WO2014141145A1 - Modified molecules resistant to proteolytic degradation - Google Patents
Modified molecules resistant to proteolytic degradation Download PDFInfo
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- WO2014141145A1 WO2014141145A1 PCT/IB2014/059750 IB2014059750W WO2014141145A1 WO 2014141145 A1 WO2014141145 A1 WO 2014141145A1 IB 2014059750 W IB2014059750 W IB 2014059750W WO 2014141145 A1 WO2014141145 A1 WO 2014141145A1
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- containing molecule
- modified
- pseudolysin
- infections
- amino acid
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/522—CH1 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to modified Fc-containing molecules which are resistant to proteolytic degradation compared to wild-type Fc-containing molecules.
- the present invention relates to modified IgG or IgA molecules which are resistant to proteolytic degradation by pseudolysin/LasB secreted by any strain of Pseudomonas aeruginosa.
- Pseudomonas aeruginosa is an opportunistic pathogen responsible for 10-15% of nosocomial infections worldwide (Blanc et al., 1998); it is of increasing clinical relevance due to natural and acquired resistance to clinically-relevant antibiotics (Strateva & Yordanov, 2009; Pechere & Kohler, 1999).
- the greatest burden of morbidity and mortality is in immunocompromised patients, in patients suffering from chronic respiratory conditions such as cystic fibrosis (CF), and in patients with acute respiratory conditions such as ventilator-associated pneumonia (VAP).
- CF cystic fibrosis
- VAP ventilator-associated pneumonia
- aeruginosa virulence factor is a secreted endometalloprotease known as pseudolysin (also known as Pseudomonas elastase/PAE, or LasB), which is capable of cleaving immunoglobulins such as IgG and IgA, cytokines, and other host cell factors (Horvat et al., 1989; Kevin et al., 2003; Parmely et al., 1990; Theander et al., 1988; Schad et al., 1987; Diebel et al., 2009; Schultz & Miller, 1974; Jacquot et al., 1985).
- pseudolysin also known as Pseudomonas elastase/PAE, or LasB
- IgG or IgA molecules for the treatment of diseases associated with P. aeruginosa infection is therefore potentially compromised by the activity of this protease, since antibody therapeutics in this setting may rely on effector mechanisms such as antibody- dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC), which require the integrity of the antibody molecule to be maintained.
- ADCP antibody- dependent cellular phagocytosis
- CDC complement-dependent cytotoxicity
- physical separation of the antigen-binding variable domain from the Fc (fragment crystallisable) region by proteolytic cleavage in the antibody heavy chain may compromise mechanisms of bacterial clearance mediated by immune effector cells such as macrophages and neutrophils, in addition to compromising complement-mediated mechanisms.
- separation of the Fc region from the remaining antigen- binding fragment may also reduce the pharmacokinetics of this fragment due to reduced or abrogated FcRn (neonatal Fc receptor)-mediated recycling of IgG or FcaRI-mediated recycling of IgA.
- Pseudomonas aeruginosa is an opportunistic pathogen with a high rate of morbidity and mortality in immunocompromised hosts, and in patients with acute or chronic conditions caused, or exacerbated by, infection with this bacterial species.
- Diseases associated with P. aeruginosa include systemic infectious diseases caused by a P. aeruginosa infection including a multidrug resistant P. aeruginosa infection, for example, septicemia, meningitis, and endocarditis.
- aeruginosa include: otitis media and sinusitis in the otolaryngologic field; pneumonia, chronic respiratory tract infection, and catheter infection in the pulmonological field; postoperative peritonitis and postoperative infection in a biliary duct or the like in the surgical field; abscess of eyelid, dacryocystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, and orbital infection in the opthalmological field; and urinary tract infections (including complicated urinary tract infection), catheter infection, and abscess around the anus in the urologic field. Further examples thereof include burns (including a serious burn and a burn of the respiratory tract), decubital infection, and cystic fibrosis.
- P. aeruginosa secretes a variety of virulence factors in order to persist in the external environment, and in human and animal hosts.
- P. aeruginosa secretes a protease (which in the case of P. aeruginosa is interchangeably referred to as pseudolysin, LasB or Pseudomonas elastase/PAE), that cleaves a variety of host cell proteins.
- Pseudolysin directly destroys host tissues and damages cellular functions (Heck et al., 1986; Komori et al., 2001), or may indirectly interfere with host defence mechanisms by cleaving human IgG and IgA (Doring et al.
- Pseudolysin-mediated cleavage of antibodies targeting bacterial epitopes may result in reduced or abrogated antibody effector function, as well as reducing the half-life of the antibody by preventing recycling of the antibody.
- Pseudolysin was originally isolated and characterized as a zinc metalloprotease (Morihara et al. 1965); the full-length gene was subsequently cloned and sequenced from P. aeruginosa strain PA01 (Bever & Iglewski,1988) amongst others.
- P. aeruginosa strain PA01 Bacillus-encoded neutral proteases
- thermolysin thermolysin
- the present invention discloses the unexpected location of the pseudolysin cleavage site on a representative antibody molecule (human IgGl), and methods to resist such proteolytic cleavage in therapeutic immunoglobulin molecules.
- One embodiment of the present invention is the discovery of the pseudolysin cleavage site on hlgGl and the use of that information to produce modified molecules resistant to degradation; treatment of purified hlgGl with P. aeruginosa culture supernatant or purified pseudolysin, followed by N-terminal sequencing of the cleavage products, revealed the amino acid sequence of the cleavage site in the antibody heavy chain.
- P. aeruginosa culture supernatant or purified pseudolysin followed by N-terminal sequencing of the cleavage products, revealed the amino acid sequence of the cleavage site in the antibody heavy chain.
- bacterially-expressed proteases cleave in the hinge or lower hinge/C H 2 region; however, unexpectedly, the actual cleavage site was located in an alpha helix within the C H 1 domain.
- Another embodiment of the present invention is that it is now possible to develop antibody therapeutics targeting P. aeruginosa that are not susceptible to pseudolysin-mediated proteolysis, on the basis of the experimentally- defined specificity of the protease (Rawlings et al., 2012). Therefore, the present invention discloses that pseudolysin-mediated cleavage of hlgGl and related molecules occurs unexpectedly in the C H 1 region; mutant constructs which resist this cleavage event, which therefore represent an approach to improve the efficacy of therapeutic anti- >. aeruginosa antibody therapeutics, are described.
- a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease.
- a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase.
- a bacterial protease for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase.
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM.
- a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is human IgGl.
- modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is mouse IgG or rat IgG.
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is mouse IgGl or rat IgGl.
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces any amino acid in SEQ ID NO: 2 with any amino acid that resists proteolytic cleavage by pseudolysin based on its substrate specificity, such as that described in the MER
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces a hydrophobic amino acid residue at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with a hydrophilic amino acid residue.
- a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid.
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces any amino acid within the C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2) which resists proteolytic cleavage by pseudolysin based on substrate specificity.
- PSSSLGTQ SEQ ID NO: 2
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is human IgGl, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by
- a modified Fc -containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule
- the modified Fc-containing molecule comprises a modified C H I domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is is human IgGl.
- a method of treating or preventing Pseudomonas aeruginosa-aggvavated diseases or conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or I
- a method of treating or preventing Pseudomonas aeruginosa-aggvavated cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA,
- a method of treating or preventing Pseudomonas aeruginosa-aggvavated ventilator-associated pneumonia (VAP), hospital-acquired pneuomonia (HAP), or health care-associated pneumonia (HCAP) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elasta
- a pharmaceutical composition comprising a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, and a pharmaceutically acceptable carrier or excipient.
- a bacterial protease for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase
- a pharmaceutical composition comprising a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, and
- a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, in the manufacture of a medicament for the treatment or prevention of a disease selected from respiratory tract infections, cystic fibrosis, ventilator-associated pneumonia, leukopenia, neutropenia, urinary tract infections, gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, and severe burns.
- a disease selected from respiratory tract infections, cystic fibrosis, ventilat
- a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild- type Fc-containing molecule
- the modified Fc-containing molecule comprises a modified C H 1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule C H 1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified C H 1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, in the manufacture of
- Figure 1 is a SDS-PAGE gel of purified recombinant hlgGl treated with either bacterial supernatant from P. aeruginosa strain PA01 grown overnight in MHII culture media or the same purified recombinant hlgGl treated with purified recombinant pseudolysin.
- Lanes 1 and 6 Molecular weight markers
- Lane 2 and 7 Supernatant from PA01 culture alone, or purified pseudolysin alone, respectively
- Lane 3 and 8 hlgGl incubated only with PBS buffer
- Lane 4 and 9 hlgGl incubated with either PA01 supernatant, or purified pseudolysin, respectively.
- the arrows show the heavy chain ( ⁇ 50 kDa) and light chain ( ⁇ 25 kDa) of the antibody. Asterisks indicate the products formed after proteolytic cleavage of the antibody.
- Figure 2 is a PVDF blot of purified recombinant hlgGl treated with either PA01 bacterial supernatant grown in LB media (lanes 1 and 2) or purified pseudolysin (lanes 3 and 4) with bands marked for N-terminal sequencing.
- Figure 3 is the results of Edman sequencing of the bands marked in Figure 2.
- Figure 4 is the amino acid sequence of the pseudolysin cleavage site on hlgGl, which is the identical sequence in hIgG3, and hIgG4; the black arrow marks the experimentally-defined cleavage site as determined by N-terminal sequencing of the fragment marked in bold type which is formed by incubation of pseudolysin with hlgGl.
- Figure 6 is a list of potential mutations in hlgG to assess resistance to pseudolysin; amino acid positions refer to that described in Figure 4 and SEQ ID NO: l. Mutations in bold were assessed experimentally.
- Figure 7 is a SDS-PAGE gel of purified IgG molecules containing mutations designed to mediate resistance to pseudolysin.
- HC parental hlgGl antibody.
- IB L197P
- 2B S196Q/G198Q
- 3F L197P/S196Q/G198Q
- 4A S194L/S195Q/S196Q/ L197W/G198R/T199Q/Q200G (ie. within SEQ ID NO: 1, sequence PSSSLGTQ (SEQ ID NO: 2) from parental hlgGl swapped with PLQQWRQG from hlgD sequence).
- the purified antibody was assessed in non-denaturing and denaturing buffer (lanes 1, 3, 5, 7 and 9, non-reduced samples; lanes 2, 4, 6, 8 and 11, reduced samples).
- IB L197P
- 2B S196Q/G198Q
- 3F L197P/S196Q/G198Q
- 4A S194L/S195Q/S196Q/ L197W/G198R/T200Q/Q201G (ie. within SEQ ID NO: 1, sequence PSSSLGTQ (SEQ ID NO: 2) from parental hlgGl swapped with PLQQWRQG from hlgD sequence).
- mutants IB and 3F successfully blocked pseudolysin-mediated cleavage of hlgGl suggesting that mutation of leucine at position 197 is particularly effective in mediating resistance to pseudolysin.
- the swapped hlgD sequence was cleaved more effectively than the parental hlgGl sequence.
- Figure 9 is an example of a human IgGl VH-C H l-hinge-C H 2-C H 3 amino acid sequence with the pseudolysin cleavage site highlighted in bold (SEQ ID NO: 1), an example of a the pseudolysin cleavage site in the human IgGl C H 1 domain sequence that could be mutated to resist degradation by a bacterial protease (SEQ ID NO: 2), an example of a partial C H 1 domain sequence that results in a human IgGl that resists degradation by a bacterial protease encoded by P.
- SEQ ID NO: 1 an example of a the pseudolysin cleavage site in the human IgGl C H 1 domain sequence that could be mutated to resist degradation by a bacterial protease
- SEQ ID NO: 2 an example of a partial C H 1 domain sequence that results in a human IgGl that resists degradation by a bacterial protease encoded by P.
- aeruginosa SEQ ID NO: 3
- SEQ ID NO: 4 an example of a partial human IgD sequence corresponding to the pseudolysin cleavage site on hlgGl
- the experimentally-defined N-terminal sequence of hlgGl treated with either purified pseudolysin or supernatant from an overnight culture of P. aeruginosa strain PA01 SEQ ID NO: 5
- an additional experimentally-defined N- terminal sequence observed with hlgGl treated with supernatant from an overnight culture of P. aeruginosa strain PA01 but not purified pseudolysin SEQ ID NO: 6
- the invention therefore represents one method to improve the efficacy of such molecules by retaining the effector function of the antibody, critical for bacterial clearance via mechanisms such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP), and by maintaining the normal pharmacokinetics of the therapeutic antibody by retaining Fc-mediated recycling.
- CDC complement-dependent cytotoxicity
- ADCP antibody-dependent cellular phagocytosis
- ADCC Antibody dependent cell mediated cytotoxicity
- CDC Complement-dependent cytotoxicity
- ADCP antibody dependent cellular phagocytosis
- FcR Fc receptors
- FcR FcyRI
- FcyRII CD32
- FcyRIII CD16
- Effector function can be measured in a number of ways including for example via binding of the FcyRIII to Natural Killer cells or via FcyRI to monocytes/macrophages to measure for ADCC effector function.
- an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al, 2001 The Journal of Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of Biological Chemistry, Vol 268, p25124- 25131; Lazar et al, 2006 PNAS, 103; 4005-4010.
- pseudolysin cleaves hlgGl further embodiments will involve constructing viable antibody molecules that contain a modified amino sequence surrounding the cleavage site. This involves altering the amino acid sequence in that particular region to try and prevent it from being open to cleavage via single or multiple-point mutations, or additions or deletions of amino acid residues in this region. Such potentially pseudolysin-resistant molecules would then be tested by incubation with pseudolysin, based on assay methods discussed herein, for example, incubation with purified bacterial protease or bacterial supernatant followed by gel electrophoresis.
- a modified Fc -containing molecule such as an IgGl antibody according to the present invention is administered as a medicament to a subject, such as a human
- a human antibody is preferably used in terms of reducing side effects.
- the present invention provides the use of modified Fc-containing molecules to treat or prevent Pseudomonas aeruginosa-aggvavated diseases or conditions. Treatment may be therapeutic or prophylactic.
- the present invention also provides modified Fc-containing molecules alone or in combination with additional therapeutic agents for use in the treatment or prevention of Pseudomonas aerug/nosa-aggravate0 diseases or conditions.
- Fc-containing molecules include but are not limited to, binding proteins, antibodies, monoclonal antibodies, antigen-binding fragments, their humanised, human or chimaeric variants, and analogues thereof.
- the present invention may be used in a method of treatment of Pseudomonas aeruginosa-aggvavated diseases or conditions, the method comprising administering a safe and effective dose of the Fc- containing molecules of the present invention to a patient in need thereof.
- the Fc-containing molecules may be a human IgGl antibody which comprises a modified C H 1 domain wherein the sequence [193]- PSSSLGTQ (SEQ ID NO: 2) is mutated with an alternative amino acid that resists degradation by a bacterial protease.
- resistant to proteolytic degradation or “resists proteolytic cleavage” as used herein is meant to describe, for example, a modified Fc-containing molecule which is partially or completely resistant to proteolytic degradation compared to a wild- type Fc-containing molecule as judged by reduced formation of proteolytic breakdown products by standard qualitative analysis techniques, such as SDS-PAGE.
- the method of specifically quantifying the proteolytic degradation of IgG or related molecules in the presence of pseudolysin may be done by analysing specific regions on the SDS-PAGE gel (eg. monitoring reduction in heavy chain band at 50kDa, or increase in the two major breakdown products at 20 and 35kDa) using a standard imaging systems, such as a LI- COR ® Imaging System.
- Percent or comparable resistance could be calculated using either reduction in heavy chain band at 50kDa, or increase in the two major breakdown products at 20 and 35kDa, relative to wild type - sufficient replicates would be used to ensure statistical significance.
- the invention provides numbering of all amino acid sequences contained herein refer directly to numbering as described in SEQ ID NO: l.
- One skilled in the art would recognize that different numbering schemes may be used for the numbering of the modified Fc-containing molecules of the present invention, for example Kabat (Kabat 1991) and Eu (Edelman 1969).
- antigen binding protein refers to antibodies and other protein constructs, such as domains, which are capable of binding to an antigen.
- antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., VH, VHH, VL, domain antibody (dAbTM), antigen binding antibody fragments, Fab, F(ab 2 , Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies,
- immunoglobulin-like domain for example IgG, IgM, IgA, IgD or IgE
- a single variable domain e.g., VH, VHH, VL, domain antibody (dAbTM)
- Fab fragment antigen binding antibody fragments
- F(ab 2 , Fv, disulphide linked Fv single chain F
- TANDABSTM etc. and modified versions of any of the foregoing (for a summary of alternative "antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).
- domain refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
- Alternative antibody formats include alternative scaffolds in which the one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
- a suitable non-immunoglobulin protein scaffold or skeleton such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
- the invention provides a method of treating Pseudomonas aeruginosa diseases comprising administering to a subject in need thereof a therapeutically effective amount of a modified Fc-containing molecule and an additional therapeutic agent.
- a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody.
- Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).
- An immortalized cell producing the desired antibody may be selected by a conventional procedure.
- the hybridomas may be grown in culture or injected intra peritonea I ly for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
- the modified Fc-containing molecules of the present invention may be "human antibodies".
- Human antibodies may be produced by a number of methods known to those of skill in the art. Human antibodies can be made by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines see Kozbor J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal Antibody Production Techniques and Applications, pp51-63 (Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice both of which utilize human V region repertories (see Winter G, (1994), Annu.Rev.Immunol 12,433-455, Green LL (1999), J. Immunol. methods 231, 11-23).
- mice Several strains of transgenic mice are now available wherein their mouse immunoglobulin loci has been replaced with human immunoglobulin gene segments (see Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature Biotechnol. 14,845- 851, Mendez MJ, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing a repertoire of human antibodies from which antibodies of interest can be selected.
- Phage display technology can be used to produce human antibodies (and fragments thereof), see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD et al (1994) EMBO 13:3245-3260.
- the modified Fc-containing molecules of the present invention may be produced by methods known to the man skilled in the art.
- Antibodies of the present invention may be produced in transgenic organisms such as goats (see Pollock et al (1999), J. Immunol. Methods 231: 147-157), chickens (see Morrow KJJ (2000) Genet. Eng. News 20: 1-55), mice (see Pollock et al ibid) or plants (see Doran PM, (2000) Curr.Opinion Biotechnol. 11, 199-204, Ma JK-C (1998), Nat.Med. 4; 601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al, (2000) Plant Mol.Biol. 42:583-590).
- Antibodies may also be produced by chemical synthesis. However, antibodies of the invention are typically produced using recombinant cell culturing technology well known to those skilled in the art.
- a polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for further propagation or expression in a host cell.
- a replicable vector such as a plasmid for further propagation or expression in a host cell.
- One useful expression system is a glutamate synthetase system (such as sold by Lonza Biologies), particularly where the host cell is CHO or NS0 (see below).
- Polynucleotide encoding the antibody is readily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes).
- Vectors that may be used include plasmid, virus, phage, transposons, minichromsomes of which plasmids are a typical embodiment.
- such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription termination sequences operably linked to the light and/or heavy chain polynucleotide so as to facilitate expression.
- Polynucleotide encoding the light and heavy chains may be inserted into separate vectors and introduced (e.g. by transformation, transfection, electroporation or transduction) into the same host cell concurrently or sequentially or, if desired both the heavy chain and light chain can be inserted into the same vector prior to such introduction.
- the modified Fc-containing molecules may be administered to an individual in order to prevent the onset of one or more symptoms of the disease or condition.
- the subject may be asymptomatic.
- the subject may have a genetic predisposition to the disease.
- a prophylactically effective amount of the modified Fc- containing molecules is administered to such an individual.
- a prophylactically effective amount is an amount which prevents the onset of one or more symptoms of a disease or condition.
- a therapeutically effective amount of the modified Fc-containing molecules is an amount effective to ameliorate one or more symptoms of a disease or condition.
- the individual to be treated is human.
- the Fc-containing molecules of the invention may be administered to the subject by any suitable means.
- the Fc-containing molecules of the invention may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes.
- the Fc-containing molecules of the invention may be administered to the subject in such a way as to target therapy to a particular site.
- Fc-containing molecules of the invention may be administered directly to the site of a Pseudomonas aeruginosa infection.
- the Fc-containing molecules of the invention may be injected locally, for example intra-articularly or in one or more joints.
- the formulation of any of the Fc-containing molecules of the invention mentioned herein will depend upon factors such as the nature of the Fc-containing molecules of the invention and the condition to be treated.
- the Fc-containing molecules of the invention may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermal ⁇ or by infusion techniques.
- the Fc-containing molecules of the invention may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
- the Fc-containing molecules of the invention are formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art.
- the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
- solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
- starches arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
- Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
- Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
- the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
- Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
- the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
- Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
- binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
- Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropyl methyl cellulose.
- compositions suitable for delivery by needleless injection may also be used.
- a therapeutically effective amount of Fc -containing molecules of the invention is administered.
- the dose may be determined according to various parameters, especially according to the Fc -containing molecules of the invention used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
- a pharmaceutical composition comprises Fc- containing molecules of the invention and a pharmaceutically acceptable carrier or diluents.
- Such pharmaceutical compositions may further comprise pharmaceutically acceptable salts (and salts of the Fc-containing molecules of the invention) and optionally additional buffers, tonicity adjusting agents, pharmaceutically acceptable vehicles, and/or stabilizers.
- Pharmaceutically acceptable carriers or diluents are used to improve the tolerability of the composition and allow better solubility and better bioavailability of the active ingredients. Examples include emulsifiers, thickeners, redox components, starch, alcohol solutions, polyethylene glycol or lipids.
- suitable pharmaceutical carriers or diluents depends greatly on how the composition is to be administered. Liquid or solid carriers or diluents may be used for oral administration; whereas liquid compositions are required for injections.
- the pharmaceutical composition according to the invention may further comprise buffers or tonicity-adjusting agents.
- the pH of the composition may be adjusted to correspond to physiological pH by means of buffers, and fluctuations in pH may also be buffered.
- Buffers include, but are not limited to, for example a phosphate buffer or a MES (2-(N-morpholino)ethanesulfonic acid) buffer.
- the pH of the pharmaceutical composition is between 4 and 10, such as between 5 and 9.5, such as between 6 and 9.
- Tonicity adjusting agents are used to adjust the osmolarity of the composition and may contain ionic substances, such as inorganic salts, e.g., NaCI, or nonionic substances, such as glycerol or carbohydrates.
- the pharmaceutical composition according to the invention is suitably prepared for systemic, topical, oral or intranasal administration. These modes of administration of the pharmaceutical composition allow a rapid and uncomplicated uptake of active substance.
- solid and/or liquid medications may be administered directly, or alternatively may be dissolved and/or diluted prior to administration.
- the pharmaceutical compositions are liquid, in particular aqueous compositions.
- the pharmaceutical composition according to the invention may be suitably prepared for intravenous, intra-arterial, intramuscular, intravascular, intraperitoneal or subcutaneous administration. For example, injections or transfusions may be used. Administration directly into the bloodstream has the advantage that the active substance(s) of the composition are distributed throughout the entire body for systemic therapy, and rapidly reach the target tissue(s).
- the pharmaceutical composition is prepared for intravenous administration, and may be in liquid form.
- PVDF polyvinyl idene difluoride
- the proteins at approximately 50 kDa and 25 kDa represent the heavy and light chains of hlgGl.
- Lanes 1 and 2 are identical samples (hlgGl treated with P. aeruginosa culture supernatant); similarly, lanes 3 and 4 are identical samples (hlgGl treated with purified pseudolysin). Consistent with the data described above, two major cleavage products were observed at approximately 35kDa and 20kDa; additional minor bands were observed in P. aeruginosa supernatant-treated samples only.
- N-terminal sequencing of the ⁇ 35 kDa fragment observed after treatment with both P. aeruginosa culture supernatant (marked 'Supernatant band 2' in lane 2) and purified pseudolysin (marked 'Purified band 1' in lane 3) suggested the identity of this fragment was a partial sequence in the heavy chain C H 1 domain, and, by inference from the molecular weight of the product, this partial C H 1 sequence is likely fused to the remainder of the heavy chain sequence (hinge-C H 2-C H 3).
- N-terminal sequence of this product was [197]-LGTQTYIXNV (SEQ ID NO: 5) (where, based on the known sequence of the heavy chain, X is a cysteine residue that could not be resolved by Edman degradation). This suggested that the cleavage site was further towards the N- terminus of the heavy chain than was expected based on similar IgG-cleaving proteases from other bacterial pathogens (Breski & Jordan, 2010) and an initial analysis of potential cleavage sites based on preferred substrate amino acids obtained from the MEROPS database (Rawlings et al., 2012).
- an amino acid with a small side chain such as serine, glycine or alanine at the PI site (the amino acid immediately preceding cleavage site) and the P2' site (the second amino acid following the cleavage site) was identified by analysis of the MEROPS database (Rawlings et al., 2012); conversely, amino acids with large side chains such as tryptophan or glutamine are less favoured in these positions. Therefore it was postulated that mutation of serine at PI and glycine at P2' in the hlgGl sequence to glutamine may reduce/resist pseudolysin-mediated cleavage of hlgGl and related molecules.
- the human IgD antibody crystal structure (1ZVO) suggests the presence of a similar helix to IgGl in this region, while the amino acid sequence suggests this is a poor substrate for pseudolysin. Therefore, substitution of the hlgGl sequence in this region (PSSSLGTQ (SEQ ID NO :2)- the identical sequence in hlgGl, hIgG3, and hIgG4 but differing in one amino acid in hIgG2) with the hlgD sequence (PLQQWRQG; SEQ ID NO: 4) may result in reduced or abrogated sensitivity to pseudolysin.
- mutant constructs were generated by site- directed mutagenesis. Single, double, and triple mutations were tested (L197P, S196Q/G198Q, and L197P/S196Q/G198Q) in addition to swapping the IgGl sequence with the corresponding sequence from IgD, as described in Example 4.
- Each mutant antibody was prepared by co-transfection of a plasmid capable of expressing the mutated heavy chain with a plasmid expressing the antibody light chain in human embryonic kidney (HEK) cells.
- Figure 7 shows a SDS-PAGE gel of the mutant antibodies affinity purified on protein A-sepharose (lanes 3-10; each antibody was assessed under non-denaturing and denaturing conditions); the parental antibody was expressed and purified in parallel with the mutant constructs and is shown in lanes 1 and 2 (HC).
- each mutant antibody was assessed for resistance to treatment with pseudolysin relative to the pseudolysin-sensitive parental antibody by co-incubation of bacterial supernatant as described in Example 1; as judged by the disappearance of proteolytic breakdown products at approximately 20 and 35 kDa (Figure 8), mutants IB (L197P) and 3F (L197P/S196Q/G198Q) successfully resisted pseduolysin-mediated cleavage.
- Mutant 2B (S196Q/G198Q) had no discernible effect, perhaps suggesting that in this panel of constructs, mutation of the leucine at position 197 is particularly important in permitting cleavage of IgG by pseudolysin.
- Mutant 4A (containing the IgD sequence surrounding the pseudolysin cleavage site in place of the IgGl sequence) did not resist proteolysis by pseudolysin, indicating that although this sequence is a poor substrate, it is still cleaved by pseudolysin; in fact, the swapped hlgD sequence was cleaved more effectively than the parental hlgGl sequence as judged by the reduced presence of the full-length heavy chain in lane 11 compared with the parental antibody (lane 3).
- the 20 kDa fragment is composed of the V H region fused to the remainder of the C H 1 region; this was not experimentally observed, likely due to the presence of pyroglutamate at the N-terminus of the heavy chain.
- pseudolysin cleaves at a site far removed from the hinge or lower hinge/C H 2 region.
- amino acid preferences of pseudolysin include:
- Embodiments of the invention therefore include constructs developed to focus on mutations to the PI, PI' and P2' sites, using single mutations and combinations of mutations to oppose the preferences of pseudolysin substrate specificity, such as L197P (SEQ ID NO: 3). Mutations that might be expected to compromise the folding of the antibody were avoided.
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Abstract
An object of the present invention is to provide modified Fc-containing molecules that are resistant to proteolytic degradation compared to wild-type Fc-containing molecules to be used in the prevention or treatment of diseases associated with Pseudomonas aeruginosa. According to the present invention, there is provided modified Fc-containing molecules are resistant to proteolytic degradation by pseudolysin/LasB secreted by any strain of Pseudomonas aeruginosa.
Description
MODIFIED MOLECULES RESISTANT TO PROTEOLYTIC DEGRADATION
FIELD OF THE INVENTION
The present invention relates to modified Fc-containing molecules which are resistant to proteolytic degradation compared to wild-type Fc-containing molecules. In particular, the present invention relates to modified IgG or IgA molecules which are resistant to proteolytic degradation by pseudolysin/LasB secreted by any strain of Pseudomonas aeruginosa. BACKGROUND OF THE INVENTION
Pseudomonas aeruginosa is an opportunistic pathogen responsible for 10-15% of nosocomial infections worldwide (Blanc et al., 1998); it is of increasing clinical relevance due to natural and acquired resistance to clinically-relevant antibiotics (Strateva & Yordanov, 2009; Pechere & Kohler, 1999). The greatest burden of morbidity and mortality is in immunocompromised patients, in patients suffering from chronic respiratory conditions such as cystic fibrosis (CF), and in patients with acute respiratory conditions such as ventilator-associated pneumonia (VAP). A key P. aeruginosa virulence factor is a secreted endometalloprotease known as pseudolysin (also known as Pseudomonas elastase/PAE, or LasB), which is capable of cleaving immunoglobulins such as IgG and IgA, cytokines, and other host cell factors (Horvat et al., 1989; Kevin et al., 2003; Parmely et al., 1990; Theander et al., 1988; Schad et al., 1987; Diebel et al., 2009; Schultz & Miller, 1974; Jacquot et al., 1985). The use of therapeutic IgG or IgA molecules for the treatment of diseases associated with P. aeruginosa infection is therefore potentially compromised by the activity of this protease, since antibody therapeutics in this setting may rely on effector mechanisms such as antibody- dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC), which require the integrity of the antibody molecule to be maintained. Specifically, physical separation of the antigen-binding variable domain from the Fc (fragment crystallisable) region by proteolytic cleavage in the antibody heavy chain may compromise mechanisms of bacterial clearance mediated by immune effector cells such as macrophages and neutrophils, in addition to compromising complement-mediated mechanisms. Furthermore, separation of the Fc region from the remaining antigen- binding fragment may also reduce the pharmacokinetics of this fragment due to reduced
or abrogated FcRn (neonatal Fc receptor)-mediated recycling of IgG or FcaRI-mediated recycling of IgA.
Pseudomonas aeruginosa is an opportunistic pathogen with a high rate of morbidity and mortality in immunocompromised hosts, and in patients with acute or chronic conditions caused, or exacerbated by, infection with this bacterial species. .Examples of the "diseases" associated with P. aeruginosa include systemic infectious diseases caused by a P. aeruginosa infection including a multidrug resistant P. aeruginosa infection, for example, septicemia, meningitis, and endocarditis. Alternative examples of the diseases associated with P. aeruginosa include: otitis media and sinusitis in the otolaryngologic field; pneumonia, chronic respiratory tract infection, and catheter infection in the pulmonological field; postoperative peritonitis and postoperative infection in a biliary duct or the like in the surgical field; abscess of eyelid, dacryocystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, and orbital infection in the opthalmological field; and urinary tract infections (including complicated urinary tract infection), catheter infection, and abscess around the anus in the urologic field. Further examples thereof include burns (including a serious burn and a burn of the respiratory tract), decubital infection, and cystic fibrosis.
P. aeruginosa secretes a variety of virulence factors in order to persist in the external environment, and in human and animal hosts. As for many other bacterial pathogens, P. aeruginosa secretes a protease (which in the case of P. aeruginosa is interchangeably referred to as pseudolysin, LasB or Pseudomonas elastase/PAE), that cleaves a variety of host cell proteins. Pseudolysin directly destroys host tissues and damages cellular functions (Heck et al., 1986; Komori et al., 2001), or may indirectly interfere with host defence mechanisms by cleaving human IgG and IgA (Doring et al. 1981; Holder & Wheeler, 1984) amongst other host cell factors. Pseudolysin-mediated cleavage of antibodies targeting bacterial epitopes may result in reduced or abrogated antibody effector function, as well as reducing the half-life of the antibody by preventing recycling of the antibody.
Pseudolysin was originally isolated and characterized as a zinc metalloprotease (Morihara et al. 1965); the full-length gene was subsequently cloned and sequenced from P. aeruginosa strain PA01 (Bever & Iglewski,1988) amongst others. One of the features of this particular enzyme is its large active site cleft which may in part explain the lack of specificity of the enzyme (Thayer et al., 1991). Pseudolysin appears to be an
evolutionarily distant relative of Bacillus-encoded neutral proteases such as thermolysin (Thayer et al., 1991).
SUMMARY OF THE INVENTION
The present invention discloses the unexpected location of the pseudolysin cleavage site on a representative antibody molecule (human IgGl), and methods to resist such proteolytic cleavage in therapeutic immunoglobulin molecules.
One embodiment of the present invention is the discovery of the pseudolysin cleavage site on hlgGl and the use of that information to produce modified molecules resistant to degradation; treatment of purified hlgGl with P. aeruginosa culture supernatant or purified pseudolysin, followed by N-terminal sequencing of the cleavage products, revealed the amino acid sequence of the cleavage site in the antibody heavy chain. Based on known cleavage sites for other bacterial pathogens (Breski & Jordan, 2010), bacterially-expressed proteases cleave in the hinge or lower hinge/CH2 region; however, unexpectedly, the actual cleavage site was located in an alpha helix within the CH1 domain.
Using this information, another embodiment of the present invention is that it is now possible to develop antibody therapeutics targeting P. aeruginosa that are not susceptible to pseudolysin-mediated proteolysis, on the basis of the experimentally- defined specificity of the protease (Rawlings et al., 2012). Therefore, the present invention discloses that pseudolysin-mediated cleavage of hlgGl and related molecules occurs unexpectedly in the CH1 region; mutant constructs which resist this cleavage event, which therefore represent an approach to improve the efficacy of therapeutic anti- >. aeruginosa antibody therapeutics, are described.
In one embodiment of the present invention a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease.
In another embodiment of the present invention a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in
the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase.
In yet another embodiment of the present invention there is a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM.
In one embodiment of the present invention a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is human IgGl.
Other embodiments of the present invention include a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is mouse IgG or rat IgG.
In yet another embodiment of the present invention there is a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ
(SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is mouse IgGl or rat IgGl.
In another embodiment of the present invention there is a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces any amino acid in SEQ ID NO: 2 with any amino acid that resists proteolytic cleavage by pseudolysin based on its substrate specificity, such as that described in the MEROPS database (Rawlings et al., 2012).
In yet another embodiment of the present invention there is a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces a hydrophobic amino acid residue at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with a hydrophilic amino acid residue.
In another embodiment of the present invention there is a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM,
and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid.
In yet another embodiment of the present invention there is a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces any amino acid within the CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2) which resists proteolytic cleavage by pseudolysin based on substrate specificity.
In one embodiment of the present invention, there is a use of a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is human IgGl, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In another embodiment of the present invention, there is a use of a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In yet another embodiment of the present invention a modified Fc -containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc- containing molecule, wherein the modified Fc-containing molecule comprises a modified CHI domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is is human IgGl.
In one embodiment of the present invention, there is a method of treating or preventing Pseudomonas aeruginosa-aggvavated diseases or conditions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule which is resistant to proteolytic degradation by pseudolysin.
In another embodiment of the present invention, there is a method of treating or preventing Pseudomonas aeruginosa-aggvavated diseases or conditions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In another embodiment of the present invention, there is a method of treating or preventing Pseudomonas aeruginosa-aggvavated cystic fibrosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain
sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In another embodiment of the present invention, there is a method of treating or preventing Pseudomonas aeruginosa-aggvavated ventilator-associated pneumonia (VAP), hospital-acquired pneuomonia (HAP), or health care-associated pneumonia (HCAP) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc- containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In another embodiment of the present invention, there is a method of treating or preventing Pseudomonas aeruginosa-aggvavated respiratory tract infections, leukopenia, neutropenia, urinary tract infections, gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, or severe burns in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any
isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
In another embodiment of the present invention there is a pharmaceutical composition comprising a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, and a pharmaceutically acceptable carrier or excipient.
In yet another embodiment of the present invention there is a pharmaceutical composition comprising a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc- containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, and a pharmaceutically acceptable carrier or excipient.
In one embodiment of the present invention, there is a use of a modified Fc- containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, in the manufacture of a medicament for the treatment or prevention of a disease selected from respiratory tract infections, cystic fibrosis, ventilator-associated pneumonia, leukopenia, neutropenia, urinary tract infections,
gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, and severe burns.
In another one embodiment of the present invention, there is a use of a modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild- type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease, for example, Pseudomonas aeruginosa pseudolysin, also known as LasB or Pseudomonas elastase, wherein the Fc-containing molecule is any isotype of human IgG, IgA, or IgM, and further wherein the substitution replaces leucine at position 5 of SEQ ID NO: 2 (or 197 of SEQ ID NO: 1) with proline or any other hydrophillic amino acid, in the manufacture of a medicament for the treatment or prevention of a disease selected from respiratory tract infections, cystic fibrosis, ventilator-associated pneumonia, leukopenia, neutropenia, urinary tract infections, gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, and severe burns.
It is to be understood that both the foregoing summary description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention as claimed.
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in, and constitute a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a SDS-PAGE gel of purified recombinant hlgGl treated with either bacterial supernatant from P. aeruginosa strain PA01 grown overnight in MHII culture media or the same purified recombinant hlgGl treated with purified recombinant pseudolysin. Lanes 1 and 6: Molecular weight markers; Lane 2 and 7: Supernatant from PA01 culture alone, or purified pseudolysin alone, respectively; Lane 3 and 8: hlgGl incubated only with PBS buffer; Lane 4 and 9: hlgGl incubated with either PA01 supernatant, or purified pseudolysin, respectively. The arrows show the heavy chain
(~50 kDa) and light chain (~25 kDa) of the antibody. Asterisks indicate the products formed after proteolytic cleavage of the antibody.
Figure 2 is a PVDF blot of purified recombinant hlgGl treated with either PA01 bacterial supernatant grown in LB media (lanes 1 and 2) or purified pseudolysin (lanes 3 and 4) with bands marked for N-terminal sequencing.
Figure 3 is the results of Edman sequencing of the bands marked in Figure 2. Figure 4 is the amino acid sequence of the pseudolysin cleavage site on hlgGl, which is the identical sequence in hIgG3, and hIgG4; the black arrow marks the experimentally-defined cleavage site as determined by N-terminal sequencing of the fragment marked in bold type which is formed by incubation of pseudolysin with hlgGl.
Figure 5 marks the pseudolysin cleavage site in the CH1 domain of hlgGl on a crystal structure deposited in the PDB database (1HZH); dark grey ball and stick = P4- Pl; light grey ball and stick = Pl'-P4'. It can be seen that the cleavage site sits in the middle of an alpha helix.
Figure 6 is a list of potential mutations in hlgG to assess resistance to pseudolysin; amino acid positions refer to that described in Figure 4 and SEQ ID NO: l. Mutations in bold were assessed experimentally.
Figure 7 is a SDS-PAGE gel of purified IgG molecules containing mutations designed to mediate resistance to pseudolysin. HC = parental hlgGl antibody. IB = L197P, 2B = S196Q/G198Q, 3F = L197P/S196Q/G198Q, and 4A = S194L/S195Q/S196Q/ L197W/G198R/T199Q/Q200G (ie. within SEQ ID NO: 1, sequence PSSSLGTQ (SEQ ID NO: 2) from parental hlgGl swapped with PLQQWRQG from hlgD sequence). In each case the purified antibody was assessed in non-denaturing and denaturing buffer (lanes 1, 3, 5, 7 and 9, non-reduced samples; lanes 2, 4, 6, 8 and 11, reduced samples).
Figure 8 is a SDS-PAGE gel of purified mutant IgG molecules treated with bacterial supernatant; HC = parental hlgGl antibody. IB = L197P, 2B = S196Q/G198Q, 3F = L197P/S196Q/G198Q, and 4A = S194L/S195Q/S196Q/ L197W/G198R/T200Q/Q201G (ie. within SEQ ID NO: 1, sequence PSSSLGTQ (SEQ ID NO: 2) from parental hlgGl swapped with PLQQWRQG from hlgD sequence). As can be seen in this figure, mutants IB and 3F successfully blocked pseudolysin-mediated cleavage of hlgGl suggesting that mutation of leucine at position 197 is particularly effective in mediating resistance to pseudolysin. Unexpectedly, the swapped hlgD sequence was cleaved more effectively than the parental hlgGl sequence.
Figure 9 is an example of a human IgGl VH-CHl-hinge-CH2-CH3 amino acid sequence with the pseudolysin cleavage site highlighted in bold (SEQ ID NO: 1), an example of a the pseudolysin cleavage site in the human IgGl CH1 domain sequence that could be mutated to resist degradation by a bacterial protease (SEQ ID NO: 2), an example of a partial CH1 domain sequence that results in a human IgGl that resists degradation by a bacterial protease encoded by P. aeruginosa (SEQ ID NO: 3), an example of a partial human IgD sequence corresponding to the pseudolysin cleavage site on hlgGl (SEQ ID NO: 4), the experimentally-defined N-terminal sequence of hlgGl treated with either purified pseudolysin or supernatant from an overnight culture of P. aeruginosa strain PA01 (SEQ ID NO: 5), and an additional experimentally-defined N- terminal sequence observed with hlgGl treated with supernatant from an overnight culture of P. aeruginosa strain PA01 but not purified pseudolysin (SEQ ID NO: 6), representing further clipping of a fragment with N-terminus as defined in SEQ ID NO: 5. DETAILED DESCRIPTION OF THE INVENTION
Pseudolysin-mediated cleavage of purified recombinant IgG molecules was confirmed in vitro and the cleavage products analysed to determine the amino acid sequence of the cleavage site; ultimately, this approach was taken to define antibody molecules with an altered sequence that may be resistant to proteolytic degradation caused by pseudolysin when used in pre-clinical or clinical setting involving infection with P. aeruginosa. The invention therefore represents one method to improve the efficacy of such molecules by retaining the effector function of the antibody, critical for bacterial clearance via mechanisms such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP), and by maintaining the normal pharmacokinetics of the therapeutic antibody by retaining Fc-mediated recycling.
The term "Effector Function" as used herein is meant to refer to one or more of Antibody dependent cell mediated cytotoxicity (ADCC), Complement-dependent cytotoxicity (CDC), Fc-mediated phagocytosis or antibody dependent cellular phagocytosis (ADCP) and antibody recycling via the FcRn or FcaRI receptor.
The interaction between the constant region of an antigen binding protein and various Fc receptors (FcR) including FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) is believed to mediate the effector functions of the antigen binding protein. Significant biological effects can be a consequence of effector functionality. Usually, the ability to
mediate effector function requires binding of the antigen binding protein to an antigen and not all antigen binding proteins will mediate every effector function.
Effector function can be measured in a number of ways including for example via binding of the FcyRIII to Natural Killer cells or via FcyRI to monocytes/macrophages to measure for ADCC effector function. For example an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al, 2001 The Journal of Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of Biological Chemistry, Vol 268, p25124- 25131; Lazar et al, 2006 PNAS, 103; 4005-4010.
Examples of assays to determine CDC function include that described in 1995 J Imm
Meth 184:29-38.
Since the present invention has established where pseudolysin cleaves hlgGl, further embodiments will involve constructing viable antibody molecules that contain a modified amino sequence surrounding the cleavage site. This involves altering the amino acid sequence in that particular region to try and prevent it from being open to cleavage via single or multiple-point mutations, or additions or deletions of amino acid residues in this region. Such potentially pseudolysin-resistant molecules would then be tested by incubation with pseudolysin, based on assay methods discussed herein, for example, incubation with purified bacterial protease or bacterial supernatant followed by gel electrophoresis.
When a modified Fc -containing molecule, such as an IgGl antibody according to the present invention is administered as a medicament to a subject, such as a human, a human antibody is preferably used in terms of reducing side effects.
The present invention provides the use of modified Fc-containing molecules to treat or prevent Pseudomonas aeruginosa-aggvavated diseases or conditions. Treatment may be therapeutic or prophylactic.
The present invention also provides modified Fc-containing molecules alone or in combination with additional therapeutic agents for use in the treatment or prevention of Pseudomonas aerug/nosa-aggravate0 diseases or conditions.
The term "Fc-containing molecules", include but are not limited to, binding proteins, antibodies, monoclonal antibodies, antigen-binding fragments, their humanised, human or chimaeric variants, and analogues thereof. The present invention may be used in a method of treatment of Pseudomonas aeruginosa-aggvavated diseases or conditions, the method comprising administering a safe and effective dose of the Fc-
containing molecules of the present invention to a patient in need thereof. In one embodiment of the present invention the Fc-containing molecules may be a human IgGl antibody which comprises a modified CH1 domain wherein the sequence [193]- PSSSLGTQ (SEQ ID NO: 2) is mutated with an alternative amino acid that resists degradation by a bacterial protease.
The terms "resistant to proteolytic degradation" or "resists proteolytic cleavage" as used herein is meant to describe, for example, a modified Fc-containing molecule which is partially or completely resistant to proteolytic degradation compared to a wild- type Fc-containing molecule as judged by reduced formation of proteolytic breakdown products by standard qualitative analysis techniques, such as SDS-PAGE. The method of specifically quantifying the proteolytic degradation of IgG or related molecules in the presence of pseudolysin may be done by analysing specific regions on the SDS-PAGE gel (eg. monitoring reduction in heavy chain band at 50kDa, or increase in the two major breakdown products at 20 and 35kDa) using a standard imaging systems, such as a LI- COR® Imaging System. These imaging systems use software that allow one skilled in the art to quantify the intensity of specific proteins at defined molecular weights. Percent or comparable resistance could be calculated using either reduction in heavy chain band at 50kDa, or increase in the two major breakdown products at 20 and 35kDa, relative to wild type - sufficient replicates would be used to ensure statistical significance.
In another aspect, the invention provides numbering of all amino acid sequences contained herein refer directly to numbering as described in SEQ ID NO: l. One skilled in the art would recognize that different numbering schemes may be used for the numbering of the modified Fc-containing molecules of the present invention, for example Kabat (Kabat 1991) and Eu (Edelman 1969).
The term "antigen binding protein" as used herein refers to antibodies and other protein constructs, such as domains, which are capable of binding to an antigen.
The term "antibody" is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., VH, VHH, VL, domain antibody (dAb™), antigen binding antibody fragments, Fab, F(ab 2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies,
TANDABS™, etc. and modified versions of any of the foregoing (for a summary of alternative
"antibody" formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).
The term "domain" refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
Alternative antibody formats include alternative scaffolds in which the one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
In another aspect, the invention provides a method of treating Pseudomonas aeruginosa diseases comprising administering to a subject in need thereof a therapeutically effective amount of a modified Fc-containing molecule and an additional therapeutic agent.
A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497). An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intra peritonea I ly for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
The modified Fc-containing molecules of the present invention may be "human antibodies". Human antibodies may be produced by a number of methods known to those of skill in the art. Human antibodies can be made by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines see Kozbor J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal Antibody Production Techniques and Applications, pp51-63 (Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice both of which utilize human V region repertories (see Winter G, (1994), Annu.Rev.Immunol 12,433-455, Green LL (1999), J. Immunol. methods 231, 11-23).
Several strains of transgenic mice are now available wherein their mouse immunoglobulin loci has been replaced with human immunoglobulin gene segments (see
Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature Biotechnol. 14,845- 851, Mendez MJ, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing a repertoire of human antibodies from which antibodies of interest can be selected.
Phage display technology can be used to produce human antibodies (and fragments thereof), see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD et al (1994) EMBO 13:3245-3260.
The modified Fc-containing molecules of the present invention may be produced by methods known to the man skilled in the art. Antibodies of the present invention may be produced in transgenic organisms such as goats (see Pollock et al (1999), J. Immunol. Methods 231: 147-157), chickens (see Morrow KJJ (2000) Genet. Eng. News 20: 1-55), mice (see Pollock et al ibid) or plants (see Doran PM, (2000) Curr.Opinion Biotechnol. 11, 199-204, Ma JK-C (1998), Nat.Med. 4; 601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al, (2000) Plant Mol.Biol. 42:583-590). Antibodies may also be produced by chemical synthesis. However, antibodies of the invention are typically produced using recombinant cell culturing technology well known to those skilled in the art. A polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for further propagation or expression in a host cell. One useful expression system is a glutamate synthetase system (such as sold by Lonza Biologies), particularly where the host cell is CHO or NS0 (see below). Polynucleotide encoding the antibody is readily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes). Vectors that may be used include plasmid, virus, phage, transposons, minichromsomes of which plasmids are a typical embodiment. Generally such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription termination sequences operably linked to the light and/or heavy chain polynucleotide so as to facilitate expression. Polynucleotide encoding the light and heavy chains may be inserted into separate vectors and introduced (e.g. by transformation, transfection, electroporation or transduction) into the same host cell concurrently or sequentially or, if desired both the heavy chain and light chain can be inserted into the same vector prior to such introduction.
The modified Fc-containing molecules may be administered to an individual in order to prevent the onset of one or more symptoms of the disease or condition. In this embodiment, the subject may be asymptomatic. The subject may have a genetic
predisposition to the disease. A prophylactically effective amount of the modified Fc- containing molecules is administered to such an individual. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of a disease or condition.
A therapeutically effective amount of the modified Fc-containing molecules is an amount effective to ameliorate one or more symptoms of a disease or condition. Preferably, the individual to be treated is human.
The Fc-containing molecules of the invention may be administered to the subject by any suitable means. The Fc-containing molecules of the invention may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes.
The Fc-containing molecules of the invention may be administered to the subject in such a way as to target therapy to a particular site. For example, Fc-containing molecules of the invention may be administered directly to the site of a Pseudomonas aeruginosa infection. The Fc-containing molecules of the invention may be injected locally, for example intra-articularly or in one or more joints.
The formulation of any of the Fc-containing molecules of the invention mentioned herein will depend upon factors such as the nature of the Fc-containing molecules of the invention and the condition to be treated. The Fc-containing molecules of the invention may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermal^ or by infusion techniques. The Fc-containing molecules of the invention may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
Typically the Fc-containing molecules of the invention are formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating
agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropyl methyl cellulose.
Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermal^, may also be used.
A therapeutically effective amount of Fc -containing molecules of the invention is administered. The dose may be determined according to various parameters, especially according to the Fc -containing molecules of the invention used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
According to the present invention, a pharmaceutical composition comprises Fc- containing molecules of the invention and a pharmaceutically acceptable carrier or diluents. Such pharmaceutical compositions may further comprise pharmaceutically acceptable salts (and salts of the Fc-containing molecules of the invention) and optionally additional buffers, tonicity adjusting agents, pharmaceutically acceptable vehicles, and/or stabilizers. Pharmaceutically acceptable carriers or diluents are used to improve the tolerability of the composition and allow better solubility and better bioavailability of the active ingredients. Examples include emulsifiers, thickeners, redox components, starch, alcohol solutions, polyethylene glycol or lipids. The choice of suitable pharmaceutical carriers or diluents depends greatly on how the composition is to be administered. Liquid or solid carriers or diluents may be used for oral administration; whereas liquid compositions are required for injections.
The pharmaceutical composition according to the invention may further comprise buffers or tonicity-adjusting agents. The pH of the composition may be adjusted to correspond to physiological pH by means of buffers, and fluctuations in pH may also be buffered. Buffers include, but are not limited to, for example a phosphate buffer or a MES (2-(N-morpholino)ethanesulfonic acid) buffer. In one embodiment, the pH of the pharmaceutical composition is between 4 and 10, such as between 5 and 9.5, such as between 6 and 9. Tonicity adjusting agents are used to adjust the osmolarity of the composition and may contain ionic substances, such as inorganic salts, e.g., NaCI, or nonionic substances, such as glycerol or carbohydrates.
The pharmaceutical composition according to the invention is suitably prepared for systemic, topical, oral or intranasal administration. These modes of administration of the pharmaceutical composition allow a rapid and uncomplicated uptake of active substance. For example, for oral administration, solid and/or liquid medications may be administered directly, or alternatively may be dissolved and/or diluted prior to administration. In one embodiment, the pharmaceutical compositions are liquid, in particular aqueous compositions.
The pharmaceutical composition according to the invention may be suitably prepared for intravenous, intra-arterial, intramuscular, intravascular, intraperitoneal or subcutaneous administration. For example, injections or transfusions may be used. Administration directly into the bloodstream has the advantage that the active substance(s) of the composition are distributed throughout the entire body for systemic therapy, and rapidly reach the target tissue(s). In one embodiment, the pharmaceutical composition is prepared for intravenous administration, and may be in liquid form.
EXAMPLES
The invention is further illustrated by way of the following examples which are intended to elucidate the invention. These examples are not intended, nor are they to be construed, as limiting the scope of the invention. Numerous modifications and variations of the present invention are possible in view of the teachings herein and, therefore, are within the scope of the invention. The examples below are carried out using standard techniques, and such standard techniques are well known and routine to those of skill in the art, except where otherwise described in detail.
Example 1: Confirmation of Pseudolysin-mediated IgG-cleaving activity
Supernatant harvested from an overnight culture of Pseudomonas aeruginosa strain PAOl (ATCC number: 47085), or purified pseudolysin (Calbiochem), was used to assess proteolytic cleavage of purified recombinant hlgGl. Dulbecco's Phosphate Buffer Saline (DPBS) with CaCI2 and MgCI2 (GIBCO) was used as a standard buffer for all dilutions and reconstitutions. In the example given in Figure 1, incubation of PA01 bacterial supernatant or purified pseudolysin with purified hlgGl and subsequent separation by SDS-PAGE, revealed two major cleavage products at 35kDa and 20kDa.
Example 2: N-terminal sequencing of IgG cleavage products
Proteolytic cleavage products formed by treatment of purified recombinant hlgGl with pseudolysin, and subsequent separation by SDS-PAGE, were transferred by blotting onto a polyvinyl idene difluoride (PVDF) membrane; the specific N-terminal amino acid sequences were then resolved by Edman degradation. By comparison with untreated samples, the proteins at approximately 50 kDa and 25 kDa represent the heavy and light chains of hlgGl. Lanes 1 and 2 are identical samples (hlgGl treated with P. aeruginosa culture supernatant); similarly, lanes 3 and 4 are identical samples (hlgGl treated with
purified pseudolysin). Consistent with the data described above, two major cleavage products were observed at approximately 35kDa and 20kDa; additional minor bands were observed in P. aeruginosa supernatant-treated samples only.
N-terminal sequencing of the ~35 kDa fragment observed after treatment with both P. aeruginosa culture supernatant (marked 'Supernatant band 2' in lane 2) and purified pseudolysin (marked 'Purified band 1' in lane 3) suggested the identity of this fragment was a partial sequence in the heavy chain CH1 domain, and, by inference from the molecular weight of the product, this partial CH1 sequence is likely fused to the remainder of the heavy chain sequence (hinge-CH2-CH3). The N-terminal sequence of this product was [197]-LGTQTYIXNV (SEQ ID NO: 5) (where, based on the known sequence of the heavy chain, X is a cysteine residue that could not be resolved by Edman degradation). This suggested that the cleavage site was further towards the N- terminus of the heavy chain than was expected based on similar IgG-cleaving proteases from other bacterial pathogens (Breski & Jordan, 2010) and an initial analysis of potential cleavage sites based on preferred substrate amino acids obtained from the MEROPS database (Rawlings et al., 2012). An additional fragment that appeared in PA01 supernatant-treated hlgGl was [198]-GTQTYIXNVN (SEQ ID NO: 6) which represents further clipping of an additional amino acid at the newly-formed N-terminus of the CH1- hinge-CH2-CH3 fragment. By inference from these data, the 20kDa product formed by incubation of pseudolysin with hlgGl is the VH domain fused to the N-terminal portion of the CH1 domain; the N-terminal sequence of this fragment was not experimentally determined by Edman degradation, likely due to the presence of a pyroglutamate residue at the extreme N-terminus of the heavy chain. The full results of N-terminal sequencing of pseudolysin-treated hlgGl are presented in Figure 3. The amino acid sequence of the pseudolysin cleavage site on hlgGl is shown in Figure 4, and the location of the cleavage site on a crystal structure of hlgGl is shown in Figure 5.
Example 3: Identification of potential pseudolysin-resistant IgG molecules
Based on the sequence specificity of pseudolysin (Rawlings et al., 2012), and analysis of the properties of amino acids surrounding the identified cleavage site, it was hypothesised that mutation of preferred amino acids with clearly defined properties to less favoured amino acids with opposing properties surrounding the cleavage site would reduce or resist pseudolysin-mediated proteolysis of hlgGl and related molecules. For example, a preference for an amino acid with a small side chain such as serine, glycine
or alanine at the PI site (the amino acid immediately preceding cleavage site) and the P2' site (the second amino acid following the cleavage site) was identified by analysis of the MEROPS database (Rawlings et al., 2012); conversely, amino acids with large side chains such as tryptophan or glutamine are less favoured in these positions. Therefore it was postulated that mutation of serine at PI and glycine at P2' in the hlgGl sequence to glutamine may reduce/resist pseudolysin-mediated cleavage of hlgGl and related molecules. Furthermore, at the PI' site (the amino acid immediately following the cleavage site), a preference for hydrophobic residues such as leucine and phenylalanine was observed. Therefore it was postulated that mutation at this site to a hydrophilic residue may reduce/resist pseudolysin-mediated cleavage of hlgGl and related molecules; based on the location of this residue buried in the CH1 domain care was taken to avoid highly hydrophilic residues which may lead to misfolding of the alpha helix. A more detailed analysis of potential sites for mutation which may reduce or resist pseudolysin cleavage of hlgGl and related molecules is shown in Figure 6.
Example 4: Additional strategy to resist pseudolysin-mediated cleavage
Analysis of human CHI sequences from different isotypes in the UniProtKB/Swiss-Prot database demonstrated that this sequence is conserved across human IgG isotypes, but differs across other isotypes. An additional strategy to produce pseudolysin-resistant antibody molecules is to substitute the pseudolysin-sensitive alpha helix sequence in IgGl with a sequence from a different isotype which should be completely pseudolysin-resistant or which contain residues that are less favoured, based on the specificity of the protease as determined by analysis of the MEROPS database (Rawlings et al. 2012). As an example, the human IgD antibody crystal structure (1ZVO) suggests the presence of a similar helix to IgGl in this region, while the amino acid sequence suggests this is a poor substrate for pseudolysin. Therefore, substitution of the hlgGl sequence in this region (PSSSLGTQ (SEQ ID NO :2)- the identical sequence in hlgGl, hIgG3, and hIgG4 but differing in one amino acid in hIgG2) with the hlgD sequence (PLQQWRQG; SEQ ID NO: 4) may result in reduced or abrogated sensitivity to pseudolysin.
Example 5: Experimental demonstration of Pseudolysin-resistant hlgGs
In order to assess the effect of altering the amino acid sequence surrounding the pseudolysin cleavage site, a number of mutant constructs were generated by site- directed mutagenesis. Single, double, and triple mutations were tested (L197P,
S196Q/G198Q, and L197P/S196Q/G198Q) in addition to swapping the IgGl sequence with the corresponding sequence from IgD, as described in Example 4. Each mutant antibody was prepared by co-transfection of a plasmid capable of expressing the mutated heavy chain with a plasmid expressing the antibody light chain in human embryonic kidney (HEK) cells. Figure 7 shows a SDS-PAGE gel of the mutant antibodies affinity purified on protein A-sepharose (lanes 3-10; each antibody was assessed under non-denaturing and denaturing conditions); the parental antibody was expressed and purified in parallel with the mutant constructs and is shown in lanes 1 and 2 (HC). Next, each mutant antibody was assessed for resistance to treatment with pseudolysin relative to the pseudolysin-sensitive parental antibody by co-incubation of bacterial supernatant as described in Example 1; as judged by the disappearance of proteolytic breakdown products at approximately 20 and 35 kDa (Figure 8), mutants IB (L197P) and 3F (L197P/S196Q/G198Q) successfully resisted pseduolysin-mediated cleavage. Mutant 2B (S196Q/G198Q) had no discernible effect, perhaps suggesting that in this panel of constructs, mutation of the leucine at position 197 is particularly important in permitting cleavage of IgG by pseudolysin. Mutant 4A (containing the IgD sequence surrounding the pseudolysin cleavage site in place of the IgGl sequence) did not resist proteolysis by pseudolysin, indicating that although this sequence is a poor substrate, it is still cleaved by pseudolysin; in fact, the swapped hlgD sequence was cleaved more effectively than the parental hlgGl sequence as judged by the reduced presence of the full-length heavy chain in lane 11 compared with the parental antibody (lane 3).
Additional studies using ex vivo (sputum) samples with a positive diagnostic test for P. aeruginosa infection from a variety of patient groups, and/or studies in Pseudomonas-challenged animal models, should provide further evidence that the approach of altering the heavy chain sequence surrounding the pseudolysin cleavage site, represented by mutants IB and 3F, mediates resistance to pseudolysin in vivo.
Example 6: Observations
Based on the evidence seen from the experiments conducted, several conclusions about the activity of pseudolysin have been deduced:
As expected, incubation of hlgGl with pseudolysin (in purified form, or contained in bacterial supernatant) results in the formation of two cleavage products, which have molecular weights of approximately 35 and 20 kDa.
As judged by N-terminal sequencing of the 35kDa product, this fragment is composed of a partial CH1 sequence fused to the remainder of the heavy chain (hinge-
The 20 kDa fragment is composed of the VH region fused to the remainder of the CH1 region; this was not experimentally observed, likely due to the presence of pyroglutamate at the N-terminus of the heavy chain.
In contrast to other bacterial pathogens which encode IgG- and IgA-cleaving proteases (Breski & Jordan, 2010), pseudolysin cleaves at a site far removed from the hinge or lower hinge/CH2 region.
Based on analysis of substrate specificity, such as that described in the MEROPS database (Rawlings et al., 2012) the amino acid preferences of pseudolysin include:
■ At PI a small charged or hydrophilic residue
■ At PI' a hydrophobic residue
■ At P2' a small charged or hydrophilic residue
Embodiments of the invention therefore include constructs developed to focus on mutations to the PI, PI' and P2' sites, using single mutations and combinations of mutations to oppose the preferences of pseudolysin substrate specificity, such as L197P (SEQ ID NO: 3). Mutations that might be expected to compromise the folding of the antibody were avoided.
As representative examples of mutations which may reduce or resist pseudolysin sensitivity of Fc-containing molecules, both L197P and L197P/S196Q/G198Q mediated pseudolysin resistance, perhaps suggesting that a leucine (or other hydrophobic amino acid residue) at position 197 (PI' site) is a particularly important component of the substrate specificity of pseudolysin on hlgGl.
This invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Claims
1. A modified Fc-containing molecule that is resistant to proteolytic degradation compared to a wild-type Fc-containing molecule, wherein the modified Fc-containing molecule comprises a modified CH1 domain with at least one substitution, deletion, or addition of another amino acid in the wild-type Fc-containing molecule CH1 domain sequence PSSSLGTQ (SEQ ID NO: 2), wherein the modified CH1 domain resists proteolytic cleavage by a bacterial protease.
2. The modified Fc-containing molecule of claim 1, wherein the bacterial protease is Pseudomonas aeruginosa pseudolysin.
3. The modified Fc-containing molecule of claim 1, wherein the Fc-containing molecule is any isotype of human IgG.
4. The modified Fc-containing molecule of claim 1, wherein the Fc-containing molecule is a corresponding mouse IgG or rat IgG.
5. The modified Fc-containing molecule of claim 1, wherein the Fc-containing molecule is human IgGl.
6. The modified Fc-containing molecule of claim 1, wherein the Fc-containing molecule is a corresponding mouse IgGl or rat IgGl.
7. The modified Fc-containing molecule of claim 1, wherein the substitution replaces any amino acid in SEQ ID NO: 2 with any amino acid that resists proteolytic cleavage by pseudolysin based on its substrate specificity.
8. The modified Fc-containing molecule of claim 1, wherein the substitution replaces a hydrophobic amino acid residue at position 5 of SEQ ID NO: 2 with a hydrophilic amino acid residue.
The modified Fc-containing molecule of claim 1, wherein the substitution repl leucine at position 5 of SEQ ID NO: 2 with proline.
10. Use of a modified Fc-containing molecule according to any one of the previous claims to resist degradation by pseudolysin secreted by a strain of Pseudomonas aeruginosa.
11. A method of treating Pseudomonas aeruginosa-aggravated diseases or conditions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that resists proteolytic degradation by pseudolysin.
12. A method of preventing Pseudomonas aerug/nosa-aggravated diseases or conditions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Fc-containing molecule that resists proteolytic degradation by pseudolysin.
13. The method according to claim 11 or 12, wherein the Pseudomonas aerug/nosa- aggravated disease is cystic fibrosis.
14. The method according to claim 11 or 12, wherein the Pseudomonas aerug/nosa- aggravated disease is ventilator-associated pneumonia (VAP), hospital-acquired pneuomonia (HAP), or health care-associated pneumonia (HCAP).
15. The method according to claim 11 or 12, wherein the Pseudomonas aerug/nosa- aggravated disease or condition is selected from the group consisting of: respiratory tract infections, leukopenia, neutropenia, urinary tract infections, gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, and severe burns.
16. Pharmaceutical composition comprising an Fc-containing molecule as defined in any one of claims 1 to 9 and a pharmaceutically acceptable carrier or excipient.
17. Use of an Fc-containing molecule as defined in any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of a disease selected from respiratory tract infections, cystic fibrosis, ventilator-associated pneumonia,
leukopenia, neutropenia, urinary tract infections, gastrointestinal infections, bone infections, joint infections, skin infections, eye infections, and severe burns.
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