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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
  • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
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  • C07K2317/565—Complementarity determining region [CDR]
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• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present disclosure relates to a method modulating the half-life of a binding domain specific to a serum carrier protein by mutating the sequence and a modulated binding domain specific to a serum carrier protein.
• HSA human serum albumin
• IgG has a similarly long half-life (21 days).
• the long serum half- lives of HSA and IgG are primarily due to protection from intracellular lysosomal degradation by the neonatal Fc receptor (FcRn).
• FcRn neonatal Fc receptor
• 4 ' 5 FcRn recycles HSA and IgG back to the cell surface following non-specific pinocytosis of plasma into vesicles by endothelial cells and hematopoietic cells lining the vascular space.
• the pinocytotic vesicles acidify by fusion with the early endosome enabling HSA and IgG to bind to FcRn in a pH dependent manner.
• Vesicles bearing membrane receptors including FcRn, and bound HSA and IgG, are recycled back to the cell surface whilst the remaining unbound material is channeled to the lysosome for degradation.
• HSA and IgG bind weakly to FcRn at neutral pH and so are released back into the circulation when the recycled vesicles are exposed to the neutral pH of the blood. 2
• HSA can be exploited in one of two ways.
• One approach is to directly couple the therapeutic protein to HSA, either genetically or chemically. 6 ' 7
• a second approach is to use an albumin binding domain.
• binding domains used to date include fatty acids (myristic acid), 8 organic molecules (Albutag), 9 synthetic peptides, 10 ' 11 bacterial albumin binding domains (Albumod Tm ), 12 ' 13 single domain antibodies (Nanobody Tm , AlbudAb Tm ) 14"17 and a Fab. 18
• Nguyen et al 2006 investigated the half- life of Fab fragments linked to a C-terminal albumin binding peptide. Nguyen concluded that reduced affinity for albumin correlated with a reduced half-life and higher rates of clearance. Figure 3 therein suggests the relationship is almost linear. This paper also went on to say that a very small difference in the fraction of the antibody that is unbound in vivo will have a profound effect on the rate of clearance.
• the present inventors have investigated the correlation between the affinity of binding domains comprising a VH and VL specific to a serum protein carrier and the in vivo half-life of the same. They have established that the duration of the half-life for binding domains is more complicated than the response for albumin binding peptides, in that large reductions in the observed affinity often translate to a moderate reduction in half-life and in some instances reduced affinity can lead to increases in half-life, which is counter-intuitive.
• a binding domain comprising a VH and VL specific to a serum carrier protein wherein the domain is mutated by a modification in the light chain variable domain (VL), in the heavy chain variable domain (VH) and a combination thereof, and the mutated binding domain has a half-life which is higher or lower than the half-life for the unmutated binding domain, for example with the proviso that the mutation is other than a mutation consisting of I50A, T56A, T95A, V96A, P97A, G98A, Y99A, S100A, TlOOAa, YlOOCa, I50A and T95A, I50A and G98A, I50A and Y99A, T56A and T95A, T56A and G98A, and T56A and Y99A of SEQ ID NO: 1.
• a binding domain comprising a VH and VL specific to a serum carrier protein wherein the domain is mutated by a modification selected from one or two amino acids substitutions in the light chain variable domain (VL), one or two mutations in the heavy chain variable domain (VH) and a combination thereof, and the mutated binding domain has a half-life which is higher or lower than the half-life for the unmutated binding domain, for example with the proviso that the mutation is other than a mutation consisting of 15 OA, T56A, T95A, V96A, P97A, G98A, Y99A, S100A, TlOOAa, YlOOCa, I50A and T95A, I50A and G98A, I50A and Y99A, T56A and T95A, T56A and G98A, and T56A and Y99A of SEQ ID NO: 1.
• the serum carrier protein is selected from, for exampe thyroxine -binding protein, transthyretin, a 1 -acid glycoprotein, transferrin, fibrinogen and albumin, or a fragment of any thereof, such as albumin, in particular human serum albumin.
• binding domain is specific to domain II of albumin.
• the mutation is a modification in the VL, for example wherein the mutation is substitution of one or two amino acids in the VL, such as a
• the mutated amino acid(s) in CDR LI is/are independently selected from a position 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35, such as position 30.
• amino acid(s) at the relevant position(s) in VL is/are replaced by a hydrophobic residue, for example selected from alanine, isoleucine, phenylalanine, valine, proline, and glycine, such as alanine.
• a hydrophobic residue for example selected from alanine, isoleucine, phenylalanine, valine, proline, and glycine, such as alanine.
• the mutations consist of modifications to the VL.
• modifications in the VL can be made which increase the Kd of the binding domain and reduces the affinity of the binding domain but increases the half-life of the molecule.
• the mutation(s) is/are in the VH domain, for example the mutation is substitution of one or two amino acids in the VH, such as mutation in a CDR selected from HI , H2, H3 and combinations thereof, in particular wherein the CDR is H2 and/or H3, more specifically wherein the CDR is H2.
• the mutated amino acid(s) in CDR H2 is/are independently selected from a position 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65 and combinations thereof, which for example is/are replaced by a hydrophobic residue, in particular
• alanine independently selected from alanine, isoleucine, phenylalanine, valine, proline, and glycine, such as alanine.
• the mutated amino acid(s) is/are in CDR is H3, and in particular the mutated amino acid(s) is/are independently selected from a position 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106 or 107, more specifically the residue 101 is mutated.
• amino acid at the relevant position(s) in CDR H3 is replaced by a hydrophobic residue, for example independently selected from alanine, isoleucine, phenylalanine, valine, proline, and glycine, such as alanine.
• the one or more amino acid substitution(s) is/are a non- conservative amino acid substitution, for example wherein the non-conserved amino acid is selection from the natural amino acids alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, threonine, asparagine, glutamine, glycine, proline, arginine, lysine, aspartic acid and glutamic acid.
• the non-conserved amino acid is selection from the natural amino acids alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, threonine, asparagine, glutamine, glycine, proline, arginine, lysine, aspartic acid and glutamic acid.
• the binding domain comprises a CDR grafted variable domain.
• the binding domain is humanised, for example the binding domain comprises a human framework in the VH and/or VL.
• the VH framework is human (for example VH3, such as VH3 1 -3 3-23) and comprises 1 , 2, 3, 4, 5 or 6 amino acid substitutions, such as amino acids which are donor residues.
• the VH comprises a sequence selected from SEQ ID NO: 2, 3, 4 and 5 or a variant of any one of the same with at least 95, 96, 97, 98 or 99% similarity or identity, such as a sequence shown in SEQ ID NO : 2, 3, 4, 5 or 6 (particularly 5 or 6).
• the VL framework is human (for example VKI, such as 2- 1 - (1) L5), for example comprising 1 , 2 or 3 amino acid substitutions, such as amino acids which are donor residues.
• the VL domain comprises a sequence selected from SEQ ID NO: 6, 7, 8 and 9 or a variant of any one of the same with at least 95, 96, 97, 98 or 99% similarity or identity.
• the VH and VL sequences are selected from the combinations SEQ ID NO: 2 & 6, 2 & 7, 2 & 8, 2 & 9, 3 & 6, 3 & 7, 3 & 8, 3 & 9, 4 & 6, 4 & 7, 4 & 8, 4 & 9, 5 & 6, 5 & 7, 5 & 8 and 5 & 9 or a variant or variants of any of the same with at least 95, 96, 97, 98 or 99% similarity or identity, in particular the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 3, respectively, or the VL and VH sequences are SEQ ID NO: 8 and SEQ ID NO: 4 respectively, or the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 5, respectively, or the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 4.
• binding domain is human.
• the affinity of the binding partners is high, 5nM or stronger, such as 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or ⁇ or stronger.
• an antibody molecule comprising a binding domain according to the present disclosure, in particular a multispecific antibody molecule, such as a bispecific.
• composition comprising a binding domain according to the present disclosure or an antibody molecule described herein.
• a method of treating a patient comprising administering a therapeutically effect amount of a binding domain according to the present disclosure, an antibody molecule described herein, or a pharmaceutical composition comprising any one of the same.
• binding domain according to the present disclosure, an antibody molecule described herein, or a pharmaceutical composition comprising any one of the same, for use in treatment.
• an antibody molecule described herein or a pharmaceutical composition comprising any one of the same for use in treatment, in particular the treatment of 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 Guill
• nephropathy idiopathic thrombocytopenic purpura
• Meniere's disease pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and cancer, including breast cancer, lung cancer, gastric cancer, ovarian cancer, hepatocellular cancer, colon cancer, pancreatic cancer, esophageal cancer, head & neck cancer, kidney, and cancer, in particular renal cell carcinoma, prostate cancer, liver cancer, melanoma, sarcoma, myeloma, neuroblastoma, placental chorio
• a binding domain for example for the treatment of 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 Guilla
• a method of selecting a serum protein carrier binding domain to provide a bespoke half-life comprising the steps of: providing a panel of VH/VL pairs specific to said serum protein carrier, and
• the serum carrier protein is selected from thyroxine-binding protein, transthyretin, a 1 -acid glycoprotein, transferrin, fibrinogen and albumin, or a fragment of any thereof, for example albumin, such as human serum albumin.
• the panel of VH/VL pairs is prepared by mutating a variable domain in a parent antibody.
• the mutation is at least one modification to the VL, for example wherein the VL modification is selected from one or two amino acids substitutions in the light chain variable domain (VL) and the mutated binding domain has a half-life which higher or lower than the half-life for the unmutated binding domain.
• VL light chain variable domain
• the mutation in VL is a mutations in CDR LI, CDRL2, and/or CDRL3, in particular CDR L2 or CDR L3.
• the mutation(s) in the binding domain consists of a modification or modifications to the VL domain.
• the mutation is at least one modification to the VH.
• the mutation is one or two mutations in the heavy chain variable domain (VH), wherein the mutated binding domain has a half-life which higher or lower than the half-life for the unmutated binding domain (also referred to herein as parent antibody).
• VH heavy chain variable domain
• a mutation(s) is in CDR HI, CDRH2, and/or CDRH3, in particular CDR H2 or CDR H3.
• the method according to the disclosure further comprises the step of replacing histidine residues in a VH and/or VL (from the panel or for the panel) with an alternative amino acid residue.
• the method further comprises the step of assessing the properties of one or more binding domains, at two or more biologically relevant pH's, such as about pH 5 and pH7.
• the mutations increase the numerical value of the Kd.
• a crystal structure of the serum carrier protein with an antibody such as the parent antibody is employed in deciding which residues to modify/mutate.
• a method of selecting a serum protein carrier binding domain to provide a bespoke half-life comprising the steps of: providing a panel of VH/VL pairs specific to said serum protein carrier, and
• the serum carrier protein is selected from thyroxine-binding protein, transthyretin, a 1 -acid glycoprotein, transferrin, fibrinogen and albumin, or a fragment of any thereof.
• VL modification is selected from one or two amino acids substitutions in the light chain variable domain (VL) and the mutated binding domain has a half-life which higher or lower than the half-life for the unmutated binding domain.
• VH heavy chain variable domain
• a method according to any one of paragraphs 1 to 12 which further comprises the step of replace histidine residues in a VH and/or VL employed in the panel with an alternative amino acid residue.
• a method according to any one of paragraphs 1 to 13 which further comprises the step of assessing the properties of one or more binding domains, at two or more biologically relevant pH's, such as about pH 5 and pH7.
• the present inventors have found that the sequences of the VH and VL can be mutated and the corresponding half-life does not necessarily correspond to the affinity of the binding domain. In particular modifications can be made to the VL where the affinity is maintained or decreased and the half-life is increased.
• half-life allows the half-life to be designed and controlled for molecule to provide a half-life which is relevant to the therapeutic indication.
• long-half may be desirable.
• a moderate/medium half-life may be appropriate.
• a relatively short half-life may be appropriate.
• a bespoke half-life as employed herein is a half-life which has been specifically designed for the binding domain by modifying the same.
• binding domain or site is the part of the antibody that contacts the antigen.
• the binding domain contains at least one variable domain or a derivative thereof, for example a pair of variable domains or derivatives thereof, such as a cognate pair of variable domains or a derivative thereof.
• the binding domain comprises 6 CDRs and a framework and together these elements contribute to the specificity of the binding interaction of the antibody or binding fragment.
• Variable regions (also referred to herein as variable domains) generally comprise 3 CDRs and a suitable framework.
• antibody refers to an immunoglobulin molecule capable of specific binding to a target antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, peptide etc., via at least one antigen recognition site (also referred to as a binding site herein), located in the variable region of the immunoglobulin molecule.
• a target antigen such as a carbohydrate, polynucleotide, lipid, polypeptide, peptide etc.
• antigen recognition site also referred to as a binding site herein
• antibody molecule includes antibodies and binding fragments thereof. The term also extends to an antibody format comprising any one of the same.
• Parent antibody as employed herein refers to the starting antibody before the mutations to change the half-life a made.
• the parent antibody may be humanised (which may include incorporate back-mutations containing so-called donor residues) or mutated, for example to remove lysine residues from a CDR or similar. However, modifications present in the parent antibody will not be for the purpose of changing/modifying the half-life.
• Parent antibody as employed herein includes antibody binding fragments.
• Antibody fragments refer to antibody binding fragments including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies, scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1 126-1 136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217).
• Multi-valent antibodies may comprise multiple specificities e.g. bispecific or may be monospecific (see for example W092/22853, WO05/1 13605, WO2009/040562 , WO2010/035012, WO2015/197772).
• binding fragment refers to a fragment capable of binding a target peptide or antigen with sufficient affinity to characterise the fragment as specific for the peptide or antigen.
• Specificity refers where the partners in the interaction only recognise each other or have significantly higher affinity for each other in comparison to non- partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher affinity, than for example a background level of binding.
• Partners as employed herein refer to antigen and antibody binding or ligand and receptor type binding relationships.
• At least one modification as employed herein refers to substitution, addition or deletion of an amino acid, for example to change the properties of the sequence, for example the change hydrophobicity or similar.
• the residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., 1987. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues.
• the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.
• the correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence.
• the CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system.
• CDR-H1 residues 31-35
• CDR-H2 residues 50-65
• CDR-H3 residues 95-102
• the loop equivalent to CDR-H1 extends from residue 26 to residue 32.
• the loop equivalent to CDR-H1 extends from residue 26 to residue 32.
• 'CDR- ⁇ as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia's topological loop definition.
• Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL (variable light) domain and a constant domain of a light chain (CL), and a VH (variable heavy) domain and a first constant domain (CHI) of a heavy chain.
• a Fab' fragment as employed herein refers to a Fab fragment further comprising a hinge region.
• single-chain Fv refers to an antibody fragment that comprises the VH and VL antibody domains linked (for example by a peptide linker) to form a single polypeptide chain.
• the constant regions of the heavy and light chain are omitted in this format.
• Single-chain Fv as employed herein includes disulfide stabilised versions thereof wherein in addition to the peptide linker a disulfide bond is present between the variable regions.
• Disulfide stabilised scFv may eliminate the propensity of some variable region to dynamically breath, which relates to variable regions separating and coming together again.
• single domain antibody refers to an antibody fragment consisting of a single monomeric variable antibody domain.
• single domain antibodies include VH or VL or VHH.
• the constant region domains may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required.
• the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains.
• human IgG constant region domains may be used, especially of the IgGl and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required.
• IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required. It will be appreciated that sequence variants of these constant region domains may also be used.
• IgG4 molecules in which the serine at position 241 has been changed to proline as described in Angal et al., 1993, Molecular Immunology, 1993, 30: 105-108 may be used.
• the antibody in the embodiment where the antibody is an IgG4 antibody, the antibody may include the mutation S241P.
• the antibody binding fragment does not comprise an Fc region.
• Does not comprise an Fc region refers to the lower constant domains, such as CH2, CH3 and CH4 are absent. However, constants domains such as CHI, CKappa/Clambda may be present.
• antibodies may undergo a variety of posttranslational modifications.
• the type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions.
• modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation.
• a frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995). Accordingly, the C-terminal lysine of the antibody heavy chain may be absent.
• the antibody heavy chain comprises a CHI domain and the antibody light chain comprises a CL domain, either kappa or lambda.
• the antibody heavy chain comprises a CHI domain, a CH2 domain and a CH3 domain and the antibody light chain comprises a CL domain, either kappa or lambda.
• Multispecific molecule refers to a molecule with the ability to specifically bind at least two distinct antigens, for example different antigens.
• the multispecific molecule is a bispecific, trispecific or tetraspecific molecule, in particular a bispecific molecule.
• multispecific formats include those known in the art and those described herein, such as wherein the molecule format is selected from the group comprising or consisting of: diabody, scdiabody, triabody, tribody, tetrabodies, tandem scFv, FabFv, Fab'Fv, FabdsFv, Fab-scFv, diFab, diFab', tandem scFv-Fc, scFv-Fc-scFv, scdiabody-Fc, scdiabody-CH3, Ig-scFv, scFv-Ig, V-Ig, Ig-V, Duobody and DVDIg, which are discussed in more detail below.
• Molecule as employed herein is used in the biochemistry sense to refer to a group of atoms that form an organic, in particular proteinaceous mass, which includes a complex suitable for handling as a single entity under appropriate conditions once the complex has been formed.
• Molecule and construct are used interchangeably herein, unless the context indicates otherwise.
• construct may be employed more often to refer to a polynucleotide molecule and molecule may be employed more often to refer an entity primarily comprising an amino acid sequence.
• Antigens of interest which may be targeted by an binding domain in the antibody molecule of the present disclosure, may also be any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands.
• cell surface proteins include: adhesion molecules, integrins such as ⁇ integrins (e.g. VLA-4), E-selectin, P selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CDl la, CDl lb, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CDW52, CD69, CD 134 (OX40), ICOS, BCMP7, CD 137, CD27L, CDCP1, DPCR1, DPCR1, dudulin2, FLJ20584, FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2, nectin-like2, NKCC1, PTK7, RAIG1, TCAM1 , SC6, BCMPlOl, BCMP84, BCMPl l, DTD, carcinoembryonic antigen (CEA), human milk fat globulf
• Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16 or IL-17, IL-21, 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-a, 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
• FIG. 1 Humanization and affinity reduction of antibody CA645.
• the heavy and light chain sequences of antibody CA645 are aligned with human germline acceptor framework sequences VH3 1-3 3-23/JH4 and VKI 2-l-(l) L5/JK4. Rabbit residues are in red, human residues are in black and CDRs are in blue (J- region CDR residues are shown but acceptor V-region CDRs are not).
• the grafted VH (gH) and VL (gL) sequences are shown below their corresponding human acceptor germline frameworks. Framework sequence differences between the rabbit and human framework sequences are shown with asterisks. Rabbit framework residues retained in the humanized grafts are highlighted in bold.
• gL4gH5 Fab Binding to HSA in absence of Fab (red circle), binding to HSA in presence of Fab (red triangle), binding to MSA in absence of Fab (blue square), binding to MSA in presence of Fab (blue triangle).
• HSA anti-human serum albumin
• Table 3 X-ray data collection and refinement statistics. Values in parentheses are for highest-resolution shell.
• mice (Ml-3)/group were dosed intravenously at 10 mg/kg with each CA645 graft. Mean and standard deviation (SD) of each group is shown, ⁇ measured by steady state.
• Table 5 Shows affinity for various grafts.
• Chrompure Human Albumin was pre-incubated for 1 hour with a 25 ⁇ solution of albumin binders; warfarin, ibuprofen, myristic acid, and copper chloride (all individually sourced from Sigma Aldrich).
• HSA was replaced with rat serum albumin (Sigma Aldrich) that had been labelled using Alexa Fluor 647 ® monoclonal antibody labelling kit (Molecular Probes).
• V region genes of single cells were amplified by RT-PCR and cloned into UCB mammalian expression vectors containing rabbit heavy CHI and rabbit light CK regions, respectively. Following transient expression in HEK293 cells, anti-HSA recombinant Fabs were further screened in SPR binding assays against HSA and MSA.
• Albumin specific antibodies were humanized in silico by grafting the CDRs from antibody V-regions onto the VKI and VH3 human germline antibody V-region frameworks.
• the CDR's grafted from the donor to the acceptor sequence were as defined by Kabat et al., 32 with the exception of CDR-H1 (residues 26-35) where the combined definitions of Kabat et al, and loop structure was used. 23 Where a framework residue differed between the donor rabbit sequence and the acceptor human sequence in a position that was considered to be important for retention of antigen binding, then the donor residue was included in the initial conservative graft. 21
• the conservative graft genes were chemically synthesized by
• Heavy chain graft genes (gHl) were cloned into two UCB expression vectors, one containing human ⁇ CHI domain and another containing the full human ⁇ constant region.
• Light chain graft genes (gLl) were cloned into a UCB expression containing human kappa constant region (Km3 allotype). These constructs were subsequently modified by oligonucleotide-directed mutagenesis to create a number of different variants of both the heavy and light chain grafts.
• Heavy and light chain vectors were co-transfected into HEK293 cells and the recombinant Fab or IgG molecules screened using a SPR binding assay to measure affinity for HSA, MSA, RSA, CSA, RbSA and BSA.
• Antibodies were transiently expressed in either HEK-293 cells using 293Fectin lipid transfection (Life Technologies, catalog #12347-019, according to the manufacturer's instructions) or CHO-S XE cells, a CHO-K1 derived cell line, 33 using electroporation.
• HEK- 293 cells were used for small scale expression ( ⁇ 100 ml) to prepare antibodies for SPR analysis.
• CHO-S XE cells were used for large scale expression (1 litre) to prepare antibodies for crystallography and in vivo pharmacokinetic studies.
• Affinity chromatography was used to purify Fab protein from culture supernatants.
• the binding affinities and kinetic parameters for the interactions of antibodies were determined by surface plasmon resonance (SPR) conducted on either a Biacore T200 or Biacore 3000 using CM5 sensor chips (GE Healthcare Bio-Sciences AB) and HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v P20, pH7.4) running buffer.
• SPR surface plasmon resonance
• the capture surface was regenerated with two 1 min injections of 40 mM HC1 followed by 30 s of 5 mM NaOH.
• the flow rates used were 10 ⁇ /min for capture, 30 ⁇ /min for both the association and dissociation phases, and 10 ⁇ /min for regeneration.
• a blank flow-cell was used for reference subtraction and buffer-blank injections were included to subtract instrument noise and drift.
• Kinetic parameters were determined by simultaneous global-fitting of the resulting sensorgrams to a standard 1 : 1 binding model using Biacore T200 Evaluation software v2.0.1 and BIAE valuation software v4.1.1 , with the exception of CA645 gL5gH47 which was fitted in prism using steady state affinity model.
• a Biacore3000 instrument was used with a CM5 chip prepared by immobilisation of HSA and MSA on separate flow cells to levels of 270 RU and 247 RU respectively.
• FcRn samples were prepared over the range 50 nM to 50 ⁇ in running buffer, (100 mM MES, 150 mM NaCl, 0.05% v/v P20, pH 5.5) and they also contained either zero or ⁇ CA645 Fab.
• Each assay cycle was run at a flow rate of 10 ⁇ / min and consisted of either a 5 min injection of 100 nM CA645 Fab to pre-saturate immobilised albumin, followed by a 5 min injection of one of the above FcRn solutions prepared in the presence of CA645 Fab, or a 5 min injection of running buffer followed by a 5 min injection of one of the above FcRn solutions in the absence of CA645 Fab. In either case a third 5 min injection followed immediately at the end of the second injection, using the 'coinject' mode, comprising respectively, buffer or 100 nM CA645 Fab.
• a blank flow-cell was used for reference subtraction and blank cycles, where FcRn was replaced with buffer, were included to subtract drift and noise. Cycle regeneration was as above. Blank corrected plateau binding levels of FcRn were plotted in Prism and fitted to a steady state model.
• Binding kinetics of wild type and mutant CA645 Fabs at pH 5.5 were also investigated in reverse format on the Biacore3000 using the immobilised albumin chip. In this case cycles were run where Fab solutions over the range 5 to 5000 nM were injected with 5 min association and dissociation phases. Buffer blank cycles were also included to correct for drift. Crystallography
• CA645 Fab and fatty acid-free HSA were mixed in a molar ratio of 1 : 1 and incubated overnight at 4 °C.
• Both CA645 Fab and the complex were purified by size exclusion chromatography over a HiLoad 16/60, Superdex 200 column (GE Healthcare) equilibrated with 50 mM NaCl, 25 mM Tris, 5% (v/v) glycerol.
• Fractions containing either CA645 Fab or the complex were pooled and concentrated to 10 mg/ml and 70 mg/ml, respectively. Conditions suitable for crystal growth were identified by the sitting drop vapour diffusion method using commercially available crystallization screens (Qiagen).
• CA645 Fab To generate diffraction quality crystals, hanging drop vapour diffusion method was used where ⁇ of protein solution was mixed with ⁇ of reservoir solution.
• the reservoir contained 500 ⁇ 2 M DL-Malic acid. Crystals were harvested and flash frozen in liquid nitrogen without additional cryoprotectant. Diffraction data to 2.68 A was collected from a single crystal on the 104 beamline at Diamond Light Source, Oxford, UK and processed using MOSFLM and SCALA. 34"36
• the structure of CA645 Fab was solved by molecular replacement with Phaser, 37 using coordinates of an in-house Fab structure as a search model.
• the reservoir contained 500 ⁇ 0.1 M Citric acid pH 4.4, 0.1 M di-Sodium hydrogen phosphate, 38% v/v Ethanol and 5% v/v Polyethylene glycol 1000 (PEG 1000).
• the crystals were cryoprotected by multiple additions to the drop of 1 ⁇ reservoir buffer containing 25% (v/v) PEG1000, until the concentration of PEG1000 in the drop reached 20%>. To minimise crystal stress, each addition was spaced at least 1 hour apart. Crystals were harvested and flash frozen in liquid nitrogen. Diffraction data to 3.58 A was collected from a single crystal on the 102 beamline at Diamond Light Source, Oxford, UK and processed using XDS.
• [Tag] concentration of albumin (600 ⁇ )
• variable regions were cloned into expression vectors containing rabbit heavy CHI and light chain constant regions and then the DNA sequenced. This revealed that the antibody sequences were unique with the exception of CA645 and CA646 which had identical heavy chain sequences. Given CA645 and CA646 must bind to the same epitope through the heavy chain it is unclear why CA646 binding was more affected by the presence of ligands. Also determined from the sequencing was that the complementarity determining regions (CDRs) of all of the antibodies lacked histidine residues.
• CDRs complementarity determining regions
• the conservative grafts were expressed as human IgGl antibodies and analysed by SPR for binding to HSA, MSA and RSA.
• the humanized IgGs displayed the same trend in binding to HSA and MSA as observed with the recombinant parental rabbit Fabs (Table 1).
• the affinities for HSA of CA647, CA648 and CA649 were similar to those of CA645 and CA646 but they showed a 6- to 10-fold reduction in affinity for MSA by comparison.
• the affinities for RSA of CA648 and CA649 also showed a 5- to 10-fold reduction in comparison with CA645 and CA646.
• CA646 exhibited marginally stronger affinities for HSA, MSA and RSA than CA645 but the transient expression yields were 4-fold lower at 35 mg/ml compared with 161 mg/ml (Table 1). Based on the near maximal retention of binding to HSA in the presence of known albumin binders, the consistent binding activity for albumin across multiple species and good yields in transient expression, CA645 was selected as our lead candidate for further progression. Further graft variants of CA645 gLlgHl were generated by replacing rabbit donor residues with human acceptor residues and filling the gap in framework three of the heavy chain with the equivalent human residues. The graft variants were assessed on affinity for HSA and transient yield expression (data not shown). The final graft pairing selected was gL4 and gH5 (Fig.l).
• CA645 gL4gH5 Fab The affinities of CA645 gL4gH5 Fab for HSA, MSA, RSA and rabbit serum albumin (RbSA) were shown to be 4.6, 7.1, 54 and 162 nM, respectively (Table 2). Significantly for utility of CA645 gL4gH5 Fab in cynomolgus monkey toxicology studies and disease models, the affinity for cynomolgus serum albumin (CSA) was very similar to that of HSA at 3.3 nM. In addition, CA645 Fab failed to bind to bovine serum albumin.
• CSA cynomolgus serum albumin
• CA645 gL4gH5 Fab was likely to remain bound to albumin in the acidic environment of the early endosome and be recycled to the cell surface.
• the affinities of CA645 gL4gH5 Fab for HSA at pH 5.0, pH 5.5, pH 6.0, and pH 7.0 were 7.1, 10.7, 12.5 and 13.3 nM indicating that binding is largely unaffected within this physiologically relevant pH range.
• CA645 Fab-HSA complex To identify where CA645 binds to HSA, the crystal structure of CA645 Fab-HSA complex was determined.
• the CA645 Fab-HSA complex protein preparation was concentrated to 70mg/ml, 24 and crystallized using ethanol and PEG 1000 as precipitants.
• To aid solving the structure of the complex with molecular replacement we also determined the structure of unbound CA645 Fab.
• the structure of free Fab was refined to 2.68 A with a final Rwork value of 21.14% and Rfree value of 25.13%.
• the structure of the complex was refined to 3.6 A with a final R wor k value of 21.38%) and Rfree value of 25.23%.
• the crystal structure of the CA645 Fab-HSA complex showed that CA645 binds to domain II of HSA (Fig.3A).
• Superimposition of the crystal structure of FcRn in complex with HSA (PDB code 4N0F), 25 showed that CA645 does not block binding of HSA to FcRn (Fig.3B).
• HSA contains seven fatty acid (FA) binding sites. Sites FA7 and FA3/FA4 are the two main drug binding sites. 26 Drugs also bind at sites FA1, FA5 and FA6 but with weaker affinity.
• Metal ion binding sites are located between domains I and II and at a site at the N-terminus.
• 27 Superimposition of the complex with the crystal structures of HSA in complex with warfarin (PDB code 2BXD), 28 ibuprofen (PDB code 2BXG) 28 and myristic acid (PDB code 1BJ5) 29 showed that CA645 binds close to site FA6 and does not occlude the main drug (FA7 and FA3/FA4), fatty acid or metal ion binding sites (Fig.3C).
• the binding kinetics of CA645 gL4gH5 Fab to HSA in comparison with those for MSA, CSA, RSA and RbSA may be explained by close visual inspection of the crystal structure.
• the epitope on HSA is formed by residues F206, G207, R209, C316, K317, AEAKD 320-324, K351, E354, E358, K359, C361, A362 and A364.
• the affinities of CA645 for CSA (3.3 nM) and MSA (7.1 nM) are very similar to the affinity for HSA (4.6 nM). This is likely due to the presence in CSA and MSA of the same residues that form the epitope in HSA.
• Position 364 is located at the tip of a short loop (positions 362-365) that links two a-helices (positions 366- 398 and 342-361) together (Fig.4A). This short loop is bound by CDR's 1 and 2 of the
• CA645 heavy chain The affinity of CA645 for RSA is approximately 10-fold lower than for HSA. It is possible that the absence of the alanine side chain increases the flexibility of the loop, compared with that of HSA, and alters the binding kinetics. RbSA shares all of the HSA epitope residues except positions 320, 358 and 364.
• Position 358 in RbSA is lysine and it clashes with Y99 of CDRH3.
• the weaker affinity of CA645 for RbSA compared with HSA is entirely due to an 18-fold reduction in the association rate (Table.2). This is likely to be caused by the presence in RbSA of the larger side chains at positions 320 and 358, and possibly 364.
• Oligonucleotide-directed mutagenesis was used to generate twenty variants across six residue positions of the heavy chain and twenty seven variants across six residue positions of the light chain (Tables 4, S2A, S2B and S2C).
• BALB/c mice were dosed by a single intravenous injection at 10 mg/kg with CA645 gL4gH5 Fab and a subset of four of the Fab variants, gL5gH5, gL4gH37, gL5gH37 and gL5gH47.
• Blood sera were sampled over 103 hours and the level of Fab quantified by ELISA.
• CA645 gL5gH37 showed no detectable binding to HSA by SPR and was cleared rapidly with a serum half-life of only 0.48 ⁇ 0.06 h (Table 4). This is in line with the short half-life (0.7 h) of an anti-TNF Fab observed in rats. 19 In contrast gL4gH5 exhibited a significantly extended half-life of 84 ⁇ 4.6 h.
• the variant with the weakest affinity for which there was no difference in pharmacokinetic profile from gL4gH5 was gL5gH5 (Fig. 5).
• gL5gH5 contained a single mutation in the light chain, W30A, and its affinity was 453 nM.
• the mutants were designed and selected on the basis of affinity for HSA but the
• the affinity for MSA of gL5gH5 Fab is 316 nM. It has a pharmacokinetic profile and half-life that matches that of gL4gH5, and is calculated to have a similarly low level of free Fab at 0.05 %. We were unable to measure the affinities of gL4gH37 and gL5gH47 Fabs for MSA. However, as the affinities of gL4gH5 and gL5gH5 were both 1.2-fold weaker for MSA than for HSA (Table 5), it is reasonable to predict that the affinities of gL4gH37 and gL5gH47 will be proportionately 1.2-fold weaker. Therefore, with predicted affinities for MSA of 1146 nM and 62.4 ⁇ , it is calculated that 0.17 % of gL4gH37 and 8.57 % of gL5gH47, respectively, are potentially free in blood.
• Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor. Diabetes Obes Metab 2010; 12: 655-673.
• Flanagan RJ Jones AL. Fab antibody fragments. Drug Safety 2004; 27: 1115-1133.
• Smith BJ Popplewell A, Athwal, D, Chapman AP, Heywood S, West SM, Carrington B, Nesbitt A, Lawson AD, Antoniw P, Eddelston A, Suitters A. Prolonged in vivo residence times of antibody fragments associated with albumin. Bioconjug Chem 2001; 12: 750- 756.

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