WO2020127968A1

WO2020127968A1 - Protein-drug conjugate comprising a monomeric form of proteinase 3

Info

Publication number: WO2020127968A1
Application number: PCT/EP2019/086675
Authority: WO
Inventors: Stephen F. MARINO
Original assignee: Marino Stephen F
Priority date: 2018-12-20
Filing date: 2019-12-20
Publication date: 2020-06-25

Abstract

The invention relates to a protein-drug conjugate (PDC), comprising a monomeric form of proteinase 3 (PR3) conjugated to a toxin. The invention further relates to a pharmaceutical composition comprising the PDC, a kit for producing the PDC comprising at least a monomeric form of proteinase 3 (PR3) and reagents for conjugating a toxin to said PR3, and to the PDC for use as a medicament in treating autoimmune disease, wherein the autoimmune disease is preferably associated with the presence of anti-PR3 autoantibodies.

Description

PROTEIN-DRUG CONJUGATE COMPRISING A MONOMERIC FORM OF PROTEINASE 3

DESCRIPTION

The invention relates to the field of protein-drug conjugates and medical uses thereof targeting B cells that produce pathogenic immunoglobulins in the treatment of autoimmune disease.

BACKGROUND OF THE INVENTION

A fundamentally important constituent of the human immune system comprises antibody molecules (immunoglobulins) that freely circulate in the bloodstream. These antibodies, primarily immunoglobulin type G (IgG), are produced by B cells that have been previously exposed to target molecules - called antigens - that have been identified as 'foreign' to the body. Antibody molecules have two functional parts - antigen binding sites that are tailored for binding to a specific target, and an effector part, that can be thought of as a 'tag' and can be recognized by other components of the immune system which then destroy the target to which the antibody is bound. After exposure of B cells to these antigens via their binding to B cell receptors (a membrane bound immunoglobulin) on the cells' surface, a series of maturation steps takes place resulting in a B cell population producing soluble antigen binding molecules (IgGs) with optimized binding properties against the antigen that provoked the response.

These B cells can develop into so-called 'memory B cells' which can persist for many years in the body, continually producing the optimized IgG molecules. These molecules themselves bind to their antigen upon further exposure, thereby marking the organisms containing the antigenic species for elimination by the immune system. This comprises a substantial part of an individual's humoral immunity. Thus, B cells produce antibodies that not only target invading pathogens for immediate immune system attack, but also continually produce optimized versions to extend this protection for years after initial exposure.

The process of B cell activation and optimization of antibodies has evolved to eliminate B cells producing antibody molecules that can recognize antigens present in a patient's own body, so- called 'self-antigens'; this normally prevents that the immune system mounts an attack on the body itself. However, there are a number of diseases - autoimmune diseases - that are indeed caused by such 'auto-antibodies'; it is not clear why some B cells activated by self-antigens escape elimination, but the results of this immune system "error" can be quite severe.

In patients having such an autoimmune disease, the immune system mounts an attack on the body's own molecules, resulting in tissue damage that can produce severe disability and death. Because it is often the case that only the specific affected tissue - and not the specific antigen - is known, the standard treatment for most autoimmune diseases consists of general immune system suppression. Such therapy can have positive effects on a patient's condition but obviously negatively effects the normal functions of the immune system, rendering the patient much more susceptible to infections.

An optimal solution to this problem would require a way to eliminate only the damage inducing immunoglobulins while leaving the rest of the humoral immunity intact. It would be ideal to be able to directly eliminate only the B cells producing the pathogenic immunoglobulins, thus removing their source and allowing the majority nonpathogenic B cells to continue providing protection against infectious agents. No such precise targeting method for B cell subsets currently exists.

Considering the difficulties outlined above, there exists a significant need in the field of treating autoimmune conditions to develop improved means for selectively eliminating pathogenic immunoglobulins while leaving the rest of the humoral immune system intact.

SUMMARY OF THE INVENTION

In light of the prior art, the technical problem underlying the present invention is to provide alternative and/or improved means for the treatment of autoimmune medical conditions. One objective of the invention is therefore the provision of means for selectively eliminating pathogenic immunoglobulins while leaving the rest of the humoral immune system intact. The present invention seeks to provide such means while avoiding the disadvantages known in the prior art.

The problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a protein-drug conjugate (PDC), comprising a monomeric form of proteinase 3 (PR3) conjugated to a toxin.

The initial step in the development of a naive B cell into one producing immunoglobulins against a defined antigen is the binding of an antigen to the B cell receptor. The recognition domain of the receptor is essentially a membrane bound immunoglobulin, similar in structure to those that will eventually be secreted by the activated B cell. Upon antigen binding, the B cell receptor/antigen complex is then internalized by the B cell as the initial step in the adaptive immune response. This antigen-initiated internalization is the key process exploited by the current invention. If the autoantigen underlying the specific disease state is known, this antigen can be used to target those B cells having a B cell receptor able to bind it. This includes both immature and mature, memory, B cells.

If the recognized antigen is modified to carry a toxic payload by means of a conditionally stable linker, i.e. a linkage that is stable extracellularly, but is broken intracellularly to release the toxin, then the B cells internalizing the construct are killed due to the effects of the toxin. That is, any cell that can internalize the modified antigen will be killed by the released toxin, thereby eliminating the source of the pathogenic antibodies without the need for general immune sup pression and the associated negative side effects. Since only those B cells having surface re ceptors able to bind the antigen can be recognized, the majority of B cells in the body remain unaffected by the toxic construct.

The current Invention is based on the neutrophil serine protease proteinase 3 (PR3) which is a major autoantigen in a group of incurable autoimmune diseases called ANCA (anti-neutrophil cytoplasmic antibody) vasculitides.

In the course of the invention, a modified PR3 variant has been developed that is recognized by anti-PR3 antibodies but does not adhere to the extracellular membranes of mammalian cells, as does the wt PR3. This variant, if linked to a cell-killing toxin, therefore binds specifi cally to the B cell receptors an B cells producing anti-PR3 antibodies, and upon internalization, exerts its toxic effect on these cells, killing them and thereby eliminating the production of the pathogenic immunoglobulins. For this reason, no successful immune response against the PR3 variant used for targeting can be expected.

In a preferred embodiment, the specific targeting construct is a modified form of human PR3 having no enzymatic activity. wtPR3 is a protease and a single point mutation (S195A) is suffi cient to eradicate its proteolytic activity without influencing the folding and stability of the mole cule. Using an inactive variant will eliminate any possibility of side effects due to construct me diated proteolysis. Furthermore, wtPR3 forms large oligomeric assemblies. wtPR3 readily ad heres to the extracellular membrane surfaces of neutrophils and also to those of nonmyeloid cells after brief incubation.

In preferred embodiments, the modified PR3 variant used as the targeting construct does not oligomerize (it is primarily a monomer) and does not adhere to the surfaces of cells with which it is incubated and, therefore, any non-specific interactions with human tissues should be mini mal.

In preferred embodiments, the very minor differences between wtPR3 and the targeting vari ant do not affect known ANCA epitope stretches and, based on the current data (production yield, size exclusion chromatography, FRET-based proteolysis assay, SDS-PAGE) do not ap preciably affect the native conformation of the active protein, nor do the mutations eliminating the proteolytic activity. ELISA experiments using anti-wtPR3 monoclonal antibodies show that the modified construct is recognized as efficiently as wtPR3 (see examples and figures below). The variant is therefore a dose mimic of the native protein without the undesirable activity, membrane association or aggregation properties.

In the course of analyzing the known PR3 aggregation properties, the inventor has prepared variants of PR3 that exist primarily as monomers in solution and show substantially reduced aggregation behavior.

In some embodiments, this fundamental change in the properties of PR3 is due to minor changes in the primary sequence of the PR3 protein. In some embodiments, a single amino acid substitution is sufficient to reduce oligomerization. In other embodiments, one or more changes in amino acid sequence from the wild type PR3 protein are envisaged in order to provide a monomeric form of PR3.

In one embodiment, the invention relates to a PDC as described herein, wherein the monomeric form of proteinase 3 (PR3) is soluble and preferentially forms monomers compared to oligomers when present in physiological conditions, i.e. when the protein is free in a physiological solution. Physiological solutions are known to a skilled person, such as in PBS, or other similar buffers.

In one embodiment, the monomeric form of PR3 refers to a PR3 protein that does not comprise the wild type human PR3 sequence. In some embodiments, the monomeric PR3 protein comprises a mutant PR3 sequence, otherwise termed a PR3 variant, or variant sequence.

The monomeric form of PR3 enables easier handling, more reliable recombinant production, reduced difficulties in isolating sufficient PR3 quantities, reduced difficulty in manufacturing the PDC, more reliable conjugation to the toxin and more effective and specific B cell toxicity due to the avoidance of aggregates.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises at least a mutation at Ne221.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises at least a mutation at Ne221 and/or Trp222.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises at least mutations at Ne221 and Trp222.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises the Me221 Ala and/or Trp222Ala mutations.

In some embodiments, the wt human PR3 sequence of SEQ ID NO 1 is used as a reference sequence for the amino acid sequence changes mentioned herein, in obtaining PR3 monomeric variants.

A skilled person would not have expected that such minor changes in primary amino acid sequence would lead to the combined beneficial properties of preferential monomer formation compared to wt PR3 protein, thereby leading to improved solubility and ease of handling, combined with maintenance of a suitable structure to present epitopes to anti-PR3 autoantibodies of clinical relevance, and furthermore maintaining binding to the natural binding partner CD 177. This combination of beneficial properties enables the until now unsuggested approach of using monomeric PR3 protein, preferably modified PR3 amino acid sequences, in a PDC according to the present invention.

In some embodiments, the variants are proteolytically active (and therefore properly folded), interact with the only confirmed binding partner of PR3, the neutrophil receptor CD 177 and are recognized as effectively by multiple anti-PR3 monoclonal antibodies as the wildtype PR3 molecule. These properties enable the employment of essentially monomeric PR3 as described herein in a PDC configured to bind CD177 and target autoantibody producing B cells. A skilled person would not have expected that disrupting aggregation via mutation would still enable correct epitope formation for clinically relevant ANCA. The present invention therefore represents a surprising success based on a combination of beneficial properties.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises a mutation that reduces or abolishes protease activity.

In some embodiments, monomeric form of proteinase 3 (PR3) that comprises a mutation that is selected from one or more mutations at His71 , Asp1 18 and/or Ser203, more preferably His71 Glu, Asp1 18Ala and/or Ser203Ala.

In these embodiments, the variant incorporates a further mutation(s) that replaces the catalytic serine, e.g. for alanine (S203A), in order to eliminate the proteolytic activity of the enzyme, thereby also eliminating any possibility of proteolytic activity of the PDC. In some embodiments, this mutation does not affect the proper folding of the protein or its recognition by AN- CAs or CD177 binding.

In some embodiments, the invention therefore relates to PR3 proteins with one or more of the following changes in amino acid sequence and their employment in a PDC, i.e. changes at positions:

Ne221 , Ne221 + Trp222, Ne221 + one or more changes at His71 , Asp1 18 and/or Ser203, or Ne221 + Trp222 + one or more changes at His71 , Asp1 18 and/or Ser203.

In the above embodiments any amino acid change at the given position is envisaged that leads to the properties described herein, i.e. preferably an essentially monomeric form of PR3, with improved solubility in physiological conditions, compared to the wild type human PR3, without significant detrimental effects on PR3-ANCA autoantibody binding or CD177 binding. PR3 sequences of the invention may comprise one or more additional amino acid sequence changes if the above properties are evident.

In some embodiments, the invention therefore relates to PR3 proteins with one or more of the following changes in amino acid sequence and their employment in PDCs, i.e. the particular changes:

Me221 Ala, Me221 Ala + Trp222Ala, Me221 Ala + one or more of His71 Glu, Asp1 18Ala and/or Ser203Ala, or Me221 Ala + Trp222Ala + one or more of His71 Glu, Asp1 18Ala and/or

Ser203Ala.

In some embodiments of the invention, lysine residues of PR3 sequence (K1 15, K195 and/or K253) may be maintained as in the WT PR3 sequence, or modified, in order to modify binding properties to the toxin. Lysine residue modification will depend on the linkage chemistry used to immobilize the monomeric PR3 to the toxin. Details on linking modes and chemistries are described in more detail below. In some embodiments, when amine chemistry (essentially any chemistry exploiting amine linkages) is used, then lysines function as linkage points to the toxin. In some embodiments, one or more lysine residues may be modified to prevent immobilization over the modified lysines, thereby directing linkage properties of the molecule.

In some embodiments, there is no difference in the behavior of the three lysines concerning conjugation to the toxin (i.e. no specific lysine is preferred and therefore the PR3 variants linked in this way are conjugated in all possible orientations allowed by the three lysine locations on the molecule surface).

However, considering that two of the lysines are located close together on the same face of the molecule, in some embodiments conjugation is not uniform, when considering the whole collection of molecules attached to the toxin.

In other embodiments, one or more lysine residues are mutated (K1 15, K195 and/or K253 changed over the wt PR3 sequence), preferably to Arginine, thereby maintaining the positive charge and thereby not substantially altering the properties of any possible epitopes that include this residue.

In one embodiment, the PDC as described herein comprises a monomeric form of proteinase 3 (PR3) that comprises one or more changes at K1 15, K195 and/or K253, preferably K1 15R, K195R and/or K253R.

In some embodiments, PR3 sequences of the invention may comprise one or more additional amino acid sequence changes if the above properties are evident, i.e. preferably an essentially monomeric form of PR3, with improved solubility in physiological conditions, compared to the wild type human PR3, without significant detrimental effects on PR3-ANCA autoantibody binding or CD177 binding.

In embodiments of the invention, the PR3 protein comprises or consists of a variation in the amino acid sequence according to SEQ ID NO. 1 (see below) leading to monomeric properties.

In preferred embodiments, the monomeric PR3 protein is SEQ ID NO 1 , in which the particular amino acid changes mentioned herein (e.g. Me221 Ala, Me221 Ala + Trp222Ala, Me221 Ala + one or more of His71 Glu, Asp1 18Ala and/or Ser203Ala, I Ie221 Ala + Trp222Ala + one or more of His71 Glu, Asp1 18Ala and/or Ser203Ala) have been carried out.

In some embodiments, preferred sequences of monomeric PR3 relate to SEQ ID NO 2, 3, 4,

5, 6 or 7.

In some embodiments, the PR3-similar sequences employed are functionally equivalent to these PR3 sequences, in other words, such functional equivalence is defined by the ability to bind ANCA and exhibit monomeric properties.

Variation in length of the amino acid sequences as described herein is also encompassed by the present invention. A skilled person is capable of providing amino acid sequence variants that are longer or shorter than SEQ ID NO 2, 3, 4, 5, 6 or 7, which will still exhibit sufficient similarity to the monomeric PR3 variant described herein in order to provide the outcomes desired. For example, shorter variants of SEQ ID NO 2, 3, 4, 5, 6 or 7 comprising 10, 20, 30, 40, 50 or up to 100 amino acids less than the full-length form may also enable effective CD177 binding, as described herein. Fragments of PR3 are therefore also considered. Additionally, longer variants of SEQ ID NO 2, 3, 4, 5, 6 or 7 comprising 10, 20, 30, 40, 50 or up to 100 amino acids any given additional sequence more than the natural PR3 sequence may also enable effective outcomes, as described herein.

In other embodiments of the invention, the PR3 protein employed may comprise or consist of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity to SEQ ID NO 2, 3, 4, 5, 6 or 7. Preferably the sequence variant comprises at least 80%, 90%,

91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO 2, 3, 4, 5, 6 or 7 and preferably exhibits functional analogy to the monomeric PR3 proteins described herein. In preferred embodiments the monomeric PR3 protein employed comprises least 80%, 90%, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO 2, 3, 4, 5, 6 or 7 and comprises at least one amino acid difference over SEQ ID NO 1 , preferably by one or more amino acid substitutions at Ne221 , Ne221 + Trp222, Ne221 + optionally one or more substitutions at His71 , Asp1 18 and/or Ser203, or Ne221 + Trp222 + optionally one or more substitutions at His71 , Asp1 18 and/or Ser203, and optionally one or more changes at K1 15, K195 and/or K253, preferably K1 15R, K195R and/or K253R.

In one embodiment, the protein-drug conjugate (PDC) as described herein is characterized in that the toxin is cytotoxic to B cells expressing B cell receptors that can bind PR3.

In some embodiments, the toxin is selected from the group consisting of enzyme (RNA-Pol II) inhibitors from toxic mushroom species of the genus Amanita (amatoxins including alpha-ama- nitin and derivatives); cytoskeletal disrupting compounds, for example auristatin and derivatives (monomethyl-auristatins E and F, vincristine, vinblastine and their derivatives), maitansine and other maytansinoid compounds (ansamitosin, mertansine/emtansin, ravtansin/soravtansin, etc., and their derivatives); cytotoxic antibiotics including the enediynes (calicheamicin and derivatives), the anthracyclines (including daunoribucin, doxorubicin, epiru- bicin, idarubicin, sabarubicin, valrubicin, pixantrone, etc., and their derivatives) the bleomicins and their derivatives, whose mechanisms of action include inhibition of nucleic acid synthesis and/or topoisomerase enzymes, induction of nucleic acid strand breaks, generation of oxygen free radical species and interference with histone-DNA interactions.

In one embodiment, the protein-drug conjugate (PDC) as described herein is characterized in that the conjugation between toxin and PR3 is a linkage that is stable extracellularly post-ad- ministration but is broken intracellularly to release the toxin.

In one embodiment, the conjugation between toxin and PR3 is a covalent linkage, a fusion between a protein toxin and PR3, where the link with PR3 is mediated by reaction of the linker- derivatized toxin with the sulfur atom of cysteine via a thiol-reactive group (for example, bromo. acetamide, iodo acetamide, 4,6-dichloro-1 ,3,5-triazin-2-ylamino, 4-[5-(methylsulfonyl)-1 H-tetrazole-1-yl]phenyl, 2-(methylsulfonyl)-benzo[d]thiazole-5-yl, 4-[5-(methylsulfonyl)-1 ,3,4- oxadiazol-2-yl]-phenyl, 2-pyridyldithio-, 2-(5-nitro-pyridiyl)dithio-, maleimide methylthiosulfonyl or their derivatives), or via the primary amine group of lysine or the PR3 amino-terminus via an amine-reactive group (for example an N-hydroxysuccinimidyl- (NHS) ester, sulfo-NHS ester or their derivatives; imidoesters, aryl-halides, isocyanates, isothiocyanates, aldehydes, car- bodiimides, ayides, anhydrides, fluorobenzenes, carbonates, fluorophenyl esters, epoxides and derivatives thereof); or via the introduction of specifically modified amino acids (referred to as non-natural amino acids) during the recombinant production of the PDC, wherein the modification comprises a chemical group that functions as the reactive site for reaction with the linker-derivatized toxin; or via a reactive C-terminus generated in the PDC by modifying the PR3 polypeptide to include a recognition site for a modifying enzyme, for example Sortase A, which catalyzes the exchange of the Sortase A recognition peptide (the Sortag) for any chemical group containing a small amine nucleophile to resolve the Sortase-PR3 intermediate, including toxins or linkers containing such a small amine nucleophile; the linker-derivatized toxins should preferably also contain a selectively cleavable linkage to enable release of the toxin after internalization by the target cells, for example hemisuccinate linkers sensitive to intracellular esterases; disulfide linkers which are cleavable by intracellular reducing agents like glutathione or enzymes like protein disulfide isomerase (PDI); acid-sensitive hydrazones hydrolyzed in the acidic lysosomal compartment; lysosomal protease sensitive linkers, cleavable for example by Cathepsin B; linkers having a -glucuronide moiety cleavable by intracellular - glucuronidase.

The invention further relates to a pharmaceutical composition comprising the PDC as described herein with a pharmaceutically acceptable carrier.

The invention further relates to a kit for producing a PDC or pharmaceutical composition as described herein, comprising at least a monomeric form of proteinase 3 (PR3) and reagents for conjugating a toxin to said PR3, and optionally one or more toxins suitable for conjugating to said PR3 using said reagents for conjugating.

In a further aspect, the invention relates to the PDC as described herein for use as a medicament in the treatment of an autoimmune medical condition.

In one embodiment, the autoimmune medical condition is associated with anti-neutrophil cytoplasmic antibodies (ANCA). The invention therefore further relates to methods of treating medical conditions associated with anti-neutrophil cytoplasmic antibodies (ANCA) using the PDC described herein. According to one aspect, the present invention provides a method of treating or ameliorating at least one symptom of such a disorder.

In a further embodiment, the medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCAs) to be treated is an autoimmune disease associated with the presence of anti-PR3 autoantibodies, preferably selected from the group consisting of anti-neutrophil cytoplasmic antibody (ANCA) vasculitides, such as granulomatosis with polyangiitis (GPA, formerly known as Wegener^'s granulomatosis) or microscopic polyangiitis, pauci-immune crescentic glomerulonephritis or eosinophilic granulomatosis with polyangiitis. In a further embodiment, the medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCAs) to be treated is anti-neutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the insight that monomeric forms of PR3 may be used in a PDC to target B cells producing pathogenic autoantibodies in a patient in need thereof.

All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety. All terms are to be given their ordinary technical meaning, unless otherwise described herein.

Proteinase 3:

Proteinase 3 (PR3), also known as Myeloblastin, PRTN3, MBN; MBT; NP4; P29; ACPA;

AGP7; NP-4; PR-3; CANCA; C-ANCA, or Wegener autoantigen, is a serine protease that degrades elastin, fibronectin, laminin, vitronectin, and collagen types I, III, and IV, and processes lnterleukin-8 (FEBS Lett 352: 231-235), IL-1 beta (JASN 23: 470-482), kinase inhibitor p21waf1 (JBC 277:47338-47347), annexin-1 (JBC 282:29998-30004), protease-activated receptor-1 (Arterioscler Thromb Vase Biol 33: 275-284) and the C5a receptor (J. Immunol. 192:1787- 1795). PR3 is the major autoantigen in anti-neutrophil cytoplasmic autoantibody (ANCA)- associated vasculitis (Wegener's granulomatosis).

The PR3 protein is described as having a length of 256 amino acids and an estimated mass of 27807 Da. The reported wild type human PR3 sequence is recorded in public sequence databases, for example under UniProtKB - P24158 (PRTN3_HUMAN):

Wt human PR3 (SEQ ID NO 1):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS

SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPH

NICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVIWGCATRLFPDFFTRVALYVDWIRSTLRRV

EAKGRP

In some embodiments, the wt human PR3 sequence of SEQ ID NO 1 is used as a reference sequence for the amino acid sequence changes mentioned herein, in obtaining PR3 monomeric variants. Further preferred sequences relate to:

Ne221X (SEQ ID NO 2):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS

SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPH

NICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVXWGCATRLFPDFFTRVALYVDWIRSTLRR

VEAKGRP wherein X is any amino acid other than lie, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr, more preferably is Ala.

Ne221X + T rp222Z (SEQ ID NO 3):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS

SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPH

NICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVXZGCATRLFPDFFTRVALYVDWIRSTLRRV

EAKGRP

wherein X is any amino acid other than lie, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr, more preferably is Ala, and

wherein Z is any amino acid other than Trp, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn,

Ser, Thr, more preferably is Ala.

Ne221X + one or more changes at His71 , Asp118 and/or Ser203 (SEQ ID NO 4):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAU1CLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNU2VLLIQ

LSSPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCR

PHNICTFVPRRKAGICFGDU3GGPLICDGIIQGIDSFVXWGCATRLFPDFFTRVALYVDWIRST

LRRVEAKGRP

wherein U1 is any amino acid other than His, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr, more preferably is Glu, and/or

wherein U2 is any amino acid other than Asp, preferably is Ala, Pro, Gly, Glu, Gin, Asn, Ser, Thr, more preferably is Ala, and/or

wherein U3 is any amino acid other than Ser, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn, Thr, more preferably is Ala.

Ne221X + Trp222Z + one or more changes at His71 , Asp118 and/or Ser203 (SEQ ID NO 5):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAU1CLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNU2VLLIQ

LSSPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCR

PHNICTFVPRRKAGICFGDU3GGPLICDGIIQGIDSFVXZGCATRLFPDFFTRVALYVDWIRSTL

RRVEAKGRP

wherein X is any amino acid other than lie, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr, more preferably is Ala, and wherein Z is any amino acid other than Trp, preferably is Ala, Pro, Gly, Glu, Asp, Gin, Asn,

Ser, Thr, more preferably is Ala, and

Ne221Ala + Ser203Ala (SEQ ID NO 6):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS

SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPH

NICTFVPRRKAGICFGDAGGPLICDGIIQGIDSFVAWGCATRLFPDFFTRVALYVDWIRSTLRR

VEAKGRP

Ne221 Ala + Trp222Ala + Ser203Ala (SEQ ID NO 7):

MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIH

PSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS

SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPH

NICTFVPRRKAGICFGDAGGPLICDGIIQGIDSFVAAGCATRLFPDFFTRVALYVDWIRSTLRR

VEAKGRP

The PR3 protein is known to be processed, producing a signal peptide (aa 1 - 25), a propep tide (aa 26 - 27), a chain peptide (aa 28 - 248) and a propeptide (aa 249 - 256).

The various functionally equivalent fragments are therefore encompassed in the invention. Re moving signal peptides and/or propeptides may result in functional monomeric PR3 variants.

The PR3 protein appears to be highly conserved in Rhesus monkey, cow, mouse, rat, mos quito, and frog, the PR3 sequences of which are hereby incorporated by reference.

Alternative recombinant PR3 variants have also been described previously, such as in the de scription of the crystal structure of PR3, as described in Fujinaga et al (J Mol Biol. 1996 Aug 16;261 (2):267-78). Numbering of amino acids may also differ in the prior art, for example as in Fujinaga et al (J Mol Biol. 1996 Aug 16;261 (2):267-78) and in Jerke et al (2017, Scientific Re ports 7:43328). A skilled person is capable of determining sequence numbering and correct reference sequences as is required based on appropriate databases, such as NCBI and Uni- ProtKB. In some embodiments, a PR3 protein with substantially the same or a similar amino acid sequence to SEQ ID NO 2, 3, 4, 5, 6, or 7, or fragments thereof, and comprising amino acid sequence changes leading to monomeric properties, may be employed. Examples are fragments of PR3, such as naturally occurring PR3 fragments, homologues or derivatives with essentially the same properties or functional analogy to the examples of monomeric PR3 as described herein.

As used herein, the term“substantially the same or similar amino acid sequence” includes an amino acid sequence that is similar, but not identical to, the reference amino acid sequence. For example, an amino acid sequence, i.e., polypeptide, that has substantially the same amino acid sequence as PR3 in SEQ ID NO 2, 3, 4, 5, 6, or 7, and comprises one or more modifications, such as amino acid additions, deletions, or substitutions relative to the amino acid sequence of SEQ ID NO 2, 3, 4, 5, 6, or 7, may be employed, provided that the modified polypeptide retains substantially at least one biological activity of PR3 such as those described above, i.e. a preferentially monomeric form of PR3, with improved solubility in physiological conditions compared to the wild type human PR3, without significant detrimental effects in PR3-ANCA autoantibody binding compared to wtPR3.

A particularly useful modification of a polypeptide of the present invention, or a fragment thereof, is a modification that confers, for example, increased stability or reactivity. Incorporation of one or more D-amino acids is a modification useful in increasing stability of a polypeptide or polypeptide fragment. Similarly, deletion or substitution of lysine residues can increase stability by protecting the polypeptide or polypeptide fragment against degradation.

The amino acid sequences may also comprise 0 to 100, 2 to 50, 5 to 20, or for example 8 to 15, or any value from 0 to 20, amino acid additions or deletions at either the N- and/or C-termi- nus of the proteins. The termini may also be modified with additional linker sequences, or removal of sequences, as long as the autoantibody binding properties and immunoreactivity of the protein is essentially maintained and the ANCA autoantibodies as described herein bind in an analogous manner to the PR3 sequence provided, in addition to preferably an essentially or preferentially monomeric form of PR3, with improved solubility in physiological conditions, compared to the wild type human PR3.

Various ways of preparing functionally analogous peptides have been disclosed in the prior art. Peptides designed starting from the peptides of the invention using such methods are included in the teaching according to the invention. For example, one way of generating functionally analogous peptides has been described in PNAS USA 1998, Oct. 13, 95(21 ), 12179- 84; WO 02/38592; the above teachings are hereby incorporated in the disclosure of the invention. That is, all peptides, peptide fragments or structures comprising peptides generated using the methods mentioned above - starting from the peptides of the invention - are peptides according to the invention, provided they accomplish the object of the invention and, in particular, interact with the pathogenic autoantibodies and show improved monomeric properties over wtPR3. As used herein, the term“monomeric form of proteinase 3 (PR3)” relates to any PR3 protein that forms monomers in physiological solution to a greater extent than wild type PR3.

In some passages of the specification, the term“PR3” is used without explicit reference to “monomeric PR3”. A skilled person is capable of deducing whether wtPR3 or monomeric PR3 variants are intended.

Preferred conditions for assessing monomer properties are in an experimental setting in a buffer of 20 mM HEPES, 150 mM NaCI buffer, at pH 7.5. Alternatively, a buffer comprising 20 mM HEPES, 150 mM NaCI, 0.02% lauryl-maltoside, pH 7.4 may be employed to assess mon omer properties. Alternatively, a plasma, blood or serum condition could be used. Whether a PR3 forms monomers to a greater or lesser extent than wild type PR3 can be assessed using techniques established in the art, for example size exclusion chromatography as described be low in the examples. Other techniques for assessing the monomeric properties of PR3 relate to dynamic light scattering combined with Gel Filtration, native gel electrophoresis, small angle X-ray scattering (SAXS) and/or mass spectrometry based on techniques known to a skilled person.

The monomeric PR3 of the invention is preferentially monomeric compared to oligomeric when in solution. This property differs from wild-type PR3 protein, which preferentially forms aggre gates or oligomers when present in physiological conditions. References to a monomeric form of PR3 therefore refer to the property of the molecule itself when in physiological conditions. In some embodiments, the monomericity or solubility of the monomeric PR3 form when conju gated to the toxin may differ compared to when the monomeric form of PR3 is present free in solution.

In some embodiments, the sequence of PR3 employed is a mutated, or not a naturally occur ring sequence, for example a sequence not according to SEQ ID NO 1.

Diseases to be treated:

The term“autoimmune disease” as used herein refers to any given disease associated with and/or caused by the presence of autoantibodies.

Autoimmune diseases arise from an abnormal immune response of the body against sub stances and tissues normally present in the body (autoimmunity). This may be restricted to certain organs or involve a particular tissue.

The term“medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCA)” as used herein refers to any medical condition in which ANCA have been detected, preferably those with an established pathological contribution to disease.

ANCAs are associated with small vessel vasculitides, including granulomatosis with polyan giitis, microscopic polyangiitis, primary pauci-immune necrotizing crescentic glomerulonephri tis (a type of renal-limited microscopic polyangiitis), eosinophilic granulomatosis with polyan giitis (previously known as Churg-Strauss syndrome) and drug induced vasculitides. PR3 directed c-ANCA is present in 80-90% of granulomatosis with polyangiitis, 20-40% of microscopic polyangiitis, 20-40% of pauci-immune crescentic glomerulonephritis and 35% of eosinophilic granulomatosis with polyangiitis. c-ANCA (atypical, a kind of PR3 ANCA) is present in 80% of cystic fibrosis (with BPI as the target antigen) and also in inflammatory bowel disease, primary sclerosing cholangitis and rheumatoid arthritis (with antibodies to multiple antigenic targets). Atypical ANCA is associated with drug-induced systemic vasculitis, inflammatory bowel disease and rheumatoid arthritis (Savige, J et al (2000) Antineutrophil cytoplasmic antibodies and associated diseases: a review of the clinical and laboratory features". Kidney International. 57 (3): 846-62).

The term“anti-neutrophil cytoplasmic antibody (ANCA) vasculitides” as used herein refers to a group of diseases exhibiting PR3 ANCA characterized by destruction and inflammation of small vessels. Examples, without limitation, are granulomatosis with polyangiitis (GPA, formerly known as Wegener^'s granulomatosis) or microscopic polyangiitis, pauci-immune crescentic glomerulonephritis or eosinophilic granulomatosis with polyangiitis. One example is antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV).

According to one embodiment of the invention, patients requiring treatment using the PDC described herein comprising monomeric PR3 are identified using standard methods, for example by assessing the blood or a sample derived from the blood of a patient for the presence of ANCA that bind PR3.

Methods suitable for diagnosing a disease associated with ANCA include for example ELISA or indirect immunofluorescent tests. Detection of ANCAs is a well-established diagnostic test used to evaluate suspected necrotizing vasculitis of small blood vessels. The diagnostic utility of ANCA testing depends on the type of assay performed and on the clinical setting and can be adjusted to the conditions of the patient. Most laboratories worldwide use standard indirect immunofluorescence tests (IFT) to screen for ANCA and then confirm positive IFT results with antigen-specific tests for proteinase 3 (PR3) and myeloperoxidase (MPO). Developments such as automated image analysis of immunofluorescence patterns, so-called third-generation PR3-ANCA and MPO-ANCA ELISA, and multiplex technology may also be employed

(Csernok et al, Nat Rev Rheumatol. 2014 Aug;10(8):494-501 ).

According to one embodiment of the invention, the diagnostic and monitoring methods described herein are used to monitor the subject during therapy or to determine effective therapeutic dosages or to determine the number and frequency of treatments needed.

According to another aspect, the method is provided to patients that additionally show either acute renal impairment or failure or are dependent on chronic renal replacement therapy. Patients with ANCA and who show renal impairment are a target group of patients to be handled.

Anti-neutrophil cytoplasmic antibodies:

As used herein, the term“anti-neutrophil cytoplasmic antibodies (ANCAs)” refer to autoantibodies, primarily but not exclusively of the IgG type, that bind to antigens in the cytoplasm of neutrophil granulocytes (the most common type of white blood cell) and monocytes. ANCA can be divided into four patterns when visualized by immunofluorescence; cytoplasmic ANCA (c-ANCA), C-ANCA (atypical), perinuclear ANCA (p-ANCA) and atypical ANCA (a- ANCA), also known as x-ANCA. c-ANCA shows cytoplasmic granular fluorescence with central interlobular accentuation. c-ANCA (atypical) shows cytoplasmic staining that is usually uniform and has no interlobular accentuation. p-ANCA has three subtypes, classical p-ANCA, p- ANCA without nuclear extension and granulocyte specific-antinuclear antibody (GS-ANA). Classical p-ANCA shows perinuclear staining with nuclear extension, p-ANCA without nuclear extension has perinuclear staining without nuclear extension and GS-ANA shows nuclear staining on granulocytes only. a-ANCA often shows combinations of both cytoplasmic and perinuclear staining (Advanced atlas of autoantibody patterns. Birmingham: The Binding Site. ISBN 0704485109).

The so-called c-ANCA antigen is specifically proteinase 3 (PR3). The term“PR3 ANCA” may refer to any autoantibody binding PR3.

p-ANCA antigens include myeloperoxidase (MPO) and bacterial permeability increasing factor (BPI). Classical p-ANCA occurs with antibodies directed to MPO. p-ANCA without nuclear extension occurs with antibodies to BPI, cathepsin G, elastase, lactoferrin and lysozyme. Other less common antigens include HMG1 (p-ANCA pattern), HMG2 (p-ANCA pattern), alpha eno- lase (p and c-ANCA pattern), catalase (p and c-ANCA pattern), beta glucuronidase (p-ANCA pattern), azurocidin (p and c-ANCA pattern), actin (p and a-ANCA) and h-lamp-2 (c-ANCA)

Linkers/Toxins:

Typically, the PDC comprises a linker region between the therapeutic agent and the PR3 protein or derivative thereof. As noted supra, in typical embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment. For example, in some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a ly- sosome or endosome or caveolae). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or en- dosomal protease. In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art (See for example

Wawrzynczak et ai, In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935.)

Typically, the linker is not substantially sensitive to the extracellular environment. In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (i.e., in the milieu of the linker-therapeutic agent moiety of the PDC as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the therapeutic agent and the PR3 or derivative thereof (i.e., in the milieu of the PDC as described herein). A variety of linkers that can be used with the present compositions and methods are described in WO 2004010957 entitled "Drug Conjugates and Their Use for Treat ing Cancer, An Autoimmune Disease or an Infectious Disease" filed July 31 , 2003 (the disclo sure of which, and any US counterparts, is incorporated by reference herein).

In certain embodiments, the immunoconjugate comprises a protein agent targeting a specific protein, i.e. PR3 targeting B-cell receptors that bind PR3, as described herein, including but not limited to, PR3 and a chemotherapeutic agent or other toxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active frag ments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phy- tolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconju- gated PR3.

In some embodiments, the RNA polymerase II inhibiting toxins from Amanita mushroom spe cies (e.g. amatoxins and their derivatives) may be used. These toxins have been shown to be effective in killing cells targeted by antibody-drug conjugates (ADCs) comprising them (see for example Moldenhauer G, Salnikov AV, Liittgau S, Herr I, Anderl J, Faulstich H. Therapeutic potential of amanitin-conjugated anti-epithelial cell adhesion molecule monoclonal antibody against pancreatic carcinoma. J Natl Cancer Inst. 2012; 104(8):622-34). A PR3 based PDC comprising such an amatoxin or derivative, attached in one or more copies to PR3 or, prefer entially, at the C-terminus of PR3, would also be expected to be effective in killing ANCA- producing B-cells harbouring receptors capable of binding PR3. The use of conditionally stable linkage moieties to promote stability of the PDC in serum while facilitation cleavage of the toxin from the internalized PDC is also envisioned. See for example WO2017149077 and US20180185509.

PR3 of the present invention may also be conjugated to one or more toxins, including, but not limited to, a calicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity. Suitable cytotoxic agents include, but are not limited to, an auristatin including dovaline-valine-dolaisoleunine-dolapro- ine-phenylalanine (MMAF) and monomethyl auristatin E (MMAE) as well as ester forms of MMAE, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an ene- diyne, a lexitropsin, a duocarmycin, a taxane, including paclitaxel and docetaxel, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1 , DM-4, netropsin. Other suitable cytotoxic agents include anti-tubulin agents, such as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolas tatin. Antitubulin agent include dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p- phenylened- iamine (AFP), MMAF, MMAE, auristatin E and F, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM- 1 , DM-4 or eleutherobin.

In some embodiments, the immunoconjugate comprises PR3 conjugated to dolastatins or do- lostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001 ) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin (which are pentapeptide derivatives of dolastatins) drug moiety may be attached to PR3 through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172). Exemplary auristatin embodiments include the N-terminus linked

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

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, "The Peptides," volume 1 , pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.

Maytansinoids may be used as an active agent coupled to the PR3 or fragment thereof according to the invention. Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896, 1 1 1 ). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4, 151 ,042). Highly cytotoxic maytansinoid drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. PR3-maytansinoid conjugates are prepared by chemically linking an PR3 to a maytansinoid molecule without significantly diminishing the biological activity of either the PR3 or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per PR3 molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the PR3, although even one molecule of toxin/PR3 would be expected to enhance cytotoxicity over the use of naked PR3. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources.

Selected examples of the calicheamicin family of antibiotics may be used as an active agent coupled to the PR3 or fragment thereof according to the invention. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos.

5,712,374, 5,714,586, 5,739, 1 16, 5,767,285, 5,770,701 , 5,770,710, 5,773,001 , 5,877,296. Another anti-tumor drug that the PR3 can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through PR3 mediated internalization greatly enhances their cytotoxic effects.

Other antitumor agents that can be conjugated to the antibodies include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.

5,877,296). The present invention further contemplates an immunoconjugate formed between an PR3 and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase). For selective destruction of the tumor, the PR3 may comprise a highly radioactive atom.

Compositions/Administration:

In some embodiments, the invention relates to a pharmaceutical composition for administration to a subject in need thereof comprising the PDC, preferably also comprising a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier in the sense of the present invention may be any non-toxic material that does not significantly interfere in a detrimental sense with the effectiveness of the biological activity of the antibodies of the present invention. Evidently, the characteristics of the carrier will depend on the route of administration. Such a composition may contain, in addition to the active substance and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Formulation of phar- maceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intra-muscular administration and formulation.

The medicament, otherwise known as a pharmaceutical composition, containing the active ingredient (PDC) may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated. The present invention also refers to a pharmaceutical composition for topical application, oral ingestion, inhalation, or cu taneous, subcutaneous, or intravenous injection. A skilled person is aware of the carriers and additives required for particular application forms.

When a therapeutically effective amount of the active substance (PDC) of the invention is ad ministered by intravenous, cutaneous or subcutaneous injection, the active substance may be in the form of a pyrogen-free, parenterally acceptable aqueous solution.

The invention also relates to administration of a therapeutically relevant amount of PDC as de scribed herein in the treatment of a subject who has the medical disorders as disclosed herein. As used herein, the term“therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit. The amount of active substance in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Larger doses may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.

The preparation of such parenterally acceptable solutions, having due regard to pH, isotonic ity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the active substance, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

The dose of the PDC administered evidently depends on numerous factors well-known in the art such as, e.g., the chemical nature and pharmaceutical formulation of the PDC, and of body weight, body surface, age and sex of the patient, as well as the time and route of administra tion. For an adult, the dose may exemplarily be between 0.001 pg and 1 g per day, preferably between 0.1 pg and 100 mg per day. In a continuous infusion, the dose may exemplarily be between 0.01 pg and 100 mg, preferably between 1 pg and 10 mg per kilogram body mass per minute.

FIGURES

The invention is further described by the following figures. There are intended to represent a more detailed illustration of a number of preferred non-limiting embodiments or aspects of the invention without limiting the scope of the invention described herein.

Figure 1 : Comparison of wtPR3 and monomeric PR3 properties by size exclusion chromatog raphy. (A) shows a size exclusion chromatogram (S200) with the elution profile of wtPR3. The protein does not elute in a discrete peak but rather as a collection of large aggregates with approximate sizes ranging from monomeric (ca. 26kD) to over 600 kD. (B) shows the analogous chromatogram of a monomeric Trp222Ala PR3 variant showing a more uniform elution primarily at the predicted retention time of a monomeric species.

Figure 2: Comparison of wtPR3 and monomeric PR3 properties by SPR and ELISA. (A) shows a raw SPR sensorgram of the binding interaction of a concentration series of soluble monomeric Trp222Ala PR3 variant to immobilized CD 177. The affinity of the interaction was measured to be 5.7 x 10-8M. (B) shows an ELISA assay using two different anti-PR3 antibodies (anti-PR3 40 and anti-PR3 81 ) purified from hybridoma supernatants. The antibodies each recognize distinct, non-overlapping epitopes on PR3. wtPR3 (left bar) and the monomeric Trp222Ala PR3 variant (right bar) are equally well recognized by the antibodies.

EXAMPLES

The invention is further described by the following examples. These are intended to present support for the workability of a number of preferred non-limiting embodiments or aspects of the invention without limiting the scope of the invention described herein.

Example 1 : Production of monomer PR3 protein variants

Monomeric PR3 protein variants were created essentially as described in Jerke et al (2017, Scientific Reports 7:43328). The proteins were produced from plasmid pTT5 as C-terminal fusions with a human Ig 1 Fc, secreted from 293_6E EBNA cells (NRC, Canada) via an N-termi- nal Ig 1 secretion signal peptide and purified from the culture supernatant by passage over immobilized Protein A. Protein A eluted material was immediately neutralized with 1 M HEPES pH 7.5 and Fc removed by addition of TEV protease and incubation overnight at 4 °C. The flowthrough of a subsequent Protein A trap column was concentrated and passed over a Su- perdex 200 size exclusion in 20 mM HEPES, 150 mM NaCI, pH 7.5 for CD177 and in the same buffer plus 0.02% LM for PR3 variants. PR3 variants were activated by enterokinase removal of an N-terminal FLAG peptide and passage over anti-M2 agarose.

Example 2: Comparison of wtPR3 and monomeric PR3 properties by size exclusion chromatography

PR3 proteins obtained as described above were assessed using size exclusion chromatography for molecular weight. PR3 proteins were diluted in suitable buffer (500 pi @ 1.2 mg/ml, 20 mM HEPES, 150 mM NaCI, 0.02% lauryl-maltoside, pH 7.4), passed through a 0.45 pm filter and subsequently applied to a Superdex S200 gel filtration column at a flow rate of 0.5 ml/min, at 4 degrees C and applied to a gel filtration column. High molecular weight aggregates of wtPR3 eluted at approx. 8.8 mL elution volume (Figure 1A), whereas the Trp222Ala PR3 variant eluted primarily at approx. 16.5 mL elution volume, consistent with a monomeric form of the protein (Figure 1 B). Similar results are obtained for Me221 Ala, Me221 Ala + Trp222Ala, Ne221Ala + Ser203Ala, and Ne221Ala + Trp222Ala + Ser203Ala. Further PR3 mu tants are being assessed.

Example 3: Comparison of wtPR3 and monomeric PR3 properties by SPR.

Experiments were performed on a ProteOn XPR36 instrument (BioRad) with proteins immobi lized to GLH sensor chips (BioRad) using standard amine chemistry. The binding interaction of a concentration series of soluble monomeric Trp222Ala PR3 variant with immobilized CD 177 was assessed (Figure 2A). The affinity of the interaction was measured to be 5.7 x 10-8M, in dicating that the PR3 variant shows no loss in biding to its natural protein target CD 177.

Example 4: Comparison of wtPR3 and monomeric PR3 properties by ELISA.

PR3 ELISA was used to quantitatively assess PR3 amounts. Briefly, anti-PR3 capture antibod ies (anti-PR3 40 and anti-PR3 81 ) were coated, blocked and incubated with PR3 samples of WT or monomeric variants. After washing, biotinylated anti-PR3 detection mab was added. A streptavidin-HRPO conjugate and OPD substrate were used to visualize binding. The absorb ance was determined at 405 nm in a plate reader. Figure 2B shows that wtPR3 (left bar) and the monomeric Trp222Ala PR3 variant (right bar) are equally well recognized by the antibod ies.

Example 5: Generation and validation of a PR3 Protein-Drug Conjugate

A preferred embodiment of the invention comprises a PR3 variant modified to maximize its solubility, minimize its propensity to form large multimers and eliminate its native enzymatic activity without disrupting the three-dimensional structure of the molecule, thereby preserving the majority of its surface epitopes.

The ability of such a PR3 variant to be recognized by antibodies (ANCAs) and also to bind to the receptors of human B-cells that produce ANCAs (B-cell receptors, BCRs) is to be exploited by the attachment to the PR3 variant of a cytotoxic species (a small molecule or toxic peptide) so that the PR3 variant acts as a specificity module to bring the toxin into the target B-cells with the goal of killing them - thereby eliminating the source of the pathogenic antibodies.

The toxin(s) to be used could be linked to the PR3 variant by any of a number of established methods, including by reaction with specific side chain moieties.

In one example, amine-based chemistry is employed to target surface lysine side chains.

In one example, maleimides targeting the thiol groups of cysteine residues are employed.

In one example, genetic fusion with a toxic protein, peptide or oligonucleotide that either itself exhibits toxicity or serves as a generalized coupling module to allow the subsequent linkage of suitably modified toxin molecules is employed.

In one example, a PR3 variant is conjugated with a C-terminal Sortag, allowing site-specific reaction with the Sortase enzyme. The PR3-Sortase covalent intermediate is then resolved by addition of a toxic moiety having a small amine substituent that can substitute the attached Sortase, thus introducing the toxic moiety at the desired location and facilitating the further purification of the PR3-toxin conjugate from the PR3-Sortase intermediate. An internal Sortag engineered between the PR3 variant sequence and affinity tag (for example, as in the construct described in Jerke, et al. 2017, Scientific Reports 7:43328), would be effective in both eliminating the affinity tag and introducing the covalently attached toxin moiety, thus simplifying production of the final PDC.

Given that the native PR3 molecule as present on the surfaces of human neutrophils is attached via a C-terminal GPI domain, the preferred location of toxin attachment is also at the C- terminus, thereby allowing full access to the most common (known) PR3 surface epitopes.

In one example, the attachment of the toxin to the PR3 variant is performed using a conditionally stable linker, for example, one that is stable in the bloodstream but can be easily broken inside of the target cells, thus allowing efficient release of the toxin moiety into the cells that endocytose the conjugate.

The ability of such a PR3-toxin conjugate (PR3-Tx) to target and kill PR3 specific B-cells is tested in culture using hybridoma cells and also in B-cell preparations from PR3-ANCA donors.

Hybridoma cells producing anti-PR3 antibodies possess PR3-binding BCRs on their surfaces, while unrelated B-cell (or hybridoma) cultures) have BCRs with no substantial affinity for PR3. Testing the functionality of the PR3-Tx construct is performed by first linking a detection module to the PR3 variant (for example, a fluorescent dye) and then adding the construct to hybridoma cultures (cultivated under standard conditions, i.e. Dulbeccos Modified Eagle^'s Medium (DMEM) supplemented with 20% fetal bovine serum, glutamine and sodium pyruvate).

Binding of substrates to their BCRs results in internalization of the substrate-receptor complex, thereby bringing the substrate into the cell^'s interior. After internalization, the cells are washed to remove unbound conjugate and investigated via fluorescence microscopy. Cells to which the conjugate specifically bind exhibit fluorescence, since the dye carried by the conjugate is internalized. Washed cells are visualized at different timepoints in order to monitor the binding of the conjugate to the cell surface and the internalization - the observation of fluorescence initially localized in endocytotic vesicles and, finally, dispersed throughout the cell, particularly when the linker attaching the dye is unstable in the cell interior. Cells - even unrelated hybrid- omas - that have no specific PR3 binding partners on their surfaces exhibit neither surface fluorescence nor internalized fluorescent molecules. The identical experiments can be performed on B-cells obtained from PR3-ANCA patients and compared to those obtained from non- ANCA patients. The B-cells can either be specifically enriched from whole blood or leucocyte preparations or assayed without specific enrichment by simultaneously staining them with fluo- rescently labelled antibodies recognizing B-cell specific surface antigens (for example, CD20). Preparations from both types of donor are incubated with B-cell antigen specific fluorescently labelled antibodies to label all B-cells and with the fluorescently labelled PR3 variant to label those B-cells whose BCR can recognize the PR3 variant. The preparations are then analysed via FACS, allowing identification of dual labelled cells and verifying the lack of background interaction of the PR3 variant with non-B-cell targets.

These experiments are also performed as a cytotoxicity test by linking a toxin moiety to the PR3 variant instead of a fluorescent (or other detection) marker. In this case, the readout is cell death, which is assessed in a number of standard ways (incorporation of dye molecules added extraneously, metabolic assays, etc.). Cells displaying no specific PR3 binders are unaffected by the conjugate while cells harbouring BCRs are effectively killed once sufficient amounts of conjugate are internalized (depending on the potency/mechanism of cell killing, the concentration of conjugate applied, etc.). Tests employing different detection and toxin molecules, assaying different concentrations, culture conditions, timepoints, etc., all according to standard experimental procedures, are employed to determine the optimal cargo (toxin) and the optimal conditions under which it should be used.

In vivo, an optimized construct is employed to bind to ANCA producing B-cells and be internalized by them, as in the in vitro proof of principle assays. These assays enable B-cells to take up the bound conjugate, thereby bringing the cytotoxic cargo into the cell^'s interior where it exerts its toxic effects (either in the context of the conjugate or, preferably, freely diffusible after cleavage of its conditionally stable linker inside the cell). Only B-cells able to specifically bind PR3 (i.e. those with PR3-specific BCRs) are affected while all other B-cells - and tissues - remain unaffected. The toxins thereby introduced exert their effects only on cells producing pathogenic antibodies, leaving the remainder intact and having no detrimental effect either on humoral immunity or on healthy tissues.

PR3-Tx conjugates employed this way specifically decrease the population of ANCA- producing pathogenic B-cells, which is tested by extraction of B-cells from the blood of conjugate-treated patients and binding/internalization assays using PR3-dye conjugates.

Testing the B-cell population in this way before and after conjugate application shows a difference in the numbers of B-cells able to bind and internalize PR3 constructs. Furthermore, as more of the ANCA-producing B-cells are eliminated by the toxic conjugate, the ANCA titer in the blood of treated patients decreases. This decrease is monitored by application of standard enzyme-linked immunosorbent assay (ELISA) methods, well known in the art. Blood samples taken at specific timepoints are directly compared by the ability of the antibodies they contain to bind to immobilized PR3 constructs in vitro, showing a decrease in the amount of PR3 binding over time as the pathogenic ANCAs are removed from circulation by normal metabolic processes but are not replaced due to the killing of the B-cells that produced them.

Claims

1. A protein-drug conjugate (PDC), comprising a monomeric form of proteinase 3 (PR3) conjugated to a toxin.

2. The protein-drug conjugate (PDC) according to the preceding claim, wherein the monomeric form of proteinase 3 (PR3) is soluble and does not form oligomers in physiological conditions (for example in 20 mM HEPES, 150 mM NaCI buffer, at pH 7.5) and/or does not adhere to the extracellular membranes of mammalian cells independent of CD177-expression.

3. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the monomeric form of proteinase 3 (PR3) comprises at least one mutation at Ne221 and/or Trp222.

4. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the monomeric form of proteinase 3 (PR3) comprises at least mutations at Ne221 and Trp222.

5. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the monomeric form of proteinase 3 (PR3) comprises the Me221 Ala and/or Trp222Ala mutations.

6. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the monomeric form of proteinase 3 (PR3) comprises a mutation that reduces or abolishes protease activity, preferably selected from one or more mutations at His71 , Asp1 18 and/or Ser203, more preferably His71 Glu, Asp1 18Ala and/or

Ser203Ala.

7. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the toxin is cytotoxic to B cells expressing B cell receptors that can bind PR3.

8. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the toxin is selected from the group consisting of enzyme (RNA-Pol II) inhibitors from toxic mushroom species of the genus Amanita (amatoxins including alpha- amanitin and derivatives); cytoskeletal disrupting compounds, for example auristatin and derivatives (monomethyl-auristatins E and F, vincristine, vinblastine and their derivatives), maitansine and other maytansinoid compounds (ansamitosin,

mertansine/emtansin, ravtansin/soravtansin, etc., and their derivatives); cytotoxic antibiotics including the enediynes (calicheamicin and derivatives), the anthracyclines (including daunoribucin, doxorubicin, epirubicin, idarubicin, sabarubicin, valrubicin, pixantrone, etc., and their derivatives) the bleomicins and their derivatives, whose mechanisms of action include inhibition of nucleic acid synthesis and/or topoisomerase enzymes, induction of nucleic acid strand breaks, generation of oxygen free radical species and interference with histone-DNA interactions.

9. The protein-drug conjugate (PDC) according to any one of the preceding claims, wherein the conjugation between toxin and PR3 is a linkage that is stable extracellu- larly post-administration, but is broken intracellularly to release the toxin.

10. The protein-drug conjugate (PDC) according to any one of the preceding claims,

wherein the conjugation between toxin and PR3 is a covalent linkage, a fusion between a protein toxin and PR3, where the link with PR3 is mediated by reaction of the linker-derivatized toxin with the sulfur atom of cysteine via a thiol-reactive group (for example, bromo. acetamide, iodo acetamide, 4,6-dichloro-1 ,3,5-triazin-2-ylamino, 4-[5- (methylsulfonyl)-l H-tetrazole-1-yl]phenyl, 2-(methylsulfonyl)-benzo[d]thiazole-5-yl, 4- [5-(methylsulfonyl)-1 ,3,4-oxadiazol-2-yl]-phenyl, 2-pyridyldithio-, 2-(5-nitro-pyridiyl)di- thio-, maleimide methylthiosulfonyl or their derivatives), or via the primary amine group of lysine or the PR3 amino-terminus via an amine-reactive group (for example an N-hydroxysuccinimidyl- (NHS) ester, sulfo-NHS ester or their derivatives; im- idoesters, aryl-halides, isocyanates, isothiocyanates, aldehydes, carbodiimides, ayides, anhydrides, fluorobenzenes, carbonates, fluorophenyl esters, epoxides and derivatives thereof); or via the introduction of specifically modified amino acids (referred to as non-natural amino acids) during the recombinant production of the PDC, wherein the modification comprises a chemical group that functions as the reactive site for reaction with the linker-derivatized toxin; or via a reactive C-terminus generated in the PDC by modifying the PR3 polypeptide to include a recognition site for a modifying enzyme, for example Sortase A, which catalyzes the exchange of the Sortase A recognition peptide (the Sortag) for any chemical group containing a small amine nucleophile to resolve the Sortase-PR3 intermediate, including toxins or linkers containing such a small amine nucleophile; the linker-derivatized toxins should preferably also contain a selectively cleavable linkage to enable release of the toxin after internalization by the target cells, for example hemisuccinate linkers sensitive to intracellular esterases; disulfide linkers which are cleavable by intracellular reducing agents like glutathione or enzymes like protein disulfide isomerase (PDI); acid-sensitive hydrazones hydrolyzed in the acidic lysosomal compartment; lysosomal protease sensitive linkers, cleavable for example by Cathepsin B; linkers having a b-glucuronide moiety cleavable by intracellular b-glucuronidase.

1 1. A pharmaceutical composition comprising the PDC according to any one of the preceding claims with a pharmaceutically acceptable carrier.

12. A kit for producing a PDC or pharmaceutical composition according to any one of the preceding claims, comprising at least a monomeric form of proteinase 3 (PR3) and reagents for conjugating a toxin to said PR3, and optionally one or more toxins suitable for conjugating to said PR3 using said reagents for conjugating.

13. The protein-drug conjugate (PDC) according to any one of the preceding claims for use as a medicament in the treatment of autoimmune disease.

14. The protein-drug conjugate (PDC) for use as a medicament according to any one of the preceding claim, wherein the autoimmune disease is associated with the presence of anti-PR3 autoantibodies such as the anti-neutrophil cytoplasmic antibody (ANCA) vasculitides, including granulomatosis with polyangiitis (GPA, formerly known as We gener^'s granulomatosis) and microscopic polyangiitis; also pauci-immune crescentic glomerulonephritis, eosinophilic granulomatosis with polyangiitis.

15. The protein-drug conjugate (PDC) for use as a medicament according to any one of the preceding claims, wherein the autoimmune disease is anti-neutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV).