds were administrated once a week intraperitoneally at 2.5 mg/kg. Tumor volume and body weight were measured twice a week. Vehicle-treated and Gemcitabine-treated (150 mg/kg) PDX mice were used as negative and positive control groups, respectively. Results are shown in FIG. 18.

(156) Location of vcPABA linker alone in R1 position (ADC-551) generated conjugates with much less cytotoxic activity in vitro in comparison with conjugates utilizing the R4 position (ADC-471) (FIG. 17; Table 7) and no anti-tumoral activity in vivo (FIG. 18).

(157) Increasing the number of ethylene-glycol groups as spacer to vcPABA linker in R4 position (ADC-471 (n=0) versus ADC-553 (n=1) and ADC-558 (n=3)) was shown to increase FAP-specific cytotoxic activity in vitro (FIG. 17) and anti-tumoral effect in vivo (FIG. 18). The TAM552 conjugate (ADC-552), having a 3 ethylene glycol spacer, but no vcPABA present in the linker was found to exhibit minimal or no in vivo anti-tumoral activity (data not shown). While ADC-471 and ADC-553 showed low and no FAP-specific cytotoxic activity (10 nM and 100 nM IC.sub.50 range, respectively) with no difference between HT1080-WT and FAP cells nor anti-tumoral effect in vivo, ADC-558 presented a 1 nM range FAP-specific cytotoxic activity with a specificity ratio of 500 between FAP(+) and FAP() HT1080 cells, and a 40% tumor growth inhibition effect at 2.5 mg/kg dose in PDX mouse model for pancreas cancer. No weight loss, nor toxic effect was observed for none of the candidates at this dose (not shown).

(158) TABLE-US-00012 TABLE 7 IC.sub.50 values obtained in Proliferation Arrest Assay (nM) Compound HT1080-WT HT1080-FAP TAM334 1.04 0.77 ADC-471 (HPS-157-039-001) 5.6 10.33 ADC-551 (HPS-157-039-002) 964 552 ADC-553 (HPS-157-039-004) 90 108 ADC-558 (HPS-157-039-005) 555 0.96

(159) Further investigation was carried out using ADC-558. Maximum tolerated dose (MTD) was performed in normal mice and ADC-558 was found to be non-toxic within 2.5 to 25 mg/kg dose range with a weekly treatment for 3 weeks. Doses from 20, 10, and 5 mg/kg were then administrated weekly for 4 weeks to a PDX mouse model (Panc185) with high FAP expression level and stroma expansion to confirm tumor growth inhibition and full regression efficacy of the ADC-558 conjugate.

(160) All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

(161) The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

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