Patent Application
20180100008
The present invention relates to polypeptides comprising an immunoglobulin chain variable domain (or ‘ICVD’) which binds to Clostridium difficile toxin B (‘TcdB’ or ‘toxin B’) as well as to constructs and pharmaceutical compositions comprising these polypeptides. The present invention also relates to nucleic acids encoding such polypeptides, to methods for preparing such polypeptides, to cDNA and vectors comprising nucleic acids encoding such polypeptides, to host cells expressing or capable of expressing such polypeptides and to uses of such polypeptides, pharmaceutical compositions or constructs.
The present application is a continuation application of PCT/EP2016/057034 filed Mar. 31, 2016 which claims priority from EP 15162117.4 filed Mar. 31, 2015, the contents of each of which are incorporated herein by reference in their entirety.
Clostridium difficile, a spore forming anaerobic bacillus is the causative agent of C. difficile infection. The hospital environment and patients undergoing antibiotic treatment provide a discrete ecosystem where C. difficile persists and selected virulent clones thrive. Consequently, C. difficile is the most frequent cause of nosocomial diarrhoea worldwide. Given the continued use of broad-spectrum antibiotics and the rising numbers of immunocompromised and elderly patients, the problems associated with C. difficile infection are unlikely to recede.
The pathology of C. difficile is mediated by two toxins, toxin A and toxin B and it has been demonstrated that C. difficile ribotypes which lack these toxins do not cause pathogenic infection. Each toxin alone is capable of causing the symptoms of disease. It is believed that the toxins mediate their effect locally by entering the epithelial cells lining the lumen of the colon resulting in cell death, with consequent fluid loss and diarrhoea. There is no consistent pathology associated with toxin entering the systemic circulation and therefore neutralisation of the toxins in the lumen of the colon may provide an effective therapy for this debilitating condition.
Alarmingly, in the last 10 years a new group of highly virulent C. difficile ribotypes has emerged to cause outbreaks of increased disease severity. The most prevalent of the hypervirulent ribotypes is ribotype 027 (strain R20291, Stabler et al 2009 Genome Biology 10:R102) and patients infected with this ribotype exhibit more severe diarrhoea, higher mortality and more recurrences. In Canada, 027 ribotypes were undetected in 2000 but reached 75.2% of all ribotypes isolated by 2003. The 027 ribotype also caused the outbreak of C. difficile at the Stoke Mandeville hospital in the UK. In less than three years a total of 498 patients acquired C. difficile at the hospital of whom 127 died.
The burden of C. difficile disease represented by ribotype 027, other newly emerging hypervirulent ribotypes such as PCR-ribotype 078 (strain ‘M120’, He et al 2010. PNAS. 107 (16) Table 51) and ribotypes such as 087 (strain VPI 10463, Zaib et al 2009 BMC Microbiology 9:6), 017 (strain M68, He et al 2010 PNAS. 107 (16)), 106 (strain Liv22) and 001 (strain Liv24) continue to be a major concern in hospitals throughout the developed world.
Current therapy for C. difficile involves a course of either vancomycin or metronidazole, which in the majority of patients resolves the symptoms and diarrhoea. Unfortunately on cessation of these antibiotics, 20-30% of patients relapse and a vicious cycle of further antibiotic courses and relapses can follow. Recurrence rates are worse with patients infected with the hypervirulent 027 ribotype.
Whilst there are new treatments in development for C. difficile, effectiveness against multiple ribotypes remains a challenge. In 2011 a new antibiotic, Fidaxomicin, the first new treatment for C. difficile in more than 20 years, was approved by the FDA and EMEA. Although Fidaxomicin demonstrated a reduced rate of recurrence in non-027 infected patients, it did not show any superiority versus vancomycin for patients infected with the 027 hypervirulent ribotype.
Merck have completed a Phase 2 trial of a combination of anti toxin A and anti toxin B antibodies administered systemically. These antibodies were originally developed by Medarex. Comparative experiments provided herein demonstrate that constructs of the present invention have a higher potency than Medarex-developed antibody mAb 124 against toxins of multiple ribotypes including 027.
There is, therefore, an overwhelming unmet need for therapies that will reduce relapse rates in patients infected with C. difficile and especially those infected with the hypervirulent 027 ribotype. Safe and effective therapeutics must be developed which are capable of neutralising TcdB from a broad range of C. difficile ribotypes, including TcdB from ribotype 027. This is particularly challenging due to the significant sequence variation of toxins produced by different ribotypes of C. difficile (see Table 1B below).
WO 2006/121422 discloses antibodies that specifically bind to toxins of C. difficile, antigen binding portions thereof, and methods of making and using said antibodies and antigen binding portions. WO 2011/130650 discloses regents, compositions and therapies with which to treat C. difficile infection and related disease conditions and pathologies, including in particular antibodies or antigen-binding fragments thereof that bind specifically to TcdA and/or TcdB of C. difficile and neutralise the activities of these toxins. WO 2012/055030 discloses C. difficile toxin-specific antibodies, compositions and uses thereof, which toxin-specific antibodies may be specific for either TcdA or TcdB.
Polypeptides of the present invention may, in at least some embodiments, have one or more of the following advantages compared to anti-TcdB substances of the prior art:
Advantages (i) to (xiv) above may potentially be realised by the polypeptides of the present invention in a monovalent format or in a multivalent format such as a bihead format (for example homobihead or heterobihead formats) or a quadrahead format.
The present inventors have produced surprisingly advantageous polypeptides comprising immunoglobulin chain variable domains which bind to TcdB. These polypeptides, in preferred embodiments, benefit from surprisingly high potency against TcdB from multiple ribotypes of C. difficile and more particularly remain stable on exposure to proteases such as trypsin, chymotrypsin and/or proteases present in the small and large intestine. In one embodiment, these polypeptides have undergone further enhancement by engineering. It is expected that these further enhanced polypeptides benefit from the above advantages, retain their TcdB-neutralising activity during passage through the intestinal tract and further resist degradation and/or inactivation by proteases present in the intestinal tract. It may be expected that these polypeptides have particular utility in the prevention or treatment of C. difficile infection, particularly when administered orally.
The present invention provides a polypeptide comprising an immunoglobulin chain variable domain which binds to Clostridium difficile toxin B, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein:
(a) CDR1 comprises a sequence sharing 40% or greater sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence sharing 55% or greater sequence identity with SEQ ID NO: 2 and CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3; or
(b) CDR1 comprises a sequence sharing 40% or greater sequence identity with SEQ ID NO: 4, CDR2 comprises a sequence sharing 55% or greater sequence identity with SEQ ID NO: 5 and CDR3 comprises a sequence sharing 60% or greater sequence identity with SEQ ID NO: 6.
In order to facilitate understanding of the invention and with no limiting effect, option (a) relates to the Q10F1arm ICVD sequence and option (b) relates to the Q31 B1 arm and/or Q35H8 ICVD sequences.
SEQ ID NO: 1—Polypeptide sequence of B10F1arm CDR1
SEQ ID NO: 2—Polypeptide sequence of B10F1arm CDR2
SEQ ID NO: 3—Polypeptide sequence of B10F1arm CDR3
SEQ ID NO: 4—Polypeptide sequence of Q31B1arm CDR1
SEQ ID NO: 5—Polypeptide sequence of Q31B1arm CDR2
SEQ ID NO: 6—Polypeptide sequence of Q31B1arm CDR3
SEQ ID NO: 7—Polypeptide sequence of Q35H8 CDR1
SEQ ID NO: 8—Polypeptide sequence of Q35H8 CDR2
SEQ ID NO: 9—Polypeptide sequence of Q35H8 CDR3
SEQ ID NO: 10—Polypeptide sequence of B10F1arm
SEQ ID NO: 11—Polypeptide sequence of Q31B1arm
SEQ ID NO: 12—Polypeptide sequence of Q35H8
SEQ ID NO: 13—Polypeptide sequence of ID1B
SEQ ID NO: 14—Polypeptide sequence of ID2B
SEQ ID NO: 15—Polypeptide sequence of ID3B
SEQ ID NO: 16—Polypeptide sequence of ID11B
SEQ ID NO: 17—Polypeptide sequence of ID12B
SEQ ID NO: 18—Polypeptide sequence of ID20B
SEQ ID NO: 19—Polypeptide sequence of ID21B
SEQ ID NO: 20—Polypeptide sequence of ID22B
SEQ ID NO: 21—Polypeptide sequence of ID24B
SEQ ID NO: 22—Polypeptide sequence of ID25B
SEQ ID NO: 23—Polypeptide sequence of ID27B
SEQ ID NO: 24—Polypeptide sequence of ID41B
SEQ ID NO: 25—Polypeptide sequence of ID43B
SEQ ID NO: 26—Polypeptide sequence of ID45B
SEQ ID NO: 27—Polypeptide sequence of ID46B
SEQ ID NO: 28—Polypeptide sequence of ID49B
SEQ ID NO: 29—Polypeptide sequence of B10F1
SEQ ID NO: 30—Polypeptide sequence of Q31 B1
SEQ ID NO: 31—Polynucleotide sequence of 3′ primer mentioned in Preparative Methods section
SEQ ID NO: 32—Polynucleotide sequence of M13.rev used in Example 2
SEQ ID NO: 33—Polynucleotide sequence of M13.fw used in Example 2
SEQ ID NO: 34—Polynucleotide sequence encoding ID11B
SEQ ID NO: 35—Polynucleotide sequence encoding ID12B
SEQ ID NO: 36—Polynucleotide sequence encoding ID41B
SEQ ID NO: 37—Polynucleotide sequence encoding ID43B
SEQ ID NO: 38—Polynucleotide sequence encoding B10F1arm (in ID43B)
SEQ ID NO: 39—Polynucleotide sequence encoding Q31B1arm (in ID43B)
SEQ ID NO: 40—Polynucleotide sequence encoding Q35H8arm (in ID12B)
SEQ ID NO: 41—Polypeptide sequence of ID1C
SEQ ID NO: 42—Polypeptide sequence of ID3C
SEQ ID NO: 43—Polypeptide sequence of ID5C
SEQ ID NO: 44—Polypeptide sequence of ID6C
SEQ ID NO: 45—Polypeptide sequence of ID7C
SEQ ID NO: 46—Polypeptide sequence of ID8C
SEQ ID NO: 47—Polypeptide sequence of ID11C
SEQ ID NO: 48—Polypeptide sequence of Q34A3 (anti-TcdA ICVD)
SEQ ID NO: 49—Polypeptide sequence of B4F10 (anti-TcdA ICVD)
SEQ ID NO: 50—Polypeptide sequence of ID33A (anti-TcdA bihead)
SEQ ID NO: 51—Polypeptide sequence of ID17A (anti-TcdA bihead)
SEQ ID NO: 52—Polypeptide sequence of TcdB from C. difficile ribotype 087
SEQ ID NO: 53—Polypeptide sequence of TcdB from C. difficile ribotype 078
SEQ ID NO: 54—Polypeptide sequence of TcdB from C. difficile ribotype 017
SEQ ID NO: 55—Polypeptide sequence of TcdB from C. difficile ribotype 027
Polypeptides Including Antibodies and Antibody Fragments Including ICVDs such as the VH And VHH
A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domains are abbreviated herein as VHC, and the light (L) chain variable domains are abbreviated herein as VLC. These domains, domains related thereto and domains derived therefrom, are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDRs”), interspersed with regions that are more conserved, termed “framework regions” (“FRs”). The framework and complementarity determining regions have been precisely defined (Kabat et al 1991 Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, NIH Publication Number 91-3242, herein incorporated by reference in its entirety). In a conventional antibody, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with the heavy and the light immunoglobulin chains inter-connected by e.g. disulfide bonds, and the heavy chains similarity connected. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be kappa or lambda types. The overall structure of immunoglobulin-gamma (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan 1994 Mol Immunol 31:169-217).
An exception to conventional antibody structure is found in sera of Camelidae. In addition to conventional antibodies, these sera possess special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), are devoid of the L chain polypeptide and lack the first constant domain (CH1). At its N-terminal region, the H chain of the homodimeric protein contains an immunoglobulin chain variable domain, referred to as the VHH, which serves to associate with its cognate antigen (Muyldermans 2013 Annu Rev Biochem 82:775-797, Hamers-Casterman et al 1993 Nature 363(6428):446-448, Muyldermans et al 1994 Protein Eng 7(9):1129-1135, herein incorporated by reference in their entirety).
An antigen-binding fragment (or “'antibody fragment” or “immunoglobulin fragment”) as used herein refers to a portion of an antibody that specifically binds to TcdB (e.g. a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to TcdB). Examples of binding fragments encompassed within the term antigen-binding fragment include:
The total number of amino acid residues in an immunoglobulin chain variable domain such as a VHH or VH may be in the region of 110-130.
Immunoglobulin chain variable domains of the invention may for example be obtained by preparing a nucleic acid encoding an immunoglobulin chain variable domain using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained According to a specific embodiment, an immunoglobulin chain variable domain of the invention does not have an amino acid sequence which is exactly the same as (i.e. shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide such as a VH or VHH domain of a naturally occurring antibody.
Substituting at least one amino acid residue in the framework region of a non human immunoglobulin variable domain with the corresponding residue from a human variable domain is humanisation. Humanisation of a variable domain may reduce immunogenicity in humans.
Suitably the polypeptide of the present invention consists of an immunoglobulin chain variable domain. Suitably, the polypeptide of the present invention is an antibody or an antibody fragment. Suitably the antibody fragment is a VHH, a VH, a VL, a V-NAR, a Fab fragment, a VL or a F(ab′)2 fragment (such as a VHH or VH, most suitably a VHH).
Specificity, affinity, avidity and cross-reactivity
Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind. The specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognise a particular antigen as a unique molecular entity and distinguish it from another.
Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (Kd), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding polypeptide: the lesser the value of the Kd, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide (alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1/Kd). Affinity can be determined by known methods, depending on the specific antigen of interest.
Avidity is the measure of the strength of binding between an antigen-binding polypeptide and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding polypeptide and the number of pertinent binding sites present on the antigen-binding polypeptide.
Suitably, antigen-binding polypeptides of the invention will bind with a dissociation constant (Kd) of 10−6 to 10−12M, more suitably 10−7 to 10−12M, more suitably 10−8 to 10−12 M and more suitably 10−9 to 10−12M.
Any Kd value less than 10−8 is considered to indicate binding. Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.
An anti-TcdB polypeptide, a polypeptide which interacts with TcdB, or a polypeptide against TcdB, are all effectively polypeptides which bind to TcdB. A polypeptide of the invention may bind to a linear or conformational epitope on TcdB. The term “binds to TcdB” means binding to any one or more of the N-terminal, hydrophobic or C-terminal domains of TcdB.
Suitably, the polypeptide of the invention is capable of neutralising TcdB from multiple ribotypes of C. difficile. More suitably, the polypeptide of the invention is capable of neutralising TcdA from ribotypes 087, 027, 078, 017, 106 and 001. More suitably, the polypeptide of the invention will neutralise TcdB from ribotype 027.
Suitably the polypeptide of the invention is directed against one or more epitopes on TcdB such that said polypeptide of the invention, upon binding to TcdB, is capable of inhibiting or reducing the cytotoxic effect that is mediated by said TcdB. Suitably, the polypeptide of the invention binds to the cell binding domain of Clostridium difficile toxin B.
The polypeptides of the present invention bind to one or more epitope(s) on TcdB. In one aspect of the invention there is provided a polypeptide which binds to the same epitope on TcdB as B10F1, Q31B1, Q35H8, ID1B, ID2B, ID3B, ID11B, ID12B, ID20B, ID21B, ID22B, ID24B, ID25B, ID27B, ID41B, ID43B, Q31B1arnn, B10F1arnn, ID45B, ID46B or ID49B.
Suitably, the polypeptide of the invention is isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally-occurring polypeptide of the invention is isolated if it is separated from some or all of the coexisting materials in the natural system.
Potency, Inhibition and Neutralisation
Potency is a measure of the activity of a therapeutic agent expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent evokes a greater response at low concentrations compared to an agent of lower potency that evokes a smaller response at low concentrations. Potency is a function of affinity and efficacy. Efficacy refers to the ability of therapeutic agent to produce a biological response upon binding to a target ligand and the quantitative magnitude of this response. The term half maximal effective concentration (EC50) refers to the concentration of a therapeutic agent which causes a response halfway between the baseline and maximum after a specified exposure time. The therapeutic agent may cause inhibition or stimulation. It is commonly used, and is used herein, as a measure of potency.
A neutralising polypeptide for the purposes of the invention is a polypeptide which defends a cell from the effects of TcdB by, for example, inhibiting the biological effect of TcdB. The effectiveness of an anti-TcdB therapeutic agent can be ascertained using a neutralisation assay. A particularly suitable neutralisation assay is the measurement of Vero cell viability using Alamar Blue (Fields and Lancaster American Biotechnology Laboratory 1993 11(4):48-50). Using a range of known concentrations of anti-TcdB polypeptide, this assay can be performed to ascertain the ability of an anti-TcdB polypeptide to neutralise the effects of TcdB cytotoxicity by producing a dose-response curve and/or by ascertaining the half maximal effective concentration (EC50) of the anti-TcdB polypeptide. This standard Vero cell assay is used herein and detailed further in the Examples section below.
Suitably the polypeptide or construct of the invention neutralises TcdB cytotoxicity (such as TcdB ribotype 087, 027, 078, 017, 106 and 001) in the standard Vero cell assay with an EC50 of 50000 pM or less, such as 40000 pM or less, such as 30000 pM or less, such as 20000 pM or less, such as 10000 pM or less, such as 5000 pM or less, such as 4000 pM or less, such as 3000 pM or less, such as 2000 pM or less, such as 1000 pM or less, such as 500 pM or less, such as 250 pM or less, such as 100 pM or less, such as 80 pM or less, such as 60 pM or less, such as 40 pM or less, such as 30 pM or less, such as 20 pM or less, such as 10 pM or less.
In one aspect of the invention there is provided a VH or VHH which specifically binds to and has neutralising activity against Clostridium difficile toxin B. More suitably there is provided a
VH or VHH which specifically binds to and has neutralising activity against toxin B of more than one strain of C. difficile. More specifically there is provided a VH or VHH which specifically binds to and has neutralising activity against toxin B of two or more of C. difficile ribotypes 027, 087, 078, 106, 001 and 017.
Polypeptide and Polynucleotide Sequences
For the purposes of comparing two closely-related polypeptide sequences, the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0, using standard settings for polypeptide sequences (BLASTP). For the purposes of comparing two closely-related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0, using standard settings for nucleotide sequences (BLASTN).
Polypeptide or polynucleotide sequences are said to be the same as or identical to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C-terminus for polypeptides; from 5′ to 3′ terminus for polynucleotides.
A “difference” between sequences refers to an insertion, deletion or substitution of a single amino acid residue in a position of the second sequence, compared to the first sequence. Two polypeptide sequences can contain one, two or more such amino acid differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the identical sequences are 17 amino acid residues long, two substitutions in the second sequence results in a sequence identity of 88.2%. If the identical sequences are 7 amino acid residues long, three substitutions in the second sequence results in a sequence identity of 57.1%. If first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity). If first and second polypeptide sequences are 17 amino acid residues long and share 16 identical residues, the first and second polypeptide sequences share greater than 94% identity (the first and second polypeptide sequences share 94.1% identity). If first and second polypeptide sequences are 7 amino acid residues long and share 3 identical residues, the first and second polypeptide sequences share greater than 42% identity (the first and second polypeptide sequences share 42.9% identity).
Alternatively, for the purposes of comparing a first, reference polypeptide sequence to a second, comparison polypeptide sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one amino acid residue into the sequence of the first polypeptide (including addition at either terminus of the first polypeptide). A substitution is the substitution of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. A deletion is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletion at either terminus of the first polypeptide).
For the purposes of comparing a first, reference polynucleotide sequence to a second, comparison polynucleotide sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one nucleotide residue into the sequence of the first polynucleotide (including addition at either terminus of the first polynucleotide). A substitution is the substitution of one nucleotide residue in the sequence of the first polynucleotide with one different nucleotide residue. A deletion is the deletion of one nucleotide residue from the sequence of the first polynucleotide (including deletion at either terminus of the first polynucleotide).
A “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group:
As used herein, numbering of polypeptide sequences and definitions of CDRs and FRs are as defined according to the Kabat system (Kabat et al 1991 Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, NIH Publication Number 91-3242, in conjunction with the methods for analysis of antibody sequence and structure described in Martin 2010 ‘Protein sequence and structure of antibody variable domains’, Antibody Engineering volume 2, both herein incorporated by reference in their entirety). A “corresponding” amino acid residue between a first and second polypeptide sequence is an amino acid residue in a first sequence which shares the same position according to the Kabat system with an amino acid residue in a second sequence, whilst the amino acid residue in the second sequence may differ in identity from the first. Suitably corresponding residues will share the same number (and letter) if the framework and CDRs are the same length according to Kabat definition. Alignment can be achieved manually or by using, for example, a known computer algorithm for sequence alignment such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.
Suitably, the polynucleotides used in the present invention are isolated. An “isolated” polynucleotide is one that is removed from its original environment. For example, a naturally-occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of its natural environment or if it is comprised within cDNA.
In one aspect of the invention there is provided a polynucleotide encoding the polypeptide or construct of the invention. Suitably the polynucleotide comprises or consists of a sequence sharing 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any one of SEQ ID NOs: 34-40. More suitably the polynucleotide comprises or consists of any one of SEQ ID NOs: 34-40. In a further aspect there is provided a cDNA comprising said polynucleotide.
In one aspect of the invention there is provided a polynucleotide comprising or consisting of a sequence sharing 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any one of the portions of any one of SEQ ID NOs: 34-40 which encodes CDR1, CDR2 or CDR3 of the encoded immunoglobulin chain variable domain.
Suitably, the polypeptide sequence of the present invention contains at least one alteration with respect to a native sequence. Suitably, the polynucleotide sequences of the present invention contain at least one alteration with respect to a native sequence. Suitably the alteration to the polypeptide sequence or polynucleotide sequence is made to increase stability of the polypeptide or encoded polypeptide to proteases present in the intestinal tract.
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGWVQAGDSLRLSCVASGAPLS SFTMG WFRQAPEKEREFLG GKSRDGRTTYYSNSVKG
DVQLQESGGGLVQAGDSLRLSCAASGRTLS SYTMG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGGSLRLSCAASGRTFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGWVQAGDSLRLSCVASGRPLS SFTMG WFRQAPEKEREFLG GKSRDGRTTYYSNSVKG
DVQLQESGGGLVQAGGSLRLSCAASGRTFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA GSSHDGHTNYYANSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA AINGSGGNHISADSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA GSSRDGRTNYYANSVKG
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
Suitably the polypeptide of the invention comprises an immunoglobulin chain variable domain which binds to Clostridium difficile toxin B, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein:
(a) CDR1 comprises a sequence sharing 60% or greater, such as 80% or greater sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence sharing 60% or greater, such as 70% or greater, such as 75% or greater, such as 80% or greater, such as 85% or greater, such as 90% or greater sequence identity with SEQ ID NO: 2 and CDR3 comprises a sequence sharing 60% or greater, such as 65% or greater, such as 75% or greater, such as 80% or greater, such as 90% or greater sequence identity with SEQ ID NO: 3; suitably any residues of CDR1, CDR2 or CDR3 differing from their corresponding residues in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, are conservative substitutions with respect to their corresponding residues; suitably CDR1, CDR2 and/or CDR3 are devoid of K or R; suitably CDR1, CDR2 and/or CDR3 have been mutated to replace one or more R or K residues with an H residue or
(b) CDR1 comprises a sequence sharing 60% or greater, such as 80% or greater sequence identity with SEQ ID NO: 4, CDR2 comprises a sequence sharing 60% or greater, such as 70% or greater, such as 75% or greater, such as 80% or greater, such as 85% or greater, such as 90% or greater sequence identity with SEQ ID NO: 5 and CDR3 comprises a sequence sharing 65% or greater, such as 70% or greater, such as 75% or greater, such as 85% or greater, such as 90% or greater sequence identity with SEQ ID NO: 6; suitably any residues of CDR1, CDR2 or CDR3 differing from their corresponding residues in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively, are conservative substitutions with respect to their corresponding residue; suitably CDR1, CDR2 and/or CDR3 are devoid of K or R; suitably CDR1, CDR2 and/or CDR3 have been mutated to replace one or more R or K residues with an H residue.
Suitably the polypeptide of the invention comprises an immunoglobulin chain variable domain which binds to Clostridium difficile toxin B, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises or consists of SEQ ID NO: 1, CDR2 comprises or consists of SEQ ID NO: 2 and CDR3 comprises or consists of SEQ ID NO: 3; or CDR1 comprises or consists of SEQ ID NO: 4, CDR2 comprises or consists of SEQ ID NO: 5 and
CDR3 comprises or consists of SEQ ID NO: 6; or CDR1 comprises or consists of SEQ ID NO: 7, CDR2 comprises or consists of SEQ ID NO: 8 and CDR3 comprises or consists of SEQ ID NO: 9.
Suitably the polypeptide of the invention comprises an immunoglobulin chain variable domain which binds to Clostridium difficile toxin B, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: CDR1 comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 1, CDR2 comprises or more suitably consists of a sequence having no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 2 and CDR3 comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 3; or
CDR1 comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 4, CDR2 comprises or more suitably consists of a sequence having no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 5 and CDR3 comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 6; or
CDR1 comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 7, CDR2 comprises or more suitably consists of a sequence having no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 8 and CDR3 comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to SEQ ID NO: 9.
Suitably the polypeptide of the invention comprises an immunoglobulin chain variable domain which binds to Clostridium difficile toxin B, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 is devoid of K or R, more suitably CDR1, CDR2 and CDR3 are devoid of K or R. Suitably, CDR1, CDR2 and/or CDR3 have been mutated to replace one or more R or K residues with an H residue.
Suitably, FR1, FR2, FR3 and FR4 each comprise a sequence sharing 40% or greater, such as 60% or greater, such as 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater sequence identity with FR1, FR2, FR3 and FR4 of SEQ ID NO 10, respectively; or
FR1, FR2, FR3 and FR4 each comprise a sequence sharing 40% or greater, such as 60% or greater, such as 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater sequence identity with FR1, FR2, FR3 and FR4 of SEQ ID NO 11, respectively; or
FR1, FR2, FR3 and FR4 each comprise a sequence sharing 40% or greater, such as 60% or greater, such as 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater sequence identity with FR1, FR2, FR3 and FR4 of SEQ ID NO 12, respectively.
Suitably FR1 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 15, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR1 of SEQ ID NO 10; FR2 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR2 of SEQ ID NO 10; FR3 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 15, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR3 of SEQ ID NO 10; and FR4 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR4 of SEQ ID NO 10.
Alternatively FR1 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 15, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR1 of SEQ ID NO 11; FR2 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR2 of SEQ ID NO 11; FR3 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 15, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR3 of SEQ ID NO 11; and FR4 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR4 of SEQ ID NO 11.
Alternatively FR1 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 16, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR1 of SEQ ID NO 12; FR2 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR2 of SEQ ID NO 12; FR3 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 15, more suitably no more than 10, more suitably no more than 7, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR3 of SEQ ID NO 12; and FR4 of the polypeptide of the invention comprises or more suitably consist of a sequence having no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s), deletion(s) and/or substitutions(s) compared to FR4 of SEQ ID NO 12.
Suitably residue 1 of FR1 is D, E or Q; and/or residue 5 of FR1 is V in the inventive polypeptide or each polypeptide of a multimeric construct.
Suitably the polypeptide of the invention comprises or more suitably consists of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ
ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30.
Suitably the Clostridium difficile toxin B bound by the polypeptide of the invention comprises or more suitably consists of a sequence sharing 40% or greater, such as 60% or greater, such as 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 100% sequence identity with any one of SEQ ID NOs: 52-55.
Linkers and Multimers
A construct according to the invention comprises multiple polypeptides and therefore may suitably be multivalent. Such a construct may comprise at least two identical polypeptides according to the invention. A construct consisting of two identical polypeptides according to the invention is a “homobihead”. In one aspect of the invention there is provided a construct comprising two or more identical polypeptides of the invention.
Alternatively, a construct may comprise a polypeptide of the invention and at least one further polypeptide which is different, but still a polypeptide according to the invention (a “heterobihead”). Suitably, the different polypeptide in such a construct is selected from: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. Suitably such a construct is selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24 or SEQ ID NO: 25.
Alternatively, such a construct may comprise (a) at least one polypeptide according to the invention and (b) at least one polypeptide such as an antibody or antigen-binding fragment thereof, which is not a polypeptide of the invention (also a “heterobihead”). The at least one polypeptide of (b) may bind TcdB (for example via a different epitope to that of (a)), or alternatively may bind to a target other than TcdB. Suitably the different polypeptide (b) binds to Clostridium difficile toxin A.
Constructs can be multivalent and/or multispecific. A multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides therefore presents two or more sites at which attachment to one or more antigens can occur. An example of a multivalent construct could be a homobihead or a heterobihead. A multispecific construct (such as a bispecific construct) comprises two or more different binding polypeptides which present two or more sites at which either (a) attachment to two or more different antigens can occur or (b) attachment to two or more different epitopes on the same antigen can occur. An example of a multispecific construct could be a heterobihead. A multispecific construct is multivalent.
A construct of the invention may comprise, or more suitably consist of, one or more polypeptides according to the invention and suitably additionally comprise, or more suitably consist of, one, two, three, four, five, six, seven, eight, nine or more further polypeptides wherein each of the further polypeptides binds to a target, such as a target selected from the list consisting of Clostridium difficile toxin A and Clostridium difficile toxin B.
A construct consisting of a total of four polypeptides which each bind a target is known as a ‘quadrahead’. The format of such a construct according to the invention may be, from N- to C-terminal, suitably A-A-A-A, A-A-A-B, A-A-B-B, A-B-B-B, B-A-A-A, B-B-A-A, A-B-A-B, B-A-B-A, A-B-B-A or B-A-A-B, more suitably A-A-B-B, B-B-A-A, A-B-A-B, B-A-B-A, A-B-B-A or B-A-A-B, wherein A is a polypeptide which binds to Clostridium difficile toxin A and B is a polypeptide which binds to Clostridium difficile toxin B, wherein the polypeptides which bind to Clostridium difficile toxin A are identical or different and the polypeptides which bind to Clostridium difficile toxin B are identical or different.
Suitably the construct is of the format A-A′-B-B′, A-B-B′-A′ or B-A-A′-B′, wherein B is a polypeptide according to the invention, B′ is a different polypeptide according to the invention, A is a polypeptide which binds to Clostridium difficile toxin A and A′ is a different polypeptide which binds to Clostridium difficile toxin A. Suitably the construct is selected from: SEQ ID NOs: 41-47.
Suitably, the polypeptides comprised within the construct are antibody fragments. More suitably, the polypeptides comprised within the construct are selected from the list consisting of: a VHH, a VH, a VL, a V-NAR, a Fab fragment and a F(ab')2 fragment. More suitably, the polypeptides comprised within the construct are VHs or VHHs.
The polypeptides of the invention can be linked to each other directly (i.e. without use of a linker) or via a linker. The linker is suitably a polypeptide and will be selected so as to allow binding of the polypeptides to their epitopes. If used for therapeutic purposes, the linker is suitably non-immunogenic in the subject to which the polypeptides are administered. Suitably the linkers are of the format (G4S)x. Most suitably x is 6.
Vectors and Hosts
The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication defective retroviruses. adenoviruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. The invention also relates to nucleotide sequences that encode polypeptide sequences or multivalent and/or multispecific constructs. The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.
In one aspect of the invention there is provided a vector comprising the polynucleotide encoding the polypeptide or construct of the invention or cDNA comprising said polynucleotide. In a further aspect of the invention there is provided a host cell transformed with said vector, which is capable of expressing the polypeptide or construct of the invention. Suitably the host cell is a bacteria such as E. coli or a yeast such a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris.
Stability
Suitably, the polypeptide or construct of the present invention substantially retains neutralisation ability and/or potency when delivered orally and after exposure to the intestinal tract (for example, after exposure to proteases present in the small and/or large intestine, C. difficile—specific proteases and inflammatory proteases). Such proteases include enteropeptidase, trypsin and chymotrypsin. Proteases present in, or produced in, the small and/or large intestine include proteases sourced from intestinal tract commensal microflora and/or pathogenic bacteria, for example wherein the proteases are cell membrane-attached proteases, secreted proteases and proteases released on cell lysis). Most suitably the proteases are trypsin and chymotrypsin. Suitably the polypeptide or construct of the invention is substantially resistant to one or more proteases.
Suitably the intestinal tract is the intestinal tract of a human. The small intestine suitably consists of the duodenum, jejunum and ileum. The large intestine suitably consists of the cecum, colon, rectum and anal canal.
The polypeptide or construct of the present invention substantially retains neutralisation ability when suitably 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% of the original neutralisation ability of the polypeptide of the invention or construct is retained after exposure to proteases present in the small and/or large intestine such as trypsin or chymotrypsin.
Suitably the polypeptide or construct of the invention substantially retains neutralisation ability after exposure to proteases present in the small and/or large intestine such as trypsin or chymotrypsin for, for example, up to at least 15, more suitably up to at least 30, more suitably up to at least 45, more suitably up to at least 60 minutes at 37 degrees C.
Suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more of the neutralisation ability of the polypeptide or construct of the invention is retained after up to 1 or more suitably up to 4 hours of exposure to conditions of the intestinal tract, more suitably the small or large intestine, more suitably human faecal supernatant.
Therapeutic Use and Delivery
A therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention, is an amount which is effective, upon single or multiple dose administration to a subject, in neutralising TcdB to a significant extent in a subject. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the polypeptide, pharmaceutical composition or construct to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the polypeptide of the invention, pharmaceutical composition or construct are outweighed by the therapeutically beneficial effects. The polypeptide or construct of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The polypeptide or construct of the invention can be in the form of a pharmaceutically acceptable salt.
A pharmaceutical composition of the invention may suitably be formulated for oral, intramuscular, subcutaneous, intravenous, intrarectal or enema delivery. The pharmaceutical compositions of the invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Solid dosage forms are preferred. The polypeptide of the invention, pharmaceutical composition or construct may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
Typically, the pharmaceutical composition comprises a polypeptide or construct of the invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the invention. Pharmaceutical compositions may include antiadherents, binders, coatings, disintegrants, flavours, colours, lubricants, sorbents, preservatives, sweeteners, freeze dry excipients (including lyoprotectants) or compression aids. Suitably, the polypeptide or construct of the invention is lyophilised before being incorporated into a pharmaceutical composition.
A polypeptide of the invention may also be provided with an enteric coating. An enteric coating is a polymer barrier applied on oral medication which protects the polypeptide from the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers which are insoluble at the highly acidic pH found in the stomach, but which dissolve rapidly at a less acidic pH. Thus, suitably, the enteric coating will not dissolve in the acidic juices of the stomach (pH ˜3), but will do so in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH-dependent release polymer is selected such that the polypeptide or construct of the invention will be released at about the time that the dosage reaches the small intestine.
A polypeptide, construct or pharmaceutical composition of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilisers, isotonic agents, suspending agents, emulsifying agents, stabilisers and preservatives. Acceptable carriers, excipients and/or stabilisers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol.
For all modes of delivery, the polypeptide, pharmaceutical composition or construct of the invention may be formulated in a buffer, in order to stabilise the pH of the composition, at a concentration between 5-50, or more suitably 15-40 or more suitably 25-30 g/litre. Examples of suitable buffer components include physiological salts such as sodium citrate and/or citric acid. Suitably buffers contain 100-200, more suitably 125-175 mM physiological salts such as sodium chloride. Suitably the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.
Exemplary polypeptide or construct concentrations in a pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL.
An aqueous formulation of the polypeptide, construct or pharmaceutical composition of the invention may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.
The tonicity of the pharmaceutical composition may be altered by including a tonicity modifier. Such tonicity modifiers can be charged or uncharged chemical species. Typical uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol). Typical charged tonicity modifiers include salts such as a combination of sodium, potassium or calcium ions, with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulphate); or amino acids such as arginine or histidine. Suitably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 1 mM to 500 nM. Suitably, at least one isotonic agent is included in the composition.
A surfactant may also be added to the pharmaceutical composition to reduce aggregation of the formulated polypeptide or construct and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, and polysorbate 80. Exemplary concentrations of surfactant may range from about 0.001% to about 10% w/v.
A lyoprotectant may also be added in order to protect the polypeptide or construct of the invention against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose and trehalose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 mM.
The dosage ranges for administration of the polypeptide of the invention, pharmaceutical composition or construct of the invention are those to produce the desired therapeutic effect. The dosage range required depends on the precise nature of the polypeptide of the invention, pharmaceutical composition or construct, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
Suitable daily dosages of polypeptide of the invention, pharmaceutical composition or construct of the invention are in the range of 50ng-50 mg per kg, such as 50ug-40 mg per kg, such as 5-30 mg per kg of body weight. The unit dosage can vary from less than 100 mg, but typically will be in the region of 250-2000 mg per dose, which may be administered daily or more frequently, for example 2, 3 or 4 times per day or less frequently for example every other day or once per week.
In one aspect of the invention there is provided the use of the polypeptide, pharmaceutical composition or construct of the invention in the manufacture of a medicament for the treatment of C. difficile infection. In a further aspect of the invention there is provided a method of treating C. difficile infection comprising administering to a person in need thereof a therapeutically effective amount of the polypeptide, pharmaceutical composition or construct of the invention.
The word ‘treatment’ is intended to embrace prophylaxis as well as therapeutic treatment. Treatment of infection also embraces treatment of exacerbations thereof and also embraces treatment of patients in remission from infection symptoms to prevent relapse of symptoms.
Combination Therapy
A pharmaceutical composition of the invention may also comprise one or more active agents (e.g. active agents suitable for treating C. difficile infection). It is within the scope of the invention to use the pharmaceutical composition of the invention in therapeutic methods for the treatment of C. difficile infection as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of C. difficile infection, such as antibiotics.
Possible combinations include combinations with, for example, one or more active agents selected from the list comprising C. difficile toxoid vaccine, ampicillin, amoxicillin, vancomycin, metronidazole, fidaxomicin, linezolid, nitazoxanide, rifaximin, ramoplanin, difimicin, clindamycin, cephalosporins (such as second and third generation cephalosporins), fluoroquinolones (such as gatifloxacin or moxifloxacin), macrolides (such as erythromycin, clarithromycin, azithromycin), penicillins, aminoglycosides, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, imipenem, meropenem, antibacterial agents, bactericides, or bacteriostats. Possible combinations also include combinations with one or more active agents which are probiotics, for example Saccharomyces boulardii or Lactobacillus rhamnosus GG.
Hence another aspect of the invention provides a pharmaceutical composition of the invention in combination with one or more further active agents, for example one or more active agents described above.
In a further aspect of the invention, the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above. In a further aspect of the invention, the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with fecal microbiota transplantation (i.e. fecal bacteriotherapy, fecal transfusion, fecal transplant, stool transplant, fecal enema, human probiotic infusion).
Similarly, another aspect of the invention provides a combination product comprising:
The invention also encompasses a kit of parts comprising components:
Component (i) of the kit of parts is thus component (A) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component (B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. The one or more other active agents (i.e. component (B) above) may be, for example, any of the agents mentioned above in connection with the treatment of C. difficile infection. If component (B) is more than one further active agent, these further active agents can be formulated with each other or formulated with component (A) or they may be formulated separately. In one embodiment component (B) is one other therapeutic agent. In another embodiment component (B) is two other therapeutic agents. The combination product (either a combined preparation or kit-of-parts) of this aspect of the invention may be used in the treatment or prevention of C. difficile infection.
Suitably the polypeptide, pharmaceutical composition or construct of the invention is for use as a medicament and more suitably for use in the treatment, prevention, diagnosis and/or detection of C. difficile infection, most suitably for use in the treatment of C. difficile infection.
Preparative Methods
Polypeptides of the invention can be obtained and manipulated using the techniques disclosed for example in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th Edition Cold Spring Harbour Laboratory Press.
Monoclonal antibodies can be produced using hybridoma technology, by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis (Köhler and Milstein 1975 Nature 256:495-497 and Nelson et al 2000 Molecular Pathology 53(3):111-117, herein incorporated by reference in their entirety).
A monoclonal antibody directed against a determined antigen can, for example, be obtained by:
Alternatively, the use of a hybridoma cell is not required. Accordingly, monoclonal antibodies can be obtained by a process comprising the steps of:
Methods for immunizing camelids, cloning the VHH repertoire of B cells circulating in blood (Chomezynnski and Sacchi 1987 Anal Biochem 162:156-159), and isolation of antigen-specific VHHs from immune (Arbabi-Ghahroudi et al 1997 FEBS Lett 414:521-526) and nonimmune (Tanha et al 2002 J Immunol Methods 263:97-109) libraries using phage, yeast, or ribosome display are known (WO92/01047, Nguyen et al 2001 Adv Immunol 79:261-296 and Harmsen et al 2007 Appl Microbiol Biotechnol 77(1):13-22). These references are herein incorporated by reference in their entirety.
Antigen-binding fragments of antibodies such as the scFv and Fv fragments can be isolated and expressed in E. coli (Miethe et al 2013 J Biotech 163(2):105-111, Skerra et al 1988 Science 240(4855):1038-1041 and Ward et al Nature 1989 341:544-546, herein incorporated by reference in their entirety).
Mutations can be made to the DNA or cDNA that encode polypeptides which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae, are known.
Modification of polypeptides can be achieved for example by substitutions, additions or deletions to a nucleic acid encoding the polypeptide. The substitutions, additions or deletions to a nucleic acid encoding the polypeptide can be introduced by many methods, including for example error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al 1997 Anal Biochem 254(2):157-178, herein incorporated by reference in its entirety), gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination of these methods. The modifications, additions or deletions to a nucleic acid can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
In particular, artificial gene synthesis may be used (Nambiar et al 1984 Science 223:1299-1301, Sakamar and Khorana 1988 Nucl. Acids Res 14:6361-6372, Wells et al 1985 Gene 34:315-323 and Grundstrom et al 1985 Nucl. Acids Res 13:3305-3316, herein incorporated by reference in their entirety). A gene encoding a polypeptide of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma and Eckstein 1998 Annu Rev Biochem 67:99-134)
Expression of immunoglobulin chain variable domains such as VHs and VHHs can be achieved using a suitable expression vector such as a prokaryotic cell such as bacteria, for example E. coli (for example according to the protocols disclosed in WO94/04678, which is incorporated herein by reference and detailed further below). Expression of immunoglobulin chain variable domains such as VHs and VHHs can also be achieved using eukaryotic cells, for example insect cells, CHO cells, Vero cells or suitably yeast cells such as yeasts belonging to the genera Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably S. cerevisiae is used (for example according to the protocols disclosed in WO94/025591, which is incorporated herein by reference and detailed further below).
Specifically, VHHs can be prepared according to the methods disclosed in WO94/04678 using E. coli cells by a process comprising the steps of:
Alternatively, immunoglobulin chain variable domains such as VHs and VHHs are obtainable by a process comprising the steps of:
Further, immunoglobulin chain variable domains such as VHHs or VHs can be produced using E. coli or S. cerevisiae according to the methods disclosed in Frenken et al 2000 J Biotech 78:11-21 and WO99/23221 (herein incorporated by reference in their entirety) as follows:
After taking a blood sample from an immunised llama and enriching the lymphocyte population via Ficoll (a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions - Pharmacia) discontinuous gradient centrifugation, isolating total RNA by acid guanidium thiocyanate extraction (Chomezynnski and Sacchi 1987 Anal Biochem 162:156-159), and first strand cDNA synthesis (e.g. using a cDNA kit such as RPN 1266 (Amersham)), DNA fragments encoding VHH and VH fragments and part of the short or long hinge region are amplified by PCR using the specific primers detailed on pages 22 and 23 of WO99/23221. Upon digestion of the PCR fragments with Pstl and Hindlll or BstEll, the DNA fragments with a length between about 300 and 450 bp are purified via agarose gel electrophoresis and ligated in the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al 1991 Nucl Acid Res 19:4133-4137, herein incorporated by reference in its entirety) and contains the laclq gene and unique restriction sites to allow the cloning of the llama VHH and VH genes. pUR4548 is derived from pSY1 (Harmsen et al 1993 Gene 125:115-123, herein incorporated by reference in its entirety). From this plasmid, the BstEll site in the leu2 gene is removed via PCR and the cloning sites between the SUC2 signal sequence and the terminator are replaced in order to facilitate the cloning of the VH/VHH gene fragments. The VH/VHHs have the c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies are transferred to 96 well microtiter plates containing 150 ml 2TY medium supplemented with 1% glucose and 100 mg L−1 ampicillin. After overnight growth (37 degrees C.), the plates are duplicated in 2TY medium containing 100 mg L−1 ampicillin and 0.1 mM IPTG. After another overnight incubation and optionally freezing and thawing, cells are centrifuged and pelleted and the supernatant can be used in an ELISA. Individual S. cerevisiae colonies are transferred to test tubes containing selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with the essential amino acids and bases) and are grown for 48 h at 30 degrees C. Subsequently, the cultures are diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacto peptone and 5% galactose). After 24 and 48 h of growth, the cells are pelleted and the culture supernatant can be analysed in an ELISA. Absorbance at 600 nm (OD600) is optionally measured.
Further, immunoglobulin chain variable domains such as VH/VHHs can be produced using S. cerevisiae using the procedure as follows:
Isolate a naturally-occuring DNA sequence encoding the VH/VHH or obtain a synthetically produced DNA sequence encoding the VH/VHH, including a 5′-UTR, signal sequence, stop codons and flanked with Sacl and Hinlll sites (such a synthetic sequence can be produced as outlined above or for example may be ordered from a commercial supplier such as Geneart (Life Technologies)).
Use the restriction sites for transfer of the VH/VHH gene to the multi-copy integration (MCI) vector pUR8569 or pUR8542, as follows. Cut the DNA sequence encoding the VHH optionally contained within a shuttle vector, cassette or other synthetic gene construct and the MCI vector with Sacl and Hindlll using: 25 ul VHH DNA (Geneart plasmid or MCI vector), 1 ul Sacl, 1 ul Hindlll, 3 ul of a suitable buffer for double digestion such as NEB buffer 1 (New England Biolabs) overnight at 37 degrees C. Run 25 ul of digested DNA encoding the VHH and 25 ul of digested MCI vector on a 1.5% agarose gel with 1×TAE buffer and then perform gel extraction for example using QlAquick Gel Extraction Kit (Qiagen)). Set-up a ligation of digested MCI vector and digested DNA encoding the VH/VHH as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul 10× ligase buffer, 1 ul T4 DNA ligase, and ddH2O. Then perform ligation overnight at 16 degrees C.
Next transform the E. coli cells. For chemical competent XL-1 blue cells, thaw 200 ul heat competent XL-1 blue cells and add 5 ul ligation mix on ice for about 30 minutes followed by heat shock for 90 seconds at 42 degrees C. Then add 800 ul Luria-Bertani low salt medium supplemented with 2% glucose and recover cells for 2 hours at 37 degrees C. Plate cells on Luria-Bertani agar and ampicillin (100 ug/ml) plates and keep overnight at 37 degrees C. For electro competent TG1 E. coli cells, use an electroporation cuvette. In the electroporation cuvette: thaw 50 ul electro competent TG1 cells and 1 ul ligation mix on ice for about 15 minutes. Place the cuvette in the holder and pulse. Add 500 ul of 2TY medium and recover cells for 30 minutes at 37 degrees C. Plate 100 ul of cells on Luria-Bertani, agar, containing ampicillin (100 ug/ml) and 2% glucose plates. Keep plates at 37 degrees C. overnight.
After cloning of the VH/VHH gene into E. coli as detailed above, S. cerevisiae can be transformed with the linearized MCI vector. Before transformation is carried out, some steps are performed: (i) the DNA should be changed from circular to linear by digestion or else the DNA cannot be integrated into the yeast genome and (ii) the digested DNA should be cleaned of impurities by ethanol precipitation. Also, during the transformation process, the yeast cells are made semi-permeable so the DNA can pass the membrane.
Preparation for yeast transformation: perform a Hpal digestion of the midi-prep prepared from the selected E. coli colony expressing the VH/VHH gene as follows. Prepare a 100 ul solution containing 20 ng of midi-prep, 5 ul Hpal, 10 ul of appropriate buffer such as NEB4 buffer (BioLabs), and ddH2O.
Cut the DNA with the Hpal at room temperature overnight. Next perform an ethanol precipitation (and put to one side a 5 ul sample from Hpal digestion). Add 300 ul ethanol 100% to 95 ul Hpal digested midiprep , vortex, and spin at full speed for 5 minutes. Carefully decant when a pellet is present, add 100 ul of ethanol 70%, then spin again for 5 minutes at full speed. Decant the sample again, and keep at 50-60 degrees C. until the pellet is dry. Re-suspend the pellet in 50 ul ddH2O. Run 5 ul on a gel beside the 5 ul Hpal digested sample.
Yeast transformation: prepare YNBglu plates. Use 10 g agar+425 ml water (sterilised), 25 ml filtered 20× YNB (3.35 g YNB (yeast nitrogen base) in 25 ml sterilized H2O) and 50 ml sterile 20% glucose and pour into petri dishes. Pick one yeast colony from the masterplate and grow in 3 ml YPD (Yeast Extract Peptone Dextrose) overnight at 30 degrees C. Next day prepare about 600 ml YPD and use to fill 3 flasks with 275 ml, 225 ml and 100 ml YPD. Add 27.5 ul yeast YPD culture to the first flask and mix gently. Take 75 ml from the first flask and put this in the second flask, mix gently. Take 100 ml from the second flask and put in the third one, mix gently. Grow until reaching an OD660 of between 1 and 2. Divide the flask reaching this OD over 4 Falcon tubes, ±45 ml in each. Spin for 2 minutes at 4200 rpm. Discard the supernatant.
Dissolve the pellets in two Falcon tubes with 45 ml H2O (reducing the number of tubes from 4 to 2). Spin for 2 minutes at 4200 rpm. Dissolve the pellets in 45 ml H2O (from 2 tubes to 1). Spin for 2 minutes at 4200 rpm. Gently dissolve the pellets in 5 ml lithium acetate (LiAc) (100 mM), and spin for a few seconds. Carefully discard some LiAc, but retain over half of the LiAc in the tube. Vortex the cells, boil carrier DNA for 5 minutes and quickly chill in ice-water. Add to a 15 ml tube containing: 240 ul PEG, 50 ul cells, 36uLiAc (1M), 25 ul carrier DNA, 45 ul ethanol precipitated VH/VHH. Mix gently after each step (treat the blank sample the same, only without ethanol precipitated VH/VHH). Incubate for 30 minutes at 30 degrees C., gently invert the tube 3-4 times, then heat shock for 20-25 minutes at 42 degrees C. Spin up to 6000 rpm for a brief time. Gently remove the supernatant and add 250 ul ddH2O and mix. Streak all of it on an YNBglu plate until plates are dry and grow for 4-5 days at 30 degrees C. Finally, prepare YNBglu plates by dividing plates in 6 equal parts, number the parts 1 to 6, inoculate the biggest colony and streak out number 1. Repeat for other colonies from big to small from 1 to 6. Grow at 30 degrees C. for 3-4 days large until colonies are produced. The VH/VHH clones are grown using glucose as a carbon source, and induction of VH/ VHH expression is done by turning on the Galactose-7-promoter by adding 0.5% galactose. Perform a 3 mL small scale culture to test the colonies and choose which one shows the best expression of the VH or VHH. This colony is then used in purification.
Purification: the VH/VHH is purified by cation exchange chromatorgraphy with a strong anion resin (such as Capto S). On day 1, inoculate the selected yeast colony expressing the VH/VHH in 5 ml YPD medium (YP medium+2% glucose) and grow the cells in 25 mL sealed sterile tubes at 30 degrees C. overnight (shaking at 180 rpm). On day 2, dilute the 5 ml overnight culture in 50 mL freshly prepared YP medium+2% glucose+0.5% galactose, grow the cells in 250 ml aerated baffled flasks at 30 degrees C. for two nights (shaking at 180 rpm). On day 4, spin the cells down in a centrifuge at 4200 rpm for 20 min. Cation exchange purification step using a strong anion resin: adjust the pH of the supernatant containing the ligand to 3.5. Wash 0.75 ml resin (+/−0.5 mL slurry) per of 50 mL supernatant with 50 mL of ddH2O followed by three washes with binding buffer. Add the washed resin to the supernatant and incubate the suspension at 4 degrees C. on a shaker for 1.5 hours. Pellet the resin-bound VH/VHH by centrifugation at 500 g for 2 minutes and wash it with wash buffer. Decant supernatant and re-suspend the resin with 10 mL of binding buffer. Put a filter in a PD-10 column, pour the resin in the column and let the resin settle for a while, then add a filter above the resin. Wait until all binding buffer has run through. Elute the VH/VHH with 6×0.5 ml elution buffer. Collect the elution fractions in eppendorf tubes. Measure the protein concentration of the 6 eluted fractions with a Nanodrop. Pool the fractions that contain the VHH and transfer the solution into a 3,500 Da cutoff dialysis membrane. Dialyze the purified protein solution against 3 L of PBS overnight at 4 degrees C. On day 5, dialyze the purified protein solution against 2 L of fresh PBS for an additional 2 hours at 4 degrees C. Finally, calculate the final concentration by BCA.
Although discussed in the context of the VH/VHH, the techniques described above could also be used for scFv, Fab, Fv and other antibody fragments if required.
Multiple antigen-binding fragments (suitably VH/VHHs) can be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by Blattler et al Biochemistry 24:1517-1524 (herein incorporated by reference in its entirety). Alternatively, the antigen-binding fragments may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more antigen-binding fragments. One way of joining multiple antigen-binding fragments via the genetic route is by linking the antigen-binding fragment coding sequences either directly or via a peptide linker. For example, the carboxy-terminal end of the first antigen-binding fragment may be linked to the amino-terminal end of the next antigen-binding fragment. This linking mode can be extended in order to link antigen-binding fragments for the construction of tri-, tetra-, etc. functional constructs. A method for producing multivalent (such as bivalent) VHH polypeptide constructs is disclosed in WO 96/34103 (herein incorporated by reference in its entirety).
Suitably, the polypeptide of the invention can be produced in a fungus such as a yeast (for example, S. cerevisiae) comprising growth of the fungus on a medium comprising a carbon source wherein 50-100 wt % of said carbon source is ethanol, according to the methods disclosed in WO02/48382. Large scale production of VHH fragments in S. cerevisiae is described in Thomassen et al 2002 Enzyme and Micro Tech 30:273-278 (herein incorporated by reference in its entirety).
In one aspect of the invention there is provided a process for the preparation of the polypeptide or construct of the invention comprising the following steps:
The present invention will now be further described by means of the following non-limiting examples.
Llama immunisations were carried out using two different immunisation protocols to optimise the chance of obtaining potent cross-strain neutralising antibodies against TcdB.
Under the first protocol, two llamas were primed with 100 ug of TcdB toxoids prepared by formalin inactivation of purified TcdB from a C. difficile 027 strain, as well as with 107 formalin inactivated spores from C. difficile strain 017 (M68) using Specol adjuvant. Llama 2 was boosted at 7, 14, 21, 28, 35 days with the same antigens, except that from day 14, gamma irradiated spores rather than formalin inactivated spores were used. In addition, the adjuvant was changed to IMS1312 for the last two boosts. Llama 1 had a similar immunisation protocol except that two further boosts were given on days 42 and 49. For llama 1, formalin inactivated spores were used on days 0, 7, 14 and 21, and Specol was the adjuvant. However, thereafter the adjuvant was changed to IMS1312 and gamma irradiated spores were used. Intramuscular injections were used for priming and boosting both llamas, except for the last boost which was administered subcutaneously. Blood samples were taken on days 28 (both llamas), 39 (llama 2, terminal sample), 42 (llama 1) and 53 (llama 1, terminal sample). On days 53 and 39 llamas 1 and 2 respectively were culled and lymph nodes removed. Lymphocytes were prepared from lymph nodes and blood samples to maximise the diversity of the immune response being sampled.
Under the second protocol, a llama was primed with TcdB toxoid and then boosted with 100 μg/injection of C.difficile 630 recombinant TcdB cell binding domain (CBD) on days 14, 28 and 35. Antigens were dissolved in 1 ml PBS+1 ml of Stimune and were injected intramuscularly. Blood samples were taken on days 0, 28 and 43. The llama was rested for 2.5 months then re-immunised three further times at 14 day intervals with TcdB toxoid prepared from C. difficile 027. Two days after the final boost, blood was removed from the llama for lymphocyte preparation.
RNA extracted from the llama lymphocytes was transcribed into cDNA using a reverse transcriptase kit. The cDNA was cleaned on PCR cleaning columns. IgH (both conventional and heavy chain) fragments were amplified using primers annealing at the leader sequence region and at the CH2 region. Two DNA fragments (˜700 bp and 900 bp) were amplified representing VHHs and VHs, respectively. The 700 bp fragment was cut from the gel and purified. A sample was used as a template for nested PCR. The amplified fragment was cleaned on a column and eluted. The eluted DNA was digested with BstEll and Sfil, and the 400 bp fragment was isolated from the gel. The fragments were ligated into the phagemid pUR8100 and transformed into E. coli TG1. Bacteria from overnight grown cultures of the libraries were collected and stored. The optical density at 600 nm (OD600) of these stocks was measured. The insert frequency was determined by picking multiple different clones from each of the library transformations and running colony PCR.
Phages were rescued from the bacteria containing libraries from the llamas by inoculating in medium containing glucose and ampicillin. When cultures were at log-phase, helper phage was added to infect the cultures and produce phages. Next day, produced phages were precipitated from the culture supernatant using a PEG/NaCI solution. The number of phages was determined by titration of the solution and infecting log-phase E. coli TG1 with the different phage dilutions. TcdB from ribotype 027 and strain VPI10463 were coated into wells of maxisorb plates, overnight. The following amounts were used: 500 ng, 167ng, 55 ng and 0 ng (non-coated well). Next day wells of the maxisorb plates were blocked with 4% Marvel in PBS, then phage from the libraries were added to the wells. After extensive washing with PBS-Tween and PBS, bound phages were eluted using alkaline pH shock and neutralized with 1 M Tris-HCl pH7.5. About half the eluted phages were rescued by infecting log-phase E. coli TG1 and selecting in medium containing ampicillin and glucose.
The sequences of the selected ICVDs are provided in Table 1A above. Selected ICVDs were B10F1, Q31B1 and Q35H8.
Selected variable domains were subcloned from the phagemid vector into the expression plasmid pMEK222 (pMEK222 is a gene3 deleted version of the phagemid pUR8100, and where the cloned variable domain is followed by FLAG-6His tags, two stop codons and the M13 terminator sequence (see WO2013/064701)). The variable domain genes were digested with Sfil and Eco91 I (or BstEll) and ligated into pMEK222 cut with the same restriction enzymes. E. coli strain BL21 DE3 was transformed by the ligations and plated on LB-agar plates supplemented with ampicillin and 2% glucose. Transformants were screened using colony PCR. Amplifications using the primers M13.rev (SEQ ID NO: 32) and M13.fw (SEQ ID NO: 33) led to the generation of plasmids containing inserts of 700 bp and of ˜350 bp (empty plasmids) observed by PCR.
Variable domains were produced from pMEK222 by inoculation of a fresh overnight grown culture at 1/100 dilution in 800 ml 2× YT, 0.1% glucose and 100 ug/ml ampicillin and grown for 2 h at 37 degrees C. Subsequently, 1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) was added and the culture was grown for an additional 5 h at 37 degrees C. Bacteria were harvested by centrifugation and resuspended into 30 mL PBS. Bacteria were frozen by incubation at −20 degrees C. overnight. Bacteria were thawed at room temperature and fractionated by centrifugation. To the soluble fraction, which contains the variable domain, Co2+ agarose beads, for example, Talon resin (Thermo Scientific) were added to bind His-tagged variable domain. After washing the beads, bound variable domains were eluted with PBS supplemented with 150 mM imidazole. Finally, fractions containing the variable domains were dialyzed against PBS to remove the imidazole.
A series of modified anti-TcdB ICVDs were produced by yeast expression of DNA constructs (see Preparative Methods section, above). Heterobiheads were linked using a [Gly4Ser]4 amino acid linker. The modified anti-TcdB ICVDs were the following:
The sequences of these modified ICVDs are provided in Table 1A.
4.1—Protocol for Preparing the Cytotoxicity Assay
Culture and Maintenance of Vero Cells Prior to Use
Routine subculture of Vero cells can be achieved as follows:
Preparing Plates for the Vero Cell Cytotoxity Assay (Day -1)
Ideally, plates should be prepared the day before use in the cytotoxicity assay. However, plates may also be prepared on the day of use if necessary. If the latter is the case, prepare plates in the morning (for use in the afternoon) and ensure that at least 3 hours are allowed for cell attachment to the microplate prior to use. A fully confluent flask of Vero cells should be used to make the cell suspension for plating.
Setting Up the Assay (Day 0)
Note: All solutions described in this section are prepared in Vero cell culture medium. You should calculate the required final volume of each toxin and ICVD to cover the number of plates/combinations before starting the assay. Mix all solutions well (by vortexing and/or multiple inversions) between dilution steps.
Processing the Assay (Day 3)
Preliminary Work: Determining the Optimal Amount of Toxin to Use in the Main Neutralisation Assay
For ease of interpretation in the main assay, the appropriate concentration of toxin to use should be determined beforehand by conducting a toxin dose-response experiment on Vero cells. Prepare 10 serial dilutions of toxin in a 12 well dilution trough. Use the remaining two wells for 0.01% Triton and a medium only control. Prepare a minimum of 330 μL of each solution in the dilution trough (this allows three replicates at 100 μl each). If there is no indication of how potent the toxin preparation is in advance, choose a broad dilution range for the preliminary experiment. This can be repeated over a finer concentration range, if necessary. Apply these solutions to Vero cells in a flat-bottomed microplate, incubate and process the plate as described above.
To assay an ICVD, or full antibody, for neutralisating activity against a given concentration of toxin, the minimum concentration of each toxin preparation capable of inducing the maximum reduction in cell viability is selected. An exemplary toxin dose-response curve on Vero cells is provided in
4.2—Potency Determination
Standard Vero cell assays were carried out using the protocol detailed under 4.1 above. Neutralisation curves were produced (Figures) and IC50 values were generated in Graphpad prism using the % neutralisation data and ‘log(inhibitor) vs. response—Variable slope (four parameters)’ to fit curves and generate IC50s. In some cases, IC50 was calculated by interpolation of an entered 50% value using the fitted curve. The calculated IC50 values from the different assays were as follows:
A hyphen denotes an assay which was not performed. It can be seen that the three unmodified ICVDs achieved good potency against both TcdB ribotypes 027 and 087, but B10F1 and Q31B1 were more potent against ribotype 027 than ribotype 087 (Table 2 and
Genes encoding ICVDs and ICVD constructs were designed based on B10F1 and Q31B1 in which R to H substitutions and an R to F substitution were introduced into the CDRs. These constructs were as follows:
5.1—Impact on Potency
Neutralising activities of these ICVDs and ICVD constructs were measured using standard Vero cell assays. These data show that the substitutions made in ID24B, ID25B and ID27B with respect to ID1 B and the substitutions made in ID20B, ID21 B and ID22B with respect to ID2B resulted in slightly reduced potency against ribotypes 027 and 087 (Table 3 and
ID41B and ID43B, although including single histidine substitutions in CDR3 of their Q31B1 component and a single histidine substitution in CDR3 (ID41 B) or a double histidine substitution in CDR3 (ID43B) of their B10F1 components, were extremely potent against ribotypes 027, 087, 078, 106, 001 and 017. The impact of the second histidine substitution in CDR3 of ID43B was limited (Table 3 and
The potencies of ID46B and ID49B, although including CDR3 arginine to histidine or phenylalanine substitutions respectively, were only slightly reduced compared to ID45B (Table 3 and
5.2—Impact on Protease Stability—Exposure to Trypsin and Chymotrypsin
ID11B and ID43B were incubated with trypsin or chymotrypsin beads for intervals of 0 (untreated control), 15, 30, 45 and 60 minutes. These data demonstrate that ID11B was digested by trypsin after between 15 and 30 minutes. ID43B however remained substantially undigested in trypsin and chymotrypsin (
5.3—Impact on Protease Stability—Exposure to Faecal Supernatant
ID11B and ID43B were incubated in faecal supernatants. The faecal supernatant pools were each produced from 5 faecal samples from either C. difficile positive patients (CD+) or C. difficile negative patients (CD−). The constructs were digested in the supernatants for 1 hour or as a separate experiment for 4 hours and then measured using an ELISA. ID43B showed much greater % survival than ID11 B after incubation for 1 hour (
A standard Vero cell assay was conducted to assess the potencies of ID11B, ID12B and ID43B relative to Medarex Mab124 (PCT Publication WO 2006/121422, in the name of University of Massachusetts and Medarex, Inc.) against TcdB produced by multiple C. difficile ribotypes.
The results show that ID11B, ID12B and ID43B are more potent than Mab124 against all ribotypes of TcdB tested here using the standard Vero cell assay. The difference in potencies between ID11B (
Quadrahead constructs comprising two different anti-TcdA ICVDs (based on Q34A3 and B4F10) and two different anti-TcdB ICVDs (based on Q31B1 and B10F1) were produced in yeast using the methodology detailed in the Preparative Methods section above. Each ICVD in each quadrahead was connected by a [Gly4Ser]4 linker. The format of these quadraheads is summarised in Table 4 below:
It can be seen from Table 4 above that the order of ICVDs in a quadrahead influences production level, with ID3C being produced at the highest level in yeast. ID-8C is ID5C with additional CDR3 R to H substitutions. ID11C is ID7C with additional CDR3 R to H substitutions.
The potency of these quadraheads against TcdA and TcdB from various C. difficile ribotypes was analysed using the cytotoxicity assay described in Example 4 above.
7.1—Potency of ID-1 C (AA′BB') and ID-3C (BAA′B′)
ID1C and ID3C were found to potently neutralise TcdA from ribotypes 027 and 087 (
7.2—Potency of ID-5C and ID-8C (BB′AA')
ID-5C was found to potently neutralise 027 TcdA (
ID8C is effectively a combination of anti-TcdA bihead ID33A and anti-TcdB bihead ID43B. The neutralising potency of ID-8C was compared to that of: (a) constituent bihead ID33A against TcdA from five ribotypes of C. difficile (
7.3—Potency of ID-6C (ABB′A′)
ID6C was found to potently neutralise 027 TcdA (
7.4—Potency of ID-7C and ID-11C (A′BB′A)
ID7C is effectively a combination of anti-TcdA bihead ID17A and anti-TcdB bihead ID41B. The neutralising potency of ID8C was compared to that of: (a) constituent bihead ID17A against TcdA from five ribotypes of C. difficile (
ID11C (ID7C with further CDR3 R to H modifications) also demonstrated potent neutralisation of 027 and 078 TcdA (
An experiment was performed to investigate whether or not ID-1 C and ID-3C bind both TcdA and TcdB simultaneously.
Two ELISA plates were set up to investigate the binding of ID-1 C and two to investigate the binding of ID-3C. Columns 1 to 3 of each plate were coated with 50 μl anti TcdA mAb PCG4.1. Columns 4 to 9 were coated with 027 TcdB or 087 TcdB. Columns 10 to 12 were left uncoated. The plates were incubated at 4° C. overnight, after which plates were washed, then all wells were blocked with 1% BSA in PBS for approximately 1.5 hours. Columns 6 to 12 were then incubated with 50 ul of (a) ID-1C, (b) ID-3C or (c) both ID-1 C and ID-3C, for 1 hour. The Plates were then washed and incubated with various concentrations (0-20 ng/ml) of either 027 TcdA or 087 TcdA. After 2 hours of incubation, plates were washed, then incubated with rabbit anti-TcdA pAb or with rabbit polyclonal anti-ICVD for 1 hour. Plates were washed again and swine anti-rabbit—HRP was added to every well for 1 hour. After washing, plates were incubated with 100 μl TMB, allowed to develop, stopped with 50 μl of 0.5M H2SO4, then read at 450 nM in a plate reader.
The results are shown in
The 027 (
Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. All patents and patent applications mentioned throughout the specification of the present invention are herein incorporated in their entirety by reference. The invention embraces all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of groups recited above.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15162117.4 | Mar 2015 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2016/057034 | Mar 2016 | US |
| Child | 15717214 | US |
