WO2014096390A1 - Single linker fabfv antibodies and methods of producing same - Google Patents
Single linker fabfv antibodies and methods of producing same Download PDFInfo
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- WO2014096390A1 WO2014096390A1 PCT/EP2013/077758 EP2013077758W WO2014096390A1 WO 2014096390 A1 WO2014096390 A1 WO 2014096390A1 EP 2013077758 W EP2013077758 W EP 2013077758W WO 2014096390 A1 WO2014096390 A1 WO 2014096390A1
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- VHGGRTWHRJRQKU-UHFFFAOYSA-N CN(CC=C1)C1=O Chemical compound CN(CC=C1)C1=O VHGGRTWHRJRQKU-UHFFFAOYSA-N 0.000 description 1
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Definitions
- the present disclosure relates to certain multi-specific constructs, pharmaceutical formulations comprising the construct, DNA encoding the constructs and vectors comprising same.
- the disclosure also extends to a method of expressing the constructs, for example in a host cell and methods for formulating same as a pharmaceutical composition.
- the disclosure also relates to use of the constructs and formulations in treatment.
- WO2009/040562 and WO2010/035012 discloses certain bi-specific molecules useful as therapeutic agents, known as Fab-Fv or Fab-dsFv respectively.
- the molecules of this type have good binding affinity for the antigens to which they are specific and no significant occlusion of antigen binding sites occurs in the format. Whilst a high percentage of these antibody molecules are expressed as functional monomer there is a proportion that aggregates and from which the monomer needs to be purified.
- the present inventors have re-engineered the molecules concerned to provide molecules with equivalent functionality, whilst minimising aggregation at the expression stage and thus substantially increasing the yield of monomer.
- Vx represents a variable domain
- Vxx represents a variable domain
- Vy represents a variable domain
- Vyy represents a variable domain
- Cy represents a constant region domain
- v 2 represents a variable domain
- n independently represents 0 or 1 ,
- polypeptide chain of formula (I) and the polypeptide chain of formula (II) are aligned such that the constant regions Cx and Cy are paired and the variable domain Vx and Vy are paired to form a binding domain and optionally a disulphide bond is present between Vi and V 2 , in particular where a disulphide bond is present.
- Vxx and Vyy are also paired to form a binding domain.
- a disulphide bond is present between Vi and V 2 .
- bi-specific antibody molecule comprising or consisting of three polypeptides
- VH represents a heavy chain variable domain
- CH ! represents domain 1 of a heavy chain constant region.
- V ! represents a variable domain
- V L represents a light chain variable domain
- C L represents a constant region from a light chain
- v 2 represents a variable domain
- bi-specific antibody molecule comprising or consisting of three polypeptides
- VH represents a heavy chain variable domain
- CHi represents domain 1 of a heavy chain constant region
- Vi represents a variable domain
- V L represents a light chain variable domain
- C L represents a constant region from a light chain
- V 2 represents a variable domain
- a disulphide bond is present between Vi and V 2 .
- the present construct minimises the amount of aggregation seen during expression and maximises the amount of monomer obtained, for example the monomer may be 50%, 60%>, 70% or 75%> or more such as 80 or 90% or more of the protein expressed.
- Figure 1 shows various sequences for single linker Fab-Fv constructs according to the invention and comparator constructs Fabdsscfv and FabdsFv
- FIG. 2 shows SDS-PAGE analysis of the various constructs
- Figure 4 shows a diagrammatic representation of various example constructs according to the
- Figure 5 shows transient expression of single linker Fab-dsFvs expressed from triple gene plasmids in
- Figure 6 shows SDS-PAGE analysis of various single linker Fab-dsFvs expressed from triple gene plasmids.
- Figure 7 shows size exclusion analysis of single linker Fab-dsFv expressed from a triple gene
- Multi-specific antibody refers to an antibody molecule as described herein which has two or more binding domains, for example two or three binding domains.
- the construct is a tri-specific antibody.
- Tri-specific molecule as employed herein refers to a molecule with three antigen binding sites, which may independently bind the same or different antigens.
- the construct is a bi-specific antibody.
- Bi-specific molecule as employed herein refers to a molecule with two antigen binding sites, which may bind the same or different antigens.
- the domains all bind the same antigen, including binding the same epitope on the antigen or binding different epitopes on the antigen.
- each of the three binding domains bind different (distinct) antigens.
- binding domains and two binding domains bind the same antigen, including binding the same epitope or different epitopes on the same antigen, and the third binding domain binds a different (distinct) antigen.
- the present disclosure relates to a bi-specific antibody comprising or consisting of three polypeptide chains.
- the multi-specific molecules according to the present disclosure are provided as a dimer of a heavy and light chain of:
- the construct of the present disclosure has only two antigen binding sites.
- Antigen binding site refers to a portion of the molecule, which comprises a pair of variable regions, in particular a cognate pair, that interact specifically with the target antigen.
- binding site that only recognises the antigen to which it is specific or a binding site that has significantly higher binding affinity to the antigen to which is specific compared to affinity to antigens to which it is non-specific, for example 5, 6, 7, 8, 9, 10 times higher binding affinity.
- Binding affinity may be measured by standard assay, for example surface plasmon resonance, such as BIAcore.
- one or more natural or engineered inter chain (i.e. inter light and heavy chain) disulphide bonds are present in the functional Fab or Fab' fragment.
- a "natural" disulfide bond is present between a CHi and C L or corresponding components Cx and Cy in the polypeptide chains of formula (I) and (II).
- CHi may apply equally to Cx.
- C L may apply equally to Cy.
- the exact location of the disulfide bond forming cysteine in CHi depends on the particular domain actually employed. Thus, for example in human gamma- 1 the natural position of the disulfide bond is located at position 233 (Kabat numbering 4 th edition 1987).
- the position of the bond forming cysteine for other human isotypes such as gamma 2, 3, 4, IgM and IgD are known, for example position 127 for human IgM, IgE, IgG2, IgG3, IgG4 and 128 of the heavy chain of human IgD and IgA2B.
- a disulfide bond or bond(s) in the constant region of the molecule may be in addition to the optional disulfide bond between a variable domain pair Vi and V 2 .
- the multi-specific antibody according to the disclosure has a disulfide bond in a position equivalent or corresponding to that in the naturally occurring between CHi and C L .
- a constant region comprising CHi and a constant region such as C L has a disulfide bond which is in a non-naturally occurring position. This may be engineered into the molecule by introducing cysteine(s) into the amino acid chain at the position or positions required. This non-natural disulfide bond is in addition to or as an alternative to the natural disulfide bond present between CHi and C L .
- engineered cysteines can be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis or cassette mutagenesis (see, generally, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-lnterscience, NY, 1993).
- Site-directed mutagenesis kits are commercially available, e.g. QuikChange® Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).
- Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be made by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.
- a disulfide bond between CHi and C L is completely absent, for example the interchain cysteines may be replaced by another amino acid, such as serine.
- the interchain cysteines may be replaced by another amino acid, such as serine.
- n is 1 in the polypeptide chain of formula (I).
- n is 1 in the polypeptide chain of formula (II).
- n is 1 in the polypeptide chain of formula (I) and (II).
- n is 0 in the polypeptide chain of formula (I) and (II).
- Vxx may be derived from a heavy chain variable region, light chain variable region or a combination thereof and may comprise an amino acid linker of 1 to 20 amino acids, for example as described below.
- Vxx consists of a variable region, in particular a variable region derived from a heavy chain.
- Vxx represents a light chain variable domain.
- Vxx is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs.
- Vxx is humanised.
- Vx may be derived from a heavy chain variable region, light chain variable region or a combination thereof, in particular a variable region derived from a heavy chain.
- Vx represents a light chain variable domain.
- Vx is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs.
- Vx is humanised.
- Vx in polypeptides of formula (I) corresponds to VH in polypeptide chain (la).
- VH represents a variable domain, for example a heavy chain variable domain. In one embodiment VH represents a heavy chain variable domain. In one embodiment V H is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non- human CDRs. In one embodiment VH is humanised.
- Vi represents a variable domain, for example a heavy chain or light chain variable domain. In one embodiment Vi represents a heavy chain variable domain. In one embodiment Vi represents a light chain variable domain. In one embodiment Vi is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs. In one embodiment Vi is humanised. Vyy may be derived from a heavy chain, light chain or a combination thereof and may comprise an amino acid linker of 1 to 20 amino acids, for example as described below. In one embodiment Vyy consists of a variable region, in particular a variable region derived from a light chain. In one embodiment Vyy represents a heavy chain variable domain. In one embodiment Vyy is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non- human CDRs. In one embodiment Vyy is humanised.
- Vy may be derived from a heavy chain variable region, light chain variable region or a combination thereof, in particular a variable region derived from a light chain.
- Vy represents a heavy chain variable domain.
- Vy is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs.
- Vy is humanised.
- Vy in polypeptides of formula (II) corresponds to VL in polypeptides of formula (Ila).
- V L represents a variable domain, for example a light chain variable domain. In one embodiment V L represents a light chain variable domain. In one embodiment V L is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs. In one embodiment V L is humanised.
- V 2 represents a variable domain, for example a heavy chain or light chain variable domain. In one embodiment V 2 represents a light chain variable domain. In one embodiment V 2 represents a heavy chain variable domain. In one embodiment V 2 is a chimeric variable domain, that is to say it comprises components derived from at least two species, for example a human framework and non-human CDRs. In one embodiment Vi is humanised.
- Vxx and Vyy together form an antigen binding domain.
- Vxx and Vyy together represent a cognate pair.
- Vx and Vy together form an antigen binding domain.
- Vx and Vy together represent a cognate pair.
- the binding domain formed by VH and VL are specific to a first antigen.
- VH and VL form a cognate pair.
- Vi and V 2 together form an antigen binding domain. In one embodiment Vi and V 2 together represent a cognate pair.
- Vi and V 2 together form an antigen binding domain specific for a first antigen
- the two binding domains in the molecule may be specific to the same antigen, for example binding the same or a different epitope therein).
- Vi and V 2 together are a binding domain for human serum albumin.
- Vi and V 2 together form an antigen binding domain specific for a second antigen (i.e. the two binding domains in the molecule are specific to different antigens).
- the disulfide bond between Vi and V 2 is between two of the residues listed below (unless the context indicates otherwise Kabat numbering is employed in the list below). Wherever reference is made to Kabat numbering the relevant reference is Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA. In one embodiment the disulfide bond is in a position selected from the group comprising:
- VH37 + VL95C see for example Protein Science 6, 781 -788 Zhu et al (1997);
- VH45 + VL87 see for example Protein Science 6, 781 -788 Zhu et al (1997);
- VH100 + VL50 see for example Biochemistry 29 1362-1367 Glockshuber et al (1990);
- VH98 + VL 46 see for example Protein Science 6, 781-788 Zhu et al (1997);
- VH105 + VL43 see for example; Proc. Natl. Acad. Sci. USA Vol. 90 pp.7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994),
- variable region pair located in the molecule a position corresponding thereto in variable region pair located in the molecule.
- an engineered cysteine according to the present invention refers to where the naturally occurring residue at a given amino acid position has been replaced with a cysteine residue.
- engineered cysteines can be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis or cassette mutagenesis (see, generally, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-lnterscience, NY, 1993).
- Site-directed mutagenesis kits are commercially available, e.g. QuikChange® Site-Directed Mutagenesis kit (Stratagen, La Jolla, CA).
- Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be made by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.
- variable domain pair (Vi/V 2 ) of the present invention may be linked by a disulfide bond between two cysteine residues, one in Vi and one in V 2 , wherein the position of the pair of cysteine residues is selected from the group consisting of VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VHlOOb and VL49, VH98 and VL46, VH101 and VL46, VH105 and VL43 and VH106 and VL57.
- variable domain pair (Vi/V 2 ) of the present invention may be linked by a disulfide bond between two cysteine residues, one in Vi and one in V 2 , which are outside of the CDRs wherein the position of the pair of cysteine residues is selected from the group consisting of VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57.
- Vi is a heavy chain variable domain and V 2 is a light chain variable domain and Vi and V 2 are linked by a disulphide bond between two engineered cysteine residues, one at position VH44 of V ! and the other at VL100 of V 2 .
- VH and Vi are variable regions which are both from a heavy chain(s) or a light chain(s), in particular are both derived from two distinct heavy chain variable regions.
- VL and V 2 are variable regions which are both from a heavy chain(s) or a light chain(s), in particular are both derived from two distinct light chain variable regions.
- Cognate pair refers to a pair of variable domains from a single antibody, which was generated in vivo, i.e. the naturally occurring pairing of the variable domains isolated from a host.
- a cognate pair is therefore a VH and VL pair.
- the cognate pair bind the antigen co-operatively.
- Variable region as employed herein refers to the region in an antibody chain comprising the CDRs and a suitable framework.
- Variable regions for use in the present disclosure will generally be derived from an antibody, which may be generated by any method known in the art.
- Derived from as employed herein refers to the fact that the sequence employed or a sequence highly similar to the sequence employed was obtained from the original genetic material, such as the light or heavy chain of an antibody.
- Highly similar as employed herein is intended to refer to an amino acid sequence which over its full length is 95% similar or more, such as 96, 97, 98 or 99% similar.
- Antibodies generated against the antigen polypeptide may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir
- mice, rats, sheep, cows, camels or pigs may be immunized.
- mice, rabbits, pigs and rats are generally most suitable.
- Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).
- Antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by, for example, the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551 ; WO2004/051268 and WO2004/106377.
- the antibodies for use in the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol.
- the bi-specific molecules according to the disclosure are humanised.
- Humanised which include CDR-grafted antibodies
- CDR-grafted antibodies refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g. US 5,585,089; W091/09967). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.
- the term 'humanised antibody molecule' refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody).
- a donor antibody e.g. a murine monoclonal antibody
- acceptor antibody e.g. a human antibody
- only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34).
- only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework.
- only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.
- any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
- the humanised antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.
- human frameworks which can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra).
- KOL and NEWM can be used for the heavy chain
- REI can be used for the light chain and EU
- LAY and POM can be used for both the heavy chain and the light chain.
- human germline sequences may be used; these are available at: http://vbase.mrc-cpe.cam.ac.uk/
- the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
- the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody.
- a protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO91/09967.
- bi-specific antibodies of the present disclosure are fully human, in particular one or more of the variable domains are fully human.
- Fully human molecules are those in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, not necessarily from the same antibody.
- Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts eg. as described in general terms in EP0546073 Bl , US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US5,770,429, EP 0438474 and EP0463151.
- Cx is a constant domain from a light or heavy chain, in particular a heavy chain.
- Cx in polypeptide of formula (I) corresponds to CHi in polypeptides of formula (la). In one embodiment Cx is equivalent to CHi.
- the CHi domain is a naturally occurring domain 1 from a heavy chain or a derivative thereof. In one embodiment the CH fragment consists of a CHi domain.
- Cy is a constant domain from a light or heavy chain, in particular a light chain.
- Cy in polypeptide of formula (II) corresponds to CL in polypeptides of formula (Ila). In one embodiment Cy is equivalent to CL.
- the C L fragment, in the light chain is a constant kappa sequence or a constant lambda sequence or a derivative thereof.
- a derivative of a naturally occurring domain as employed herein is intended to refer to where one, two, three, four or five amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is/are retained.
- the peptide linker is 50 amino acids in length or less, for example 20 amino acids or less.
- the linker is selected from a sequence shown in sequence 13 to 77.
- the linker is selected from a sequence shown in SEQ ID NO: 103 or SEQ ID NO:104.
- (S) is optional in sequences 24 to 28.
- rigid linkers examples include the peptide sequences GAPAPAAPAPA (SEQ ID NO: 62), PPPP (SEQ ID NO: 63) and PPP.
- the peptide linker is an albumin binding peptide.
- albumin binding peptides examples include:
- albumin binding peptides as a linker may increase the half-life of the bi-specific antibody molecule.
- V 2 is still present in the antibody molecule and is retained therein by virtue of pairing with Vi including where a disulphide bond is present between Vi and V 2 .
- the bi-specific antibody molecules of the disclosure are capable of selectively binding two different antigens of interest.
- an antigen of interest bound by Vxx/Vyy, Vx/Vy, VH/VL and V 1 /V 2 are independently selected from a cell-associated protein, for example a cell surface protein on cells such as bacterial cells, yeast cells, T-cells, endothelial cells or tumour cells, and a soluble protein.
- Antigens of interest may also be any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands.
- Particular examples of cell surface proteins include adhesion molecules, for example integrins such as ⁇ integrins e.g.
- VLA-4 E- selectin, P selectin or L-selectin
- CEA carcinoembryonic antigen
- HMFGl and 2 human milk fat globulin
- MHC Class I and MHC Class II antigens MHC Class I and MHC Class II antigens
- VEGF vascular endothelial growth factor
- Soluble antigens include interleukins such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-16 or IL-17, IL-23, viral antigens for example respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins, such as IgE, interferons such as interferon a, interferon ⁇ or interferon ⁇ , tumour necrosis factor- ⁇ , tumor necrosis factor- ⁇ , colony stimulating factors such as G-CSF or GM-CSF, and platelet derived growth factors such as PDGF-a, and PDGF- ⁇ and where appropriate receptors thereof.
- Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza, EBV, HepA, B and C, bioterrorism agents, radionuclides and heavy metals, and snake and spider venoms and toxins.
- the antibody fusion protein of the invention may be used to functionally alter the activity of the antigen of interest.
- the antibody fusion protein may neutralize, antagonize or agonise the activity of said antigen, directly or indirectly.
- the antigen of interest bound by VH and VL is OX40.
- the Vx-Cx or VHCH1 portion of the multi-specific antibody has the sequence given in SEQ ID NO:3.
- the Vy-Cy or VL-CL portion of the multi-specific antibody has the sequence given in SEQ ID NO:7.
- an antigen of interest bound by VH/VL or Vi/V 2 provides the ability to recruit effector functions, such as complement pathway activation and/or effector cell recruitment.
- the recruitment of effector function may be direct in that effector function is associated with a cell, said cell bearing a recruitment molecule on its surface. Indirect recruitment may occur when binding of a binding domain (such as Vi/V 2 ) in the molecule according to present disclosure to a recruitment polypeptide causes release of, for example, a factor which in turn may directly or indirectly recruit effector function, or may be via activation of a signalling pathway.
- a binding domain such as Vi/V 2
- a recruitment polypeptide causes release of, for example, a factor which in turn may directly or indirectly recruit effector function, or may be via activation of a signalling pathway.
- a binding domain such as Vi/V 2
- a recruitment polypeptide causes release of, for example, a factor which in turn may directly or indirectly recruit effector function, or may be via activation of a signalling pathway.
- a binding domain such as Vi/V 2
- a recruitment polypeptide causes release of, for example, a factor which in turn may directly or indirectly recruit effector
- 'a recruitment polypeptide' includes a FcyR such as FcyRI, FcyRII and FcyRIII, a complement pathway protein such as, but without limitation, Clq and C3, a CD marker protein (Cluster of Differentiation marker) such as, but without limitation, CD68, CD115, CD16, CD80, CD83, CD86, CD56, CD64, CD3, CD4, CD8, CD28, CD45, CD19, CD20 and CD22.
- Further recruitment polypeptides which are CD marker proteins include CD 1, CD Id, CD2, CD5, CD8, CD9, CD 10, CD 11, CD 11 a, CD l ib, CD 11c,
- a recruitment polypeptide also includes immunoglobulin molecules such as IgGl, IgG2, IgG3, IgG4, IgE and IgA which possess effector function.
- a binding domain (such as Vi/V 2 ) in the molecule according to the present disclosure has specificity is a complement pathway protein, with Clq being particularly preferred.
- molecules of the present invention may be used to chelate radionuclides by virtue of a single domain antibody which binds to a nuclide chelator protein.
- fusion proteins are of use in imaging or radionuclide targeting approaches to therapy.
- one binding domain in a molecule according to the disclosure has specificity is a CD marker protein, with CD68, CD80, CD86, CD64, CD3, CD4, CD8 CD45, CD16 and CD35 being particularly preferred.
- a binding domain within a molecule according to the disclosure has specificity for a serum carrier protein, a circulating immunoglobulin molecule, or CD35/CR1 , for example for providing an extended half-life to the antibody fragment with specificity for said antigen of interest by binding to said serum carrier protein, circulating immunoglobulin molecule or CD35/CR1.
- 'serum carrier proteins' include thyroxine-binding protein, transthyretin, a 1 -acid glycoprotein, transferrin, fibrinogen and albumin, or a fragment of any thereof.
- a 'circulating immunoglobulin molecule' includes IgGl, IgG2, IgG3, IgG4, slgA, IgM and IgD, or a fragment of any thereof.
- CD35/CR1 is a protein present on red blood cells which have a half-life of 36 days (normal range of 28 to 47 days; Lanaro et al, 1971 , Cancer, 28(3):658-661).
- the protein for which Vi/V 2 has specificity is a serum carrier protein, such as a human serum carrier.
- the serum carrier protein is human serum albumin.
- bi-specific antibody molecule comprising or consisting of three polypeptides
- V H represents a heavy chain variable domain
- CH ! represents domain 1 of a heavy chain constant region.
- V L represents a light chain variable domain
- C L represents a constant region from a light chain
- v 2 represents a variable region, wherein V H and V L are a cognate pair of variable regions aligned to form a binding domain and Vi and V 2 are a cognate pair of variable regions aligned to form a binding domain optionally a disulphide bond therebetween, for example wherein Vi and V 2 are capable of binding albumin in vivo, in particular human serum albumin.
- VI or V2 comprise a sequence selected from the group consisting of SEQ ID NO: 1
- VI has the sequence given in SEQ ID NO:4 and V2 has the sequence given in SEQ ID NO:8. In one embodiment VI has the sequence given in SEQ ID NO:86 and V2 has the sequence given in SEQ ID NO:83.
- VI or V2 in particular VI , is a VH domain comprising the sequence given in SEQ ID NO:87 for CDRH-1, the sequence given in SEQ ID NO:88 for CDRH2 and the sequence given in SEQ ID NO:89 for CDRH-3.
- VI or V2 in particular VI , is a VH domain comprising the sequence given in SEQ ID NO:93 for CDRH-1 , the sequence given in SEQ ID NO:94 for CDRH2 and the sequence given in SEQ ID NO:95 for CDRH-3.
- VI or V2 in particular V2, is a VL domain comprising the sequence given in SEQ ID NO:90 for CDRL-1 , the sequence given in SEQ ID NO:91 for CDRL2 and the sequence given in
- VI or V2 for CDRL-3 is a VL domain comprising the sequence given in SEQ ID NO:96 for CDRL-1 , the sequence given in SEQ ID NO:97 for CDRL2 and the sequence given in SEQ ID NO:98 for CDRL-3.
- VI or V2 in particular VI , is a VH domain comprising the sequence given in SEQ ID NO:4, SEQ ID NO: 86, SEQ NO:99 or SEQ ID NO: 100.
- VI or V2 in particular V2, is a VL domain comprising the sequence given in SEQ ID NO: 8, SEQ ID NO: 83, SEQ NO: 101 or SEQ ID NO: 102.
- VI is a VH domain comprising the sequence given in SEQ NO:99 and V2 is a VL domain comprising the sequence given in SEQ NO: 101.
- VI is a VH domain comprising the sequence given in SEQ NO: 100 and V2 is a VL domain comprising the sequence given in SEQ NO: 102.
- polypeptide la has the sequence given in SEQ ID NO:6.
- polypeptide Ila has the sequence given in SEQ ID NO:7.
- polypeptide lb has the sequence given in SEQ ID NO:3.
- polypeptide lib has the sequence given in SEQ ID NO:5.
- polypeptide la has the sequence given in SEQ ID NO:6
- polypeptide Ila has the sequence given in SEQ ID NO:7
- V 2 has the sequence given in SEQ ID NO:8.
- polypeptide lb has the sequence given in SEQ ID NO:3
- polypeptide lib has the sequence given in SEQ ID NO:5
- Vi has the sequence given in SEQ ID NO:4.
- the invention also provides sequences which are 80%, 90%, 91 %, 92%, 93% 94%, 95% 96%, 97%,
- the present invention provides a Fab or Fab' fragment linked to a disulphide stabilised scFv, wherein the disulphide stabilised scFv (dsscFv) is linked to the C-terminus of the heavy or the light chain of the Fab or Fab' fragment directly or via a linker such as a linker described herein.
- dsscFv disulphide stabilised scFv
- the Fab-dsscFv fusion protein is bi-specific.
- the dsscFv binds a serum carrier protein such as human serum albumin.
- the dsscFv is linked to C-terminus of the heavy chain of the Fab or Fab' fragment by the linker given in SEQ ID NO:78.
- the dsscFv is linked to the C-terminus of the light chain of the Fab or Fab' fragment by the linker given in SEQ ID NO:103.
- the heavy chain of the Fab-dsscFv has the sequence given in SEQ ID NO:9 and the light chain has the sequence given in SEQ ID NO: 10.
- the heavy chain of the Fab-dsscFv has the sequence given in SEQ ID NO:l 1 and the light chain has the sequence given in SEQ ID NO: 12.
- the bi-specific antibody molecules of the present disclosure are processed to provide improved affinity for a target antigen or antigens.
- affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin.
- an antibody for use in the present invention may be conjugated to one or more effector molecule(s).
- the effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies of the present invention.
- this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule.
- Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp.
- effector molecule includes, for example, biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
- biologically active proteins for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
- effector molecules may include chelated radionuclides such as 11 lln and 90Y, Lul77,
- effector molecules include proteins, peptides and enzymes.
- Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases.
- Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumour necrosis factor, a-interferon, ⁇ - interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
- angiostatin or endostatin or, a biological response modifier such as a lymphokine, interleukin-1 (TL-l), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.
- a biological response modifier such as a lymphokine, interleukin-1 (TL-l), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.
- effector molecules may include detectable substances useful for example in diagnosis.
- detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics.
- Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 1251, 1311, l l lIn and 99Tc.
- the effector molecule may increase the half- life of the antibody in vivo, and/or reduce immunogenicity of the antibody and/or enhance the delivery of an antibody across an epithelial barrier to the immune system.
- suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO05/117984.
- the effector molecule is a polymer it may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.
- synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.
- polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
- derivatives as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like.
- the reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.
- the size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to 40000Da such as from 20000 to 40000Da.
- the polymer size may in particular be selected on the basis of the intended use of the product for example ability to localize to certain tissues such as tumors or extend circulating half-life (for review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545).
- a small molecular weight polymer for example with a molecular weight of around 5000Da.
- a higher molecular weight polymer for example having a molecular weight in the range from 20000Da to 40000Da.
- Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 15000Da to about 40000Da.
- antibodies for use in the present invention are attached to poly(ethyleneglycol) (PEG) moieties.
- the antibody is an antibody fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group.
- Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example US 5,219,996; US 5,667,425; W098/25971,
- the antibody molecule of the present invention is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy chain one or more amino acids to allow the attachment of an effector molecule.
- the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.
- PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment.
- Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulphur atom of a cysteine residue located in the fragment.
- the covalent linkage will generally be a disulphide bond or, in particular, a sulphur-carbon bond.
- thiol group is used as the point of attachment
- appropriately activated effector molecules for example thiol selective derivatives such as maleimides and cysteine derivatives may be used.
- An activated polymer may be used as the starting material in the preparation of polymer-modified antibody fragments as described above.
- the activated polymer may be any polymer containing a thiol reactive group such as an a-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
- a thiol reactive group such as an a-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
- Such starting materials may be obtained commercially (for example from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be prepared from commercially available starting materials using conventional chemical procedures.
- Particular PEG molecules include 20K methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly Shear
- a Fab or Fab' in the molecule is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. according to the method disclosed in EP 0948544 or EP 1090037 [see also “Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, “Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A.
- PEG is attached to a cysteine in the hinge region.
- a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region.
- a lysine residue may be covalently linked to the maleimide group and to each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000Da.
- the total molecular weight of the PEG attached to the Fab fragment may therefore be approximately 40,000Da.
- PEG molecules include 2-[3-(N-maleimido)propionamido]ethyl amide of ⁇ , ⁇ '- bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine, also known as PEG2MAL40K (obtainable from Nektar, formerly Shearwater).
- PEG linkers include NOF who supply GL2-400MA2 (wherein m in the structure below is 5) and GL2-400MA (where m is 2) and n is approximately 450:
- each PEG is about 20,000Da.
- an antibody which is PEGylated (for example with a PEG described herein), attached through a cysteine amino acid residue at or about amino acid 226 in the chain, for example amino acid 226 of the heavy chain (by sequential numbering).
- a polynucleotide sequence encoding a molecule of the present disclosure, such as a DNA sequence.
- Vx represents a variable domain
- Vxx represents a variable domain
- Vy represents a variable domain
- Vyy represents a variable domain
- Cy represents a constant region domain
- v 2 represents a variable domain
- n independently represents 0 or 1.
- the polynucleotide, such as the DNA is comprised in a vector.
- a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the present invention.
- Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecule of the present invention.
- Bacterial, for example E. coli, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used.
- Suitable mammalian host cells include CHO, myeloma or hybridoma cells.
- the present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell containing a vector of the present invention under conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.
- the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide.
- a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.
- the cell line may be transfected with three vectors, each encoding a polypeptide chain of an antibody molecule of the present invention.
- the cell line is transfected with three vectors each one encoding a different polypeptide selected from
- Vx represents a variable domain
- Vxx represents a variable domain
- Vy represents a variable domain
- Vyy represents a variable domain
- Cy represents a constant region domain
- v 2 represents a variable domain
- n independently represents 0 or 1 ,
- the ratio of each vector transfected into the host cell may be varied in order to optimise expression of the multi-specific antibody product.
- the ratio of vectors is 1 : 1 : 1. It will be appreciated that skilled person is able to find an optimal ratio by routine testing of protein expression levels following transfection.
- each polypeptide component can be varied by utilising different promoters for each polynucleotide encoding a polypeptide component of the present invention.
- the vector comprises a single polynucleotide sequence encoding all three polypeptide chains of the multispecific antibody molecule of the present invention under the control of a single promoter.
- the vector comprises a single polynucleotide sequence encoding all three polypeptide chains of the multispecific antibody molecule of the present invention wherein each polynucleotide sequence encoding each polypeptide chain is under the control of a different promoter.
- the antibodies and fragments according to the present disclosure are expressed at good levels from host cells. Thus the properties of the antibodies and/or fragments appear to be optimised and conducive to commercial processing.
- the antibodies of the present invention are useful in the treatment and/or prophylaxis of a pathological condition.
- the present invention also provides a pharmaceutical or diagnostic composition comprising an antibody molecule of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier. Accordingly, provided is the use of an antibody of the invention for use in treatment and for the manufacture of a medicament.
- composition will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier.
- a pharmaceutical composition of the present invention may additionally comprise a pharmaceutically-acceptable adjuvant.
- the present invention also provides a process for preparation of a pharmaceutical or diagnostic composition comprising adding and mixing the antibody molecule of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.
- the antibody molecule may be the sole active ingredient in the pharmaceutical or diagnostic composition or may be accompanied by other active ingredients including other antibody ingredients, for example anti-TNF, anti- IL- ⁇ , anti-T cell, anti-IFNy or anti-LPS antibodies, or non-antibody ingredients such as xanthines.
- suitable active ingredients include antibodies capable of inducing tolerance, for example, anti-CD3 or anti-CD4 antibodies.
- the antibody, fragment or composition according to the disclosure is employed in combination with a further pharmaceutically active agent, for example a corticosteroid (such as fluticasonoe propionate) and/or a beta-2 -agonist (such as salbutamol, salmeterol or formoterol) or inhibitors of cell growth and proliferation (such as rapamycin, cyclophosphmide, methotrexate) or alternatively a CD28 and /or CD40 inhibitor.
- a corticosteroid such as fluticasonoe propionate
- a beta-2 -agonist such as salbutamol, salmeterol or formoterol
- inhibitors of cell growth and proliferation such as rapamycin, cyclophosphmide, methotrexate
- CD28 and /or CD40 inhibitor a CD28 and /or CD40 inhibitor.
- the inhibitor is a small molecule.
- the inhibitor is an antibody specific to the target.
- compositions suitably comprise a therapeutically effective amount of the antibody of the invention.
- therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
- the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- compositions may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the invention. Compositions may be administered individually to a patient or may be administered in combination (e.g. simultaneously, sequentially or separately) with other agents, drugs or hormones.
- the dose at which the antibody molecule of the present invention is administered depends on the nature of the condition to be treated, the extent of the inflammation present and on whether the antibody molecule is being used prophylactically or to treat an existing condition.
- the frequency of dose will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (e.g. 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the antibody molecule has a long half- life (e.g. 2 to 15 days) it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 months.
- the pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.
- Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
- compositions for example mineral acid salts, such as
- hydrochlorides hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
- Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.
- Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion.
- parenteral administration e.g. by injection or infusion, for example by bolus injection or continuous infusion.
- the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents.
- the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid.
- compositions of the invention can be administered directly to the subject.
- the subjects to be treated can be animals. However, in one or more embodiments the compositions are adapted for administration to human subjects.
- the pH of the final formulation is not similar to the value of the isoelectric point of the antibody or fragment, for if the pH of the formulation is 7 then a pi of from 8-9 or above may be appropriate. Whilst not wishing to be bound by theory it is thought that this may ultimately provide a final formulation with improved stability, for example the antibody or fragment remains in solution.
- compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention.
- the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
- the antibody molecules of the present invention are administered subcutaneously, by inhalation or topically.
- Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
- the compositions can also be administered into a specific tissue of interest. Dosage treatment may be a single dose schedule or a multiple dose schedule.
- the active ingredient in the composition will be an antibody molecule. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.
- the formulation is provided as a formulation for topical administrations including inhalation.
- Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases (such as nebulisable solutions or suspensions).
- Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the above mentioned active substances with physiologically acceptable excipient.
- These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides
- oligo- and polysaccharides e.g. dextranes
- polyalcohols e.g. sorbitol, mannitol, xylitol
- salts e.g. sodium chloride, calcium carbonate
- Mono- or disaccharides are suitably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.
- Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns for example from 0.1 to 5 ⁇ , in particular from 1 to 5 ⁇ .
- the particle size of the active (such as the antibody or fragment is of primary importance).
- the propellent gases which can be used to prepare the inhalable aerosols are known in the art.
- Suitable propellent gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane.
- hydrocarbons such as n-propane, n-butane or isobutane
- halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane.
- the abovementioned propellent gases may be used on their own or in mixtures thereof.
- Particularly suitable propellent gases are halogenated alkane derivatives selected from among TG 11, TG 12, TG 134a and TG227.
- TG134a (1,1,1,2- tetrafluoroethane)
- TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.
- the propellent-gas-containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art.
- the propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0.1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to 1 % by weight of active.
- topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).
- a nebulizer for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).
- the formulation is provided as discrete ampoules containing a unit dose for delivery by nebulisation.
- the antibody is supplied in lyophilised form, for reconstitutions or alternatively as a suspension formulation.
- the antibody of the invention can be delivered dispersed in a solvent, e.g., in the form of a solution or a suspension. It can be suspended in an appropriate physiological solution, e.g., physiological saline, a pharmacologically acceptable solvent or a buffered solution.
- physiological solution e.g., physiological saline, a pharmacologically acceptable solvent or a buffered solution.
- Buffered solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about 4.0 to 5.0.
- a suspension can made, for example, from lyophilised antibody.
- the therapeutic suspensions or solution formulations can also contain one or more excipients.
- Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will generally be provided in a substantially sterile form employing sterile manufacture processes.
- buffers e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer
- amino acids e.g., amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol.
- Nebulisable formulation according to the present disclosure may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 ml, of solvent/solution buffer.
- the antibodies of the present disclosure are thought to be suitable for delivery via nebulisation. It is also envisaged that the antibody of the present invention may be administered by use of gene therapy. In order to achieve this, DNA sequences encoding the heavy and light chains of the antibody molecule under the control of appropriate DNA components are introduced into a patient such that the antibody chains are expressed from the DNA sequences and assembled in situ.
- the pathological condition or disorder may, for example be selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis such as rheumatoid arthritis, asthma such as severe asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave
- the present invention also provides an antibody molecule according to the present invention for use in the treatment or prophylaxis of pain, particularly pain associated with inflammation.
- an antibody in particular an antibody or fragment according to the invention.
- a process for purifiying an antibody comprising the steps: performing anion exchange chromatography in non-binding mode such that the impurities are retained on the column and the antibody is maintained in the unbound fraction.
- the step may, for example be performed at a pH about 6-8.
- the process may further comprise an intial capture step employing cation exchange
- the process may further comprise of additional chromatography step(s) to ensure product and process related impurities are appropriately resolved from the product stream.
- the purification process may also comprise of one or more ultra-filtration steps, such as a concentration and diafiltration step.
- Purified form as used supra is intended to refer to at least 90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.
- Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU per mg antibody product or less such as 0.5 or 0.1 EU per mg product.
- Substantially free of host cell protein or DNA is generally intended to refer to host cell protein and/or DNA content 400 ⁇ g per mg of antibody product or less such as 100 ⁇ g per mg or less, in particular 20 ⁇ g per mg, as appropriate.
- the antibody molecule of the present invention may also be used in diagnosis, for example in the in vivo diagnosis and imaging of disease states involving OX40.
- Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.
- A26Fab fusion proteins for the expression of A26Fab-645dsscFv were constructed as follows.
- a single chain Fv (scFv) was constructed by linking the N-terminus of 645vL to the C-terminus of 645vH via the flexible linker
- A26Fab light chain-645dsvL, A26Fab heavy chain-645dsvH, A26Fab light chain, A26Fab heavy chain, 645dsvL free domain, 645dsvH free domain, A26Fab light chain- 645dsscFv and A26Fab heavy chain-645dsscFv were manufactured chemically and individually cloned into mammalian expression vectors under the control of the HCMV- ⁇ promoter and SV40E polyA sequence.
- HEK293 cells were transfected with the relevant plasmids using Invitrogen's 293fectin transfection reagent according to the manufacturer's instructions. Plasmids were mixed as follows to express the different constructs; for A26Fab-645dsFv, the plasmids were A26Fab Heavy-(S, 3xG4S/T)-645dsvH and A26Fab Light-(S, 3xG4S)-645dsvL; for Single linker A26Fab-645dsFv (LC-vL linked), the plasmids were A26Fab Heavy, 645dsvH and A26Fab Light-(S, 3xG4S)-645dsvL; for Single linker A26Fab-645dsFv (HC-vH linked), the plasmids were A26Fab Heavy-(S, 3xG4S/T)-645dsvH, A26Fab Light and 645dsvL; for A26Fab- 645dsscFv
- the ratio of the plasmids used for the transfections also varied, for the 2 plasmid combinations the ratio was 1 : 1, whereas for the 3 plasmid combinations several different ratios were tested.
- a total of 5C ⁇ g of plasmid DNA was incubated with 125 ⁇ 293fectin + 4.25ml Optimem media for 20mins at RT.
- the mixture was then added to 50ml of HEK293 cells in suspension at 1 x 10 6 cells/ml and incubated with shaking at 37°C. Supernatants were harvested on day 7 by centrifugation at 1500g to remove cells and the supernatant passed through a 0.22 ⁇ filter. Expression level was determined by Protein-G HPLC.
- the level of expression of all the constructs was comparable, see table 4, covering the range 3-15 ⁇ g/ml.
- There have been reports in the literature that the expression of Fv regions that lack either a linker between the vL and vH or a dimerisation motif to bring the vL and vH together have substantially lower expression levels than linked Fv's. This is not observed in this data where there is no significant difference observed between the best expression of each type of construct.
- Binding affinities and kinetic parameters for the interactions of Fab-dsFv, Fab-dsscFv and single linker Fab- dsFv constructs were determined by surface plasmon resonance (SPR) conducted on a BIAcore T100 using CM5 sensor chips and HBS-EP (lOmM HEPES (pH7.4), 150mM NaCl, 3mM EDTA, 0.05% v/v surfactant P20) running buffer.
- Single linker Fab-dsFv samples were captured to the sensor chip surface using either a human F(ab')2-specific goat Fab (Jackson ImmunoResearch, 109-006-097) or an in-house generated anti human CHI monoclonal antibody. Covalent immobilisation of the capture antibody was achieved by standard amine coupling chemistry.
- Each assay cycle consisted of firstly capturing the Fab-dsFv, Fab-dsscFv or single linker Fab-dsFv construct using a 1 min injection, before an association phase consisting of a 3 min injection of antigen, after which dissociation was monitored for 5 min. After each cycle, the capture surface was regenerated with 2 1 min injections of 40mM HC1 followed by 30s of 5mM NaOH. The flow rates used were 1 ⁇ /min for capture, 30 ⁇ 1/ ⁇ for association and dissociation phases, and ⁇ /min for regeneration.
- kinetic assays either a titration of human serum albumin 50-6.25nM, or a single concentration of OX40 of 25nM was performed. A blank flow-cell was used for reference subtraction and buffer-blank injections were included to subtract instrument noise and drift.
- the ⁇ 50ml HEK293 supematants were concentrated -25 fold to ⁇ 2ml using lOkDa molecular weight cut off centrifugation concentrators.
- the concentrated supematants were applied to a 1ml HiTrap Protein-G FF column (GE Healthcare) equilibrated in 20mM phosphate, 40mM NaCl pH7.4.
- the column was washed with 20mM phosphate, 40mM NaCl pH7.4 and the bound material eluted with 0.1M glycine/HCl pH2.7.
- the elution peak was collected and pH adjusted to ⁇ pH7 with 2M Tris/HCl pH8.5.
- the pH adjusted elutions were concentrated and buffer exchanged into PBS pH7.4 using lOkDa molecular weight cut off
- Samples were diluted with water where required and then to 26 ⁇ 1 was added 1 ⁇ 4x Bis-Tris LDS sample buffer and A ⁇ L of 1 OX reducing agent for reduced samples.
- the samples were vortex mixed, incubated at 100°C for 3 minutes, cooled and centrifuged at 12500 rpm for 30 seconds.
- the prepared samples were loaded on to a 4-20% acrylamine Tris/Glycine SDS gel and run for 110 minutes at 125V, constant voltage.
- the gels were stained with Coomassie Blue protein stain, see Figure 2.
- the reducing SDS-PAGE gel has banding patterns in terms of both migration position and staining intensity that is constant with all the constructs being expressed correctly.
- lane 2 there should be 2 bands at ⁇ 36 and ⁇ 37kDa with roughly equivalent staining.
- lane 3 there should be 2 bands at ⁇ 51 and ⁇ 23kDa with roughly twice the staining in the upper band.
- Fab-dsscFv HC- scFv
- lane 4 there should be 2 bands at ⁇ 50 and ⁇ 26kDa with roughly twice the staining in the upper band.
- the triple gene plasmids were constructed by first generating an intermediate double gene vector from the single gene components of the single linker Fab-dsFv formats as described in example 1/ Figure 1.
- the heavy chain is either a A26 Fab heavy chain or the A26 Fab heavy linked to a 645dsvH or 648dsvH via a linker (S, 3xG 4 S), and the light chain is either a A26 Fab light linked to a 645dsvL or 648dsvL via a linker (S, 3xG 4 S) or a A26 Fab light chain, respectively.
- the fragment encoding the expression of the free cognate v region (645dsvL, 648dsvL, 645dsvH or 648dsvH), was subsequently sub-cloned at a unique restriction site downstream of the heavy chain gene in the intermediate vector.
- a mammalian selection marker was finally sub-cloned into the expression plasmids.
- CHO cells were grown in CD CHO media supplemented with lx L-glutaMAX (Life Technologies) to exponential phase with > 99 % viability. The cells were prepared by washing in Earle's balanced salt solution (Life Technologies) and plasmid DNA was electrop orated into the CHO cells according to in-house recommendations. The transfected cells were transferred to CD CHO medium supplemented with 1 x L- glutaMAX and lx anti-mycotic solution (Life Technologies) and incubated in an orbital shaker for 24 h at 37°C, 8% C0 2 , and shaking at 140 rpm.
- the level of expression from triple gene plasmids was dependent on the presence of the mammalian selection marker, see Figure 5.
- Expression titres were higher amongst single linker A26 Fab-dsFv proteins expressed from plasmids without a mammalian selection marker whereas lower but comparable levels were obtained if expressed from plasmids with the mammalian selection marker, as would be expected due to the metabolic burden exacted by expression of an extra gene. Additionally, higher expression titres were observed for proteins that contained an A26 Fab light linked to a 645dsvL or 648dsvL chain.
- the 200 ml supernatants were concentrated by ⁇ 20-fold using a lOkDa molecular weight cut off centrifugation concentrators.
- the concentrated supernatants were applied to a 1ml HiTrap Protein-G FF column (GE Healthcare) equilibrated in 20mM phosphate, 40mM NaCl pH7.4.
- the column was washed with 20mM phosphate, 40mM NaCl pH7.4 and the bound material eluted with 0.1M glycine/HCl pH2.7.
- the elution peak was collected and the pH adjusted to ⁇ pH7 with Tris/HCl pH8.5.
- Protein samples were diluted in PBS where required. To 8.25 ⁇ of the subsequent sample, 3.75 ⁇ of 4x Bis- Tris LDS sample buffer (Life Technologies), 1.5 ⁇ of 100 niM N- ethylmaleimide and 1.5 ⁇ of 10X reducing agent were added for reduced samples. The samples were vortex mixed, incubated at 100°C for 3 minutes, cooled and centrifuged at 13200 rpm for 30 seconds. The prepared samples were loaded on to a 4- 20% acrylamide Tris/Glycine SDS gel (Life Technologies) and run in Tris-glycine buffer for -150 minutes at 125V, constant voltage. An SDS-PAGE protein standard, Seeblue2 (Life Technologies) was used as the standard marker. The gels were stained with InstantBlue Coomassie blue protein stain (Expedeon) and destained with distilled water, see Figure 6.
- the reducing SDS-PAGE gel has banding patterns in terms of both migration position and staining intensity that is constant with all the constructs being expressed correctly.
- banding patterns in terms of both migration position and staining intensity that is constant with all the constructs being expressed correctly.
- staining should be roughly in the ratio of 3 :2:1 upper to lower band.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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CN201380072293.8A CN105026431B (en) | 2012-12-21 | 2013-12-20 | Single linker FabFv antibodies and methods of making the same |
RU2015129762A RU2714159C2 (en) | 2012-12-21 | 2013-12-20 | SINGLE LINKER ANTIBODIES FabFv AND METHODS FOR PRODUCING THEREOF |
ES13815506.4T ES2689470T3 (en) | 2012-12-21 | 2013-12-20 | Single-connector Fab-Fv antibodies and methods to produce them |
EP13815506.4A EP2935336B1 (en) | 2012-12-21 | 2013-12-20 | Single linker fabfv antibodies and methods of producing same |
US14/654,240 US10457748B2 (en) | 2012-12-21 | 2013-12-20 | Single linker FabFv antibodies and methods of producing same |
CA2895608A CA2895608C (en) | 2012-12-21 | 2013-12-20 | Single linker fabfv antibodies and methods of producing same |
BR112015014872-7A BR112015014872B1 (en) | 2012-12-21 | 2013-12-20 | MULTI-SPECIFIC ANTIBODY MOLECULE, MICROORGANISM HOST CELL, PROCESS AND PHARMACEUTICAL COMPOSITION |
JP2015548647A JP6411362B2 (en) | 2012-12-21 | 2013-12-20 | Single linker FabFv antibody and method for producing the same |
US16/573,893 US11401349B2 (en) | 2012-12-21 | 2019-09-17 | Single linker FabFv antibodies and methods of producing same |
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US16/573,893 Continuation US11401349B2 (en) | 2012-12-21 | 2019-09-17 | Single linker FabFv antibodies and methods of producing same |
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EP (1) | EP2935336B1 (en) |
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BR (1) | BR112015014872B1 (en) |
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ES (1) | ES2689470T3 (en) |
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Cited By (23)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2016169992A1 (en) * | 2015-04-22 | 2016-10-27 | Ucb Biopharma Sprl | Method for protein purification |
WO2017009473A1 (en) | 2015-07-16 | 2017-01-19 | Ucb Biopharma Sprl | Antibody molecules which bind cd45 |
US9969813B2 (en) | 2012-05-10 | 2018-05-15 | Bioatla, Llc | Multi-specific monoclonal antibodies |
JP2018515453A (en) * | 2015-04-22 | 2018-06-14 | ユーシービー バイオファルマ エスピーアールエル | Method for increasing the percentage of monomeric antibody Fab-dsFv multimeric species |
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US10457748B2 (en) | 2019-10-29 |
EP2935336B1 (en) | 2018-07-04 |
GB201223276D0 (en) | 2013-02-06 |
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US20160017057A1 (en) | 2016-01-21 |
RU2015129762A (en) | 2017-01-27 |
CN105026431B (en) | 2021-06-25 |
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US11401349B2 (en) | 2022-08-02 |
BR112015014872A2 (en) | 2017-08-15 |
JP2016501906A (en) | 2016-01-21 |
CA2895608C (en) | 2021-05-25 |
CN105026431A (en) | 2015-11-04 |
US20200055959A1 (en) | 2020-02-20 |
EP2935336A1 (en) | 2015-10-28 |
ES2689470T3 (en) | 2018-11-14 |
CA2895608A1 (en) | 2014-06-26 |
RU2714159C2 (en) | 2020-02-12 |
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